Identification And Function Study Of Long Noncoding RNA TSG5 In Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma(HCC)is the sixth most common and the second most lethal cancer worldwide [1],with increasing incidence in many developed countries,including China [2].The risk factors for HCC include hepatitis B and C viral infection,aflatoxin-B exposure,alcohol consumption and so on.Hepatitis viral infection currently remains the major risk factor for HCC in China.Despite major advances in the development of treatment approaches for the disease,including surgery,chemotherapy,target therapy,improvements in disease-free survival and long-term prognosis for patients with HCC remain unsatisfactory [3].Therefore,it is of great clinical significance to deeply understand the molecular mechanism of the occurrence and development of HCC in China,and to find novel molecular targets for early diagnosis and treatment.With the development of genomics,it has been found that there are a large number of noncoding genes in eukaryotic genomes with limited coding potential.Long noncoding RNAs(lncRNAs)are defined as transcripts longer than 200 nucleotides(nt)[4].lncRNAs is widely transcribed in eukaryotes,and it is not homogeneous [5].Previous studies have shown that lncRNAs plays a crucial role in tumor cell proliferation,cell cycle,apoptosis,migration,autophagy,cell stemness and other physiological processes.Dysregualtion of lncRNAs can lead to a variety of diseases,including tumor.Most lncRNAs are involved in multiple stages of tumor development,and they are associated with the malignant phenotype of tumor cells,such as abnormal proliferation,anti-apoptosis,tumor invasion and metastasis,chemoresistance,etc.However,the expression and function of lncRNAs in cancer progression are still unclear.In this study,we detected the expression of lnc RNAs in 43 pairs of HCC tissues and corresponding adjacent tissues by lncRNAs microarray,and found the long noncoding RNATSG5 is one of the most significantly down-regulated lncRNAs in HCC tissues.All results above suggest TSG5 may be involved in tumorigenesis.Here,we further analyzed its expression,functions and in-depth mechanisms in HCC.Methods:The expression of lncRNAs was screened in 43 HCC tissues as well as its corresponding normal tissues,and found the expression of TSG5 is significantly decreased in HCC.There results were further confirmed by quantificational real-time polymerase chain reaction(qRTPCR).Besides,the expression of TSG5 was verified by qRT-PCR in another cohort of HCC tissues demonstrating that TSG5 expression is lower in HCC than in normal tissues.Moreover,we obtained the full-length sequence of TSG5 by RACE(Rapid Amplification of cDNA Ends)technique.We employed lentivirus system to up-and down-regulation of TSG5 in HCC cell lines to explore its biologic function both in vivo and in vitro.Finally,we analyzed the molecular mechanism of TSG5 in HCC by RNA-pulldown,mass spectrometry,RNA immunoprecipitation(RIP)and chromatin isolation by RNA purification(ChIRP)as well as bioinformatics techniques.Results:A number of lncRNAs were aberrantly decreased in HCC by comparing tumor to peritumoral tissues using microarrays,including TSG5.Using quantitative PCR(qRT-PCR),we confirmed that TSG5 was down-regulated in 12/16 of HCC.In line with the microarray analysis,TSG5 had considerably lower expression in another cohort of HCC samples.Using 5’ and 3’ rapid amplification of cDNA ends(RACE)on RNA extracted from the HepG2 cell line,we showed that the TSG5 transcripts was 887 nt,composed of two exons with a poly(A)tail attached.5’end sequence of TSG5 was consistent with the database annotation sequence in UCSC database,while the 3’end sequence was 108 bp less than the annotation sequence.In addition,a computational analysis of the TSG5 sequence suggested no coding potential.By overexpressing the constructs in HEK293 T cells,we found that while fused TSG5 ORFs(Open Reading Frames)as well as deletes with Flag tag could be expressed as a transcript,no fused protein was produced.RNA fluorescent in situ hybridization(RNAScope)and reverse transcription(RT)-PCR of nuclear and cytoplasmic fractions suggested that TSG5 was located in the nucleus.Collectively,these results indicated that TSG5 is a long noncoding RNA which is down-regulated in HCC compared to adjacent normal tissues.To investigate the role of TSG5 in HCC,the lentivirus-mediated RNA interference(RNAi)was used to knockdown TSG5 in HepG2 cells and HCC-LM3 cells.Down-regulation of TSG5 expression can significantly enhanced the ability of colony formation,migration and invasion in both cell lines.On the contraty,overexpression of TSG5 in SMMU-7721 cell line by lentivirus system could significantly inhibited the above processes.Furthermore,downregulation of TSG5 expression could promote the ability of subcutaneous tumor formation and distant lung metastasis in HCC-LM3 nude mice model.Down-regulation of TSG5 could induce typical epithelial-mesenchymal transition(EMT)in HepG2 and HCC-LM3 cell lines.The intercellular junction was reduced and the cells were elongated.qRT-PCR revealed the expression of epithelial marker(E-cadherin)was decreased,while the mesenchymal markers(N-cadherin、 Fibronectin、slug、 snail)and EMT related transcription factors(ZEB1、IL17B、SOX4、 Spock2、 DUSP5)are increased.Immunoblot assay further confirmed the above results at protein level.RNA-pull-down assay was performed to identify proteins that are associated with TSG5.RNA-associated proteins were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis,and the bands specific to TSG5 were excised and subjected to mass spectrometry.Among all of the proteins identified by mass spectrometry,only SRSF1 was detected by western blotting from an independent RNA pull-down assay.Subsequently,RNA immunoprecipitation(RIP)with an antibody against SRSF1 was performed by using cell extracted from the HepG2 cell lines.The results of RIP showed TSG5 enrichment using the SRSF1 antibody versus a nonspecific antibody(IgG control).Pulldown of TSG5 using chromatin isolation by RNA purification(ChIRP)recovers SRSF1 protein in HepG2 cells expressing TSG5.Furthermore,downregulation of SRSF1 could paritically block TSG5 silence-induced cell migration and EMT processes,indicating that TSG5 function as a tumor suppressor at least in part by interacting with SRSF1.Conclusion:We observed a significantly decreased expression of TSG5 in HCCs.Then,results of functional experiments indicated that TSG5 controlled cell migration and invasion in vitro as well as metastasis in vivo,by forming a complex with SRSF1.Therefore,our study identifies TSG5 as a functional tumor suppressor in HCC.These findings improve our understanding of the molecular mechanisms underlying HCC progression and provide a potential target for cancer therapy.