Revolutionizing HCV Therapy: microRNA Approaches in New Era of Treatment

Abstract

Since the development and evolution of COVID-19 immunization, the use of mRNA-based technologies has led to revolutionary changes due to the potential of RNA-based therapies, which are believed to be useful in treating many infectious diseases. Information on the treatment of hepatitis C virus (HCV) following this rule highlights the potential therapeutic use of microRNAs (miRNAs). The advent of direct-acting antivirals (DAAs) has changed the paradigm of HCV treatment. However, challenges remain, particularly in the areas of viral resistance, genetic diversity, and chronic diseases. Among these, miRNAs are a sensible approach to complementing and improving existing models. The implementation of new non-coding RNAs should be investigated. This chapter discusses the potential and public awareness of non-coding RNA (ncRNA) strategies against HCV. From the modification of miRNAs to the discovery of non-coding RNA pathways and focusing on their applications, efficacy, and therapeutic potential in HCV. As the scientific community looks toward the development of antiviral drugs, this chapter demonstrates that the introduction of non-coding RNA drugs into existing health systems holds promise for addressing and providing solutions to challenges such as drug resistance, viral persistence, and more. New non-coding RNAs in HCV therapy not only expand the scope of treatment but also define the therapeutic landscape and increase flexibility and adaptability in the face of HCV challenges.

1. Introduction

1.1 The current state of HCV therapy

Hepatitis C virus (HCV) is a liver-affecting viral infection that is highly prevalent and has a significant impact on people’s health worldwide. As such, it is considered a major global health concern [1]. In 1989, American researchers made a breakthrough discovery. They were able to identify a new virus. This virus was isolated from the serum of patients with non-A and non-B hepatitis and was officially named hepatitis C or HCV [2]. Hepatitis C virus is a small, enveloped, positive-sense, single-stranded RNA virus that belongs to the Flaviviridae family and genus Hepacivirus. The untranslated regions, 3′-UTR and 5′-UTR, found at both ends of the HCV genome are crucial to both replications of the virus. The internal ribosome entry site (IRES), which is a part of the 5′-UTR, is the internal ribosome binding site that creates a polyprotein that has about 3010 amino acids (9.6 kb). This polyprotein is cleaved by cellular and viral proteases into three structural regions (Core, E1, and E2) and seven viral nonstructural proteins, including P7 and NS2, NS3, NS4 A/B, and NS5 A/B [3].

This life-threatening virus can cause a type of acute (short-term) or chronic (long-term) viral hepatitis that can be associated with liver fibrosis and failure and even some cancers such as hepatocellular carcinoma (HCC) and lymphoma [4]. Acute HCV infections are usually asymptomatic and do not cause fatalities. Within 6 months, 15–20% of infected individuals can recover without treatment. The remaining 70% of those affected (55–85%) will become chronically infected with HCV. Within 20 years, there is a 15–30% risk of cirrhosis among people with a persistent HCV infection. Around 58 million people worldwide are infected with the chronic hepatitis C virus, with an annual total of about 1.5 million new infections. Patients with chronic hepatitis C (CHC) are at a high risk of developing liver cirrhosis, progressive fibrosis, and even HCC if they are not treated [5]. Prevention of HCV infection involves reducing bloodstream transmission risk and public health measures to prevent spread in high-risk communities, including injection drug users. The most commonly reported strategy for contracting HCV infections is intravenous drug abuse [6]. Choo and colleagues sequenced HCV’s genome, proposing a comprehensive nomenclature system for genotypes and subtypes based on similarity to various genetic groups, known as genotypes (with 60% nucleotide similarity) and their subtypes (76–80% nucleotide similarity) [7]. A set of genetic variants that differ in each patient but have 90–99% nucleotide similarity is referred to as a quasi-species. The genetic composition of quasi-species results from mutations that occur during viral replication. The elimination of one quasi-species leads to the formation of another because the HCV virus might include incorrect or imperfect copies of its sequence during replication and could evade the immune system’s response through continuous mutation. For many people, this is one of the reasons for chronic hepatitis C. Quasi-species have a significant impact on the progression of the disease and how it responds to treatment [8].

There are eight primary genotypes and 86 subtypes of HCV. The distributions of HCV genotypes and subtypes differ throughout regions worldwide. Genotypes 1, 2, and 3 are widely distributed throughout the world. Genotypes 4 and 5 are found in East Africa, South Africa, and Arab countries; genotypes 6 through 8 are found in Southeast Asia, the Democratic Republic of the Congo, and Punjab (India). HCV genotypes significantly influence treatment response rates, aiding in drug selection and duration [9].

Before the discovery of HCV, scientists emphasized the effect of IFN-alpha on the treatment of chronic viral liver diseases. Immune cells naturally produce IFN-alpha in response to viral infections or other stressors. This interferon can create an “interference” against the viral replication and finally protects the host cells from spreading the infection [10]. Since 1984, an experimental study of IFN has been started on the treatment of non-A and non-B hepatitis viral hepatitis. Scientists administered daily doses of IFN-alpha to patients for 16 weeks and measured viral status and liver parameters through blood tests. This experiment brought immediate and impressive results. While some patients showed a minimal response to IFN-alpha treatment and others experienced relapse, half of the participants showed a complete response to the trial [11]. The introduction of HCV was accompanied by extensive research focused on determining the molecular structure of the virus, these studies were able to take a fundamental step in designing drugs that can target specific components of the virus and prevent its replication [12]. In additional studies, it was seen that combination therapy containing IFN-alpha and other antiviral drugs such as ribavirin has more promising results [13, 14]. By altering the nucleoside supply (by decreasing NS5B polymerase activity), ribavirin, a guanosine analog and potent inhibitor of cellular inosine dehydrogenase monophosphate, inhibits the viral replication and subsequently maintains the balance between Th1 and Th2 [15]. Another significant advance in the treatment of HCV came when scientists chemically modified IFN-alpha to increase its duration in the body, called “pegylated” IFN. Finally, the combination of “pegylated” IFN with ribavirin became the standard of care for patients with hepatitis C [16]. The combination of PEG-IFN allows the drug to create a suitable therapeutic interval between interferon injection and facilitating treatment. Combining PEG-INF and ribavirin resulted in sustained viral response levels or SVR (SVR means 100-fold reduction of the viral replication or permanent elimination of the virus) of approximately 42–46% of patients with genotypes 1 and 4 of the virus, and 76–82% for those with genotypes 2 and 3 [17]. Antiviral treatment for HCV genotypes lasts 24 to 48 weeks. If PCR-HCV becomes negative within 24–48 weeks, the patient is considered a responder; otherwise, drug resistance occurs [18].

As of right now, there is no effective vaccination to prevent all HCV genotypes due to the high antigenic variation and heterogeneity of the HCV genome; however, anti-HCV therapy is still developing. Additional virological and biological studies provided valuable insights into how the virus interacts with the immune system, and ultimately, this information served as a basis for designing more effective agents and led to the introduction of the development of multiple direct-acting antivirals (DAAs) [19]. By targeting the nonstructural proteins of HCV, DAAs inhibit the virus from replicating. Some types of DAAs are NS3/NS4A protease inhibitors, NS5A inhibitors, NS5B polymerase inhibitors, Cyp A inhibitors (Cyclophilin A), and Scavenger Receptor BI, such as ITX 5061. More than 95% of hepatitis C patients can be cured with DAAs; however, testing and therapy are not widely available [18].

The antiviral agents specifically target the HCV protease, which is a key player in viral replication [20]. When the protease inhibitors were combined with pegylated IFN and ribavirin, significant treatment response rates of up to 75% were reported, although this triple therapy also produced some side effects [12]. Then, several new anti-HCV drugs including ombitasvir, ledipasvir, daclatasvir, elbasvir, and velpatasvir were introduced during the following years, which target and block the HCV NS5A protein, while sofosbuvir and dasabuvir were two promising agents from the same family that they inhibited HCV NS5B protein. Initially, the combination of these drugs with pegylated IFN and ribavirin resulted in a higher SVR rate [21]. The most notable hope came when the results of a clinical trial showed that the combination of sofosbuvir and velpatasvir could produce an impressive SVR of 99% for all HCV genotypes [22]. New drug regimens were introduced as oral formulations and eliminated the need for injections. Finally, the combination of sofosbuvir/volpatasvir significantly reduced the duration of treatment to 12 weeks [23]. Another highly successful combination regimen consisting of sofosbuvir and ledipasavir resulted in SVR rates of 95–99% and reduced treatment duration to 8–12 weeks [24]. Although newer DAAs make it possible to treat HCV infections in many cases, there are still serious doubts about the 2030 goal of the World Health Organization (WHO) to eradicate HCV [6]. One of the major problems is the diversity of the virus’s outer proteins and the ability of the HCV’s genomics to mutate repeatedly, making it almost impossible to develop any immunity in the host against subsequent HCV infections [25]. One of the other problems is the cost of producing and distributing anti-viral drugs, which is not possible in many countries [26]. Patients in high-risk groups face the possibility of re-infection with HCV and the need for further DAA treatment courses. Although international guidelines in HCV management do not support viral resistance tests before prescribing DAAs, the risk of developing resistant viruses in individuals and society is still serious [27]. Another point is that the development of the HCV vaccine has faced significant challenges, and it seems impossible, at least in the short term [28]. Effective DAA treatment for chronic hepatitis C patients prevents re-infection with HCV; however, high mutation rates can result in resistance to the DAAs, making an effective vaccine necessary for eradication. Also, the successful treatment does not ensure any further HCV re-infection [29].

The usage of DAA-based therapy is restricted in underdeveloped nations where treatment is most in need due to its expensive cost, serious side effects, and rapid emergence of resistance mutations. Although DAA-based therapy greatly increases SVR, finding novel therapeutic approaches that are effective for all HCV genotypes and are both clinically and financially feasible is essential. One of the novel antiviral therapies is microRNAs (miRNAs), which regulate gene expression post-transcriptionally. Because miRNAs regulate the immune system, they can either promote or inhibit the propagation of infectious diseases and are effective in diagnosis, treatment, and prognosis. miRNAs play a crucial role in cellular fate by regulating development, maturation, differentiation, apoptosis, cell signaling, interactions, and homeostasis. When HCV infection occurs, dysregulated miRNAs may either directly or indirectly affect HCV replication and/or cause liver disorders [30, 31].

1.2 The role of microRNAs in HCV co-infection

Only 2% of transcripts of the human genome are translated into proteins. Of these, over 90% are non-coding RNAs (ncRNAs), which are divided into two categories based on size: small (s)ncRNAs (less than 200 nucleotides; nt) and long (l)ncRNAs (more than 200 nt). miRNAs belong to the class of small non-coding RNAs that engage in the regulation of eukaryotic posttranscriptional genes. Mature miRNAs need to undergo many stages of processing, including: 1. The initial transcripts of the miRNA genes that RNA polymerase II controls are known as pri-miRNAs, which have a 5′ cap and 3′ poly-A tail (hairpin structure). 2. Drosha (an RNaseIII-like enzyme) and DiGeorge syndrome critical region 8 (DGCR8) cleave pri-miRNAs in the nucleus to produce the precursor miRNAs, also known as pre-miRNAs, which are 70–100 bp long. 3. Pre-miRNAs are transported to the cytoplasm by Exportin 5. They are then cleaved by Dicer to produce mature miRNA duplexes with about 22 bp of 3′-overhangs. and 4. Strand separation has been done on the only mature miRNA that can be found by its target mRNAs and enter the RNA-induced silencing complex (RISC) that contains the argonaut protein (Ago2). This string serves as a functional guide strand. The other strand is destroyed and nonfunctional [32]. The coding or 3′UTR region of the target mRNAs and the 5′ ends of the miRNA, often referred to as the “seed” region, usually interact specifically through base-paring to regulate the expression of eukaryotic genes at the post-transcriptional stage. When the target mRNA and miRNA have perfect base matching, miRNA degrades the target mRNA; if there is only partial pairing, miRNA suppresses mRNA translation (Figure 1) [29, 33].

Figure 1.

The miRNA biogenesis. To sum up, a drosha-DGCR8 microprocessor processes primary-miRNA (pri-miRNA), which leads to the formation of a 70–100 nucleotide hairpin structure (precursor-miRNA, pre-miRNA). Following being transported into the cytoplasm, it matures into miRNA and is incorporated into an argonaut protein. BioRender is used to draw the figure.

It expresses over 2600 miRNAs [source: human genome database, accessed May 24, 2022; www.mirbase.org]. It is likely that multiple distinct miRNAs regulate each mRNA. MiRNAs thus appear to have a role in almost every biological function, including apoptosis, immunological response, aging, and cell division. Additionally, cellular miRNAs may influence virus replication through various interactions with viruses [34].

HCV can alter host cell functions to promote its replication, but it still needs host cells to replicate. It has been revealed that several biological factors, including miRNAs, are dysregulated during HCV infection. HCV infection modulates the expression of the cellular miRNA profile, which either directly or indirectly regulates HCV replication. Hepatocytes can alter the expression of several miRNAs to protect themselves from infection. The roles of miRNAs in HCV infection are varied. While some cellular miRNAs upregulated HCV-RNA replication (stimulation effect), others down-regulated it (inhibition effect) (Table 1) [36, 37].

	Functions	Mechanism	References
Down-regulated microRNAs
miR-196, miR-296, miR-351, miR-431, miR-448, miR-199a, let-7b, miR-181c	Suppressing on HCV replication	Directly interacts with the HCV genome	[29, 35]
miR-99a and miR-27a	Suppressing on HCV replication and packaging	Reducing on lipid metabolism
miR-548 m and miR-194	Suppressing on HCV infectivity	Targeting and suppressing CD81 receptor expression
miR-182	Suppressing on HCV infectivity	Inhibit CLDN1 expression
miR-501-3p and miR-619-3p	Suppressing on HCV infectivity	Downregulating on OGT protein expression
miR-29	Suppressing on HCV replication	—
miR-185-5p	Suppressing on HCV replication	Targeting on GALNT8
Upregulated microRNAs
miR-122	Facilitating on HCV replication	Directly interacts with the HCV genome
miR-141	Facilitating on HCV replication	Suppression of DLC-1 protein
miR-134, miR-320c, miR-483-5p	Facilitating on HCV replication	Increasing of immune evasion and cell survival
miR-491	Facilitating on HCV replication	inducing on PI3K/Akt pathway
miR-130a, miR-373	Facilitating on HCV replication	Inhibiting on IFN pathway

Table 1.

Some significant microRNAs were classified as either up- or down-regulated and implicated in many aspects of HCV replication at a glance.

1.2.1 Inhibition effect

Type I IFNs (IFN-α and IFN-β) intervention can quickly alter the expression of a lot of cellular miRNAs in HCV therapy. Using the analysis of cellular miRNA expression in cells stimulated with IFN β-induced miRNAs, about 30 miRNAs (miR-1, miR-30, miR-128, miR-196, miR-296, miR-351, miR-431, and miR-448) were discovered to have altered levels of expression [38]. Some studies showed that blocking these miRNAs with anti-miRNAs stopped IFN-β from killing HCV, but increasing their levels by transfecting miRNA mimics made the antiviral effect of IFN-β happen again in Huh7 cells. Apart from miRNAs that induce IFN-β synthesis, other additional miRNAs can reduce HCV replication (e.g., miR-181c, miR-199a or the Let family, like let-7b). For instance, overexpression of miR-199a inhibits HCV-RNA replication [39].

Certain miRNAs not only directly prevent HCV-RNA replication, but they also perform so by triggering the IFN pathway. For instance, miR-130a. There have been conflicting findings from earlier research on this microRNA. It has been demonstrated that, in cells lacking TLR3 and RIG-I, such as Huh7.5.1 cells, miR-130a may indirectly suppress HCV multiplication by reestablishing the host’s innate immune response. For type I IFN induction, TLR3, and RIG-I are two crucial mediators of HCC. By downregulating the expression of antiviral interferon-induced transmembrane protein 1 (IFITM1), a target protein for it, through miR-130a, HCV, on the other hand, evades the innate immune response. Since this miRNA interacts intricately with the host’s innate immune system, it serves several purposes and requires further study [40].

1.2.2 Stimulation effect

Around 70% of the total miRNA present in mammalian liver tissue is liver-specific miRNA, of which miR-122 is the most prevalent liver-specific miRNA. Due to its distinct function in stimulating HCV replication rather than inhibiting it, miR-122 is an attractive target for antiviral therapies. Jopling et al. were the first to report miR-122’s involvement in HCV replication. Two target sites in the 5′UTR of HCV are present in this miR-122 and are essential for HCV replication. The promotion of HCV replication essentially depends on the interaction between miR-122 and the viral 5′UTR [41, 42].

MiR-141, which HCV infection induces, may also be necessary for a robust HCV replication process. Further study is needed as the results suggest the existence of a novel mechanism of HCV infection-associated miRNA-mediated regulation of a tumor suppressor protein. Specifically, miR-141 is expressed by HCV-infected cells, and its overexpression significantly suppresses DLC-1 expression (a Rho GTPase-activating protein), mediated tumorigenic transformation [29].

1.3 The relationship between HCV and hepatocellular carcinoma

Several stages of the HCV-induced progression of HCC are associated with the viral genotype and length of the disease. Since HCV is not incorporated into the host’s DNA, it is not carcinogenic. Several studies have indicated that HCV chronic infection has a significant role in the formation of HCC [43]; nevertheless, the progression of the disease and the host’s immune responses are caused by HCC. Studies indicate that viral proteins are responsible for that. For example, core proteins may affect lipogenesis and oxidative stress. By altering cell signaling and cell cycling pathways and downregulating some tumor suppressor genes, such as p53 and retinoblastoma, other proteins can cause HCC [44].

Furthermore, HCV nonstructural proteins facilitate the activation of hepatic stellate cells (HSCs) and the promotion of transforming growth factor beta (TGF-β), both of which lead to liver fibrosis. Recently, studies have shown how crucial inflammation is to the growth of HCC. Some important components of hepatocarcinogenesis, such as transforming growth factors α and β, are upregulated in chronic inflammation. TGF-α upregulation causes hepatocyte dysplasia, cell regeneration, proliferation, and HCC. TGF-β is essential for inflammation in its later phases. Proliferation and differentiation processes are halted by inhibiting leukocyte activation [45, 46].

TGF-α is a strong proinflammatory cytokine that can accelerate the development of hepatocellular carcinoma and cause liver damage and cirrhosis. There is a correlation between elevated proinflammatory cytokine release and elevated TGF-α levels. As a result, several genes linked to cancer cell division, invasion, and metastasis are induced, and the proto-oncogene is activated [47].

In certain cases, untreated chronic hepatitis C (CHC) and, consequently, chronic inflammation can cause fibrosis, which can eventually progress to cirrhosis. Hepatic stellate cells and fibroblast activation happen because cirrhosis is the prelude to the majority of HCC-related HCV cases. Collagen is produced as a result. Complex structures are formed in the liver tissue as a result of an overabundance of extracellular matrix (ECM) proteins, such as collagen [48]. HSCs can synthesize collagen types IV and VI in liver injury situations. Reactive oxygen species (ROS) are produced by immune cells such as neutrophils and macrophages in response to HCV, according to certain studies. Excessive production of ROS and the inability of the body to detoxify them result in oxidative stress. It is demonstrated that HCV core protein may affect oxidative stress metabolism. Oxidative stress, which results from a disruption, is what causes liver fibrosis in the cellular redox balance. ROS activation stimulates hepatic stellate cells, turning them into myofibroblasts. These myofibroblasts produce collagen and play crucial roles in many different processes, such as inflammation, angiogenesis, regeneration, and tumorigenesis. In addition, HCV causes the dysfunction of mitochondria in hepatic cells, which is followed by the production of excessive ROS. Excessive exposure to ROS may result in abnormal signaling that promotes cell growth and survival and may play a role in the development of cancer [48, 49].

2. microRNAs: a new strategy in HCV therapy

2.1 Understanding the mechanism of microRNA in HCV

Alterations in host miRNA levels have been linked with viral infections and can influence viral infection both directly and indirectly. The genome of the flaviviruses includes one 3′ non-coding region (3′UTR), one 5′ non-coding region (5′UTR), and a single open reading frame (ORF). In addition to being the carrier of genetic information and having the capability of protein coding, RNAs have another important role in the regulation of the viral life cycle and its relationship with its host and vector [50]. As said before, sncRNAs are divided into small interfering RNAs (siRNAs), transfer RNA-derived small RNAs (tsRNAs), small nuclear RNAs (snRNAs), PIWI-interacting RNAs (piRNAs), vault RNAs (vtRNAs), and microRNAs (miRNAs) [51, 52]. Among them, miRNAs have been proven to affect thousands of genes. They regulate the translation of more than 60% of protein-coding genes [53] in the stable parts or tumor-associated regions of the genome [54, 55]. Compared to normal cells, miRNA expression differs in different diseases, like in hepatoma cells infected with HCV and HCC patients [56, 57, 58]. Hepatic fibrosis is followed by inflammation, which occurs as a result of the excessive accumulation of lipids in the liver [59]. During inflammation, inflammatory mediators such as TNF-α, IL-1, and IL-6 are produced by the macrophages and endothelial cells. These mediators are important for the regulation of inflammation [60, 61]. Here, we reviewed some of the miRNAs that are responsible for HCV pathogenesis (Table 2).

miRNA	Mechanism	References
miR-548 m	Suppression the expression of CD81	[62]
miR-99a	Decreases the intracellular lipid accumulation	[63]
miR-194	Suppression the expression of CD81	[64]
miR-501-3p and miR-619-3p	Suppresses the expression of OGT	[65]
miR-182	Suppresses the expression of CLDN1	[66]
miR-122	5′UTR of HCV interaction, Suppression the expression of OCLN	[67, 68]
miR-200c	Suppression the expression of OCLN	[69]
miR-181c	E1 and NS5A coding regions of HCV interaction	[70]
miR-196, miR-296, miR-351, miR-431 and miR-448	HCV genome interaction	[38]
let-7b	NS5B coding region and 5′-UTR of HCV interaction	[71]
miR-199a	5′UTR of HCV genome interaction	[72]
miR-21	TGF-β-mediated fibrosis enhancing	[73]
miR-141	Promote the replication of HCV	[74, 75]

Table 2.

miRNAs responsible for the HCV pathogenesis.

2.1.1 Lipid metabolism

According to the liver’s function in maintaining lipid homeostasis and controlling the metabolism of triglycerides and fatty acids, infections with HCV can lead to the accumulation of fatty acids and triglycerides in the hepatic cells [76]. Several miRNAs have been reported to be responsible for the metabolism of cholesterol and fatty acids. In this era, some evidence reveals the role of miR-34a and miR-122 in sustaining the liver’s metabolic homeostasis [76]. Some others that modulate the levels of cholesterol and lipids are miR-103, miR-307, miR-33, and miR-104 [77]. miR-122 account for 70% of the miRNA content of the liver, approximately is equal to more than 66,000 copies in each liver cells [59]. It is well-known for being a regulator of replication, translation, and a stabilizer of HCV through binding to the two highly conserved binding sites located in the 5′ UTR of the RNA of this virus [78, 79]. To some extent, the stimulation of translation by miR-122 occurs via the internal ribosome entry site (IRES) of HCV [80]. This miRNA stimulates lipid synthesis and storage by upregulating fatty acid and cholesterol biosynthesis enzymes, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and fatty acid synthase (FASN) [81, 82]. In addition, miR-122 promotes lipid accumulation in HCV-infected hepatocytes. This is done by promoting the formation of lipid droplets, which leads to a suitable environment for viral replication [83]. For instance, in healthy mice, the expression of two genes encoding key enzymes related to lipogenesis has been observed to decrease following the inhibition of miR-122 [59]. On top of that, we know that HNF-4α controls different pathways in the liver that are involved in disease, such as lipid metabolism, through its effect on miR-122. In addition, it is important to know that HNF-4α controls these pathways by affecting miR-122, which is involved in a number of liver pathobiology pathways, such as lipid metabolism. Studies have demonstrated that HCV regulates these pathways through its impact on HNF-4α and subsequent modulation of miR-122 [84]. HCV uses its proteins, nonstructural proteins (NS5A) and core proteins, to downregulate the expression of HNF4α [85].

miR-34a is another miRNA that plays a crucial role in the hepatic inflammation in HCV-infected patients. This miRNA, regulates the metabolism by affecting hepatic sirtuin 1. Hepatic sirtuin 1 changes the way energy is used by turning on two proteins, the liver X receptor (LXR) and the peroxisome proliferator-activated receptor (PPAR) [86, 87]. In fact, miR-34a increases the proliferation of hepatic cells by targeting the RXRα, ACSL1, CASP2, and SIRT1 genes [88, 89]. Another study reported the regulation of caspase-2 levels followed by miR-34a induction, which leads to liver steatosis in mice and humans [90]. Other miRNAs, such as miR-307, miR-33, miR-104, and miR-103, are reported to modulate cholesterol and lipid regulatory genes [77]. Some miRNAs, including miR-33a and miR-33b, are crucial to the regulatory lipid metabolism of the liver. This regulation occurs via sterol regulatory element-binding protein (SREBP) genes [91]. It has been reported that in nonalcoholic fatty-liver disease (NAFLD) patients, the inhibition of miR-34a contributes to increasing the levels of SIRT1, which results in the activation of AMP-activated protein kinase (AMPK) and PPARα and thus hepatic steatosis improvement [92].

2.1.2 Inflammation of hepatocytes

miR-155, miR-132, and miR-122 are associated with liver inflammation regarding their important roles in innate and adaptive immunity [76]. In addition to its important role in the lipid metabolism of the liver, MiR-122 has a crucial function in the inflammation of the liver. In fact, as shown by using miR-122-deficient mice, the function of this miRNA seems to be anti-inflammatory. This process is linked to the activation of the cancer-causing pathway and involves the infiltration of inflammatory cells into the liver, releasing pro-tumorigenic mediators like IL-6 and TNF. Moreover, miR-122 strongly hinders the formation of tumors [60, 61]. miR-132, which is reported to mediate inflammation and chronic liver diseases. This process has been done during malnutrition and via the enhancement of the interaction between inflammatory cells and adipocytes. miR-132 induces the progression of this interaction through the deacetylation of p65 and the inhibition of SIRT1. It is revealed that overexpression of miR-132 stimulates the production of IL-8 and MCP-1, the transfer of nuclear factor-κB (NF-κB) into the nucleus, and the acetylation of p65 [93]. Conversely, a partial decrease in the levels of IL-8 and MCP-1 and a reduction in the acetylation level of p65 are the results of the loss of miR-132. So, miR-132 inhibition may have anti-inflammatory effects in the liver [94]. It is now proven that miR-155 in viral infection regulates the innate immunity [95]. This miRNA serves as a positive regulator of inflammation. In individuals infected with HCV, miR-155 is upregulated in both the serum and monocytes [96]. Studies also suggested that some of the HCV proteins, including NS5, NS3, and the HCV core protein, have the potential to increase the production of miR-155 and TNF-α in patients with chronic HCV infection. In addition, miR-155 regulates adaptive immune responses by affecting the function and differentiation of T cells, which have a crucial role in antiviral activity. Thus, it seems that miR-155 mediates anti-inflammatory and proinflammatory signals. According to recent studies, HCV upregulates the expression of miR-155, which itself induces the secretion of TNF-α and activation of the JNK signaling pathway [97]. This phenomenon occurs through the interaction of TNF-α with TNF-R1 and recruits TRAF2, TRADD, and RIP (a serine/threonine kinase) to form a complex I, which leads to the activation of the JNK/MAPK signaling pathway [98].

2.1.3 Liver fibrosis

Liver fibrosis develops when hepatocytes are severely damaged, causing an inflammatory cytokine storm and extracellular matrix exposition. It can be caused by a number of factors, including hepatitis virus infections [99]. Some miRNAs regulate the signaling pathways, thus enabling hepatic fibrosis progression and activating hepatic stellate cells (HSCs). Activation of HSCs through multiple signaling pathways leads to severe inflammation, and it is proven that miRNAs are modulators of various growth factor receptor signals [100]. miR-29 family of miRNAs use the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway to induce apoptosis [101, 102, 103] and thus suppress hepatic fibrosis, either by influencing collagen production and accumulation of the extracellular matrix (ECM) or by inhibiting the TGF pathway [104]. The study by Huang et al. looked at how miR-29a affected the signaling pathways of toll-like receptor 2 (TLR2) and TLR4 in cholestatic mice, which are two important factors in liver fibrosis. They reported that the overexpression of miR-29a in these mice resulted in the obstruction of the signaling of these mediators in Kupffer cells and HSCs in liver tissue [105].

miR-21, which is known as oncogenic miRNA, activates HSCs via the PTEN/AKT pathway. This pathway regulates another pathway called PI3K/AKT negatively. The modulation inhibits the activation of AKT via the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). AKT is located downstream of PI3K and promotes the growth, survival, and proliferation of cells when it is activated [106]. The result of one study revealed that increased miR-21 expression stimulates Krüppel-like factor 5 (KLF5) in vitro, which in turn promotes cancer cell migration and invasion [107]. Moreover, another study by Zhou and colleagues demonstrated that the activation of the pyruvate dehydrogenase kinase 1 (PDK1)/AKT pathway in hepatic stellate cells (HSCs) that are located near hepatocellular carcinoma (HCC), specifically cancer-associated fibroblasts, occurs by secreted exosomal miR-21 [108]. This activation occurs by targeting PTEN directly and encouraging the growth of cancer by causing angiogenic molecules to be secreted by HCC cells, including VEGF, bFGF, MMP2, MMP9, and TGF [94].

miR-221 is another miRNA that plays an important role in different cellular processes, including cell proliferation, apoptosis, and differentiation. miR-221, along with miR-222, are the most dysregulated miRNAs in the HCC case. According to studies, miR-221 is dysregulated in HCV-infected cells and upregulated in HCV-related HCC cases [109]. This happens via an NF-kB-dependent mechanism [110]. Like miR-122 and miR-222, miR-199a-5p and miR-199a-3p upregulate the liver fibrosis of HCV-infected individuals. In mouse primary stellate cells, miR-221/222 levels increased in LX-2 cells through a mechanism similar to the expression of α-smooth muscle actin mRNAs and α1(I) collagen [111].

Some of the other miRNAs related to the fibrosis of hepatic cells include miR-181b, which acts positively in the fibrosis progression through the TGF-β or NF-kB pathways. By contrast, miR-214-3p, miR-29b, and miR-101, through inhibition of the TGF pathway or suppression of collagen production in the extracellular matrix, inhibit hepatic fibrosis [104].

2.2 The potential of microRNA as a biomarker (diagnosis/prognosis) target for HCV

Since 90% of cases of liver cancer are hepatocellular carcinoma (HCC), which also happens to be the primary cause of cancer-related deaths globally, early diagnosis is crucial to improving operative treatment and patient survival [112, 113]. Given that miRNAs play a big part in the start and progression of hepatitis and hepatoma cancer and the fact that they can move from the liver to the serum through exosomes, viral particles, apoptosis, and necrosis, they have been looked at as useful and accurate biomarkers [114, 115]. One of these miRNAs that gets out of whack in cancer is miR-221. It has been studied a lot as a good biomarker for HCV diagnosis [115]. Because it plays a part in inflammation, chronic liver injury, and tumor growth, it is involved in growth, tumorigenesis, and apoptosis suppression by inhibiting the expression of SOCS3 [116], DKN1C/p57, and CDKN1B/p27 [117], as well as the activation of NF-kβ [118]. It is reported that in HCC patients, the levels of miR-221 overexpression were related to the shorter time to progression of the disease. According to the studies, the upregulation of miR-221 in the serum of people with HCC can be a non-invasive biomarker for early diagnosis. In addition, this miRNA has been demonstrated to be an independent potential predictor of tumor recurrence after resection [119]. One of the oncogenic miRNAs is miR-21, which can be used as a diagnostic biomarker regarding its important role in hepatocellular carcinoma. In a meta-analysis, the sensitivity of diagnosis using this miRNA was reported to be 85.2% (73.3–88.4%). The overall usefulness of circulating miR-21 was moderate, which indicates the possibility of using it as a biomarker for the early stages of HCC [114]. According to the role of miR-494-3p in cell proliferation, migration, and invasion, the use of this miRNA as a biomarker has been identified in some studies. In one study, the levels of miR-494-3p were reported to be significantly upregulated in HCC patients compared to other groups. These studies confirmed the potential use of this miRNA as a biomarker for HCV and HCC detection [120, 121].

Another miRNA with the role of regulating gene expression is miR-199a. An investigation related to the effect of this miRNA on HCV disease suggested the possibility of using that as a diagnostic biomarker. The reduction in the levels of miR-199a was reported repeatedly in the HCC patients [122, 123]. In addition, it is suggested that the levels of miR-199a in the serum of HCC patients may have the potential to predict the number and size of tumors in these patients [124].

Like miR-199a, miR-16 is responsible for regulating gene expression. Dysregulation of this miRNA may affect the expression of genes related to HCV infection pathways, immune response, cell cycle, and apoptosis [125]. El-Abd et al. reported that the levels of miR-16 decreased significantly in HCC patients compared to HCV patients. This reduction was more significant in patients with multiple tumors than in individuals with a single tumor [124]. miR-296, which is located in chromosome 20q, plays a crucial role in biological processes. miR296-3p and miR-296-5p are the names of mature miR-296. They drive from the 3′ arm and 5′ arm of precursor miR-296, respectively. Thus, these two miRNAs are two partners involved in tumor progression and tumorigenicity [126, 127]. The results show that high levels of miR-296-5p in the blood are linked to angiogenesis, a process that is essential for the growth of neoplasia [128]. Several studies have shown the upregulation of miR-296 both in the serum and tissue of HCC patients [129]. miRNA 486-5p is another biomarker of HCV-related HCC that is upregulated in these patients. Although the accurate mechanism has not been identified yet, it has been demonstrated that it is a highly sensitive biomarker for the diagnosis of the disease in HCV-related HCC patients. It is also reported that the levels of this miRNA are strongly correlated with the size of the tumor and predict the invasion of the portal vein [130]. Table 3 demonstrates other miRNAs used as biomarkers to diagnose HCV infection and related diseases.

miRNA	Expression/regulation	Etiology	Reference
miR-222	Up	HCV (diagnosis)	[131, 132]
miR-21	Up	HCV (diagnosis)	[133]
miR-99a	Down	HCV (diagnosis)	[134]
miR-122	Down	HCV (diagnosis)	[135]
miR-124	Down	HCV (diagnosis)	[136]
miR-125a-5p	Up	HCV (diagnosis)	[137]
miR-135a	Up	HCV (diagnosis)	[138]
miR-199b	Down	HCV (diagnosis)	[139]
miR-221	Up	HCV (diagnosis)	[131]
miR-122	Up	HCV (poor prognosis)	[140]
miR-148a	Up	HCV (poor prognosis)	[140]
miR-638	Down	HCV (poor prognosis)	[141]
miR-1246	Up	HCV (poor prognosis)	[140]
let-7a	Down	HCC1	[142, 143]
let-7b	Down	HCC	[144]
let-7c	Down	HCC	[145, 146, 147]
let-7d	Down	HCC	[142]
let-7f-1	Down	HCC	[142]
let-7 g	Down	HCC	[148, 149, 150]
miR-1	Down	HCC	[151]
miR-7	Down	HCC	[152]
miR-10a	Down	HCC	[153]
miR-10b	Down	HCC	[154]
miR-15a/16	Down	HCC	[155]
miR-15b	Down	HCC	[156]
miR-21	Down	HCC	[157]
miR-26a	Down	HCC	[158, 159]
miR-29a	Down	HCC	[160]
miR-29b	Down	HCC	[161]
miR-29c	Down	HCC	[162]
miR-31-5p	Down	HCC	[163]
miR-33b	Down	HCC	[164]
miR-34a	Down	HCC	[165, 166]
miR-98	Down	HCC	[167]
miR-99a	Down	HCC	[168]
miR-100	Down	HCC	[168]
miR-101	Down	HCC	[169, 170]
miR-122	Down	HCC	[171]
miR-124	Down	HCC	[172]
miR-125a	Down	HCC	[173, 174]
miR-125b	Down	HCC	[173, 175]
miR-126	Down	HCC	[176]
miR-137	Down	HCC	[177]
miR-139	Down	HCC	[132, 178]
miR-139-5p	Down	HCC	[145]
miR-140-5p	Down	HCC	[179, 180]
miR-141	Down	HCC	[75]
miR-142	Down	HCC	[181, 182]
miR-142-3p	Down	HCC	[183]
miR-144	Down	HCC	[184]
miR-145	Down	HCC	[185, 186]
miR-148a	Down	HCC	[187, 188, 189]
miR-152	Down	HCC	[190]
miR-187-3p	Down	HCC	[191]
miR-195	Down	HCC	[192, 193]
miR-194	Down	HCC	[194]
miR-199a-5p	Down	HCC	[195]
miR-200a	Down	HCC	[196]
miR-200b	Down	HCC	[145]
miR-200c	Down	HCC	[157]
miR-212	Down	HCC	[197]
miR-203	Down	HCC	[198]
miR-206	Down	HCC	[199]
miR-214	Down	HCC	[200, 201]
miR-219-5p	Down	HCC	[202]
miR-222	Down	HCC	[157]
miR-223	Down	HCC	[203]
miR-224	Down	HCC	[204]
miR-296	Down	HCC	[205]
miR-302b	Down	HCC	[206, 207]
miR-337	Down	HCC	[208]
miR-338-3p	Down	HCC	[209]
miR-340	Down	HCC	[210]
miR-345	Down	HCC	[211]
miR-363-3p	Down	HCC	[212]
miR-370	Down	HCC	[213]
miR-375	Down	HCC	[214, 215]
miR-376a	Down	HCC	[216]
miR-429	Down	HCC	[217]
miR-449	Down	HCC	[218]
miR-450a	Down	HCC	[219]
miR-451	Down	HCC	[220]
miR-495	Down	HCC	[221]
miR-497	Down	HCC	[222]
miR-520b/e	Down	HCC	[223, 224]
miR-539	Down	HCC	[225]
miR-612	Down	HCC	[226]
miR-637	Down	HCC	[227]
miR-10a	Up	HCC	[153, 228]
miR-10b	Up	HCC	[229]
miR-17-5p	Up	HCC	[230]
miR-18a	Up	HCC	[231]

Table 3.

miRNAs listing as a biomarker (diagnostic/prognosis) in HCV infection and related diseases.

¹

HCC: hepatocellular carcinoma.

2.3 The potential of microRNA as a therapeutic target for HCV

Due to the recent discoveries, there is a chance to design therapeutic mechanisms based on gene therapy for HCV and HCC-related HCV diseases. One of these mechanisms is based on using miRNAs that regulate many of these mechanisms [232]. The latest ways include host immune response modulation, direct targeting of HCV genes, liver-specific delivery to the liver (the specific cite of infection), immunomodulation and tolerance induction, and combination therapies. Although much effort and considerable progress have been made in this field, due to some problems like toxicity, many of the miRNA-based treatments have not reached clinical development [233]. Due to this circumstance, two types of miRNA drugs, including miRNA inhibitors (antagomirs) and miRNA mimics (antimirs), are trying to be developed using miRNAs [234, 235]. In other words, miRNA mimics try to treat diseases by restoring them, and antagomir corrects the patterns of miRNA expression [236]. Some examples of using miRNA as therapeutic agents in clinical trials include Miravirsen (SPC3649), which is the first miRNA used in therapeutic ways. Targeting miR-122, Roche Pharmaceuticals and Santaris Pharma are presenting it [237].

2.4 Overcoming obstacles in microRNA therapy development

Due to its high molecular weight and negative charge, miRNA delivery for therapeutic purposes faces difficulties such as limited cellular uptake, bloodstream degradation, determining the best routes of administration, controlling internal stability, focusing on particular tissues and cell types, and fast renal clearance. miRNA therapeutics use synthetic miRNAs (miRNA mimics), recombinant expression vectors, and oligonucleotide-based miRNA inhibitors (anti-miRs) to reverse pathological miRNA expression changes by enhancing or blocking suppressor and driver endogenous miRNAs. Systemic miRNA delivery faces significant obstacles in the bloodstream because of RNases’ rapid degradation and renal elimination. Although systemic administration induces an immune response and causes RNA accumulation in the reticuloendothelial system (RES), chemical modifications can protect RNase digestion. Encapsulation may decrease RES absorption, prevent degradation, and extend its half-life [238].

Biocompatible, biodegradable, nonimmunogenic, stable in circulation, able to reach the target location, aid in cellular uptake, prevent lysosomal degradation, facilitate endosomal escape, and escape quickly renal clearance are all desirable qualities in a delivery system. A number of strategies, involving lipid- or polymer-based nanoparticles (e.g., chitosan and phosphatidylcholine), have been developed to successfully deliver RNAi molecules in vivo. The miRNA molecule is encapsulated in nanoparticle form via several delivery techniques, such as artificial expression by viral transduction, customizing therapeutic oligonucleotides by adding biomolecule conjugates or introducing chemical modifications. The nonviral miRNA delivery methods provided by nanocarriers include complexation (lipid vesicles, polymer-carriers, and gold nanoparticles), encapsulation (PLGA nanoparticles and silica nanoparticles), and conjugation; each has distinct advantages in terms of biocompatibility, specificity, targeting ability, intracellular trafficking, and miRNA release/activation processes. When you combine miRNA therapies with chemical changes, biomolecule conjugation, or carrier technology, you can make them more site-directed and effective at targeting cells. Before beginning any in vivo targeting, an in-depth risk assessment of miRNA treatments is necessary to reduce off-target effects and prevent miRNA overdose [239].

The following are some suggestions to overcome this treatment’s obstacles [234]:

1. Small compounds developed with the use of bioinformatics innovations or chemical compound screening can be employed to get around therapy obstacles. As shown in phase II clinical trial, using the miR-122 inhibitor Miravirsen/SPC3649 (NCT01200420, NCT01872936) to overcome HCV treatment resistance can be achieved by combining miRNA-based strategies with traditional drugs.

2. A possible way to increase the therapeutic efficiency of miRNAs is by the use of amiRNAs, which are artificially generated miRNA constructs that combine siRNA sequences and miRNA primary transcript scaffolds.

3. miRNA sponges are RNA constructs that have multiple binding sites that sequester endogenous miRNAs to regulate the level of miRNAs in cells. Apart from the conventional way of administration, plant-derived miRNA-based drugs can also be taken orally; however, details regarding their absorption, function, target gene regulation, and origin in plant foods remain uncertain. To completely focus on the therapeutic potential of miRNAs, more study is required, and dose recommendations for particular application methods are still unfulfilled.

Since miRNA therapy is still in its infancy, there is not a broad spectrum of tested carriers, and many have only been studied in vitro so far. In conclusion, it can be stated that there are two approaches to using miRNAs for therapeutic purposes. The first strategy involves utilizing miRNA antagonists, such as anti-miRNAs or LNAs, to suppress miRNAs that have carcinogenic qualities. Sequences complementary to endogenous miRNA are present in these oligonucleotides. Chemical changes increase their affinity for the target miRNA, and help trap the miRNA in a configuration that RISC is unable to process. These antagonists have the potential to cause endogenous miRNA degradation despite their ongoing controversy. For animal activity and cell culture, modifications that stabilize miRNA mimics and anti-miRNA to nuclease degradation and increase target RNA affinity are essential. Effective stabilizers include oligonucleotides with backbone modifications of 2′-sugar and phosphorothioate. The mentioned changes increase the binding affinity of anti-miRNA oligonucleotides to their equivalent miRNAs and confer nuclease resistance.

A second strategy makes use of miRNA replacement to make up for a tumor suppressor miRNA’s lost functionality. This method introduces a fresh approach to miRNA therapy.

3. Regulatory aspects

3.1 Current clinical trials focusing on microRNA therapy in HCV

Over 60 siRNA drugs are currently in clinical trials, with two approvals, compared to less than 20 miRNA therapeutics that have not progressed to phase III trials. Endogenous miRNAs target multiple genes, but exogenous siRNAs only target specific genes. Whereas siRNAs have one to three targets, miRNAs have between 30 and 250 targets. Sequence complementarity ranges between endogenous miRNAs (30–90%) and siRNAs (100%). Consequently, a significant obstacle to the development of miRNA therapies is the diverse targets of miRNAs. To effectively use miRNA therapies, it is necessary to identify biologically significant miRNAs, regulate gene expression, and minimize nonspecific interactions [240].

Current studies for the miRNA class of their preclinical and clinical research applications as FDA-approved small RNA therapeutics start to make progress into clinical medicine. In clinical trials, miRNAs have demonstrated efficacy as miRNA therapeutics, medical intervention drugs, biomarkers, and improvements for attenuated viral vaccines [241]. Regarding this, the following two validations ought to be carried out for such treatments:

1. Preclinical validation via in silico analysis: before research in animal models, several of the functional roles of potential miRNAs can be assessed bioinformatically and/or in vitro. miRNA bioinformatics systems can simplify therapeutic candidate selection and assessment, clinical research data sources and predicting regulatory targets. Databases like ViTa (prediction of host microRNAtargets on viruses), TargetScan, miRbase, and others are available for this purpose, enhancing research efficiency. Links to most of the databases now in circulation can be found on websites like https://tools4mirs.org/ [242].

2. Validation in vitro and in vivo: cell culture platforms like primary cells, immortalized cell lines, and induced pluripotent stem (IPS) are used to evaluate the mechanisms, toxicity, and therapeutic efficacy of miRNA candidates in vitro and in vivo, supporting preclinical miRNA research and clinical trials. Clinical trials for miRNA-based therapies have been completed in the “ClinicalTrials.gov” database to date. Clinicaltrials.gov currently lacks phase III trial candidates for miRNA drugs, but early phase trials are underway to explore new candidates, including miR-122/miravirsen studies [241, 243].

Miravirsen, an LNA-based anti-miRNA ASO (15-nt LNA-PS modified ASO) developed by Santaris Pharma and Hoffman-La Roche, is currently in the clinical trial as a treatment for HCV infection, sequesters miR-122, a crucial factor for HCV replication [244]. One important miRNA for in vivo therapy is miRNA-122, which is involved in several physiological and pathological processes related to the liver. miR-122, which regulates HCV replication, is highly expressed in the liver. At present, phase IIa of the Miravirsen trial focuses on investigating the drug’s effectiveness and long-term safety in patients suffering from persistent HCV genotype 1 infection. According to the findings, patients receiving Miravirsen did not develop any major side effects or mutations in the HCV-miR-122 RNA binding site. Effectively decreased HCV-RNA in individuals with chronic HCV genotype 1 infection (after five subcutaneous doses of Miravirsen). The reduction was dose-dependent. Miravirsen is undergoing trials and could be the first drug based on miRNA to be commercialized [245].

Mice and primates had lower liver plasma cholesterol levels when miRNA-122 was used as an antisense blockade in vivo. Also, binding to certain sites within the HCV-5′UTR contributes to the infection of hepatitis C. However, there is no correlation between serum levels of HCV and miRNA-122, indicating the significance of miRNA-122 in the identification of potential inhibitors [246]. Studies on this miRNA that have produced conflicting results highlight how crucial it is to manipulate miR-122 in a tissue- and time-specific approach [60, 247, 248]. According to each of these studies, depending on the cell and developmental stage, miRNA-122 levels may have highly diverse effects on a range of activities (growth, embryonic development, fibrosis, cancer development, and hepatitis C infection).

The moieties differ and include sugars, aptamers, peptides, and antibodies. To improve absorption efficiency, miRNA mimetics or inhibitors can potentially be conjugated with different moieties and then packaged into nanoparticles. miR-122, in conjunction with N-acetylgalactosamine (GalNAc), was developed to treat hepatitis C virus-infected liver (Miravirsen), with endocytosis of GalNAc-conjugated oligonucleotide drugs mediated by the asialoglycoprotein receptor. Hepatocyte cell surface asialoglycoprotein receptors are the particular binding site for GalNAc. Hepatocytes express the asialoglycoprotein receptor, enabling them to selectively absorb GalNAc-conjugated oligonucleotides. One of the main obstacles of miRNA therapy is the delivery of miRNA to the right tumor or infection site in vivo. To reach disease sites, miRNA mimics or inhibitors need to overcome nuclease degradation in the extracellular site. To address this problem and improve delivery efficiency, oligonucleotide modifications by chemical processes have been developed [249, 250]. miRNA-based therapy has been shown in mice, primates, and early humans to be promising for therapeutic applications. Nonetheless, complications like off-target impacts are still concerning. Since miRNAs are internal regulatory molecules, more research is necessary to fully understand their therapeutic potential [251]. Up until recently, miRNAs have been chosen as therapeutic targets due to their physiological roles and disease-specific dysregulation. For instance, in a profiling analysis conducted on a substantial group of human samples, miR-26 was initially linked to liver cancer; then, the potential of miRNA-26a was studied as an in vivo liver cancer treatment based on the earlier discovery demonstrating miRNA-26a downregulation in liver malignancies. Adeno-associated virus (AAV) vectors (AAVs) were used to deliver miRNA to liver cells, demonstrating tissue-dependent expression [252].

In conclusion, microRNA therapy application in public health requires a multidisciplinary strategy that takes ethical, therapeutic, legal, and scientific factors into account. To move in this direction, investments should be allocated to research and development to find microRNAs related to disease, create effective delivery techniques, and enhance the safety and efficacy profiles of treatments—the two key elements of cooperative collaboration and partnerships with regulatory bodies to set specific requirements for It demands the creation, assessment, and verification of treatments based on microRNA.

In addition to research and development, well-planned clinical trials should be carried out to assess the efficacy of microRNA therapies in various patient populations. microRNA therapy must also be integrated into the healthcare system to support therapeutic approaches and eventually clarify to patients the potential advantages as well as the risks and limitations. Finally, it is important to think about ethical issues (like getting informed consent, protecting privacy, being able to take part in research, and making sure that benefits and risks are shared equally) and cost-effectiveness (comparing the cost-effectiveness of microRNA therapy to other treatments). Table 4 lists some microRNA-based therapies for HCV infection that have been studied in clinical trials [253].

miR-based therapeutics	Target disease	Clinical trial phases
Miravirsen (AntimiR-122)	Chronic hepatitis C (CHC)	Phase I, completed: NCT01646489; Phase II, completed: NCT01200420; Phase II, ongoing: NCT02508090
RG-101 (AntimiR-122)	Chronic hepatitis C (CHC)	Phase I, completed and Phase II, ongoing
MRX34 (miR-34 mimic)	Multiple solid tumors (HCC)	Phase I, terminated: NCT01829971

Table 4.

The list of clinical trials examining microRNA-based therapies for HCV infection.

3.2 Personalized HCV therapy with microRNA

Hepatitis-related miRNAs, which are differentially expressed in patients, are linked to pathogenesis and treatment effectiveness, with significant differences observed between patients achieving sustained virologic response (SVR) and those not. miRNAs are a viable tool for personalized medicine in the future since they can improve therapy efficacy, reduce side effects, and improve patient well-being when handled appropriately. Despite their inexperience and the need for additional research, miRNAs have a promising future in precision medicine, with major advancements predicted in the years ahead. microRNAs, a class of small non-coding RNAs, have rapidly emerged as a potential biomarker for personalized medicine, guiding physicians’ clinical decisions. Personalized medicine enhances disease prediction, infection or cancer prevention, and chemoresistance by selecting effective drugs for each patient, saving time, improving cost-effectiveness, and enhancing patient quality of life by limiting adverse effects [254].

With the beginning of FDA-approved small RNA treatments in clinical trials, recent research on miRNAs has increased their use in preclinical and clinical medicine. miRNAs can control drug resistance, treat a variety of diseases, and act as disease biomarkers for prediction, prognosis, and therapeutic agents. Since disorders of multifactorial origin currently have no effective treatments, the pleiotropic characteristics associated with non-protein-coding RNAs make them fascinating therapeutic targets. The field of diagnostic and interventional medicine is expected to continue evolving as a result of the examination of candidate miRNAs in phases III and IV of clinical research. There is potential for this unique miRNA signature to be a predictive and diagnostic tool. Physicians might then quickly and accurately diagnose diseases by matching patient profiles to databases of known miRNA signatures associated with particular diseases [255].

4. Conclusion

The possibility of novel miRNA-based therapies, diagnostics and prognostics, and vaccinations related to it is becoming notably closer as the extent of studies on miRNAs increases. miRNA-based therapies, such as miravirsen, are in phase IIb trials, indicating their potential for clinical applications. Research on the interaction between miRNA and HCV is ongoing, with the potential for manipulating miRNAs as a therapeutic option in HCV treatment still in its early stages. Identifying miRNAs involved in the HCV viral life cycle and liver disease progression opens new drug discovery frontiers against chronic HCV infections. microRNA-based therapies present a viable approach for personalized medicine and next-generation treatments by offering accuracy, efficacy, and minimal side effects in the treatment of HCV. The unique viral profiles and host characteristics of these treatments enable them to be tailored to individual patients, enhancing their potency and specificity. The use of microRNA therapy in HCV therapy provides great promise for a revolutionary change in patient care. Targeting different phases of the HCV lifecycle improves the effectiveness of antiviral medications by reducing virus replication and altering immune responses. On the other hand, issues including delivery obstacles, off-target effects, safety concerns, and an increase in viral resistance are still pressing issues that call for more research and development. Finally, as already mentioned, HCV induces liver cirrhosis to HCC in a variety of ways. Virus-induced inhibition of human tumor suppressor genes, excessive production of reactive oxygen species (ROS), and disruption of oxidative stress metabolism are a few of these strategies. Furthermore, HCV modifies these pathways by regulating the expression of miRNAs associated with proliferation and the cell cycle. Therefore, using miRNA profiles should be noticed during diagnosis and treatment.

Acknowledgments

We must first give thanks and honor to Almighty God for His favors during our search. We are grateful to dear Dr. Georgios Tsoulfas, who heads the Department of Transplantation Surgery at Aristotle University School of Medicine and is a professor of transplantation surgery. He gave us the opportunity to write and contribute a valuable chapter in the book Hepatocellular Carcinoma: A Multidisciplinary Approach about the significance of the most recent HCV treatment strategy. Dear readers, we would like to say something to each one of you. We dedicate this section of the book to the men and women who love freedom worldwide, and we sincerely thank you all.

Conflicts of interest

The authors report no conflicts of interest.

Authors’ contributions

Designing the chapter’s outline (M.Sh), drafting the chapter (M.Sh, M.Z, and MR.S), collecting relevant information, and editing the initial chapter (M.Sh, M.A, and MR.S), and finalizing the chapter (M.Sh). All authors have read and agreed to the published version of this chapter.

Abbreviations

AAV	adeno-associated virus (AAV) vectors
ACSL1	long-chain acyl CoA synthetase 1
AGO2	argonaute-2 protein
amiRNA	artificial microRNA
AMPK	AMP-activated protein kinase
ASO	antisense oligonucleotide
bFGF	basic fibroblast growth factor
CASP2	caspase-2 protein
CDKN1C/P57	cyclin-dependent kinase inhibitor
CHC	chronic hepatitis C
DAAs	direct-acting antivirals
DGCR8	DiGeorge syndrome critical region 8
DLC1	deleted liver cancer 1
ECM	extracellular matrix
FASN	fatty acid synthase
FDA	United States Food and Drug Administration
GalNAc	N-acetylgalactosamine
HCC	hepatocellular carcinoma
HCV	hepatitis C virus
HMGCR	hydroxy-3-methylglutaryl-coenzyme A reductase
HNF4alpha	hepatocyte nuclear factor 4alpha
HSCs	hepatic stellate cells
IFITM1	interferon-induced transmembrane protein 1
IFNs	interferons
IL	interleukin
IPS	induced pluripotent stem
IRES	internal ribosome entry site
ISRE	interferon-stimulated response element
JNK/MAPK	c-Jun N-terminal kinase/mitogen-activated protein kinases
KLF5	Krüppel-like factor 5
Lnas	locked nucleic acids
LX-2	human hepatic cell line
LXR	liver X receptor
miRNAs	microRNAs (miRs)
MMP	matrix metalloproteinases
NAFLD	nonalcoholic fatty-liver disease
ncRNA	non-coding RNA
NF-κB	nuclear factor-κB
NSP	non-structural proteins
ORF	open reading frame
OGT	O-GlcNAc transferase
OCLN	occludin protein
PCR	polymerase chain reaction
PDK	phosphoinositide-dependent kinase-1
PEG-IFN	pegylated-interferon
PI3K/AKT	phosphatidylinositol 3-kinase/protein kinase B
piRNAs	PIWI-interacting RNAs
PLGA	poly lactic-co-glycolic acid
PPAR	peroxisome proliferator-activated receptor
Pre-miRNA	precursor-miRNA
Pri-miRNA	primary-miRNA
PTEN	phosphatase and tensin protein
RES	reticuloendothelial System
RIG-1	retinoic acid-inducible gene I
RIP	receptor-interacting protein
RISC	RNA-induced silencing complex
ROS	reactive oxygen species
RXRα	retinoid X receptor α
SIR1	silent information regulator 1
siRNAs	small interfering RNAs
SIRT1	Hepatic Sirtuin 1
snRNAs	small nuclear RNAs
SOCS	suppressor of cytokine signaling
SREBP	sterol regulatory element-binding protein
SVR	sustained viral response
TGF-β	transforming growth factor-β
TLR	Toll-like receptor response
TNFR1	tumor necrosis factor receptor 1
TNF-α	tumor necrosis factor-alpha
TRADD	TNFR1-associated death domain protein
TRAF2	TNF receptor associated factor 2
TRBP	TAR RNA binding protein
tsRNAs	transfer RNA-derived small RNAs
UTR	untranslated regions
VEGF	vascular endothelial growth factor
vtRNAs	vault RNAs
WHO	World Health Organization