| Gastric cancer is one of the common cancer with high morbidity and mortality inChina.Studies have shown that the occurrence of gastric cancer is affected by variousfactors including genetic and epigenetic alternation, but the mechanism is still not fullyunderstand. The alternation of gene expression may occur in the initial stage of the cancer.The epigenetic changes are usually one of important factores in regulating gene expression.This change may affect transcription of oncogene or tumor suppressor gene, and furtheraffect the development of gastric cancer. In previous work, we found that the transcriptionfactor NFIX may be methylated in gastric cancer. The transcription factor is one of theimportant regulator in gene expression and it can activate or inhibit many target genes.Therefore, the methylation of NFIX may play a key role in occurrence of gastric cancer. Inthis study, we will carry out a serious of studies to investigate the methylation status, theexpression and function of NFIX in gastric cancer, and meanwhile the underlyingmechanisms will also study. This study may provide a new target for clinical intervertionof gastric cancer. 【Objective】1. To identify the methylation status of NFIX in gastric cancer and adjacent normaltissues, and examine the expression of the NFIX in gastric cancer and study therelation ship between expression and methylation.2. To discover the biological effect of NFIX on the progression of gastric cancer.3. To explore the underlying mechanisms of NFIX in development of gastric cancer.【Methods】1. By using BSP to evaluate the methylation status of the NFIX promoter in gastric cacerand matched normal mucosa tissue specimens.2. mRNA and protein levels of DHRS3were determined using real-time qPCR andimmunohistochemical staining, respectively in in gastric cacer and matched normalmucosa tissue specimens.3. Lentivirus expressing NFIX and control were used to infect gastric cell line SGC7901,4. Real-time PCR was used to confirm the expression of NFIX in stable infected cell.5. High content screening, colony formation, transwell, invasion assay, cell cycle wereused to examine biological functions of NFIX in gastric cancer.6. Immunoprecipitation was used to enrich NFIX protein, western blot and silver stainingwere used to identify the enrichment. Q-Exactive mass spectrometry was used to findthe interacting protein of NFIX.【Results】1. The promoter of NFIX was hypermethylated in gastric cancer samples compared tonormal samples, in consistence, mRNA and protein of NFIX was significantlydownregulated in gastric cancer samples as compared with normal mucosa.2. Treatment of GC cells with the demethylating agent5-Aza increased the expression ofNFIX. It is thought to be that the methylation of NFIX gene promoter may beassociated with down-regulation of NFIX expression. 3. We used LV-NFIX and LV-Control to infect SGC7901cell line. Real-time PCRshowed a significant over-expression of NFIX by LV-NFIX infection.4. Ectopic expression of NFIX in SGC7901cells inhibited cell proliferation, reducedcolony formation in vitro, decreased the migration and invasion ability, and inducedearly cell apoptosis and arrested cells in the G1phase.5. We used LV-NFIX-Flag and LV-NC-Flag to infect SGC7901cell line. Real-time PCRand Western blot showed a significant over-expression of NFIX-Flag byLV-NFIX-Flag infection.6. Immunoprecipitation enriched NFIX-Flag protein successfully, and Q-Exactive massspectrometry analysis indicated that RPS6KA1was the one candidate interactingprotein of NFIX.【Conclusion】Our data demonstrated that the expression of NFIX is down-regulated and thepromoter of NFIX is hypermethylated in gastric cancer patients, suggesting that NFIX is atumor suppressor; silencing NFIX may accelerate tumor progression in gastric cancerpatients. Over-expression of NFIX inhabit the progression growth and invasion of gastriccancer cells in vitro. RPS6KA1may be the one candidate interacting protein of NFIX.RPS6KA1may phosphorylate NFIX, then enhance its ability to regulate downstreamgene. |
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