Contaminated food or water acts as a source for spreading Hepatitis A and E. Transmission of hepatitis B, C and D generally takes place through the infected body fluids. These viruses are usually transmitted through transfusion of infected blood, use of contaminated equipment during surgery, and sexual contact. HBV is also transmitted from mother to child during parturition. Acute infection may be symptomatic or non-symptomatic. Symptoms include yellow colouration of eyes, skin and urine, intense weakness, abdominal pain, nausea, and vomiting [14Report WHO. 2016.What is Hepatitis? ]. The HBV infection involves an initial step that is attachments of mature virion to the host cell surface, with the help of preS domain of the surface protein [15Klingmüller U, Schaller H. Hepadnavirus infection requires interaction between the viral pre-S domain and a specific hepatocellular receptor. J Virol 1993; 67(12): 7414-22.
[PMID: 8230462] ]. Various factors have been suggested to act as receptors for viruses in the cell. However, only carboxypeptidase D mediated viral entry has been revealed during duck HBV infection [16Breiner KM, Urban S, Schaller H. Carboxypeptidase D (gp180), a Golgi-resident protein, functions in the attachment and entry of avian hepatitis B viruses. J Virol 1998; 72(10): 8098-104.
[PMID: 9733850] ]. Disassembly of the virus and mechanism of intracellular nuclear transport for the viral genome are not clearly understood and nucleocapsid core protein modification has been implicated in the process [17Kang HY, Lee S, Park SG, Yu J, Kim Y, Jung G. Phosphorylation of hepatitis B virus Cp at Ser87 facilitates core assembly. Biochem J 2006; 398(2): 311-7.
[http://dx.doi.org/10.1042/BJ20060347] [PMID: 16740137] ]. After nuclear import viral DNA is converted to the covalently closed circular DNA (cccDNA) (Fig. 2) [18Beck J, Nassal M. Hepatitis B virus replication. World J Gastroenterol 2007; 13(1): 48-64.
[http://dx.doi.org/10.3748/wjg.v13.i1.48] [PMID: 17206754] ]. The cccDNA transcripts do not undergo splicing and have a polyadenylated structure with a 5' cap. Two different 5' ends are present in the genomic transcripts (3.5kb) which consist of two species i.e., the pregenomic RNA (pgRNA) and the precore RNA. The pgRNA serves as messenger RNA for core and polymerase as well as the template for reverse transcription. The pre-core RNA is translated into the pre-core gene products. Through ribosomal scanning mechanism of the pgRNA, the pol start codon initiates the polymerase translation [19Jean-Jean O, Weimer T, de Recondo AM, Will H, Rossignol JM. Internal entry of ribosomes and ribosomal scanning involved in hepatitis B virus P gene expression. J Virol 1989; 63(12): 5451-4.
[PMID: 2585611] ]. The 2.4kb subgenomic RNA produces large HBsAg protein, 2.1kb RNAs produce the middle HBsAg (M-HBsAg) and small HBsAg (S-HBsAg) proteins, and 0.7kb RNA is translated to the HBxAg protein.

Fig. (2) Replication cycle of HBV.

Loss of cell cycle control is a general feature observed in all cancerous cells. This leads to an increased multiplicative tendency, hyperplasia, and subsequent tumour formation. Normal liver cells primarily live in the G0 phase (quiescent phase) of the cell cycle and renewed slowly. However, they possess the strong regenerative ability and after getting mitogenic signals, they enter the cell cycle and proceed to cell division [21Michalopoulos GK. Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas. Am J Pathol 2010; 176(1): 2-13.
[http://dx.doi.org/10.2353/ajpath.2010.090675] [PMID: 20019184] ]. Advancement through the eukaryotic cell cycle phases is governed by the combined actions of cyclins and cyclin-dependent kinases (Cdk). Cytokines and growth factors promote de-novo expression of cyclin D1 gene which is responsible for the transition of quiescent hepatocytes into the cell cycle [22Sharma AK, Kumar S, Chashoo G, Saxena AK, Pandey AK. Cell cycle inhibitory activity of Piper longum against A549 cell line and its protective effect against metal-induced toxicity in rats. Ind J Biochem Biophys 2014; 51(5): 358-64.
[PMID: 25630105] , 23Boylan JM, Gruppuso PA. D-type cyclins and G1 progression during liver development in the rat. Biochem Biophys Res Commun 2005; 330(3): 722-30.
[http://dx.doi.org/10.1016/j.bbrc.2005.03.042] [PMID: 15809057] ]. Many regulatory checkpoints apply the brake on free proliferation and avert quiescent hepatocytes from entry in the cell cycle (Fig. 3). As depicted in Fig. (3), retinoblastoma (pRb) and other proteins bind to and seize E2F transcription factors and thereby repress its activity [24Henley SA, Dick FA. The retinoblastoma family of proteins and their regulatory functions in the mammalian cell division cycle. Cell Div 2012; 7(1): 10.
[http://dx.doi.org/10.1186/1747-1028-7-10] [PMID: 22417103] ]. Entry into the cell cycle is also prevented by the Ink4 family of Cdk inhibitors (p15/16/18/19) by binding to Cdk4/6 kinases and inhibiting the formation of cyclin D-Cdk4/6 complex [25Ortega S, Malumbres M, Barbacid M. Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 2002; 1602(1): 73-87.
[PMID: 11960696] ]. Binding of CDK interacting protein (Cip)/Kinase inhibitory protein (Kip)family inhibitory proteins p21/27/57 with Cdk/cyclin complexes inactivates it and inhibits cell cycle advancement [26Besson A, Dowdy SF, Roberts JM. CDK inhibitors: cell cycle regulators and beyond. Dev Cell 2008; 14(2): 159-69.
[http://dx.doi.org/10.1016/j.devcel.2008.01.013] [PMID: 18267085] ]. A “proliferation cluster” has been identified in gene expression profiles of HCC samples which accounted for increased expression of proliferation-associated genes [27Chen X, Cheung ST, So S, et al. Gene expression patterns in human liver cancers. Mol Biol Cell 2002; 13(6): 1929-39.
[http://dx.doi.org/10.1091/mbc.02-02-0023] [PMID: 12058060] , 28Xu XR, Huang J, Xu ZG, et al. Insight into hepatocellular carcinogenesis at transcriptome level by comparing gene expression profiles of hepatocellular carcinoma with those of corresponding noncancerous liver. Proc Natl Acad Sci USA 2001; 98(26): 15089-94.
[http://dx.doi.org/10.1073/pnas.241522398] [PMID: 11752456] ]. Abnormalities that decrease the expression levels of p16/pRb genes or hamper their protein functions eventually cause tumorigenesis because p16/pRb pathway manages entry into cell cycle. Altered expression of the pRb is a universal phenomenon in HCC [29Murakami Y, Saigo K, Takashima H, et al. Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut 2005; 54(8): 1162-8.
[http://dx.doi.org/10.1136/gut.2004.054452] [PMID: 16009689] ]. It has been reported that the expression levels of CIP/KIP family member proteins p21/27 are frequently reduced in HCC samples [30Tannapfel A, Grund D, Katalinic A, et al. Decreased expression of p27 protein is associated with advanced tumor stage in hepatocellular carcinoma. Int J Cancer 2000; 89(4): 350-5.
[http://dx.doi.org/10.1002/1097-0215(20000720)89:4<350::AID-IJC6>3.0.CO;2-3] [PMID: 10956409] ].

Fig. (3) Cell cycle pathway and common checkpoints. The downward arrows indicate decreased expression of Ink4 family of Cdk inhibitor (p16) and Cip/Kinase inhibitory protein (Kip) family inhibitor (p21/27) during hepatocarcinogenesis. Release of E2F transcription factor leads to G1 to S transition.

Senescence is a type of irreversible growth inhibition of cells in cell culture showing distinct morphological alterations [31Hayflick L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 1965; 37(3): 614-36.
[http://dx.doi.org/10.1016/0014-4827(65)90211-9] [PMID: 14315085] ]. In hepatocytes, mechanism of senescence is not clearly understood. Replicative senescence controls partial proliferative ability of liver cells by a gradual decrease in the telomeric segment [32Paradis V, Youssef N, Dargère D, et al. Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas. Hum Pathol 2001; 32(3): 327-32.
[http://dx.doi.org/10.1053/hupa.2001.22747] [PMID: 11274643] ]. Telomere-independent mechanisms have also been suggested for hepatocyte senescence monitored in severe chronic liver diseases and these include free radical and oncogene-dependent senescence The resulting DNA damage activates ATM/Chk/p53 pathway and arrests cells at G1 phase. Alternatively, the p16/pRb pathway also performs the same function. Anomalies in DNA damage checkpoint and cell cycle regulatory pathway paved a way for the unlimited proliferation of genetically altered hepatic cells at the senescent phase and subsequently to malignant transformation. (Fig. 4).

Fig. (4) The proposed model of hepatocellular carcinoma development.

In human HCC, the p53 pathway has an effect on many levels i.e., (a) about 50% aflatoxin-mediated HCC cases exhibit p53 mutations while 20–30% cases of non-aflatoxin mediated HCC show p53 mutations; (b) microdeletions of p14^ARF rarely occurs in HCC with p53 mutation while it is reported in 15-20% of human HCC; (c) human HCC also shows elevated Mdm2 expression; (d) over expression of gankyrin, an oncoprotein, is commonly observed in human HCC, which imposes restriction on the pRb and p53 [33Higashitsuji H, Higashitsuji H, Itoh K, et al. The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53. Cancer Cell 2005; 8(1): 75-87.
[http://dx.doi.org/10.1016/j.ccr.2005.06.006] [PMID: 16023600] ].

pRb pathway anomalies (p16, p15 or RB1 genes) are observed in more than 80% of human HCC. The anomalies include p16/15 promoter methylation and deletion or mutation of RB1 gene. Promoter methylation causing p16 repression is the most common anomaly [34Azechi H, Nishida N, Fukuda Y, et al. Disruption of the p16/cyclin D1/retinoblastoma protein pathway in the majority of human hepatocellular carcinomas. Oncology 2001; 60(4): 346-54.
[http://dx.doi.org/10.1159/000058531] [PMID: 11408803] ]. Telomerase activation occurs during the transformation of precancerous lesions to HCC. Telomere-dependent senescence arrest in hepatocytes is frequently observed in cirrhosis. Reactivation of Telomerase Reverse Transcriptase (TERT) acts as a bypass for HCC growth. TERT is absent in normal hepatocytes, hence 90% human HCC show telomerase activation, a rate-limiting step for the commencement of cell immortality [35Llovet JM, Chen Y, Wurmbach E, et al. A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology 2006; 131(6): 1758-67.
[http://dx.doi.org/10.1053/j.gastro.2006.09.014] [PMID: 17087938] ]. Deregulation of TERT expression by integration of HBV DNA into TERT gene is a rare phenomenon [29Murakami Y, Saigo K, Takashima H, et al. Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut 2005; 54(8): 1162-8.
[http://dx.doi.org/10.1136/gut.2004.054452] [PMID: 16009689] ]. Besides, HBV surface proteins (viral X and PreS2) and HCV core protein may increase the activity of telomerase [36Liu H, Luan F, Ju Y, et al. In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase. Biochem Biophys Res Commun 2007; 355(2): 379-84.
[http://dx.doi.org/10.1016/j.bbrc.2007.01.160] [PMID: 17307151] ]. The above-mentioned facts indicate the cooperation between the anomalies in telomerase activity and senescence controlling genes (p53) during the hepatocarcinogenesis.

Cell death resulting from liver injury may be either accidental (necrotic), programmed (apoptotic), or uncontrolled. Extrinsic or intrinsic pathways initiate apoptosis by activating caspases 3, 6 and 7 [22Sharma AK, Kumar S, Chashoo G, Saxena AK, Pandey AK. Cell cycle inhibitory activity of Piper longum against A549 cell line and its protective effect against metal-induced toxicity in rats. Ind J Biochem Biophys 2014; 51(5): 358-64.
[PMID: 25630105] , 37Eguchi A, Wree A, Feldstein AE. Biomarkers of liver cell death. J Hepatol 2014; 60(5): 1063-74.
[http://dx.doi.org/10.1016/j.jhep.2013.12.026] [PMID: 24412608] ]. Death receptors mediate resistance towards apoptosis in HCC cells. The majority of the HCCs show one or more alterations in the Fas pathway molecules, which inhibit Fas-mediated apoptosis. HCC cells or tissues become unresponsive to Fas by downregulating Fas expression resulting in reduced expression of FADD or FLICE or increased expression of cellular FLICE/caspase-8-inhibitory protein (cFLIP), or by upregulation of nuclear factor-kappa B (NF-κB), Bcl-2 or Bcl-XL and Mcl-1 [38Okano H, Shiraki K, Inoue H, et al. Cellular FLICE/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma. Lab Invest 2003; 83(7): 1033-43.
[http://dx.doi.org/10.1097/01.LAB.0000079328.76631.28] [PMID: 12861043] -40Ranjan K, Pathak C. FADD regulates NF-κB activation and promotes ubiquitination of cFLIPL to induce apoptosis. Sci Rep 2016; 6: 22787.
[http://dx.doi.org/10.1038/srep22787] [PMID: 26972597] ]. Pro-apoptotic proteins (Bax or Bcl-XS) are downregulated in HCC. The TGF-β pathway is regularly stimulated at the cirrhosis stage and promotes apoptosis by activating Smad3 mediated Bcl2 downregulation and thereby reducing the susceptibility towards HCC development [41Yang YA, Zhang GM, Feigenbaum L, Zhang YE. Smad3 reduces susceptibility to hepatocarcinoma by sensitizing hepatocytes to apoptosis through downregulation of Bcl-2. Cancer Cell 2006; 9(6): 445-57.
[http://dx.doi.org/10.1016/j.ccr.2006.04.025] [PMID: 16766264] ]. Insulin-receptor signalling and activation of the PI3K-Akt pathway might also be involved in resistance towards apoptosis [42Chen RH, Su YH, Chuang RL, Chang TY. Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway. Oncogene 1998; 17(15): 1959-68.
[http://dx.doi.org/10.1038/sj.onc.1202111] [PMID: 9788439] ]. The insulin-like growth factor 2 receptor (IGF2R) reduces cell division by stimulating TGF-β signalling and breakdown of the IGF2 mitogen [43Dennis PA, Rifkin DB. Cellular activation of latent transforming growth factor beta requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor. Proc Natl Acad Sci USA 1991; 88(2): 580-4.
[http://dx.doi.org/10.1073/pnas.88.2.580] [PMID: 1846448] ]. During the initial phase of human hepatocarcinogenesis heterozygosity in IGF2R locus is frequently lost [44Yamada T, De Souza AT, Finkelstein S, Jirtle RL. Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis. Proc Natl Acad Sci USA 1997; 94(19): 10351-5.
[http://dx.doi.org/10.1073/pnas.94.19.10351] [PMID: 9294214] ]. In human HCCs loss of IGF2R and overexpression of IGF2 growth factor are common features. Stimulation of the Akt signalling and reduced expression of a negative regulator of Akt i.e., phosphatase and tensin homolog (PTEN) have been described in 40-60% HCC cases [45Hu TH, Huang CC, Lin PR, et al. Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer 2003; 97(8): 1929-40.
[http://dx.doi.org/10.1002/cncr.11266] [PMID: 12673720] ].

Most of the studies suggest that liver injury in viral hepatitis does not result from the direct cytopathic effects of viruses but caused by the viral protein-mediated host immune response [46Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995; 13: 29-60.
[http://dx.doi.org/10.1146/annurev.iy.13.040195.000333] [PMID: 7612225] ]. Animal studies have provided ample proof that viral hepatitis is triggered by an antigen-specific intrahepatic cellular response that set in motion a series of antigen-nonspecific cellular and molecular effector systems. Cellular and humoral limbs of the immune system work towards viral clearance by three different mechanisms: firstly, the virus-specific T-cell mediated direct destruction of infected hepatocytes; secondly, the removal of free viral particles from the circulation by the antibody response; and thirdly, non-cytopathic viral inactivation in infected hepatocytes by some inflammatory cytokines produced by activated mononuclear cells [47Ferrari C, Chisari FV. In: The liver biology and pathobiology. Arias, I.M., (ed.), Lippincott Williams & Wilkins, Philadelphia. 2001; pp. 763-82.]. Recent evidence suggests that NF-κB signalling mediated inflammation plays an essential role in commencement, promotion and development of tumours [48Naugler WE, Karin M. NF-kappaB and cancer-identifying targets and mechanisms. Curr Opin Genet Dev 2008; 18(1): 19-26.
[http://dx.doi.org/10.1016/j.gde.2008.01.020] [PMID: 18440219] ].

HCC initiation and progression is associated with genetic alteration. The permanent genetic abnormalities build up in hepatocytes and cause disrupted gene expression which ultimately leads to cancerous transformation. Genetic alterations include large chromosomal translocation, amplification, single nucleotide variation, small fraction loss and deletion. The genetic changes frequently cause the loss of function or activation of oncogenes or tumour suppressor genes. Contrary to genetic alterations, no change in the genome sequence is found in epigenetic regulations but it influences the chromatin structure and transcription of the gene. Gene products are affected at transcriptional and post-transcriptional levels during epigenetic regulations which include DNA methylation, histone modification, and lncRNA. This provides greater diversity to the gene regulation [86Singh AK, Bishayee A, Pandey AK. Targeting histone deacetylases with natural and synthetic agent: An emerging anticancer strategy. Nutrients 2018; 10(6): 731.
[http://dx.doi.org/10.3390/nu10060731] [PMID: 29882797] ].

5.2.1. Chromosomal Instability

In HCC, chromosomal instability is the most frequently observed genetic changes. It could be promoted by either error during mitosis or disruption in DNA replication and repair processes. The chromosome abnormalities include amplification/deletion of small chromosomal segments or gain/loss of whole chromosome arms. Comparative genomic hybridization data in HCC represent frequent amplification of chromosome 1q and 8q, while the frequent loss of chromosome 1p, 4q, 6q, 9p, 16p, 16q, and 17p (Table 1) [87Guan XY, Fang Y, Sham JST, et al. Recurrent chromosome alterations in hepatocellular carcinoma detected by comparative genomic hybridization. Genes Chromosomes Cancer 2000; 29(2): 110-6.
[http://dx.doi.org/10.1002/1098-2264(2000)9999:9999<::AID-GCC1022>3.0.CO;2-V] [PMID: 10959090] ]. Chromosome 1q amplification in HCC is a characteristic feature of chromosome abnormalities. In a large number of HCC patients, the chromosome 1q21 region containing CHD1L (an oncogene) was reported to be amplified [110Ma NF, Hu L, Fung JM, et al. Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma. Hepatology 2008; 47(2): 503-10.
[http://dx.doi.org/10.1002/hep.22072] [PMID: 18023026] ]. CHD1L is associated with oncogenic functions during hepatocarcinogenesis such as anti-apoptotic, mitosis regulation, and stimulating cell epithelial-to-mesenchymal transition [88Chen L, Chan THM, Yuan YF, et al. CHD1L promotes hepatocellular carcinoma progression and metastasis in mice and is associated with these processes in human patients. J Clin Invest 2010; 120(4): 1178-91.
[http://dx.doi.org/10.1172/JCI40665] [PMID: 20335658] , 111Chen L, Hu L, Chan THM, et al. Chromodomain helicase/adenosine triphosphatase DNA binding protein 1-like (CHD1l) gene suppresses the nucleus-to-mitochondria translocation of nur77 to sustain hepatocellular carcinoma cell survival. Hepatology 2009; 50(1): 122-9.
[http://dx.doi.org/10.1002/hep.22933] [PMID: 19441106] ]. In HCC chromosome, 8q24 region is another highly amplified region which contains oncogenes including c-Myc and PTK2 [98Santoni-Rugiu E, Jensen MR, Thorgeirsson SS. Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c-myc and transforming growth factor alpha. Cancer Res 1998; 58(1): 123-34.
[PMID: 9426068] , 99Okamoto H, Yasui K, Zhao C, Arii S, Inazawa J. PTK2 and EIF3S3 genes may be amplification targets at 8q23-q24 and are associated with large hepatocellular carcinomas. Hepatology 2003; 38(5): 1242-9.
[http://dx.doi.org/10.1053/jhep.2003.50457] [PMID: 14578863] , 112Wang Y, Wu MC, Sham JS, Zhang W, Wu WQ, Guan XY. Prognostic significance of c-myc and AIB1 amplification in hepatocellular carcinoma. A broad survey using high-throughput tissue microarray. Cancer 2002; 95(11): 2346-52.
[http://dx.doi.org/10.1002/cncr.10963] [PMID: 12436441] ]. SGK3A, a serine/threonine kinase, having similarity with AKT is commonly amplified in HCC which provides AKT independent oncogenic roles [100Liu M, Chen L, Chan THM, et al. Serum and glucocorticoid kinase 3 at 8q13.1 promotes cell proliferation and survival in hepatocellular carcinoma. Hepatology 2012; 55(6): 1754-65.
[http://dx.doi.org/10.1002/hep.25584] [PMID: 22262416] ]. Segmental loss of chromosome 1p35-36 region containing many tumour suppressors (14-3-3 σ and Rb-interacting zinc finger 1) is also commonly found in HCC. Short arm loss of chromosome 8 (a minimal region of 8p21-22 containing DLC-1) is a common feature in HCC. Because of promoter hypermethylation and allele loss, DLC-1 is recurrently deleted in HCC tissues [113Wong CM, Lee JM, Ching YP, Jin DY, Ng IO. Genetic and epigenetic alterations of DLC-1 gene in hepatocellular carcinoma. Cancer Res 2003; 63(22): 7646-51.
[PMID: 14633684] ]. DLC-1 expression restoration in hepatoma cells could induce cell apoptosis, and inhibit tumour growth [114Zhou XL, Thorgeirsson SS, Popescu NC. Restoration of DLC-1 gene expression induces apoptosis and inhibits both cell growth and tumorigenicity in human hepatocellular carcinoma cells. Oncogene 2004; 23(6):1308-13. [https://doi.org/10.1038/sj.onc.1207246][PMID: 14647417].

Table 1
Chromosomal aberration in hepatocellular carcinoma.

5.2.2. Circulating miRNAs

MicroRNA (miR), a class of non-coding RNAs, has been identified as important regulators of gene expression at post-transcriptional levels. Role of circulating miRNAs in serum as cancer biomarkers were described in 2008 and overexpression of miR-155, miR-21 and miR-210 were observed in B-cell lymphoma patients [115Lawrie CH, Gal S, Dunlop HM, et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol 2008; 141(5): 672-5.
[http://dx.doi.org/10.1111/j.1365-2141.2008.07077.x] [PMID: 18318758] ]. Abnormal expression of HCC development and progression related miRNAs and their role is under investigation. miR-122 and miR-221 regulate the cell cycle by modulating cyclins or cdk [116Gramantieri L, Ferracin M, Fornari F, et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Res 2007; 67(13): 6092-9.
[http://dx.doi.org/10.1158/0008-5472.CAN-06-4607] [PMID: 17616664] , 117Kumar S, Pandey AK. Oxidative stress-related microRNAs as diagnostic markers: A newer Insight in diagnostics. In: Maurya P, Chandra P. (eds.) Oxidative Stress: Diagnostic Methods and Applications in Medical Science. Springer, Singapore. 2017; 113-125.
[http://dx.doi.org/10.1007/978-981-10-4711-4_6] ]. Pro-apoptotic proteins (Bmf) are targets of some miRNAs (miR-221) which help HCC cells to avoid apoptosis [118Gramantieri L, Fornari F, Ferracin M, et al. MicroRNA-221 targets Bmf in hepatocellular carcinoma and correlates with tumor multifocality. Clin Cancer Res 2009; 15(16): 5073-81.
[http://dx.doi.org/10.1158/1078-0432.CCR-09-0092] [PMID: 19671867] ]. However, some miRNA (miR29) can promote HCC apoptosis by targeting the Bcl-2 and Mcl-1, the anti-apoptotic proteins [119Xiong Y, Fang JH, Yun JP, et al. Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma. Hepatology 2010; 51(3): 836-45.
[PMID: 20041405] ]. The most important characteristics of HCC i.e., invasion and metastasis are also regulated by miRNAs. Cell migration and spreading in HCC is promoted by pro-metastatic miRNAs e.g., miR-106b induces cell migration and invasion in HCC by activating epithelial-mesenchymal transition process [120Yau WL, Lam CSC, Ng L, et al. Over-expression of miR-106b promotes cell migration and metastasis in hepatocellular carcinoma by activating epithelial-mesenchymal transition process. PLoS One 2013; 8(3): e57882.
[http://dx.doi.org/10.1371/journal.pone.0057882] [PMID: 23483935] ]. Metastasis and HCC progression are suppressed by let-7g, miR-139, and miR-195 [121Wang R, Zhao N, Li S, et al. MicroRNA-195 suppresses angiogenesis and metastasis of hepatocellular carcinoma by inhibiting the expression of VEGF, VAV2, and CDC42. Hepatology 2013; 58(2): 642-53.
[http://dx.doi.org/10.1002/hep.26373] [PMID: 23468064] ]. Unusually expressed miRNAs and their roles are given in Table 2.

Table 2
Aberrantly expressed miRNA and their reported target genes.

Sorafenib (BAY43-9006, Nexavar) is multikinase inhibitor with dual inhibitory activity against RAF/MEK/ERK (Raf-1, B-Raf) in the tumour cell and vascular growth factor inhibitor family (VEGFR1, VEGFR2) and platelets derived growth factor receptor (PDGFR, c-Kit) which promote tumour progression and angiogenesis. Therefore, sorafenib acts either directly on the tumour or on angiogenesis and inhibits tumour growth [146Wilhelm S, Carter C, Lynch M, et al. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 2006; 5(10): 835-44.
[http://dx.doi.org/10.1038/nrd2130] [PMID: 17016424] ]. Sunitinib malate (SU11248, Sutent; Pfizer, NY, USA) and Linifanib (ABT-869) are also oral multikinase inhibitors that act on growth factors and receptor tyrosine kinases involved in angiogenesis and HCC progression [147Teramoto K, Ohshio Y, Fujita T, Hanaoka J, Kontani K. Simultaneous activation of T helper function can augment the potency of dendritic cell-based cancer immunotherapy. J Cancer Res Clin Oncol 2013; 139(5): 861-70.
[http://dx.doi.org/10.1007/s00432-013-1394-4] [PMID: 23411688] , 148Wörns MA, Schuchmann M, Düber C, Otto G, Galle PR, Weinmann A. Sunitinib in patients with advanced hepatocellular carcinoma after progression under sorafenib treatment. Oncology 2010; 79(1-2): 85-92.
[http://dx.doi.org/10.1159/000320363] [PMID: 21071995] ].

C-MET is a protein, encoded by MET oncogene, possesses tyrosine kinase activity involved in tumour development and metastasis [149Bottaro DP, Rubin JS, Faletto DL, et al. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991; 251(4995): 802-4.
[http://dx.doi.org/10.1126/science.1846706] [PMID: 1846706] ]. Tivantinib (ARQ 197) and cabozantinib are MET inhibitors that act by binding to its dephosphorylated state which is responsible for inhibition of growth and apoptosis in human tumour cell line [150Gherardi E, Birchmeier W, Birchmeier C, Vande Woude G. Targeting MET in cancer: rationale and progress. Nat Rev Cancer 2012; 12(2): 89-103.
[http://dx.doi.org/10.1038/nrc3205] [PMID: 22270953] , 151Fallahi P, Ferrari SM, Di Bari F, et al. Cabozantinib in thyroid cancer. Recent Patents Anticancer Drug Discov 2015; 10(3): 259-69.
[http://dx.doi.org/10.2174/1574892810666150708110816] [PMID: 26152149] ].

HCC is characterized by hyper vasculature resulting from higher expression of angiogenesis promoting factors viz., angiopoietin 2, PDGF, and VEGF [152Kumar S, Ahmad MK, Waseem M, Pandey AK. Drug targets for cancer treatment: An overview. Med Chem 2015; 5: 115-23. [https://doi.org/10.4172/2161-0444.1000252].]. Bevacizumab (Avastin; Genentech, CA, USA) is a humanised monoclonal antibody (mAb) acting on VEGF and one of the important drugs for colorectal cancer and liver metastasis of colorectal cancer [153Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350(23): 2335-42.
[http://dx.doi.org/10.1056/NEJMoa032691] [PMID: 15175435] ]. Brivanib (BMS-582664), an inhibitor of VEGF and FGF signalling has shown efficacy as a first-line treatment for advanced HCC patients [154Chou T, Finn RS. Brivanib: A review of development. Future Oncol 2012; 8(9): 1083-90.
[http://dx.doi.org/10.2217/fon.12.104] [PMID: 23030483] ]. Ramucirumab (Cyramza), a mAb, is an inhibitor of VEGFR-2 [155Cheng AL, Kang YK, Chen Z, et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol 2009; 10(1): 25-34.
[http://dx.doi.org/10.1016/S1470-2045(08)70285-7] [PMID: 19095497] ].

Immunohistochemistry has shown that approximately 50% of HCC patients have activated mTOR pathway. This activation may be the result of increased signalling due to overexpression of ligands (EGF, IGF1, and IGF2) or may be due to mutant oncogenes (PI3KCA) or tumour suppressor genes (PTEN). Temsirolimus and Everolimus, an analogue of rapamycin are the inhibitors of mTOR [156Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology 2008; 48(4): 1312-27.
[http://dx.doi.org/10.1002/hep.22506] [PMID: 18821591] ].