MicroRNA Differential Expression Profile In The SMMC-7721 Liver Cancer Cell Line

Background and objectiveMicroRNA (miRNA) are a class of non-coding small RNA (nod-coding small RNA), They are widely distributed in plants and animals with length of about 19nt～23nt single-stranded RNA, its function is a negative regulator of gene expression. Now, with the deepening research of miRNA and many of its un known functions were found, including regulation of cell proliferation, differen tiation and apoptosis, mainly by regulating the expression of signaling molecul es to achieve. These signaling molecules including cytokines, growth factors, tr anscription factor, pro-apoptotic and anti-apoptotic factor. By activating the exp ression of proto-oncogenes and tumor suppressor gene mutation deletionWhich led to the development of tumors. MiRNA are closely related to t he occurrence, development, and prognosis of cancerHCC (hepatocellular carcinoma, HCC) is a common malignant tumor in C hina, it's mortality rate is second highest in the malignant tumor .The occurre nce and development of HCC is a multi-factor, multi-step complex process. Lo oking for occurrence and development related genes of HCC, in order to un derstand the molecular genetic mechanism of hepatocellular carcinoma so as to HCC diagnosis, treatment and providing a theoretical foundation is the current research focus. The research of miRNA provides a new idea and hope for ca ncer researchBut so far, the occurrence of liver cancer and development related to the molecular mechanism of miRNA is still not very clear, and further explore m olecules mechanism of the miRNA in liver cancer will improving the diagnos is and treatment of liver cancer and this is very important, the present study was screening differentially expressed miRNAs between hepatoma cell line SM MC-7721 and human embryo liver cell line CCC-HEL-1 by miRNA microarra y, and to explore the relationship between the occurrence and development of liver cancer.It will clarify the molecular mechanisms of liver cancer.the present study was screening differentially expressed miRNAs between hepatoma cell line SMMC-7721 and human embryo liver cell line CCC-HEL-1 by miRNA microarray, and to explore the relationship between the occurrenc e and development of liver cancer.It will clarify the molecular mechanisms of liver cancer.validate differential expression of MicroRNA to explore the relationship be tween differential expression of miRNA and liver cancer by Real-time quantitat ive PCRMethodsHuman hepatoma cell line SMMC-7721 and human embryo liver cell line CC C-HEL-1 were purchased from Chinese Academy of Medical Sciences, Institute of Basic Medical Cell Center of Basic Medicine,Total RNA extraction by Tr izol method, Identify the quality and concentration of RNA by NanoDro p ND-1000 at 260nm and 280nm and denaturing Agarose Gel Electrophoresi s .Isolating and labeling miRNA by Hy3 and Exiqon's miRCURYTM Array Power Labeling kit, the miRNA was hybridized on miRNAarray, Scanning is performed with the Axon GenePix 4000B microarray scanner.The result of miRNA array was verified by real time PCRResultsBy means of up-regulation or down-regulation up to twice (fold change> 2) a s a screening criteria, This study showed that total 238 differential expressed miRNAs were found, including 154 over-expression and 84 low-expression mi RNAs. Four-fold increase (fold change> 4) of the miRNAs were 64,10-fold i ncrease (fold change>10) of the miRNAs were24,ranked as hsa-miR-205, hsa-m iR-16, hsa-miR-101, hsa-miR-195, hsa-miR-30b, hsa-miR-30e,hsa-miRPlus-E1209, hsa-miR-200a,hsa-miR-378,hsa-miR-15a,hsa-let-7f,hsa-miR-141,hsa-miR-424,hsa-m iR-181 a-2*,hsa-miR-24,hsa-miR-30a,hsa-let-7a,hsa-miR-26a,hsa-miR-26a-2*,hsa-mi R-130a,hsa-miR-15b,hsa-miR-23a,hsa-miR-27a,hsa-miR-193b,hsa-miRPlus-C1005, The highest fold change was up to 52 (P< 0.05). A total of 84 of the miRN As down-regulate, there were 22 miRNAs are up to 4 times (fold change> 4) , there were 10 are up to 5 times (fold change> 5), ranked as hsa-miRPl us-E1120, hsa-miRPlus-A1027, hsa-miRPlus-E1012, hsa-miR-608, hsa-miRPlus-F 1208, hsa-miRPlus-F1205, hsa-miR-767-3p, hsa-miR-373*, hsa-miR-769 -3p, hs a-miRPlus-E1245, which hsa-miRPlus-E1120 reduced 13-fold.Application Realtime PCR to detect the expression of let-7f between S MMC-7721-1 and CCC-HEL-1-4.The dissolution curve has a single peak, This indicating a good specificity of the primers .Compare to CCC-HEL-1 cell line , The expression of let-7f is increase 2.3 times in SMMC-7721 cell line.Application Realtime PCR to detect the expression of miR-205 betweenSMMC-7721-1 and CCC-HEL-1-4.The dissolution curve has a single peak. This indicating a good specificity of the primers .Compare to CCC-HEL-1 cell line. The expression of miR-25 is increase 2.7times in SMMC-7721 cell lineCouclusion 1, The result suggests that there are different miRNA between the human hepatoma cell line SMMC-7721 and human fetal liver cell line CCC-HEL-1 Established between the miRNA expression profiles.2, After validation of Realtime PCR, miR-205 and let-7f were up-regulatio n in liver cancer cell lines3, Hsa-miRPlus-E1120, hsa-miRPlus-A1027, hsa-miRPlus-E1012, hsa-miR-6 08, and other miRNA are down-regulation in liver cancer cell line.