| Background and Aims:In recent years,studies have shown that long non-coding RNAs(lncRNAs)play a key regulatory role in the biological processes of cancer.Hepatocellular carcinoma is one of the most fatal cancers in human cancer.However,there are many unknown mechanisms for the role of lncRNAs in hepatocellular carcinoma(HCC).Abnormal lipid metabolism is a major sign of cancer and the liver as a central metabolic organs,the occurrence of HCC is always accompanied by changes in lipid metabolism,fatty acid metabolism is mainly regulated by the transcription factor SREBP-1c,the target genes include ACC and FASN which are key genes in the synthesis of fatty acids.It is one of the hot spots to understand the mechanism of lncRNAs in the lipid metabolism of HCC in the field of HCC.We found that the expression of long-non-coding RNA SNHG3 in HCC was significantly increased by large-scale chip detection and data analysis.The expression of lncRNA SNHG3 was also reported to be significantly related to the malignancy and poor prognosis of HCC.Therefore,the study elucidates the role and molecular mechanism of SNHG3 in lipid metabolism of HCC,and provides the SNHG3 as a new marker for the diagnosis of HCC and as drug target for treatment.Methods:1.The mRNA and lncRNA transcription levels of cancer and adjacent tissues of 16 patients with HCC were tested by using mRNA-lncRNA hybrid microarray(repeated three times for each sample).The data were normalized and analyzed for differences in the lncRNAs whose expression are More than 2 times,and further screening about the relative expression abundance and the change multiple and the information of the sequence itself has been done.The difference between the expression of the lncRNA SNHG3 in HCC and the different stage was analyzed in TCGA,Oncomine and GEO.The expression of SNHG3 in human normal liver cell line LO2 and HCC cell line Hep3 B,Hep G2,LM3 SMCC7721,MHCC97 H were detected by qRT-PCR.2.The expression levels of SNHG3 variable splice(2238nt and 2346nt)were detected by qRT-PCR.The expression of the two snoRNA-U17 a and U17 b contents in the upstream and downstream genes which were RHS1,PHACTR4 and TRNAU1 AP and SNHG3 were detected by RNAi and qRT-PCR.It was confirmed whether SNHG3 had the function of cis.3.The expression of triglyceride and cholesterol in HepG2 cells were detected after RNAi experiment.and we detected the expression of transcription factor SREBP-1c and its target gene FASN and ACC by qRT-PCR and Western blotting.The SNHG3 overexpression plasmid pcDNA3.0-SNHG3 were prepared and the changes of triglyceride and cholesterol levels in HepG2 cells and the expression of transcription factor SREBP-1c and its target gene FASN and ACC were detected by RT-PCR.4.The expression of SNHG3 in the promoter region of SNHG3 was analyzed.The expression of SNHG3 was detected by RNAi and overexpression of c-Myc.After RNAi experiment,the expression of SRBP-1C expression changes.5.To predict and analyze the microRNAs which bound to SNHG3,and to examine the role of these microRNAs in the development and progression of HCC and the regulation of fatty acid metabolism.expression of SNHG3 gene was detected by Q-PCR.Results:1.Through the analysis of lncRNA chip,542 lncRNAs' expression was more than 2 times,and the relative expression abundance,change multiple and the information of the sequence itself were screened.The relative abundance of SNHG3 in HCC was larger,And the expression difference is more than 10 times.Consistent with the results of the chip,the expression levels of SNHG3 in HCC tissues were significantly higher in the databases TCGA,Oncomine and GEO than in adjacent tissues.QRT-PCR assay showed that the expression level of SNHG3 was significantly increased in HCC tissues and cells in 20 samples.2.SNHG3 is located on chromosome 1(chr1: 28832455-28837404)and overlaps with 3 exons of 5 'end of protein coding gene RCC1,containing polyA tail,with two variable splicing bodies in different HCC cell lines and hepatocellular carcinoma(2238 nt),the longer the size of the variable splice(2346nt)is sometimes undetectable,so we mainly have a shorter length(2238 nt)of the variable splicing body for testing and subsequent experiments.The expression of the two snoRNA-U17 a and U17 b contents who were in the upstream and downstream genes of RCC1,PHACTR4 and TRNAU1 AP and SNHG3 introns did not change significantly after knockdown of SNHG3 by RNAi.3.Gene analysis of SNHG3 expression in chip data showed that SNHG3 was mainly involved in HCC cell cycle,cell migration and Fatty acid metabolic.The data analysis showed that there was a significant positive correlation between SNHG3 and mRNA expression of SREBP-1c which is a key gene of fatty acid metabolism.the expression of SREBP-1c,FASN and ACC were significantly decreased in the cell line after SNHG3 RNA interference,and the expression of triglyceride(TG)and cholesterol was significantly decreased.After overexpression of SNHG3.The expression of SNHG3 and the levels of triglyceride(TG)and cholesterol were significantly increased,and the expression levels of SREBP-1c,FASN and ACC were significantly increased.4.SNHG3 promoter region has multiple c-Myc binding site,After RNAi or inhibitor 10058-F4 restraining c-Myc‘s' expression,SNHG3 expression decreased significantly;on the contrary,after overexpressing of c-Myc,SNHG3 expression Significantly increased,which suggests that SNHG3 is likely to be regulated by transcriptional activation of transcription factor c-Myc,SREBP-1C mRNA expression was significantly decreased by c-Myc RNAi expriment,c-Myc may be able to affect the transcription of SREBP-1C's activity.5.MiR-93,miR-205 / 205 ab,miR-214,miR-221,miR-24,miR-338,miR-10 abc,miR-128,mi R-182 can directly regulate the fatty acid metabolic pathway key transcription factor SREBP-1c or its upstream gene or downstream target gene.Conclusion:1.SNHG3 was significantly overexpressed in HCC tissues and cells,and its expression level was positively correlated with the malignancy and prognosis of HCC.There were two variable splicing bodies in the cell with a length of 2238 nt as the main form;SNHG3'expression does not affect other gene'expression which are near to SNHG3,and it doesn't have the function of cis(cis)regulation.2.SNHG3 was positively correlated with the metabolism of fatty acids and was positively correlated with the expression of SREBP-1c.SNHG3 may mediate transcription factor SREBP-1c to regulate HCC cell fatty acid metabolism.3.c-Myc may be able to affect the transcription of SREBP-1C activity.4.SNHG3 is likely to control the way through the ceRNA SREBP-1c... |
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