Study On Function Of Long Non-Coding RNA ICR In Hepatocellular Carcinoma

Background & Aim:Hepatocellular carcinoma(HCC)is a common malignant tumor with high mortality.HCC patients in China accounts for more than half of the world’s HCC patients.Because of diagnosed at an advanced stage and the heterogeneity of HCC,the prognosis for most patients was poor.It’s well known that the main risk factors for the poor prognosis of HCC patients is tumor recurrence and metastasis.As one common complication of HCC,portal vein tumor thrombosis(PVTT)is formed by the invasion of hepatoma cells to portal vein and associated with poor survival.However,the mechanism underlying PVTT formation remains largely unknown.Understanding the cause of the PVTT is conducive to find new therapeutic methods for HCC patients.LncRNAs are reported to have important epigenetic regulatory roles in diverse biologic cellular processes.Studies have indicated that lncRNAs play important regulatory roles in tumorigenesis and metastasis.Besides,studies have indicated that cancer stem cells(CSCs)are the main factors for the recurrence and metastasis of tumor.Although lncRNAs and CSCs have been found to play important roles in the development and metastasis of tumor,but whether and how they affect PVTT development remains unclear.Purpose of our study is: 1)reveal the gene expression profile of PVTT;2)investigate the role of lncRNA and CSCs in the development of PVTT and the molecular mechanism;3)investigate the possibility of using lncRNA and CSCs as therapeutic targets for HCC with PVTT.Methods1.Analysis of the gene expression in PVTT.cDNA microarray analyses were used to investigate the specially highly expressed lncRNAs and mRNAs in PVTT tissues and the corresponding tumor tissues.Then,the results were validated using the corresponding tumor cell lines.Based on the results of the differential expression genes,GO and KEGG pathway enrichment analysis was used to analyze the abnormal signal pathways and reveal the molecular mechanisms of PVTT.2.Screening of the specific and high-expressed lncRNAs in PVTT.On the basis of the results of cDNA microarray,Blast were used to explore lncRNA molecules which were complementary with the differential expressed mRNAs in the PVTT tissues and the PVTT cell lines CSQT-2.3.Validation of the differential expressed lncRNA in PVTT.SYBRGreen Realtime PCR was used to test the expression of candidate lncRNAs in tumor tissues and tumor cell lines.Combined with bioinformatics methods,RACE-PCR was used to evaluate the coding potential of candidate lncRNA.For the future analysis of its molecule function,FISH was employed to detect its molecular localization in the tumor cells.4.Analysis of the molecule function of specific and high-expressed lncRNAs in PVTT.Over-expression vector and specific target siRNA was transfected into tumor cells to up-regulated or down-regulated the expression of candidate lncRNA.Based on these results,proliferation\metastasis were used to test the function of candidate lncRNA in vitro.The mouse model with subcutaneous tumor was used to investigate the function of candidate lncRNA in vivo.5.Evaluating the molecule mechanisms of candidate lncRNA in PVTT.Combined with bioinformatics analysis,Ch IP assay was used to seek for the transcription factors binding the promoter of candidate lncRNA.Functional acquisition\deletion experiment for TFs was performed by transfected the over-expression vector or specific target siRNA.Then,SYBRGreen real-time PCR method was used to test the expression of candidate lncRNA.ResultscDNA microarray revealed that PVTT and HCC displayed unique coding RNA and lncRNA expression profile.The same results were also found in PVTT cell lineCSQT-2 and HCC cell line-Hep 3B.By the analysis of GO enrichment and KEGG pathways enrichment,the specific expressed genes were involved in series of biology process,such as redox reaction,cell proliferation,drug metabolism,cell adhesion and immune response.The results of cDNA microarray from tumor tissues and tumor cell lines showed that 32 lncRNAs and 26 mRNAs were both differently expressed.Based on these differently expressed genes,Bioinformatic software Blast analysis found that LNC24236 contained about 800 bp consecutive complementary base pairing with ICAM-1 mRNA.The following real time-PCR showed that LNC24236 and ICAM-1 were highly expressed in PVTT tissues and CSQT-2 which compared with HCC tumors and Hep3 B cell respectively.For the functional analysis,ICR expression was upregulated\down-regulated in CSQT-2,Hep3 B or Huh7,and expression of ICAM-1 showed up-regulated\down-regulated accordingly.In addition,the metastasis capability of Hep3 B was significantly enhanced after the up-regulation of ICR.Moreover,downregulation of ICR reduced the stability of ICAM-1 mRNA and its protein level in CSQT-2.And up-regulation of ICR increaced the stability of ICAM-1 mRNA and its protein level in Hep3 B.Additionally,the expression level of ICR was significantly higher in ICAM-1+ tumor cells than that in the corresponding ICAM-1-tumor cells from both tumor tissues and tumor cell lines.Down-regulation of ICR in HCC tumor cells significantly decreased the ratio of ICAM-1+ tumor cells and reduced the ability of sphere formation in sphere formation assay in vitro.In mice tumor model,the in situ injection of letivirus expressing siRNA targeting ICR into the subcutaneous tumor resulted in the decreasing expression of ICR and ICAM-1,decreasing of the ratio of ICAM-1+ tumor cells and decreasd tumor growth.Also,we found that upregulation\down-regulation of Nanog led to the up-regulated\down-regulated of ICR in HCC tumor cells.And Nanog regulated ICR expressin in tumor cells by binding to the upstream DNA sequence of ICR.ConclusionsIn this study,we identified the unique gene expression profile of PVTT tissues,HCC tumor tissues and adjacent tissues.Among the differently expressed lncRNAs,ICR was specificly highly-expressed in PVTT tissues and CSQT-2 cell lines.ICR regulated ICAM-1 expression by increasing the stability of its mRNA through RNA duplex formation.ICR can promote the metastasis of tumor cells and enhance the selfrenewal ability of ICAM-1+ HCC CSCs.Nanog is able to activate the expression of ICR in tumor cells by binding to the upstream DNA sequence of ICR.