A comprehensive systemic review was conducted on miRNAs in advanced HCC with emphasis on the biogenesis, role in liver cancer, association with cell proliferation, apoptosis, regulation of angiogenesis, invasion and metastasis in HCC. A search was conducted in PUBMED, Science Direct, Scopus, MEDLINE, Cochrane Library, National Centre for Biotechnology Information, and Google scholar. The keywords used for the search are: (“miRNA”) AND (“Hepatocellular carcinoma” OR “liver cancer” OR “HCC”) AND (“cell proliferation OR “cell growth”) AND (“apoptosis OR cell death”) AND (“regulation of angiogenesis”) AND (“regulation of invasion” OR “regulation of metastasis”).
Micronome was constructed using Cytoscape software and search tools for the retrieval of interacting genes (STRING) database. Only experimentally validated miRNA studies of cell proliferation, apoptosis, regulation of angiogenesis, invasion, and metastasis were included in the study. To study the interactions between the supplied linked genes set at the miRNA level, the micronome was built using Cytoscape 3.9.0 based on the miRNA: target interaction data generated on the literature search. The number of nodes in the network, the total number of interactions in the network, and the number of nodes with a degree larger than three were used to determine the relevance of associated gene networks.
miRNA Production
The main miRNAs are created from the microRNA sequence in the nucleus by RNA polymerase II. The microprocessor complex, which comprises of the DGCR8 gene for DiGeorge syndrome and the nuclease Drosha further breaks down the core miRNAs into miRNA precursors (pre-miRNAs), which are miRNAs that are between 60 and 70 nucleotides long. Pre-miRNAs are moved by exportin-5 from the nucleus to the cytoplasm. The final miRNA duplexes, which have 19–25 nucleotides, are created by further processing the pre–miRNAs byRNase Dicer in the cytoplasm.At last, mature single-stranded miRNAs are created and maintained in the RNA-induced silencing complex (RISC), while their passenger strand is broken down. The purpose of RISC is to cleave or inhibit mRNA.Several mRNA targets' 3′ untranslated regions are recognised and bound to by mature miRNAs, either with imperfect or ideal complementarity. However, numerous genes can be stopped from being translated by a single miRNA [17, 18].
The function of miRNAs in hepatocarcinogenesis
It is impossible to ignore the unique function played by miRNA during development and spread of liver cancer. Depending on the genes they target, microRNAs can either serve as tumour suppressors or oncogenes. Dysregulation of these miRNAs has been associated to the inhibition of growth suppressors and the promotion of proliferative signalling in cancer cells, as well as the activation of invasion and metastasis [19, 20]. One key aspect of cancer is the regulation of cell growth. MiR-17-92 inhibits E2F1 translation, a well-known regulator of cell proliferation, after being triggered by c-Myc in a way that depends on the cell cycle [21]. Ectopic expression of MiR-221/222 led to the growth of cancer cells whereas its inhibition resulted in G1 cell-cycle arrest [22]. Apoptosis is yet another critical component of cancer. By targeting the cyclin G1 and the protein that binds cytoplasmic polyadenylation elements and enhancing p53 activity, miR-122 increases cell sensitivity to the medication doxorubicin, laying the framework for the creation of combined chemo- and miRNA-based therapeutics [23, 24].
Aside from invasion, cancer also exhibits metastasis, which is another crucial feature. By specifically targeting RhoA GTPase, a crucial regulator of cellular polarity, tight junction formation, and stability, miR-155 aids in the promotion of the epithelial-mesenchymal transition (EMT). When miR-155 is silenced, TGF-induced EMT, tight junction damage, migration of cell, and invasion are downregulated [25]. Angiogenesis is a feature of cancer as well. MiR-27b and miR-128, which target VEGF-C, had been found to prevent tumour angiogenesis and development [26]. By precisely targeting and inhibiting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, miR-296 improved angiogenesis mediated ECs VEGFR-2 and PDGFR degradation [27]. Targeting BRCA1, MiR-146a stimulated the transcription of PDGFRA while suppressing tumour angiogenesis [28].
Autophagy, a defining characteristic of cancer, is essential for the development and metastasis of cancer. The cascade of PI3K-AKT-mTOR, which targets PTEN in cancer cells, is inhibited by miR-214,miR-21, miR-26b, and which has been found to be essential for autophagy. miR-9-3p, miR-30d, miR-34a, and miR-181a have all been discovered to regulate autophagy in cancer cells by specifically targeting ATG5. Several miRNAs have been reported to control autophagy in several cancer cells by binding to ATG12, including miR-23a, miR-200b,miR-23b-3p, miR-630, miR-378, and miR-146a-5p. It is possible to find useful indicators and therapeutic targets by analysing the miRNA profiles that occur during the beginning and development of HCC [4, 29].
miRNA as a therapeutic target through control of apoptosis and proliferationof cells in HCC
Recent research has shown a connection between abnormal miRNA expression and the growth and death of HCC cells. While others were repressive, some miRNAs in HCC encouraged cell division and death. Consequently, by controlling cell proliferation and death, HCC can be prevented from developing and spreading by using miRNAs as potential cancer inhibitors [30]. In the simulated transition zone of RFA, miR-103 targets PTEN to increase HCC cell migration and proliferation by activating the PI3K/Akt signalling cascade, and AKAP12 to control HCC growth [31, 32]. It has been discovered that miR-30d, miR-181a, miR-9-3p, and miR-34a directly target ATG5 to regulate autophagy in tumour cells.
Additionally, Increased expression of miR-103a boosted HCC cell motility and proliferation, and predominantly implicated in regulating the metabolism of glucose and the prevention of cell death, according to functional enrichment analysis [33]. In vitro, miR-665 targets PTPRB to increase HCC cell proliferation and cell cycle progression [34]. Exo-miR-638 overexpression inhibits apoptosis, colony formation, viability, arrest of cell cycle in the growth 1 phase, and the ability of HCC to migrate and invade by reducing potential for all these processes. Due to miR-1299's decreased expression in HCC cells compared to healthy hepatocytes, MTT experiments reveal that miR-1299 overexpression inhibited the development of HCC cells. According to cell cycle analyses, MiR-1299 control resulted in a drop in G0/G1-phase cells and an upsurge in S-phase cells[35].
By suppressing HSF1 expression and encouraging the development of BH3-only proteins, miR-644a prevents the growth of HCC tumours [36]. In HCC cells, miR-148a overexpression inhibited and miR-148a knockdown enhanced growth [37]. Huh7-1.3 and HepG2.2.15 cells' proliferative ability was markedly decreased by increased miR-325-3p expression leading to apoptosis [38]. MiR-182-5p increased the HCC cells' motility and invasiveness both in vitro and in vivo. To encourage the growth of HCC, miR-182-5p directly targets the 3′-UTR of FOXO3a and suppresses its expression [39].
Table 1 List of miRNAs which are mainly downregulated in HCC and connected to cell growth and death.
MicroRNA family
|
Targeted pathway
|
References
|
miR-1
|
ET-1
|
[40]
|
miR-10a, miR-10b
|
EphA4
|
[41]
|
let-7b
|
HMGA2
|
[42]
|
miR-15a, miR-16
|
EphA4
|
[43]
|
miR-7
|
PIK3CD
|
[44]
|
let-7g
|
Bcl-xL, c-myc, COLIA2, p16
|
[45]
|
miR-15b
|
BCL-2
|
[46]
|
miR-26a
|
CDK6, IL-6, cyclin D2, E1, E2
|
[47]
|
let-7a, let-7d, let-7c, let-7f-1,
|
Bcl-xL, c-myc, Caspase-3, STAT3
|
[48]
|
miR-29a
|
SPARC
|
[47]
|
miR-98
|
EZH2
|
[49]
|
miR-99a
|
PLK1, IGF-1R
|
[50]
|
miR-101
|
EZH2
|
[51]
|
miR-142-3p
|
LDHA
|
[52]
|
miR-194
|
MAP4K4
|
[53]
|
miR-124
|
PIK3CA
|
[54]
|
miR-223
|
STMN1
|
[15]
|
miR-206
|
Cyclin D1, CDK6
|
[55]
|
miR-125b
|
Bcl-2
|
[56]
|
miR-449
|
c-Met
|
[57]
|
miR-144
|
ZFX
|
[58]
|
miR-376a
|
PIK3R1
|
[59]
|
miR-203
|
Survivin
|
[60]
|
miR-122
|
Bcl-w, ADAM17
|
[61]
|
miR-219-5p
|
GPC3
|
[62]
|
miR-125a
|
MMP11
|
[63]
|
miR-1299
|
CDK6
|
[35]
|
miR-296
|
FGFR1
|
[64]
|
miR-429
|
Rab18
|
[65]
|
miR-450a
|
DNMT3a
|
[66]
|
Table 2 List of miRNAs, which are primarily upregulated and associated with cell proliferation and apoptosis in HCC.
MicroRNA family
|
Targeted pathway
|
References
|
miR-18a
|
ER1a
|
[67]
|
miR-221
|
p27, p57
|
[68]
|
miR-92
|
FBXW 7
|
[69]
|
miR-657
|
TLE1, NF–Κb
|
[70]
|
miR-96-5p
|
Caspase-9
|
[71]
|
miR-25
|
TRAIL
|
[72]
|
miR-519d
|
p21,
|
[73]
|
miR-26a
|
CyclinD2
|
[47]
|
miRNA as a therapeutic target through regulation of invasion and metastasis in HCC
Recent studies have shown that miRNAs can control HCC cells by stimulating or hampering invasion, EMT, and metastasis. The main problem with treating HCC is preventing tumour spread. The identification of miRNAs may open new options for anti-metastatic treatments. MiR-4325 regulateinvasion of HCC cell and their migration by specifically targeting the transcription factor GATA6. Furthermore, the knockdown of GATA6 had no effect inthe expression of miR-4325 expression in cells that expressed miR-4325 mimics, demonstrating that miR-4325 targets GATA6 [74]. MiR-129-5p overexpression accelerated HCC cells' invasion and migration, aiding in the diseases spread [75].
The HCC cell line LM9 showed less hepatic and pulmonary metastatic colonisation following tail vein injection in vivo after miR-101 transfection [76]. MiR-129-3p inhibited HCCLM3 and MHCC97-H in vitro cell invasion and migration, as well as intrahepatic and lung metastases in naked mice [77]. MiRs-137 prevented the SK-Hep1 and QGY-7703 HCC cells relocation which in turn reduced the HCC xenografts metastasis of lung and liver in mice's flanks [78]. MiR-transfected HepG2 cells were injected into the caudal veins and demonstrated reduced in vivo metastasis to the lungs, while miR-149 prevented HepG2 and MHCC97-H HCC transfectants from migrating and invading [79].
Compared to a matching reference cell line, the subcutaneous implantation of HCCLM3 cells that had been miR-503 expression vector transfected reduced lung metastases and tumour growth [80]. TGFβ decreases the expression of miR-630, which goes after SLUG, a nuclear transcription factor that binds to the CDH1 promoter's E-box region and induces the production of CDH1 mRNA [81]. In vitro, miR-139 reduced lung metastasis in MHCC97-H cells as compared to regulate cells, while it did not affect the proliferation of the SMMC-7721 and BEL-7402 [82].
Table 3 List of miRNAs which are downregulated and connected to HCC cell invasion and metastasis.
MicroRNA family
|
Targeted pathway
|
References
|
miRNA-7
|
P13K, AKT-mTOR
|
[44]
|
miRNA-34a
|
CCL2
|
[83]
|
miRNA-100
|
VETC
|
[84]
|
miRNA-101
|
ROCK2
|
[76]
|
miRNA-125b
|
ANGPTN2
|
[84]
|
miRNA-135b
|
RECK, EV15
|
[85]
|
miRNA-137
|
AKT2
|
[78]
|
miRNA-139-3p
|
ANXA2R
|
[80]
|
miRNA-148a
|
c-MET
|
[86]
|
miRNA-186
|
RUNX3
|
[87]
|
miRNA-187-3p
|
S100A4
|
[88]
|
miRNA-199a-3p
|
VEGF
|
[89]
|
miRNA-200a
|
SNAIL, AKT, FOX
|
[90]
|
miRNA-203
|
SNAIL, TWIST
|
[91]
|
miRNA-331-3p
|
AKT, GSK3B, SNAIL
|
[92]
|
miRNA-345
|
STAT3, P-AKT
|
[93]
|
miRNA-367
|
MDM2
|
[94]
|
miRNA-379
|
FAK
|
[95]
|
miRNA-422
|
FOXG1
|
[96]
|
miRNA-435a
|
MTSS1
|
[97]
|
miRNA-449a
|
c-MET
|
[98]
|
miRNA-501-3p
|
LIN7A
|
[99]
|
miRNA-503
|
ARHGEF19
|
[80]
|
miRNA-612
|
AKT2
|
[100]
|
miRNA-630
|
TGFβ
|
[81]
|
miRNA-885-5p
|
β-Catenin
|
[55]
|
miRNA-1271
|
PTP4A1
|
[101]
|
Table 4 List of miRNAs which are upregulated and connected to the invasion of the cell and their metastasis in HCC
MicroRNA family
|
Targeted pathway
|
References
|
miRNA-21
|
ARHGAP24
|
[102]
|
miRNA-23
|
MGAT3
|
[103]
|
miRNA-29a
|
SOCS1
|
[104]
|
miRNA-103
|
LATS2
|
[105]
|
miRNA-130b
|
PTEN/AKT
|
[106]
|
miRNA-143
|
NFĸB
|
[107]
|
miRNA-192-5p
|
Semaphorin 3A
|
[108]
|
miRNA-219-5p
|
CDH1
|
[109]
|
miRNA-362-5p
|
CYLD
|
[110]
|
miRNA-382-5p
|
DLC1
|
[111]
|
miRNA-425-5p
|
PI3K/AKT/GSK3β
|
[112]
|
miRNA-429
|
PI3K/AKT/GSK3β
|
[112]
|
miRNA-487a
|
SPRED2, PIK3R1
|
[113]
|
miRNA-1247-3p
|
B4GALT3
|
[114]
|
miRNA as a therapeutic target through angiogenesis in HCC
In our latest study, we integrate prediction miRNA databases (miRTarbase and Target scan) to construct a micronome involved in tumor growth and angiogenesis in patients of hepatocellular carcinoma. For the purpose of identifying the signature miRNAs implicated and identifying the genes to clarify the likely regulatory mechanism or signalling, dysregulated genes of HCC were integrated with miRNAs. Interestingly, the characteristic miRNA for positively controlling angiogenesis in HCC through VEGFA is hsa-mir-205-5p [115]. An important modulator of angiogenesis is the family of vascular endothelial growth factors (VEGF). Seven important subtypes of the VEGF family, including vascular endothelial growth factor (A-E) and placental growth factors 1 and 2 [116]. miRNAs control factors involved in tube formation, angiogenesis, proliferation, endothelial and cell migration. Pro-angiomiRs are angiomiRNAs that target angiogenesis's negative regulators to encourage neo-angiogenesis, whereas anti-angiomiRs target angiogenesis's positive regulators to stop it (anti-angiomiRs) [117].
Rat aortic ring angiogenesis was significantly reduced by miR-375-transfected hepatoma cells, and hepatoma cell pro-angiogenic activity could also be reduced by chicken CAMs and overexpression of miR-375 [118]. MiR-200b-3p levels in cancer cells are lowered in HCC, which increases the expression of endothelium ERG. In a recent study, miR-497 effect in THE angiogenesis of HCC, endothelial recruitment in vitro, and formation of capillary tube assay were investigated in Huh7 and PLC/PRF/5. The enhanced miR-497 expression was ascertained using quantitative real time PCR on HCC cells transfected with miR-497 mimics. When miR-497 expression was restored, the endothelial recruitment experiment, demonstrated that the ability of HCC cells to promote HUVEC migration was significantly reduced.These results suggested that overexpressing miR-497 could decrease the pro-angiogenic activity of HCC cells in vitro [119].