| 【Background】Hepatocellular carcinoma (HCC) is the most commonly occurring primarymalignancy of the liver in adults. Despite the therapeutic advances that have been made inHCC, such as surgical resection, liver transplantation, radiotherapy and chemotherapy, theoverall5-year survival rate of HCC patients has not significantly improved. The highmortality and poor prognosis of HCC are mainly attributed to the precise molecularmechanisms underlying liver carcinogenesis and progression, which remain poorlyunderstood. However, with the improvement of techniques in cellular and molecularbiology, many studies have indicated that hepatocarcinogenesis is a complex, multistepprocess characterized by a large number of abnormal molecules, and several HCC-relatedderegulated molecular signaling pathways have been identified. Therefore, it is ofparamount importance to explore the mechanism of the occurrence and development ofHCC from the molecular level and to identify effective therapeutic targets for HCC, whichwill open up new avenues for prevention and treatment of liver cancer. In recent years, the systematical researchers are concentrating on long non-codingRNAs (lncRNAs), which are currently defined as transcripts greater than200nucleotidesin length without obvious protein coding functions. Recent evidence suggests that theinconsequential “dark matter” of the genetic transcription may play a major role inregulating multiple biological processes, including cell growth, cell cycle progression,differentiation, and apoptosis. Furthermore, a large and growing body of literature hasindicated the involvement of lncRNAs in a variety of diseases, especially cancer. Recentstudies have demonstrated that dysregulation of several lncRNAs is related to humancancers, such as breast cancer, gastric cancer, lung cancer, prostate cancer, etc. Moreover,those lncRNAs that have been associated with cancers are often aberrantly expressed andplay functional roles in carcinogenesis, tumor growth, invasion, metastasis, and recurrence,indicating that lncRNAs may act as oncogenes or tumour suppressor genes in thedevelopment of cancers.Compared with previous studies in microRNA (miRNA), the research of lncRNAs isstill in a relatively unknown field. Currently, there is less study on lncRNA in HCC, eventhough several deregulated lncRNAs have been found to be associated with HCC.Unfortunately, the specific functions of some aberrantly expressed lncRNAs are not yetclear and the molecular mechanisms of most of lncRNAs remain largely unknown.Therefore, it is necessary to continue to search cancer-specific expressed lncRNAs andreveal their functions, and further to explore their molecular biological mechanisms,thereby enriching the research in molecular mechanisms of HCC.In the present study，we examine the differentially expressed lncRNAs betweenhuman hepatoma cell lines and normal hepatocyte cell line using a lncRNA microarrayand then screen some deregulated lncRNAs that are associated with HCC, which willprovide experimental evidence for further exploring functions and mechanisms oflncRNAs in the development of HCC in the future.【Aims】The aim of our study is to investigate aberrantly expressed lncRNAs by comparinglncRNA expression profiles using a lncRNA microarray and identify a newfound lncRNA, termed URHC (up-regulated in hepatocellular carcinoma), and then to determine thebiological functions and molecular mechanisms of URHC in tumorigenesis andprogression of HCC, thereby better elucidating the mechanisms of HCC and providingtherapeutic targets for the treatment of HCC.【Methods】1. The lncRNA expression profiles of HepG2、SMMC7721、Huh7human HCC celllines and HL-7702human immortalized normal hepatocyte cells were examined andanalyzed by a lncRNA microarray. We screened several HCC-related differentiallyexpressed lncRNAs from the aspect of expression levels, the length of lncRNAs, fullgenetic sequence, GO annotation, and KEGG pathway analyses. We detected theexpression levels of some differentially expressed lncRNAs using qRT-PCR (quantitativereal-time polymerase chain reaction) and finally determined URHC for further study.2. A total of52HCC tissues and corresponding benign liver tissues were obtainedfrom patients who underwent surgical resection. Expression level of URHC was detectedby qRT-PCR analysis. The association between URHC expression and clinicopathologicalfactors was explored, and a Kaplan-Meier survival analysis revealed the correlation ofoverall survival with URHC expression.3. We observed the function of URHC on biological behavior of HCC cells in vitro.SMMC7721cell with high expression of URHC was selected for study, and thenSMMC7721cell was transfected with specific siRNAs to down-regulate URHCexpression. MTT, EdU incorporation, cell cycle and colony formation assay were used forevaluating cell proliferation. Flow cytometry and Western-blot analysis were used foranalysis of cell apoptosis. Transwell assays were employed for investigating the cellinvasion and migration ability.4. We predicted and determined the targets of URHC by gene mapping analysis andbioinformatics prediction methods. We speculated that ZAK (sterile alpha motif andleucine zipper containing kinase AZK) might be one of the target genes of URHC.qRT-PCR was used to examine the expression of ZAK in HCC tissues, and then qRT-PCRand Western-blot were used to determine the mRNA and protein expression of ZAK while URHC was down-regulated. MTT, cell cycle and Western-blot analysis were used toinvestigate the role of URHC on ZAK in SMMC7721cell that had been transfected withURHC-siRNA, ZAK-siRNA or co-transfected with URHC-siRNA and ZAK-siRNA.5. We explored the molecular mechanisms regulated by URHC. Western-blot wasused to examine expression changes of the key proteins in MAPK signaling pathway, andprotein phosphatase inhibitor PD98059was used and then cell function tests wereperformed to investigate if ERK/MAPK participated in the process that mediated byURHC-ZAK.【Results】1. Hierarchical clustering was used to analyze the differential lncRNA expressionprofiles in HCC cell lines. Compared with normal hepatocytes,1658lncRNAs wereaberrantly expressed in3hepatoma cell lines simultaneously and were regarded asdifferentially expressed lncRNAs (fold change>2), accounting for3.9%of all lncRNAs.Among them,477lncRNAs were up-regulated at least2-fold,1181lncRNAs weredown-regulated at least2-fold，56lncRNAs were up-regulated at least5-fold and131lncRNAs were down-regulated at least5-fold. GO annotation and KEGG pathwayanalyses indicated that those aberrantly expressed lncRNAs were associated with cellularbiological processes, such as cell proliferation, cell cycle progression and apoptosis. Someof the differentially expressed lncRNAs may participate in the cell signaling pathways,such as MAPK, JAK-STAT, Wnt, etc.2. We investigated a novel lncRNA, termed URHC, and evaluated its role in thedevelopment of HCC. Expression profiling using a lncRNA microarray revealed thatURHC was highly expressed in3HCC cell lines compared to normal hepatocytes.qRT-PCR analyses confirmed that URHC expression was increased in hepatoma cells andHCC tissues, which was in accordance with the result of the lncRNA microarray.Moreover, using qRT-PCR, we confirmed that URHC expression was up-regulated in57.7%(30/52) of HCC tissues compared with adjacent benign tissues. Statistical analysesrevealed that high URHC expression levels were significantly associated with both tumorsize (χ2=8.868, P=0.003) and tumor number (χ2=6.857, P=0.009). However, we did not find any correlation between URHC expression levels and other clinicopathologicalfeatures, including gender, age, AFP level, tumor metastasis, PVTT (portal vein tumorthrombi), histological differentiation and AJCC (American Joint Committee on Cancer)stage. A Kaplan-Meier survival analysis revealed that high URHC expression wascorrelated with shorter overall survival after surgery (χ2=4.863, P=0.027).3. qRT-PCR analyses revealed that URHC expression was efficiently inhibited inSMMC7721cells. Compared with the control cells, MTT, EdU incorporation, cell cycleand colony formation assays showed that down-regulation of URHC expression resultedin a decrease in cell growth of SMMC7721cells. In addition, an analysis of apoptosisdemonstrated that URHC knockdown promoted apoptosis. SMMC7721cells in whichURHC had been down-regulated expressed higher levels of Bax and displayed an increasein Bax/Bcl-2protein levels compared with the control cells. Compared with the negativecontrols, significant up-regulation of cleaved caspase3was observed in cells that had beentransfected with URHC-siRNA, and the ratio of cleaved caspase3/caspase3wasremarkably increased. These results indicated that down-regulation expression of URHCmay play a pivotal role in inhibiting cell proliferation and inducing apoptosis. However，URHC had no effect on invasion and migration of SMMC7721cells.4. Gene mapping analysis revealed that ZAK was located near URHC, suggestingURHC may regulate the expression of gene in a close genomic proximity (cis-actingregulation) via a variety of mechanisms. We found that ZAK levels were significantlydown-regulated in HCC tissues compared with the normal controls. A negative correlationwas observed between URHC levels and ZAK mRNA levels in HCC tissues. Transfectionof SMMC7721cells with URHC-siRNA increased the ZAK mRNA levels and relativeprotein expression compared with the normal controls, suggesting that ZAK acted as apotential target for URHC and URHC negatively regulated the expression of ZAK.Through MTT assay, we demonstrated that the effects of URHC knockdown on cellproliferation were partially prevented by the presence of siRNA targeting ZAK inSMMC7721cells. In addition, cell cycle results showed that we found that ZAK silencingwas able to partially abrogate the G0/G1accumulation induced by URHC knockdown. Moreover, we observed that ZAK silencing reduced the expression levels of Bax andcleaved caspase3, and ZAK silencing incompletely abrogated URHC knockdown-mediated apoptosis in SMMC7721cells by quantitative analyses. Based on these findings,we concluded that ZAK mediated the biological effects of URHC on cell proliferation andapoptosis in human HCC.5. Western-blot indicated that ERK1/2，JNK and p38kinase phosphorylation wasdetected in URHC knockdown cells in contrast to the control cells, while URHC-silencingplus ZAK-silencing attenuated the expression levels of phosphorylated ERK1/2,phosphorylated JNK and p38, which were lower in simple ZAK-silencing group,indicating that URHC may regulate cell growth and induce apoptosis by inhibiting ZAKexpression and activity through the inactivation of the MAPK signaling pathway. Tofurther demonstrate that cell growth and apoptosis resulted from URHC-ZAK-mediatedMAPK induction, we used a specific ERK inhibitor, PD98059. An MTT assay revealedthat the inhibition of cell growth induced by URHC down-regulation could be rescued byPD98059treatment. In addition, an apoptosis assay showed that PD98059treatmentattenuated the increased ratios of Bax/Bcl-2and cleaved caspase3/caspase3that had beeninduced by URHC-siRNA. Taken together, our results indicate that URHC promotes cellgrowth and inhibits apoptosis by suppressing ZAK expression through the downstream ofERK/MAPK pathway inactivation.【Conclusions】1. We screened for a novel lncRNA, termed URHC, and found that it wasup-regulated in both hepatoma cell lines and clinical HCC tissue samples. Additionally,our data indicated that its high expression was correlated with tumor growth and poorsurvival in HCC patients, indicating that URHC may display oncogenic activity incarcinogenesis and progression of HCC.2. Decreased URHC expression inhibited hepatoma cell proliferation and promotedapoptosis in vitro, but had no effect on cell migration and invasion，indicating that URHCmay play important roles in HCC growth.3. URHC promoted cell proliferation and inhibited apoptosis by negatively regulating ZAK via inactivation of ERK/MAPK. Therefore, the URHC-ZAK-ERK/MAPK pathwaymay participate in HCC growth through regulating cell proliferation and apoptosis, andmay provide new ideas for studying the molecular mechanism of HCC and develop a newtherapeutic strategy for HCC in the future. |
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