The Role of MicroRNAs in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers, leading to the second cancer-related death in the global. Although the treatment of HCC has greatly improved over the past few decades, the survival rate of patients is still quite low. Thus, it is urgent to explore new therapies, especially seek for more accurate biomarkers for early diagnosis, treatment and prognosis in HCC. MicroRNAs (miRNAs), small noncoding RNAs, are pivotal participants and regulators in the development and progression of HCC. Great progress has been made in the studies of miRNAs in HCC. The key regulatory mechanisms of miRNAs include proliferation, apoptosis, invasion, metastasis, epithelial-mesenchymal transition (EMT), angiogenesis, drug resistance and autophagy in HCC. And exosomal miRNAs also play important roles in proliferation, invasion, metastasis, and drug resistance in HCC by regulating gene expression in the target cells. In addition, some miRNAs, including exosomal miRNAs, can be as potential diagnostic and prediction markers in HCC. This review summarizes the latest researches development of miRNAs in HCC in recent years.

Introduction

Hepatocellular carcinoma (HCC) has become the second most common cause of cancer-related death worldwide ¹, with approximately 782,500 new cases and 745,500 deaths occurring in the global during 2012 ². In the early stage of HCC, surgical resection, liver transplant, local ablation and other curative therapies can improve patient´s survival ³. However, the 5-year recurrence rate is very high, it may reach as high as 80%-90% even the HCC patients have received potentially curative therapies ⁴. It has been already advanced stage for most people when HCC was diagnosed ⁵. For the advanced stage, the small molecule targeted therapeutics drugs sorafenib and regorafenib are the standard treatments that have been approved by the US Food and Drug Administration (FDA). Sorafenib is the only standard first-line systemic therapy available for advanced HCC, but the median survival was reported only 3 months ⁶. Regorafenib is a second-line drug when HCC patients were progressing on sorafenib treatment, whereas, the median survival was still only 10.6 months according to a phase 3 clinical trial report ⁷. Even though sorafenib and regorafenib can improve overall survival of HCC patients, it is not too long. Furthermore, the worries for drug resistance and adverse action of these drugs are rising as well. Therefore, it is urgent to explore new therapies, especially seek for more accurate markers for early diagnosis, treatment and prognosis in HCC. Nucleic acid-based drugs such as microRNAs (miRNAs) may have the promising therapeutic potential for HCC treatment. MiRNAs are pivotal participants and regulators in the development and progression of HCC. And exosomal miRNAs also play important roles in the development and progression in HCC. In addition, some miRNAs, including exosomal miRNAs, can be as diagnostic and prediction markers in HCC. In this review, we summarize the latest researches development of miRNAs in HCC in recent years.

Biogenesis of miRNAs

The sequence of the human genome has been finished in 2003, and it was reported that only 20,000-25,000 genes, about 1.5% of the total human genome, can encode protein ⁸. In other words, noncoding RNAs (ncRNAs), including miRNAs, long noncoding RNAs (lncRNAs), small nuclear RNAs (snRNAs) and circularRNAs (circRNAs), are the major components of the human transcriptome ⁹. MiRNAs are the pivotal members of this noncoding RNA family ¹⁰. MiRNAs, ~ 23 nucleotides in length, act as important gene regulators in animals and plants ¹¹. MiRNAs control the expression of their target mRNAs principally by binding to the 3'-untranslated region (3'-UTR) ¹². A mature miRNA formation goes through a series of complicated process. It was described in the Figure 1. At first, miRNA genes are transcribed to primary microRNAs (pri-miRNAs) by RNA polymerase II in the nucleus ¹³. Pri-miRNAs are cleaved by the RNase III type endonuclease Drosha the next, resulting in releasing the precursor miRNAs (pre-miRNAs), which have about ~70 nucleotides and stem-loop structures ¹⁴. After that, being transported by exportin-5 from nucleus to cytoplasm, pre-miRNAs are processed by another RNase III type endonuclease Dicer to generate a miRNA protein complex with two strands ^{15, 16}. One strand will become a mature miRNA, and then the mature miRNA is bound to RNA-mediated silencing complexes (RISC) immediately ¹⁷. In the RISC, the mature miRNA targets the 3'-UTR of its target mRNAs to regulate gene posttranscriptional expression, including translational inhibition and mRNA cleavage ¹⁸. The other one will be degraded. It has been proved that miRNAs play crucial roles in multiple biological processes by regulating gene expression, and the abnormal expression of miRNAs are related to numerous cancers and many other diseases ¹⁹.

Figure 1.

The process of miRNA formation. At first, miRNA genes are transcribed to primary pri-miRNAs by RNA polymerase II in the nucleus. Pri-miRNAs are cleaved by the RNase III type endonuclease Drosha producing pre-miRNAs the next. After that, being transported by exportin-5 from nucleus to cytoplasm, pre-miRNAs are processed by another RNase III type endonuclease Dicer to generate a miRNA protein complex with two strands. One strand of the complex will become a mature miRNA, and then the mature miRNA is bound to RNA-mediated silencing complexes (RISC) immediately. In the RISC, the mature miRNA targets the 3'-untranslated regions of its target mRNAs to regulate translational inhibition or mRNA cleavage. The other one will be degraded.

miRNAs and HCC

The research in miRNAs and their relevant functional mechanisms of cancer will contribute to the development of the therapeutics. Thus, we summarize recent researches development in regulating miRNAs in HCC. Some miRNAs have been found to be upregulated in HCC, which can be seen in the Table 1 ^20-53, and some downregulated can be seen in the Table 2 ^54-146. The key regulatory mechanisms of miRNAs in these studies include proliferation, apoptosis, invasion, metastasis, epithelial-mesenchymal transition (EMT), angiogenesis, drug resistance and autophagy in the development and progression of HCC. In addition, some miRNAs can also be as potential diagnostic and prediction markers in HCC.

Table 1.

Upregulated miRNAs in HCC

MiRNA	Targets	Mechanisms	References
miR-10b	CSMD1	Migration, invasion	20
miR-21	CAMSAP1, DDX1, MARCKSL1	No mentioned	21
miR-25	RhoGDI1, TRAIL	EMT, apoptosis	22, 23
miR-32	No mentioned	Prognostic marker	24
miR-92a	FBXW7	Cell growth, prognostic marker	25
miR-96-5p	Caspase-9	Apoptosis	26
miR-107	Axin2, HMGA2, HMGCS2	Proliferation, prognostic marker	27-29
miR-135a	FOXO1	Migration, invasion	30
miR-155-5p	PTEN	Proliferation, apoptosis, invasion, migration	31
miR-181a	Atg5	Autophagy	32
miR-182	TP53INP1	Drug resistance	33
miR-197	CD82	Migration, invasion	34
miR-203a-3p.1	IL-24	Cell growth, proliferation, metastasis	35
miR-210	FGFRL1, YES1	Metastasis, angiogenesis, proliferation	36, 37
miR-214-5p	WASL	Migration, invasion, EMT	38
miR-216a/217	PTEN, SMAD7	Drug resistance	39
miR-221	No mentioned	Prognostic marker	40
miR-302d	TGFBR2	Cell growth, apoptosis, migration	41
miR-331-3p	ING5	Proliferation, apoptosis	42
miR-346	FBXL2	Proliferation, migration, invasion	43
miR-454	CHD5	Proliferation, EMT, prognostic marker	44
miR-487a	SPRED2, PIK3R1	Proliferation, metastasis, prognostic marker	45
miR-765	INPP4B	Proliferation	46
miR-873	TSLC1	Proliferation, migration, invasion	47
miR-892a	CD226	Proliferation, invasion	48
miR-1246	CADM1	Migration, invasion, diagnostic and prognostic marker	49
miR-1249	PTCH1	Cell growth, migration, invasion	50
miR-1468	CITED2, UPF1	Proliferation, apoptosis	51
miR-3910	MST1	Cell growth, migration	52
miR-4417	TRIM35	Proliferation, apoptosis	53

Table 2.

Downregulated miRNAs in HCC

MiRNA	Targets	Mechanisms	References
miR-7	mTOR, TYRO3	Autophagy, drug resistance	54, 55
miR-7/21/107	Maspin	Drug resistance, prognostic marker	56
miR-26	ULK1	Autophagy	57
miR-29a	CLDN1	Proliferation, migration	58
miR-30a-5p	AEG-1	Cell growth, apoptosis	59
miR-30e	MTA1	EMT	60
miR-31	NDRG3	Drug resistance	61
miR-31-5p	SP1	Proliferation, migration, invasion	62
miR-33a	No mentioned	Prognostic marker	63
miR-33a-5p	No mentioned	Drug resistance	64
miR-33b	SALL4	Proliferation, metastasis	65
miR-98	EZH2	Proliferation	66
miR-101	Mcl-1, RAB5A, STMN1, ATG4D	Apoptosis, autophagy, diagnostic marker	67-69
miR-105-1	NCOA1	Diagnostic and prognostic marker	70
miR-122	Snail1, Snail2, PKM2, DLX4	EMT, proliferation, apoptosis, prognostic marker	71-73
miR-124-3p	MAPK14, RELA, CDK2, CDK4, SP1	No mentioned	74
miR-126	VEGF	Angiogenesis	75
miR-137	EZH2	Proliferation, invasion	76
miR-138	Cyclin D3, SP1	Prognosis marker, Proliferation, invasion, migration	77, 78
miR-142	THBS4, TGF-β	Migration, invasion, cell growth, metastasis	79, 80
miR-142-3p	ATG5, ATG16L1, LDHA	Autophagy, drug resistance, proliferation	81, 82
miR-143	TLR2	Proliferation, invasion	83
miR-144	ZFX	Proliferation, invasion, migration	84
miR-146a	HAb18G	Metastasis, angiogenesis	85
miR-152	RTKN, DNMT1	Cell growth	86, 87
miR-186	YAP1	Migration, invasion, proliferation	88
miR-187-3p	S100A4	EMT	89
miR-194	MAP4K4	Proliferation, diagnostic and prognostic marker	90
miR-195	Wnt3a, CBX4, FGF2, VEGFA	Proliferation, metastasis, angiogenesis	91-93
miR-199	RGS17	Proliferation, migration, invasion	94
miR-199a-3p	VEGFA, VEGFR1, VEGFR2, HGF, MMP2, YAP1	Angiogenesis, proliferation, apoptosis	95, 96
miR-199a-5p	CLTC	Cell growth	97
miR-199b-5p	TGF-β1	EMT	98
miR-200a	CXCL1, GAB1	EMT, invasion, migration	99, 100
miR-203	IL-1β, Snail1, Twist1	Proliferation, metastasis	101
miR-206	CCND1, cMET, CDK6	Proliferation, apoptosis	102
miR-211	SPARC	Proliferation, migration, invasion	103
miR-212	FOXM1	Migration, cell growth	104
miR-217	MTDH	Proliferation, apoptosis, migration, invasion	105
miR-223	Rab1	Proliferation, apoptosis	106, 107
miR-296	FGFR1	Proliferation, apoptosis, prognostic marker	108
miR-320a	c-Myc	Proliferation, invasion	109
miR-337	HMGA2	Proliferation, apoptosis	110
miR-338-3p	TAZ, MACC1, β-catenin, VEGF	Proliferation, migration, angiogenesis	111, 112
miR-340	JAK1	Proliferation, invasion	113
miR-345	IRF1	Metastasis, EMT	114
miR-361-5p	VEGFA	Proliferation, invasion	115
miR-365	ADAM10	Proliferation, metastasis	116
miR-367-3p	MDM2	Drug resistance	117
miR-370	PIM1	Cell growth, invasion	118
miR-375	HMSN	Drug resistance	119
miR-377	Bcl-xL	Apoptosis	120
miR-429	RAB23	Metastasis, EMT	121
miR-451	IL-6R	Angiogenesis	122
miR-491-3p	ABCB1, Sp3	Drug resistance	123
miR-495	IGF1R	Proliferation, invasion	124
miR-497	VEGFA, AEG-1	Angiogenesis, metastasis	125
miR-503	No mentioned	Drug resistance	126
miR-506	ROCK1	Proliferation, apoptosis	127
miR-520f	TM4SF1	Proliferation, invasion, migration	128
miR-539	FSCN1	Migration, invasion, drug resistance	129, 130
miR-542-3p	FZD7, Survivin	Proliferation	131, 132
miR-613	YWHAZ	Proliferation, invasion	133
miR-634	Rab1A, DHX33	Cell growth, metastasis	134
miR-638	VEGF, SOX2	Angiogenesis, invasion, EMT	135, 136
miR-663a	HMGA2	Proliferation, invasion	137
miR-708	SMDAD3	Proliferation, migration, invasion	138
miR-874	DOR	Proliferation, metastasis	139
miR-874-3p	PIN1	Proliferation, apoptosis	140
miR-876-5p	BCORL1	Migration, invasion, EMT	141
miR-940	CXCR2	Migration, invasion, prognostic marker	142
miR-1207-5p	FASN	Cell growth, invasion	143
miR-1271-5p	FOXK2	Cell growth, prognosis marker	144
miR-1299	CDK6	Proliferation	145
miR-1301	BCL9, β-catenin, VEGFA	Migration, invasion, angiogenesis	146

MiRNAs and proliferation and apoptosis of HCC

Cell growth, proliferation and apoptosis are the significant processes that guarantee the internal stability and balance of cell number and biological functions ¹⁴. Cell proliferation is achieved through the cell cycle, a strictly and orderly controlled process of cell activity. The cyclin dependent kinases (CDKs) are the core regulators of the cell cycle ¹⁴⁷. Any cell proliferation process follows certain rules. When the cell cycle is out of control and cell unlimited proliferate, it will develop into a tumor cell ¹⁴⁸. Apoptosis is also called as programmed cell death ¹⁴⁹. Apoptosis contributes to maintain the internal balance between cell death and renewal ¹⁵⁰. Disorder of apoptosis is often associated with human diseases, for example, deficient apoptosis may lead to tumor ¹⁵¹. In short, cell unlimited proliferation and abnormal regulation of apoptosis will promote the formation of cancer, including HCC.

Recent studies have indicated that aberrant expressions of miRNAs were linked to HCC cells proliferation and apoptosis. Some miRNAs promoted cell proliferation and apoptosis of HCC, and the others were repressive. Therefore, these miRNAs can be as potential cancer inhibitors to control the development and progression of HCC by regulating cell proliferation and apoptosis.

Plenty of miRNAs were reported that they could mediate cell proliferation and apoptosis by controlling cell cycle in HCC. Overexpression of miR-1468 promoted cell cycle transition from G1 to S phase and apoptosis resistance ⁵¹. Increased expression of miR-98 arrested HCC cell cycle in G0/G1 phase to repress cell proliferation via targeting enhancer of zeste homolog 2 (EZH2) ⁶⁶. Overexpression of miR-195 induced G1 phase cell cycle arrest and promoted apoptosis by directly targeting Wnt3 in HCC ⁹¹. MiR-506 was reported to induce HCC cell cycle G1/S phase arrest and apoptosis ¹²⁷. MiR-1299 overexpression inhibited HCC cell cycle from G0/G1 phase entering into S phase, and its target cyclin dependent kinase 6 (CDK6) was the key regulator in the G0/G1 phase arrest ¹⁴⁵.

Some aberrant expression of miRNAs could promote HCC cell proliferation and apoptosis by binding to their target genes. Inhibition of miR-25 expression was showed via PTEN/PI3K/Akt/Bad signaling pathway to enhance HCC cells apoptosis caused by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) ²³. MiR-107 was observed to be upregulated in HCC. Zhang JJ et al. reported that overexpression of miR-107 contributed to HCC cells proliferation via targeting Axin2 ²⁷. However, Wang Y et al. got that repressing miR-107 by targeting high mobility group A2 (HMGA2) could increase HCC cells proliferation ²⁸. MiR-155-5p was found to elevate HCC cells proliferation ability but inhibit apoptosis ³¹. High expression of miR-203a-3p.1 could improve HCC cell proliferation by targeting interleukin-24 (IL-24) in HCC ³⁵.

In addition, other miRNAs, which abnormally express, were proved to inhibit cell proliferation and apoptosis in HCC. MiR-96-5p was observed to inhibit apoptosis by targeting caspase-9 ²⁶. MiR-122 appeared abundant and downregulated in HCC cells. Xu Q et al. reported that overexpression of miR-122 repressed proliferation but induced apoptosis by targeting pyruvate kinase muscle 2 (PKM2) in HCC ⁷². Another report found that miR-122 could downregulate the expression of oncogenic distal-less 4 (DLX4), knockdown the expression of this oncogene would inhibit HCC cells proliferation ⁷³. Overexpression of miR-137 was reported to reduce HepG2 cells proliferation by targeting EZH2 ⁷⁶. MiR-217 overexpression was revealed to inhibit cells apoptosis by targeting metadherin (MTDH) in HCC ¹⁰⁵. MiR-337 and miR-370 overexpression were also found to inhibit cell proliferation and promote apoptosis in HCC by HMGA2 and PIM1 ^{110 118}. Decreased levels of miR-377 could suppress HCC cell apoptosis through inhibiting Bcl-xL expression ¹²⁰.

The above reports have shown that miRNAs serve important roles in proliferation and apoptosis of liver cancer. In addition, it was indicated that many miRNAs participate in the development and progression of HCC by mediating proliferation and apoptosis.

MiRNAs and metastasis of HCC

Invasion and metastasis are the essential characteristics of the tumor cells. Metastasis is one of the most dominative causes of cancer death ¹⁵². And 90% of cancer deaths are because of metastasis ¹⁵³. Tumor metastasis is a very complex process; it usually undergoes these major procedures: (1) local migration and infiltration, (2) vascular invasion, (3) survival in the circulating blood, (4) homing and implantation of metastatic organs in the distant place, (5) substantial infiltration, (6) adaptation to new environment, (7) secondary tumor growth ¹⁵⁴. EMT is a biological process that epithelial cells transform into mesenchymal cells by a specific procedure ¹⁵⁵. EMT participates in cancer metastasis through empowering tumor cells with migratory and invasive biological properties ¹⁵⁶.

The latest researches have demonstrated that miRNAs can regulate HCC cells by promoting or suppressing HCC cells invasion, EMT and metastasis. How to prevent tumor metastasis has become one of the most important problems in the treatment of HCC. The discoveries of miRNAs may provide us with choices of anti-metastatic therapies.

These miRNAs dysregulated expression would contribute to HCC metastasis. MiR-25 overexpression could facilitate EMT formation by inhibiting Rho GDP dissociation inhibitor alpha (RhoGDI1) in HCC ²². Overexpression of miR-135a promoted HCC cells migration and invasion by targeting forkhead box O1 (FOXO1) ³⁰. High expression of miR-203a-3p.1 could improve HCC migration and invasion by targeting IL-24 in HCC ³⁵. Upregulated miR-892a ⁴⁸ and miR-1246 ⁴⁹ expression were observed to enhance HCC cells migration and invasion. Downregulation miR-30e was showed to heighten metastasis and EMT of HCC cells by enhancing MTA1 ⁶⁰. Additionally, loss levels of miR-345 ¹¹⁴ and miR-638 ¹³⁶ would heighten invasion and EMT of HCC cells.

Above miRNAs aberrant expression could motivate cell invasion, EMT and metastasis in HCC. Of course, some were the opposite. Downregulation of miR-197 was identified to inhibit HCC cells migration and invasion by targeting KAI1/CD82 ³⁴. Overexpression of miR-214-5p could inhibit the migration and invasion of HCC cells; besides, miR-214-5p could also suppress EMT ³⁸. MiR-212 overexpression was observed to inhibit the migration of HCC cells by targeting forkhead box M1 (FOXM1) and suppress the Wnt/β-Catenin signaling pathway ¹⁰⁴. MiR-495 and miR-613 overexpression were showed to inhibit cell proliferation and invasion in HCC by targeting IGF1R and YWHAZ ^{124 133}. Upregulation of miR-122 expression in HCC repressed cell proliferation, invasion and EMT by targeting Snail1 and Snail2 ⁷¹. Overexpression of miR-187-3p ⁸⁹, miR-199b-5p ⁹⁸ and miR-1301 ¹⁴⁶ in HCC were also reported to inhibit EMT and metastasis. Besides, some miRNAs overexpression could repress invasion, migration and metastasis in HCC, for instance, miR-137 ⁷⁶, miR-146a ⁸⁵, miR-186 ⁸⁸, miR-199 ⁹⁴, miR-365 ¹¹⁶, miR-370 ¹¹⁸, miR-520f ¹²⁸, miR-634 ¹³⁴, miR-1207-5p ¹⁴³, and so on.

Thus, miRNAs have been demonstrated to regulate metastasis of HCC. Absolutely, these miRNAs might be used to treat metastasis in HCC.

MiRNAs and angiogenesis of HCC

Abundant angiogenesis provides the necessary nutrition for tumor growth and metastasis, thus, it is essential for tumor growth and metastasis in solid tumor ¹⁵⁷. As one of the common solid tumors, HCC usually has affluent and deformed blood vessel tissue ¹⁵⁸. In the process of angiogenesis, vascular endothelial growth factor (VEGF), a highly conserved homodimeric glycoproteina, is identified as one of the most effective cytokines ¹⁵⁹. VEGF is a superfamily with seven subtypes, for example, VEGF-A, VEGF-B, VEGF-C, and so on. VEGF receptors have three types, VEGFR1, VEGFR2 and VEGFR3. VEGF family members combine with their receptors VEGFRs to induce tumor angiogenesis ¹⁶⁰. High expression of VEGF in tumor tissue or circulation blood frequently implies tumor may be invasion and metastasis ¹⁶¹.

Great deals of miRNAs were reported to regulate angiogenesis in HCC by VEGF. Overexpression of miR-146a was showed to repress HCC angiogenesis and tumor metastasis by downregulating VEGF ⁸⁵. MiR-199a-3p was proved to repress angiogenesis by directly decreasing VEGF secretion and suppressing expression of its receptors VEGFR1 and VEGFR2 on HCC cells ⁹⁵. MiR-451 could suppress VEGF production and block VEGFR2 pathway to reduce angiogenesis ¹²². Overexpression of miR-638 was reported to suppress angiogenesis and tumor growth of HCC cells by inhibiting VEGF in HCC ¹³⁵. MiR-1301 overexpression was found to inhibit HCC angiogenesis by downregulating VEGFA, BCL9, and β-catenin ¹⁴⁶.

On the contrary, some miRNAs could enhance angiogenesis by VEGF. Suppression miR-338-3p could upregulate VEGF expression to promote angiogenesis in HCC ¹¹². Furthermore, downregulating miR-497 promoted angiogenesis and metastasis by directly inhibiting VEGFA ¹²⁵.

In consequence, miRNAs were proved the vital regulators in the process of HCC angiogenesis. What's more, miRNAs could act as inhibitors of tumor angiogenesis.

MiRNAs and drug resistance of HCC

Chemotherapy is currently one of the most commonly used treatment methods, when most patients with HCC are diagnosed at advanced stages ¹⁶². Large numbers of trials that tested the efficacy of various drugs have manifested that HCC has low sensitivity to chemotherapy ¹⁶³. And several chemotherapies fail due to the intrinsic or acquired drug resistance ¹⁶⁴. Thus, how to reverse drug resistance and improve the effectiveness of chemotherapy are crucial problems to be solved urgently. Many reports have showed that miRNAs could act as regulators to promote or reverse drug resistance in HCC, indicating miRNAs might have the promising therapeutic potential for drug resistance.

Sorafenib is as known the first-line drug for advanced HCC, but its curative effect is limited due to acquired resistance, which may be the primary factor ¹⁶⁵. MiRNAs could reverse this effect. MiR-7 was proved to overcome sorafenib resistance by suppressing its target TYRO3 via PI3-Kinase/AKT pathway ⁵⁵. Overexpression of miR-216a/217 activated TGF-β pathway to induce sorafenib resistance, but interdicting TGF-β pathway would reverse this resistance in HCC ³⁹. MiR-367-3p increased sorafenib efficacy to suppress HCC metastasis through changing the MDM2/AR/FKBP5/PHLPP/ (pAKT and pERK) signals ¹¹⁷. Another report has showed that sorafenib significantly reduced miR-142-3p levels by acting on the transcription factor PU.1; however, miR142-3p upregulation could sensitize HCC cells to sorafenib through targeting autophagy-related 5 (ATG5) and autophagy-related 16-like 1 (ATG16L1) to reduce sorafenib-induced autophagy, enhance sorafenib-induced apoptosis and inhibit cell growth ⁸².

For the drug resistance induced by other chemotherapeutic drugs, miRNAs also can promote or reverse drug resistance in HCC. Upregulating miR-182 was observed to increase cisplatin resistance in HCC treatment by regulating tumor protein 53-induced nuclear protein1 (TP53INP1) ³³. Inhibition of miR-33a-5p expression could also reduce cisplatin sensitivity and increased its drug resistance in HCC ⁶⁴. MiR-7/21/107 was enhanced by HBV X protein to promote HCC cells drug resistance by directly suppressing its target maspin expression ⁵⁶. MiR-31 and its target gene NDRG3 made HCC cells sensitize to chemotherapeutic drug Adriamycin ⁶¹. MiR-375 was combined with hollow mesoporous silica nanoparticles (HMSN) to overcome doxorubicin hydrochloride resistance in HCC ¹¹⁹. Additionally, miR-539 overexpression was reported to increase sensitivity to antagonize arsenic trioxide resistance in HCC ¹³⁰.

Therefore, miRNAs were involved in drug resistance of HCC. Furthermore, miRNAs could prove a new therapeutic strategy for how to improve the effectiveness of chemotherapy when treating HCC.

MiRNAs and autophagy of HCC

Autophagy has been reported for many years ago, but it has recently gained more attention, especially the Nobel Prize in Physiology or Medicine awarding to the great discovery of autophagy in 2016 makes it a popular topic again. Autophagy, a self-digestive catabolism process ¹⁶⁶, depends on lysosomes to degrade and recycle proteins or cell organelles ¹⁶⁷. Autophagy can regulate cell survival, differentiation, senescence, death and many other biological processes ¹⁶⁸. It has been proved that autophagy has a dual regulation role in HCC occurrence and suppression ¹⁶⁹.

MiRNAs might participate in the process of HCC development and progression through autophagy. MiR-181a was reported to repress autophagy in HCC by targeting pro-autophagic protein Atg5, leading to reducing apoptosis of HCC cells and accelerate hepatoma growth ³². MiR-7 was confirmed to induce HCC cells autophagy by targeting mammalian target of rapamycin (mTOR), and inhibition of autophagy heightened the antitumor activity of miR-7 to repress HCC cells proliferation ⁵⁴. MiR-26 could improve HCC cells sensibility to chemotherapy and facilitated apoptosis of HCC cells through inhibiting autophagy initiator ULK1 ⁵⁷. MiR-101 was found to enhance cisplatin-induced apoptosis through repressing autophgy in HCC ⁶⁸.

Thus, miRNAs participate in the process of HCC tumorigenesis and development through autophagy.

To sum up, miRNAs appear to play crucial roles in modulating HCC development and progression. The aberrant expression of miRNAs in HCC was summarized in the Figure 2. These studies indicated that miRNAs have the promising therapeutic potential for HCC treatment.

Figure 2.

Summary of miRNAs in the development and progression of HCC. Red arrow means: increased expression of miRNA, blue arrow means: decreased expression of miRNA.

Exosomal miRNAs and HCC

Exosomes, one type in extracellular vesicles (EVs), are small vesicles with a size range of 40-150 nm and a lipid bilayer membrane ¹⁷⁰. Exosomes, which now considered as an additional mechanism for intercellular communication ¹⁷¹, are generated inside multivesicular endosomes or multivesicular bodies (MVBs) ¹⁷². Exosomes exist in all body fluids, such as serum, urine, and saliva ¹⁷³. Exosomes have been shown to act as shuttles between cells including RNA, proteins, miRNAs, long noncoding RNAs (lncRNAs), or DNA fragments ^174-177. Tumor-derived exosomes are recognized as a critical determinant of the tumor progression ¹⁷⁸. Studies have demonstrated the mechanism of HCC-derived exosome-mediated miRNA transfer is important in the growth and progression of HCC ¹⁷⁹. The studies of exosomal miRNAs in HCC in recent years were summarized in the Table 3 ^180-194.

Table 3.

Exosomal miRNAs in HCC

Exosomal miRNA	Targets	Mechanisms	References
miR-9-3p	HBGF-5	Proliferation	180
miR-21	No mentioned	Diagnostic marker	181
miR-26a	No mentioned	Proliferation, migration	182
miR-32-5p	PTEN	Drug resistance	183
miR-103	VE-Cad, p120 ZO-1	Metastasis	184
miR-122	ADAM10, IGF1R, CCNG1	Drug resistance	185
miR-320a	PBX3	Proliferation, migration, metastasis	186
miR-335-5p	No mentioned	Proliferation, invasion	187
miR-638	No mentioned	Prognosis marker	188
miR-718	HOXB8	Proliferation, prognostic marker	189
miR-1247-3p	B4GALT3	Metastasis	190
miR-122, miR-148a, miR-1246	No mentioned	Diagnostic marker	191
miR-18a, miR-221, miR-222, miR-224, miR-101, miR-106b, miR-122, miR-195	No mentioned	Diagnostic marker	192
miR-10b, miR-21, miR-122, miR-200a	No mentioned	Diagnostic marker	193
miR-519d, miR-21, miR-221, miR-1228	No mentioned	Diagnostic marker	194

Exosomal miRNAs were involved in proliferation, migration, metastasis, drug resistance in HCC. Exosomal miR-9-3p, lower level in HCC patients, could reduce HCC cell viability and proliferation, and additionally reduced ERK1/2 expression by targeting fibroblast growth factor 5 (HBGF-5) ¹⁸⁰. Exosomal miR-32-5p was testified to activate the PI3K/Akt pathway, and induce multidrug resistance by modulating angiogenesis and EMT in HCC ¹⁸³. Exosomal miR-103 was proved to increase vascular permeability and promote metastasis by targeting junction proteins ¹⁸⁴. Zhang Z et al. found that the expression of exosomal miR-320a in cancer-associated fibroblasts (CAFs) was lower than paracancer fibroblasts (PAFs), leading to the cancer cells towards a more malignant phenotype. Furthermore, they revealed that miR-320a could suppress HCC cell proliferation, migration and metastasis by directly targeting PBX3 ¹⁸⁶. Tumor-derived exosomal miR-1247-3p was observed to convert fibroblasts to cancer-associated fibroblasts (CAFs) via downregulating B4GALT3, to activate β1-integrin-NF-κB signaling pathway to promote lung metastasis of liver cancer ¹⁹⁰.

Therefore, exosomes can transfer miRNAs between cells, and these miRNAs play important roles in proliferation, invasion, metastasis, and drug resistance in HCC by regulating gene expression in the target cells.

Acting as diagnostic and prediction markers in HCC

When HCC are diagnosed, many patients have already been advanced stage. It would have far-reaching influence on the prevention and treatment of HCC if the cancer could be early diagnosed and detected. It is well recognized that alpha fetoprotein (AFP) is the most common used hematology diagnosis marker of HCC in the clinical. But its false negative rate may be 40% with early stage HCC ¹⁹⁵. Moreover, some non-tumor diseases, such as hepatitis and cirrhosis, these patients' serum APF levels may also elevate ¹⁹⁶ .Therefore, it is necessary to seek for some new markers to diagnose and predict HCC. MiRNAs may have the potential functions according to the above mechanisms.

Numerous researches have supported that miRNAs could act as diagnostic and prediction markers in HCC. MiRNAs could be used to diagnose and distinguish HCC. For example, miR-101 levels in the serum were found to be significantly downregulated in the HBV-related HCC patients compared with the HBV-related liver cirrhosis patients, chronic hepatitis B patients and healthy controls, indicating that miR-101 could severe as a potential hematological marker of to diagnose and distinguish HBV-related HCC ⁶⁹. MiRNAs could also diagnose tumor size and TNM stages of HCC. High miR‑32 expression was observed that large tumor size (≥ 5cm) had significantly decreased ²⁴. High expression of miR-1246 and its target gene CADM1 low expression were correlated with stage 1 of TNM stages in HCC ⁴⁹. Low expression of miR-296 in HCC patients might have large tumor size and advanced TNM stage ¹⁰⁸. Low expression of miR‑137 was significantly closely related with lymph node metastasis, vein invasion and advanced clinical stage in HCC ⁷⁶.

Besides, miRNAs were reported to be as useful prognosis markers as well. High expression of miR-92a ²⁵, miR-221 ⁴⁰, miR-487a ⁴⁵ and miR-1468 ⁵¹ might indicate poor prognosis in HCC. Low expression of miR-33a ⁶³, miR-122 ⁷², miR-137 ⁷⁶, miR-194 ⁹⁰ and miR-940 ¹⁴² in HCC patients were observed to have unfavorable prognosis. High levels of miR-7/21/107 and low expression of maspin implied poor survival of HBV-related HCC ⁵⁶. Low expression of miR-138 combined with high expression of its target cyclin D3 showed worse clinical prognosis in HCC ⁷⁷. High expression of forkhead box K2 (FOXK2) protein, the target of miR-1271-5p, had poor overall survival (OS) and disease-free survival (DFS) of HCC patients ¹⁴⁴.

Exosomal miRNAs have been as novel biomarkers for HCC diagnoses and prognosis in clinical in recent years. For example, high expression of exosomal miR-32-5p and low expression of its target PTEN were positively associated with poor prognosis ¹⁸³. HCC patients with lower levels of serum exosomal miR-638 had poor overall survival than those with higher levels of exosomal miR-638 in serum ¹⁸⁸. The expression level of serum exosomal miR-21 was significantly higher in patients with HCC than those with chronic hepatitis B (CHB) or healthy volunteers. Besides, high level of miR-21 expression correlated with cirrhosis and advanced tumor stage ¹⁸¹. MiR-122, miR-148a, and miR-1246 were significantly elevated in serum exosomes from HCC patients compared to liver cirrhosis (LC) and normal control (NC) individuals ¹⁹¹.

Taken together, all of the above researches suggested that these miRNAs, including exosomal miRNAs, could be valuable of diagnostic and prediction markers in HCC.

Conclusions and future directions

Great progress has been made in the study of miRNAs in HCC. MiRNAs are pivotal participants and regulators in the development and progression of HCC. Proliferation, apoptosis, invasion, metastasis, EMT, angiogenesis, drug resistance and autophagy of miRNAs may be the primary regulatory mechanisms in HCC. Exosomal miRNAs have been focused on in recent years, and the researches are progressing rapidly. Recent studies have shown that exosomes can transfer miRNAs between cells in proliferation, invasion, metastasis, and drug resistance of HCC. In addition, exosomal miRNAs could be as biomarkers for HCC diagnoses and prognosis. The above studies indicated miRNAs could be used valid therapeutic targets and acted as valuable early diagnostic and prediction markers in HCC. Understanding the regulatory mechanisms of miRNAs in the HCC development and progression will help us to develop more effective new therapies and molecular therapeutic drugs.

However, there are also some problems in the studies of miRNAs. Lots of studies only stay in the experimental stage and do not really be used into the clinic. Secondly, the security and reliability of miRNAs acting as HCC early diagnosis and treatment markers also need to further research. In addition, Exosomal miRNAs are mostly concentrated in the observation of the content of miRNAs in serum exosomes, but their specific mechanisms of HCC are not fully understood. And the lacks of sensitive preparatory and analytical technologies for exosomes are also big challenges to clinical translation ¹⁹⁷.

Therefore, the future studies should pay more attention to make the acquired achievement of miRNAs in HCC translate into clinical application, for instance, develop available miRNA inhibitors for clinical. Besides, the research in security and reliability of miRNAs for HCC early diagnosis and treatment should also be more concerned. For the research of exosomal miRNAs in HCC, the mechanisms of exosome-mediated miRNAs transfer should be focused on in the future studies. Meanwhile, the analytical technologies also need to be improved.