The Regulation Of BRM On HTERT Alternative Splicing And Telomerase Activity

Objectives The gastric cancer(GC)accounts for 783 000 deaths in both sexes worldwide in 2018,being the third leading cause of cancer-related death.With devastating prognosis,the gastric cancer is becoming a heavy burden as well as a threat to human health.Though promoted a lot during the past few decades,the 5-year survival of gastric cancer is still very low.Multiple factors are involved in the carcinogenesis of gastric cancer,including environmental factors and genetic factors.SWI/SNF complex is a ATPase dependent chromatin remodeling complex.The core catalytic subunits of SWI/SNF complex are BRM and BRG1,which are mutually exclusive in the same complex.The BRM is frequently down regulated in multiple types of tumor,indicating that BRM might play a role in tumor suppression despite the ATPase activity.BRM is reported to be an alternative splicing regulator to participate in the expression regulation of several tumor-related genes.hTERT,which is short for the human telomerase reverse transcriptase,is the catalytic subunit of human telomerase and a hallmark of tumor cell.hTERT is partly regulated by alternative splicing to generate multiple incompetent splicing variants.The purpose of this study is to investigate the role of BRM in the regulation of hTERT alternative splicing pattern and telomerase activity.Our research results could shed a light on the telomerase-targeting anti-cancer therapy.Methods There are two major parts of this project:Part ?.Several databases were adopted to analyze the cellular distribution and m RNA expression level and protein expression level in human normal tissue and multiple tumors,the survival analysis with gastric cancer patients,association with Helicobacter Pylori infection,interaction genes and enrichment analysis of BRM and BRG1.Part ?.Lentivirus vectors with sh RNA targeting BRM was adopted to infect gastric cancer cell line BGC-823 to construct steady BRM-knockdown cell line.RT-PCR and immunoblotting were utilized to examine the m RNA expression level and protein expression level of BRM separately.RT-PCR was applied to test the alternative splicing pattern of hTERT.The telomerase activity was tested by TRAP.The cell samples were also subjected to RNA sequencing to analyze genes which were affected upon the knockdown of BRM.ResultsPart ?.The distribution of BRM is uniform in both nucleus and cytoplasm while BRG1 is only displayed uniformly in nucleus.BRM and BRG1 are expressed generally in the majority of the human organs and tissues.The survival percentage of gastric cancer patients with high expression level of BRM is quite higher than patients with low expression level of BRM,while the low expression level of BRG1 is seemed to associated with better prognosis,suggesting the role of BRM and BRG1 in tumor progression might be different.The expression level of BRM and BRG1 in gastric cancer patients infected with H.pylori is relative high and likely to mutate.133 genes were identified to possess interaction with BRM and 213 genes were identified to possess interaction with BRG1.64 genes were shown to be overlapped between the interaction genes of BRM and BRG1.The interaction genes of BRM and BRG1 mainly enriched in the tumor-relative pathways,indicating BRM and BRG1 take important part in carcinogenesis.Part ?.BRM was knocked down efficiently by sh RNA-containing lentivirus vector both in m RNA expression level and protein expression level.The hTERT pre-m RNA alternative splicing pattern was changed upon the knockdown of BRM.The expression level of full-length hTERT transcript increased significantly.Consistently,the activity of telomerase also increased.Altogether,these results suggest that BRM could regulate the telomerase activity through modulating the alternative splicing pattern of hTERT.As a splicing regulator of hTERT,BRM is a promising target of anti-cancer therapy.Conclusion: 1.BRM and BRG1 might play a role in the progression of gastric cancer.The exact function of BRM and BRG1 in tumor might be different which means BRM is likely to be a tumor suppressor while BRG1 is more like a tumor vulnerable factor.2.The knockdown of BRM could modulate the alternative splicing pattern of hTERT and telomerase activity,indicating that BRM is potential to be developed as a target of anti-tumor therapy.