| [Objective] To obtain the differential expression profile of mi RNAs regulated by 14-3-3σ(SFN) through mi RNA microarray technology, verify the reliability of the results by RT-PCR technique, analyze the signal pathway that relates to the differential expression profile of mi RNAs by bioinformatics, clarify the role of the regulatory mechanism between mi RNA and SFN in nasopharyngeal carcinoma.[Methods] The recombinant cell line 5-8F-14-3-3σ(+) with high expression of 14-3-3σand its control cell line 5-8F-14-3-3σ(-) were built. The expression level of 14-3-3σ in cells from the two groups was detected by Western blot, so as to confirm whether there was successful transfection. Cells were collected and RNAs were extracted to detect the total RNA purity. The differential expression profile of mi RNAs regulated by SFN was detected by the mi RNA microarray. Mi Randa software was used to analyze the thermodynamic stability score(mir SVR) and sequence conservation score(Phast Cons) of the 10 target mi RNAs with the highest ratio of down regulation, so as to identify targeted mi RNAs of SFN. By mi Rwalk online software, the target mi RNAs of SFN were predicted and analyzed. By combining the results gained by gene microarray and conducting comparative analysis, the potential target mi RNAs of SFN were identified. Target Scan online software was used to predict the binding sites of target mi RNAs and SFN. The target mi RNA OF SFN was validated by RT-PCR, and compared with the results of microarray. Bioinformatics was used to analyze the signal pathway,biological process, molecular function and cellular component that relate to the differential expression profile of 14-3-3σ-related mi RNAs, thus to find out the key signal pathways.[Results] 1. As detected by western blot, expression of 14-3-3σin 5-8F-14-3-3σ(+) cells was significantly higher than in 5-8F- 14-3-3σ(-) and 5-8F cells, and there was no significant difference in its expression between 5-8F-14-3-3σ(-) and 5-8F cells.2.mi RNA microarray was used to detect the differential mi RNAs between 5-8F-14-3-3σ(+) and 5-8F- 14-3-3σ(-) cell lines and the differential expression profile of 14-3-3σ-regulated mi RNAs was obtained; the mi RNA microarray analysis showed that 239 mi RNAs were differentially expressed after high expression of 14-3-3σ, among which 58 mi RNAs were significantly up-regulated and 181 mi RNAs were significantly down- regulated. 3.Among the 10 targeted mi RNAs with the highest ratio of down regulation, there were 2 qualified mi RNAs(mir SVR≤-0.1, Phast Cons 0.4~0.7) as analyzed by the Mi Randa software. Therefore, mi R-675 and mi R-4253 may be target mi RNAs of SFN. 4. The target mi RNAs of SFN were predicted and analyzed by mi Rwalk online software, and the results showed that among the 10 softwares, 6 predicted that mi R-145 and mi R-220-c could bind with SFN and 5 predicted that in total 18 mi RNAs: mi R-597, mi R-129-3p, mi R-603, mi R-769-3p, mi R-431, mi R-608, mi R-766, mi R-31, mi R- 532-3p, mi R-675, mi R-922, mi R-329, mi R-483-3p, mi R-554, mi R-642, mi R-886- 5p, mi R-362-3p, mi R-486-3p could bind with SFN. Comparative analysis with microarray results showed that mi R-675 might be the target mi RNA of SFN. 5.Targetscan software was applied to predict the binding sites of mi R-675 and SFN, finding that the binding site was located within the 226-232 bases of SFN 3’-UTR, with the binding sequence of 5’-CCGCACC-3’, which was consistent with part of the sequence of mi R-675. mi R-675 could regulate the expression of SFN by binding to its 3`-UTR, and their binding was of specificity, which further illustrated SFN might be the reliable acting target gene of mi R-675. 6. Bioinformatics analysis found that signaling pathways related to target genes regulated by differential mi RNAs mainly included WNT, MAPK and TGF.7.RT-PCR was performed to confirm the expression levels of mi R-675 and mi R-4253. Results showed that expression levels of mi R-675 and mi R-4253 in 5-8F-14-3-3σ(+) were-3.458 and-5.098 times as in the control cell line 5-8F- 14-3-3σ(-), respectively, which was consistent with the results of mi RNA microarray, revealing that the results of gene microarray were reliable.[Conclusion] 1.Differential expression profile of mi RNAs regulated by 14-3-3σ was obtained. After the up-regulated expression of 14-3-3σin NPC cells, 58 mi RNAs were significantly up-regulated and 181 mi RNAs were significantly down- regulated. 2.Bioinformatics analysis results showed that functions involved in the expression profile of 14-3-3σ-related mi RNAs were complex, and the WNT, MAPK and TGF-β signaling pathways might be involved in development and progress of nasopharyngeal carcinoma. 3. mi R-675 was the target mi RNA of 14-3-3σand could bind to 14-3-3σ; SFN might be the target gene of mi R-675. Mutual regulation between mi R-675 and SFN might play an important role in the development and progress of nasopharyngeal carcinoma. |
PDF Full Text Request