Screen Of Livin Gene-related MiRNAs In Human Colon Cancer HT-29Cell Line

Objective:1.To investigate the impact of Livin gene inhibited by siRNA on miRNA geneexpression profiling of human colon cancer HT-29cell line.2.To screen out the Livin gene-related miRNAs by bioinformatics technologyand real-time PCR validation,.Methods:1.Experiment were divided into three groups. The transfection of Lipo2000vector-Livin-siRNA was considered as the experimental group; The transfection ofthe Lipo2000vector-nonsense siRNA sequence was considered as the negativecontrol group; The transfection of the Lipo2000was as the blank control group. TheLivin mRNA expression was measured by real-time quantitative PCR after Lipo2000transfecting48h.2.Then miRNA expression profile were observed by miRNA microarray testsbetween Livin-siRNAexperimental group and negative control group HT-29cells.3.Bioinformatics technique was used to predict the Livin gene-related miRNAs.Livin gene-related miRNAs were screened by combining with relevant literature.4.Fluorescent real-time quantitative PCR (real-time PCR,QPCR) analysis wasperformed to validate the differentially expressed miRNAs obtained by screening.Results:1.48h after the transfection of Lipo2000, Livin mRNA relative expression levelsof HT-29cells were0.365±0.072in Livin-siRNA experimental group, while therelative expression of Livin mRNA in negative control group and in blank controlgroup were1.043±0.027and1respectively. Real-time PCR result showed that,compared with the negative control group and blank control group, the Livinexpression in the experimental group decreased obviously and the difference hadstatistical significance（p<0.05）.However,the expression level of Livin mRNAhad noobvious difference between the negative control group and blank control group(p>0.05).2.The miRNA microarray results revealed that miRNA expression patterns changed significantly after the down-regulation of Livin gene expression. Comparedwith the negative control group, Livin-siRNA experimental group had71up-regulated miRNAs and46down-regulated miRNAs. The differences hadstatistical significance (all P <0.05).3.There were60Livin gene-related miRNAs predicted by bioinformaticssoftware. Seven Livin gene-related miRNAs were screened out by combining theresults of miRNA microarray detection, among which hsa-miR-486-3p, hsa-miR-4688, hsa-miR-3918, hsa-miR-762, hsa-miR-3620-5p were upregulated, whilehsa-miR-3135b, hsa-miR-148b-3p were downregulated. It was found throughliterature retrieval that hsa-miR-486-3p, hsa-miR-762, hsa-miR-148b-3p were closelyrelated to the occur and development progress of malignant tumours. So we validatedfurther by means of real-time quantitative PCR.4.48h after the transfection of Lipo2000, relative expression level ofhsa-miR-486-3p, hsa-miR-762, hsa-miR-148b-3p mRNA in HT-29cells were2.254±0.272,2.484±0.337,0.645±0.100in Livin-siRNA experimental group; therelative expression level of hsa-miR-486-3p, hsa-miR-762, hsa-miR-148b-3p mRNAin negative control group and blank control group were0.882±0.067,0.842±0.080,1.083±0.029respectively; the relative expression level of hsa-miR-486-3p,hsa-miR-762, hsa-miR-148b-3p mRNA in blank control group were1. After theinhibition of Livin gene expression, the expression level of hsa-miR-486-3p,hsa-miR-762mRNA increased (P<0.05), while the expression level of hsa-miR-148b-3p mRNA decreased (P<0.05). There was no significant difference betweennegative control group and blank control group (P>0.05).Conclusions：1. Liposome-mediated siRNA interference could effectively inhibit theexpression of Livin gene in human colon cancer HT-29cell line.2. After Livin gene expression was inhibited, miRNA expression profileschanged significantly in human colon cancer HT-29cell line.3. hsa-miR-486-3p,hsa-miR-762,hsa-miR-148b-3p were Livin gene-related threemiRNAs.