The Study About The Effect Of Brm On The Alternative Splicing Of HTERT In Immortal Gastric Mucous Epithelial Cell

Objective:The SWI/SNF (switch/sucrose non-fermentable) is a chromatin remodeling complex, which located in genetic coding region, and plays a significant role during the regulation of alternative splicing. Brm (Brahma) is a catalytic subunits of the SWI/SNF chromatin remodeling complex, and have DNA-dependent ATPase activity that drives the remodeling of nucleosomes. The catalytic subunits Brm contributes to the regulation of gene expression by altering chromatin structures, and plays many important roles during epigenetic regulation in many organisms. At the same time, Brm is also a regulator of many gene precursor mRNA (pre-mRNA) during alternative, and could favor inclusion of alternative exons in E-cadherin, BIM, and cyclin Dl, and so on. In our study, we used Brm-siRNA to transfect to immoral human gastric mucous epithelial cell line GES-1, and observed the effect of Brm on hTERT (human telomerase reserse transcriptase) alternative splicing. Our study aimed to demonstrate the molecule mechanism of alternative splicing, which indicated a new experimental method and theory basic.Methods:Immoral human gastric mucous epithelial cell line GES-1 was purchased from Heredity Institution of Tumor Hospital in Beijing. GES-1 cells were cultured in 37℃,5% CO2 for 4-5 generation, then randomly selected part of the logarithmic growth cells and transfected to Brm-siRNA. Firstly, RT-PCR method was used to detect the expression of Brm gene. Secondly, Western bolt method was used to detect the expression of BRM protein, and determined whether Brm was knockdown. Finally, we used GES-1 cDNA as the temple to detect the expression lever of hTERT alternative splicing, and observed the effect of Brm regulation on hTERT. Statistical data analysis was carried out using SPSS version17.0 and one way ANOVA test were used for a statistical analysis. Differences were considered statistically significant when P value was<0.05.Results:1 RT-PCR assay:For blank control and negative control, the expression of Brm between the two groups showed no significant difference (P>0.05). For the Brm-siRNA experimental group, the expression of Brm was obviously lower than the blank control and negative control and difference showed statistically significant (P<0.01).2 Western blot:For blank control and negative control, we could detected the expression of BRM protein between the two groups showed no significant difference (P>0.05). For the Brm-siRNA experimental group, we found the expression of BRM was obviously lower than the blank control and negative control and difference showed statistically significant (P<0.0l).3 RT-PCR assay to detect the expression of hTERT ASV:In our study, we could detect three hTERT alternative splicing variants (hTERT ASV) in each group:α+β+ hTERT ASV,α+β-hTERT ASV andα-β+hTERT ASV. Additional, we found that the expression ofα+β+hTERT ASV was significant lower than the blank control and negative control, and the expression ofα+β-hTERT ASV was significant higher than blank control and negative control, and difference showed statistically significant (P <0.05).Conclusions:After transfected Brm-siRNA to GES-1 cells, the expression of both Brm mRNA and BRM protein were lower than the blank control and negative control, and that indicated Brm-siRNA could knockdown the expression of the target gene and protein. The GES-1 hTERT mRNA could transcript three alternative splicing variants:α+β+ hTERT ASV,α+β-hTERT ASV andα-β+hTERT ASV. Besides, the expression ofα+β+ hTERT ASV showed decreased and theα+β-hTERT ASV showed increased after knockdown Brm gene. Brm could regulate the expression level of GES-1 hTERT ASV, thus changing the activity of telomerase, in this way could imply new methods for tumor clinical research and therapy.