The emergence of long non-coding RNAs in hepatocellular carcinoma: an update

Abstract

Hepatocellular carcinoma (HCC) accounting for roughly 90% of all primary liver neoplasms is the sixth most frequent neoplasm and the second prominent reason of tumor fatality worldwide. As regulators of diverse biological processes, long non-coding RNAs (lncRNAs) are involved in onset and development of neoplasms. With the continuous booming of well-featured lncRNAs in HCC from 2016 to now, we reviewed the newly-presented comprehension about the relationship between lncRNAs and HCC in this study. To be specific, we summarized the overview function and study tools of lncRNAs, elaborated the roles of lncRNAs in HCC, and sketched the molecule mechanisms of lncRNAs in HCC. In addition, the application of lncRNAs serving as biomarkers in early diagnosis and outcome prediction of HCC patients was highlighted.

Introduction

Hepatocellular carcinoma (HCC) accounts for roughly 90% of all primary liver neoplasms, and is the sixth most frequent neoplasm and the second prominent reason of tumor fatality worldwide ¹. The highest morbidity of HCC has been found in China and the Asia-Pacific area, which accounts for >50% of HCC cases in the world ². Meanwhile, liver cancer is fatal in both developed and developing countries, with the 5-year overall survival rate generally lower than 20% ³. HCC is the sole solid neoplasm that could be cured by liver transplant which can simultaneously heal the neoplasm and latent cirrhosis, and which is not influenced by the extent of liver function impairment ⁴. However, the extreme shortage of liver donors results in procrastination before transplantation, and increasing the risk of tumor progression, and transplantation failure during this period ⁵. These suggested that HCC is a devastating disease with disappointing outcomes and limited therapeutic options. Similar to other cancers, HCC is characterized by the gradual accumulation of genetic and epigenetic changes ^6-8. Among these alterations, long non-coding RNAs (lncRNAs) play crucial roles in the initiation and progression of HCC.

LncRNAs are transcripts with more than 200 bp in length, and have incrementally been recognized as the emerging star in neoplasm study as their essential function in tumor biology ^9-11. These transcripts are lack of the protein-coding potential in general and are particularly ubiquitous in almost whole livings ⁹. LncRNAs have emerged as regulators in diverse biological processes ^12-14 containing cellular proliferation, differentiation, motility, invasiveness, survival and so on ^15-17. Mounting evidences have demonstrated that lncRNAs are involved in tumor onset and development ^{18, 19}, and their expression is frequently deregulated in cancers ^20-27. LncRNAs are strikingly cell type-specificity and cancer type-specificity in expression which are relatively stable ^{18, 28}. Therefore, lncRNAs stand a chance to serve as a kind of desired indicators with underlying utilizations in neoplasm divination, early-discovery, classification and treatment.

With the continuous booming of well-featured lncRNAs in HCC from 2016 to now, a great quantity of newly-presented researches is coming into being on the relationship between lncRNAs and HCC. Here, we reviewed the present comprehension of lncRNAs in HCC. To be specific, we summarized the overview function and study tools of lncRNAs, elaborated the roles of lncRNAs in HCC, and sketched the molecule mechanisms of lncRNAs in HCC. Meanwhile, the utilization of lncRNAs as markers in HCC early-diagnosis and outcome prediction was also highlighted.

Tools for the research of lncRNAs

A mounting number of databases about lncRNAs are developed to assist in the research of lncRNAs that include the functions of lncRNAs under the physiological state and pathological conditions. Over twenty databases touching upon the biological properties of lncRNAs are outlined in Table 1. These databases will dramatically facilitate a better understanding of lncRNAs which are essential members of epigenetic regulation, and their interaction with other RNAs.

Table 1.

Main databases of long noncoding RNAs

NO.	Database name	Availability	Characteristics #	References
1	Arraystar	http://www.arraystar.com/	analysis for expression profiling and for the regulation of RNAs, especially the regulatory ncRNAs	―
2	C-It-Loci	http://c-it-loci.uni-frankfurt.de/	conserved loci and silico screening of tissue-enriched lncRNAs	83
3	ceRDB	http://www.oncomir.umn.edu/cefinder/	miRNA binding sites for a given mRNA target	84
4	CHIPBase	http://rna.sysu.edu.cn/chipbase/	the TF binding sites and motifs, co-expression patterns, ChIP-function and genome browser	85, 86
5	Co-LncRNA	http://www.bio-bigdata.com/Co-LncRNA/	GO annotations and KEGG pathways	87
6	DIANA-LncBase v.2	http://www.microrna.gr/LncBase	expression regulation and the annotation of MREs on the basis of "ceRNA hypothesis"	88
7	GEPIA	http://gepia.cancer-pku.cn/index.html	customizable functions analysis, such as expression, survival and correlation analysis	84
8	Linc2GO	http://www.bioinfo.tsinghua.edu.cn/~liuke/Linc2GO/index.html	comprehensive functional annotations on the basis of "ceRNA hypothesis"	90
9	Lnc2Cancer	http://www.bio-bigdata.net/lnc2cancer	relationships between lncRNA and human tumors	91
10	LncACTdb	http://www.bio-bigdata.net/LncACTdb/	interactions and annotations of lncRNA-miRNA-mRNA	92
11	lncATLAS	http://lncatlas.crg.eu/	subcellular localization of lncRNAs	93
12	lnCeDB	http://gyanxet-beta.com/lncedb/	lncRNAs acting potentially as ceRNAs	94
13	LNCipedia	http://www.lncipedia.org	basic transcript information and structure; protein coding potential and miRNA binding sites; downloads of lncRNA sequences and structures	95, 96
14	LncRBase	http://bicresources.jcbose.ac.in/zhumur/lncrbase/	transcript features, microarray probes and lncRNA expression	97
15	LncRNA2Function	http://mlg.hit.edu.cn/lncrna2function/	functions, annotations, lncRNA expression values, associations between lncRNAs and functional terms, as well as known functions of human lncRNAs	98
16	lncRNAdb	http://www.lncrnadb.org/	annotations of eukaryotic lncRNAs and references information about these RNAs	99, 100
17	LncRNADisease	http://cmbi.bjmu.edu.cn/lncrnadisease	lncRNA-disease association data, novel lncRNA-disease associations and curated lncRNA interactions	101
18	lncRNASNP	http://bioinfo.life.hust.edu.cn/lncRNASNP/	resources of SNPs in human/mouse lncRNAs and functional SNP selection	102, 103
19	LncRNAWiki	http://lncrna.big.ac.cn/index.php	as a component of ScienceWikis, it affords community-curated resource of lncRNA knowledge	104
20	LncRNome	http://genome.igib.res.in/lncRNome/	types, chromosomal locations, descriptions on the biological functions and disease associations, protein-lncRNA interactions, and genomic variations	105
21	miRcode	http://www.mircode.org	Affords "whole transcriptome" human miRNA target predictions based on the GENCODE gene annotation	106
22	NONCODE	http://www.noncode.org, or http://www.bioinfo.org/noncode/)	conservation annotations and lncRNAs-diseases relationships	107-112
23	ncRNA Expression Database (NRED)	http://www.nred.org/, or http://jsm-research.imb.uq.edu.au/NRED	gene expression information and ancillary data for featured ncRNAs	113
24	RegRNA2.0	http://regrna2.mbc.nctu.edu.tw/	functional RNA motifs and sites	114,115
25	StarBase v2.0	http://starbase.sysu.edu.cn/	miR-function and ceRNA-function web tools	116, 117
26	TANRIC	http://ibl.mdanderson.org/tanric/_design/basic/index.html	the expression profiles of lncRNAs in large patient cohorts of 20 cancer types including TCGA, CCLE and other independent datasets	―
27	TPGLDA	https://github.com/USTC-HIlab/TPGLDA	prediction of relationships of lncRNAs and diseases	118

Dysregulation and roles of lncRNAs in HCC

Tumor progression and recurrence

HCC is a complicated disease referred to multiple factors. A growing body of evidence suggested that lncRNAs involved in the occurrence and development of diverse neoplasm containing HCC. Importantly, some could affect the features of neoplasm, including proliferation, apoptosis, motility, invasiveness and angiogenesis. As an example, HOTAIR expression levels increased in both HCC tumor tissues and HepG2 cells, which promoted HCC progression ²⁹ and were bound up with earlier relapse ^{30, 31}. Those showed the importance of HOTAIR in the evolvement and the relapse of HCC. Furthermore, lncRNAs could be involved in tumor progression and had potential to serve as an attractive target for precision therapy in HCC.

Invasiveness and metastasis

It is well known that invasiveness is an origin in worse disease prognosis and higher recurrence of neoplasm patients ³², which was partly correlated with certain behaviors of cancerous cells. Studies have showed that lncRNAs were related to vital growth-boosting properties and their deregulation dedicated to the survival of cancerous cells. LINC00052 ³³, ZEB1-AS1 ³⁴ and LINC01225 ³⁵ was showed to accelerate cellular motility and invasiveness. In short, these lncRNAs could function as oncogenes via facilitating invasion and metastasis of HCC cells. Inversely, it was indicated that CPS1-IT1 ³⁶, lncRNA XIST ³⁷ and lncRNA FTX ³⁸ were down-regulated in HCC samples and inhibited the relapse and metastasis of HCC cells. These lncRNAs act as tumor suppressor genes via restraining invasion and metastasis in HCC.

Proliferation and apoptosis

A flood of literatures have mirrored that lncRNAs participated in the development of HCC via modulating cell proliferation and apoptosis. On the one aspect, some lncRNAs could accelerate cell proliferation while inhibit cell apoptosis in HCC. It was demonstrated that the expression of XIST ³⁹, lncRNA HOST2 ⁴⁰, HOXA-AS2 ⁴¹, CCHE1 ⁴² and AFAP1-AS1 ⁴³ was significantly elevated in HCC tumor tissues and/or cell lines, which promoted cell proliferation while protected cells from apoptosis in HCC. Conversely, lncRNA AK058003 ⁴⁴, lincRNA-p21 ⁴⁵ and lncRNA XIST ³⁷ were revealed to decrease in HCC tumors, and act as a tumor suppressor, suppressing HCC cellular multiplication and clonality while accelerating cellular apoptosis. These results suggested that lncRNAs played important roles in proliferation and apoptosis of HCC and may serve as latent therapeutic targets of HCC.

Chemo-sensitivity and radio-resistance

LncRNAs have also been reported to function in chemo-sensitivity or radio-resistance through arrest of cell cycle, suppression of apoptosis as well as strengthening of DNA injury repair ^{46, 47}. For instance, lncRNA TUC338 ⁴⁸ and MALAT1 ⁴⁹ was involved in HCC evolution and drug-resistance (such as sorafenib). These suggested that lncRNAs have the potential to serve as new targets for exploiting novel strategies of chemotherapy and radiotherapy in HCC patients.

Angiogenesis

Coupling with the increase of neoplasm sizes, angiogenesis is required. In order to supply nutrient substances and O₂, angiogenesis permits neoplasm to handle its metabolism waste with following access to blood metastasis process. Frequently, neoplasm cell leads to induction of pro-angiogenic signs or blockage of anti-angiogenic markers, which could open an “on-off” of angiogenesis. LncRNAs are mirrored to serve as a key player in the regulation of angiogenesis. LncRNAs contributed to abnormal hypervascularity of HCC and afforded a novel landscape into the mechanism of neoplasm angiogenesis.

Molecular mechanisms of lncRNAs in HCC

Epigenetic regulatory lncRNAs

LncRNAs have been shown to excert regulatory roles at nearly all stages of gene expression ⁵⁰, from targeting epigenetic modification, transcriptional regulation, interactions of lncRNAs-proteins, lncRNAs-miRNAs-mRNAs, lncRNA-lncRNA, autophagy as well as signaling pathway to modulation of mRNA stabilization. LncRNA GIHCG promoted HCC progression through epigenetically upregulating H3K27me3 levels and DNA methylation contents ⁵¹. LncRNA ZNFX1-AS1 suppressed HCC cell growth through modulating miR-9 methylation ⁵². Linc00441 promoted HCC tumorigenesis in a H3K27 modification-dependent manner ⁵³.

Transcriptional regulatory lncRNAs

Several lncRNAs could serve as transcriptional regulators. It was reported that CCAT2 ⁵⁴ and HOTAIR ⁵⁵ accelerated EMT process mediated by transcription factor (TF) Slug and Snail in HCC, respectively. LncRNA ZEB1-AS1 promoted neoplasm invasiveness and indicated an adverse clinical outcome via positively regulating the ZEB1 expression in HCC ³⁴. Meanwhile, lncRNA CPS1-IT1 suppressed HCC aggressivity via controlling HIF-1α activity ³⁶. LncRNA uc.338 was found to promote cellular growth via modulating of CDKN1A transcription in HCC ⁵⁶.

LncRNAs-proteins interactions

LncRNA HNF1A-AS1 repressed NKD1 and p21 expression via interacting with EZH2, and then promoted HCC cell proliferation ⁵⁷. LncRNA NEAT1 modulated hnRNP-A2 level to facilitate cellular multiplication and invasiveness in HCC cell lines ⁵⁸. LncRNA TUC338 targeted RASAL1 to be involved in sorafenib-sensitized HCC cells ⁴⁸. LncRNA BANCR elevated the protein level of VIM while lowered the protein content of E-cad to cripple cellular malignant degree ⁵⁹. The lncRNA EGFR-AS1, a target of GHR, up-regulated the expression of EGFR in HCC ⁶⁰. To summarize, lncRNAs-proteins interactions functioned as a vital mechanism of lncRNAs in HCC.

LncRNAs-miRNAs-mRNAs interactions

It has been demonstrated that microRNAs (miRNAs) functioned crucially in many caners, including HCC ^61-63. LncRNAs could share miRNA recognition elements (MREs) with mRNAs and modulate the function of mRNAs with miRNAs-mediated mechanisms, where lncRNAs were called competing endogenous RNAs (ceRNAs) ^64-66. For instance, lncRNA SNHG12 - miR-199a/b-5p - MLK3 - NF-κB signaling ⁶⁷ accelerated tumorigenesis and metastasis in HCC. LncRNA UCA1 - miR-203 - Slug axis ⁶⁸ and lncRNA Ftx - miR-545 - RIG-I network ⁶⁹ were involvement in HCC progression. The HIF-2α - MALAT1 - miR-216b axis regulated multi-drug resistance of HCC cells ⁴⁹. Overall, lncRNAs could serve as “miRNA sponges” to share the same MREs with mRNAs.

Others

LncRNAs - lncRNAs interactions

LncRNAs functioned vitally in a variety of biology processes. Frequently, the occurrence and development of neoplasm frequently was attributed to the interaction effects of some lncRNAs. LncRNA-lncRNA synergistic networks were beneficial to explore clinically related lncRNAs in neoplasm ⁷⁰. Reports suggested that LncRNA HULC cooperated with MALAT1 to promote CSCs proliferation in HCC ⁷¹. And the overexpression of HULC along with MALAT1 increased the binding of RNA pol II, P300, CREPT to TRF2, which triggered the upregulation, phosphorylation and SUMOylation of TRF2 ⁷¹. Indeed, lncRNAs could interact with other lncRNAs.

Autophagy

Autophagy is a cellular degradation pathway that is essential to maintain cell physiology progress, and its disruption leads to multiple diseases in humans ⁷². Recently, macro-autophagy/autophagy has emerged as a promising therapeutic target in various types of solid tumor treatment ⁷³. LncRNAs exerted crucial roles in the regulation of autophagy lately, which made them act as potential biomarkers of disease phenotypes ⁷⁴. LncRNA HULC could induce a protective autophagy ⁷⁵. LncRNA HNF1A-AS1 served as a autophagic accelerator in HCC ⁷⁶. And the lncRNA HOTAIR activated autophagy ³⁰. These studies indicated that the lncRNAs-autophagy networks are likely to supply additional insights into treatment interventions and markers evaluation in human diseases, especially in cancers.

Signaling pathway

As we all know, signaling pathways functioned considerably in the development of HCC and presented some therapeutic strategies based on in vivo and in vitro findings. LncRNA T-UCR severed as a potential growth driver gene modulated by the Wnt/β-catenin signaling in hepatobiliary carcinogenesis ^{77, 164}. LncRNA MEG3 could activate ER stress and p53 signaling related to NF-κB pathway, which followed by suppressing cell multiplication and promoting apoptosis ⁷⁸. Lnc-DILC was downregulated in liver cancer stem cells and mediated intrahepatic inflammation via governing the cross-linking of TNF-α - NF-κB pathway with IL-6 - STAT3 pathway ⁷⁹.

mRNA stabilization

Transcription activity and post-transcription mechanisms involved in lncRNAs could modulate ribonucleotide reductase followed by altering the stability of message RNAs ⁸⁰. In turn, mRNA steady state levels could directly influence their expression ⁸⁰. The lncRNA MALAT1 accelerated arsenite-induced glycolysis, which was mediated via HIF-1α mRNA stabilization in human liver L-02 cell lines ⁸¹. LncRNA AK058003 could reduce the expression of mRNA stabilizing protein HuR and act as a precursor of miR-15a to suppress γ-synuclein-mediated cell proliferation and the metastasis of HCC ⁴⁴. In addition, lncRNA HULC triggered autophagy via stabilizing Sirt1 and attenuated the chemosensitivity of HCC cells ⁷⁵. These studies suggested that some lncRNAs participating biological processes may be mediated by selective stabilization of mRNAs.

Potential clinical application of lncRNAs in HCC

LncRNAs for diagnosis of HCC

A growing amount of evidence supports that benefiting from their attractive characteristics, lncRNAs are potential indicators for human diseases including HCC. The majority of lncRNAs displayed the trait of strict tissue-specific and neoplasm-specific expression. Frequently, distinct upregulation or downregulation of lncRNA was found in neoplasm tissues in comparison with normal samples. Meanwhile, a few lncRNAs were declared to be existed in urine, blood and other body fluids, which could be easily acquired through the non-invasive way as much as possible. What's more, utilizing lncRNAs as indicators of neoplasm states was superior to protein-coding RNAs, due to the more representative expression of lncRNAs ⁸². Compared with current varying greatly biomarkers in protein coding genes, lncRNAs are likely to be advantageous indicators on the strength of their specificity and easy acquirement.

The pooled sensitivity and specificity of HCC patients predicted by lncRNAs were showed as Table 2. These lncRNAs are likely to act as valuable indicators for neoplasm diagnosis and have potential application value for clinic diagnosis of HCC patients.

Table 2.

Application as diagnostic index of lncRNA in hepatocellular carcinoma patients

LncRNAs	Functions	AUC	Sensitivity	Specificity	References
2-lncRNA signature (PVT1 and uc002mbe.2)	distinguishes HCC patients from the healthy population	0.764 (0.684-0.833)	60.56 %	90.62 %	119
CCHE1	discriminates tumor tissues from normal tissues	0.9262	―	―	42
CRNDE	distinguishes tumor tissues from adjacent normal tissues in HCC	0.699	―	―	120
DANCR	differentiates patients with HCC from HVs and patients with CHB and cirrhosis	0.868	83.8 %	72.7 %	121
	differentiates patients with HCC from CHB and cirrhosis	0.864	80.8 %	84.3 %	121
DGCR5	discriminates tumor tissues from normal tissues	0.782	63.3%	83.3%	122
LINC RP1130-1	distinguishes HCC from adjacent normal tissues	0.74	―	―	123
MALAT1 (plasma)	discriminates HCC patients and hepatic disease patients	0.66	51.1 %	89.3 %	124
JPX	discriminates between HCC patients and controls	0.814	100.0 %	52.4 %	125
JPX and AFP	discriminates between HCC patients and controls	0.905	97.1 %	72.2 %	125
SPRY4-IT1	differentiates HCC patients from controls	0.702 (0.609-0.796)	87.3 %	50.0 %	126
SPRY4-IT1 and AFP	differentiates HCC patients from controls	0.800 (0.706-0.874)	87.3 %	65.0 %	126
UCA1	discriminates HCC patients from healthy controls	0.91	91.4 %	88.6 %	127

LncRNAs as prognostic biomarkers for HCC

Accumulating reports declared that lncRNAs have potential to serve as prognostic predictors in neoplasms containing HCC patients. A variety of lncRNAs were demonstrated to exhibit abnormal expression in HCC, which were significantly correlated with the survival time and were independent outcome predictors in HCC patients (Table 3). Therefore, lncRNAs might be acted as potential and useful prognostic indicators in HCC.

Table 3.

Prognostic abilities of lncRNAs for hepatocellular carcinoma patients in Cox proportional hazards model

LncRNAs	Univariate analysis		Multivariate analysis		References
	HR (95% CI)	p	HR (95% CI)	p
OS
JPX (low vs. high)	2.283 (1.211-4.304)	0.010	2.294 (1.178-4.468)	0.015	125
XIST (low vs. high)	2.155 (1.136-4.088)	0.003	2.207 (1.152-4.225)	0.017	125
CCHE1 expression (Low vs. High)	2.246 (0.605-5.172)	0.027	0.977 (0.312-3.434)	0.041	42
lncRNA SNHG15 (High vs. Low)	3.017 (1.448-6.221)	0.018	2.247 (1.331-6.255)	0.001	128
SNHG20	4.440 (2.254-8.743)	0.000	3.985 (1.981-8.017)	0.000	129
lncRNA GAS5 (Low vs. High)	―	<0.0001	3.257 (2.418 - 8.839)	<0.001	130
CARLo-5 (Low vs. High)	3.267 (1.620-6.271)	0.014	2.981 (1.316-3.952)	0.011	131
SNHG3	4.442 (2.368-8.332)	0.000	3.464 (1.820-6.594)	0.000	132
CCAT2 expression (high vs low)	2.118 (1.245-3.603)	0.006	1.849 (1.064-3.213)	0.029	54
UC001kfo (High vs. Low)	1.876 (1.098-3.207)	0.021	1.751 (1.017-3.015)	0.043	133
lnc-DILC (low vs. high)	2.618 (1.136-6.036)	0.024	2.465 (1.03-5.9)	0.043	79
RFS
CARLo-5 (Low vs. High)	2.873 (1.669-5.852)	0.005	1.810 (1.562-5.177)	0.010	131
lnc-DILC (low vs. high)	2.142 (1.025-4.477)	0.043	1.499 (0.651-3.451)	0.342	79
PFS
UC001kfo (High vs. Low)	2.147 (1.260-3.658)	0.005	1.975 (1.149-3.394)	0.014	133

Conclusion and future prospective

In a nutshell, lncRNAs have the potential to serve as promising biomarkers for tumor progression and recurrence (lncRNA HOTAIR, SNHG1, HULC, MALAT1, CRNDE, GIHCG, UCA1, lncSox4 and lncBRM), as well as for invasion and metastasis (SNHG20, CCAT2, HOST2, Linc-cdh4-2, LINC00052, AFAP1-AS1, ZEB1-AS1, ZEB2-AS1, LINC01225, UC001kfo, SPRY4-IT1, Unigene56159, HULC, plncRNA-1, SchLAH, lncRNA-AK058003, CPS1-IT1, XIST, FTX, TUSC7 and GAS5). Besides, lncRNAs are involved in HCC through affecting on cell proliferation and apoptosis (UC001kfo, SPRY4-IT1, ZEB2-AS1, RBMY2FP, uc.338, UCA1, XIST, HOST2, HOXA-AS2, CCHE1, CCAT2, SNHG1, HNF1A-AS1, PCAT-1, AFAP1-AS1, HULC, MALAT1, lncCAMTA1, lnc-DILC, lncRNA-AK058003, ZNFX1-AS1, lincRNA-p21, XIST, FTX and GAS5), as well as angiogenesis (lncRNA TUG1 and HULC). Additionally, lncRNAs serve as a conceivable indicator for chemo-sensitivity and radio-resistance (HULC, RP11-134G8.8, RP11-363E7.4, RP1-193H18.2, TUC338 and MALAT1; Figure 1, Table S1). Meanwhile, lncRNAs exerted their efficient and effective actions in mechanisms of interaction with proteins/ miRNAs/ mRNAs/ lncRNAs, in epigenetic regulation/transcriptional regulation, as well as in regulation of autophagy, signaling pathway and mRNA stabilization in HCC on the horizon of the current studies (Figure 1, Table S1). Furthermore, these lncRNAs might be promising indicators for disease diagnosis (Table 2), prognosis and recurrence prediction (Table 3), and even be new up-and-coming targets for therapeutic intervention of HCC.

Figure 1.

Dysregulation and functional roles of lncRNAs in hepatocellular carcinoma.

Yet, the precise biological function and molecule mechanisms of lncRNAs in HCC remained uncharacterized. Thus, further exploration and validation researches are required for illuminating the intricate mechanisms (especially in epigenetic and transcriptional modulation of lncRNAs) as well as the clinical utilizations of lncRNAs in HCC.

Supplementary Material

Supplementary table.