The Role Of CENPO In The Proliferation And Apoptosis Of Gastric Cancer And Its Mechanism Study

So far,proteomics research found that CENPs contain more than 150 members and constitute a huge protein network system.Depending on the function,CENPs can be divided into several subgroups,including CENP-C,CENP-H/I/K/M,CENP-L/M,CENP-O/P/Q/R/U,and CENP-T/W,CENP-S/X,experiments confirmed that CENPs play an important role in cell mitosis.Recently,more and more reports have suggested that CENPs may play an important role in the development of human tumors,and these results have attracted our attention.In this study,the expression of CENP-O in gastric cancer was analyzed by bioinformatics method,and its expression and function were verified at the cytological level.The effect of CENP-O expression on tumor growth was detected in nude mice in vivo,and gene expression profile was utilized.The chip and bioinformatics methods may be influenced by CENP-O-mediated signaling pathways.The completion of this study may provide new therapeutic targets and prognostic indicators for the treatment of gastric cancer.Methods:1.To analyze the expression of CENP-O mRNA in gastric cancer in TCGA database,and analyze the paired data of 32 gastric cancer and adjacent normal tissues,to explore the relationship between CENP-O expression and gastric cancer.2.The expression levels of CENP-O mRNA and protein in gastric cancer,paracancerous tissues and gastric cancer cells were detected by qRT-PCR and Western Blot.The expression of CENP-O and prognosis were analyzed by tissue microarray in 94 cases of gastric cancer tissues,to explore the correlation between CENP-O expression and gastric cancer.3.Construction of CENP-O-interfering lentiviral plasmid,using gastric cancer cell line and control cells stably and lowly expressing CENP-O,MTT,clonal proliferation assay,flow cytometry experiment,observation of silencing CENP-O expression on gastric cancer cell proliferation,withering the impact of apoptosis.4.Using gastric cancer cell line stably expressing CENP-O,a nude mouse modelof subcutaneous transplantation was established,and the effect of silencing CENP-O expression on tumor growth was observed by tumor volume measurement,tumor weight measurement and imaging analysis.Histochemistry and Western Blot were used to detect the change of CENP-O expression in transplanted tumors,and further confirmed the biological effects of CENP-O expression changes in gastric cancer.5.Using gastric cancer cell lines and control cell lines with low expression of CENP-O,using the method of gene chip detection,screening the signal molecules of the downstream pathway after CENP-O expression changes,and using IPA bioinformatics analysis methods to find CENP-O expression changes.Significantly enriched signal pathways.6.Western Blot method was used to verify the downstream pathway proteins that may be affected by CENP-O,and the expression of related proteins was detected after CENP-O expression was downregulated.The signal pathway molecules of CENP-O regulating proliferation and apoptosis of gastric cancer cells were preliminarily investigated.Results:1.For CENP-O expression analysis,we used 32 pairs RNAseqV2 in the TCGA database to analyze the available data of gastric cancer.The analysis showed that the expression of CENP-O gene in gastric cancer was significantly higher than that in adjacent tissues.2.In the cell line,the expression of CENP-O in gastric cancer cells was significantly higher than that in normal gastric mucosal epithelial cell line GES-1,among which MGC-803 and AGS cell lines had the highest CENP-O expression;in24 pairs of fresh gastric cancer tissues and adjacent gastric mucosa tissues,CENP-O was also found to be highly expressed in gastric cancer.The expression of CENP-O was also detected in gastric cancer using tissue microarray containing 94 samples of gastric cancer and clinicopathological features.The results showed that high expression of CENP-O was associated with tumor TNM stage,tumor size,and lymph node metastasis,and gastric cancer patients with high CENP-O expression had shorter survival than those with low CENP-O.3.The effect of CENP-O expression on gastric cancer cells in vitro: Comparedwith the control group,after silencing CENP-O expression,MTT assay showed that the proliferation ability of MGC-803 and AGS cell lines was significantly weakened;and the ability of MGC-803 and AGS cells to clonal formation decreased;flow cytometry showed that the apoptosis was increased in MGC-803 and AGS cells with CENP-O downregulation.4.Down-regulating CENP-O expression on gastric cancer cells: Using MGC-803 gastric cancer cells to construct a subcutaneous xenograft model in nude mice,the study found that knocking-down CENP-O expression group,transplanting tumor volume and weights were lower than the control group;western-blot analysis showed that the expression of CENP-O protein in the transplanted tumor was lower than that in the control group.5.In the gastric cancer cell line with low expression of CENP-O and control cell line,gene chip detection was used to analysis the downstream gene differential expression,the results showed that 356 genes were down-regulated and 386 genes were up-regulated.IPA analysis showed that after CENP-O expression down-regulation,ATM pathway was significantly enriched,suggesting that ATM may be involved in the mechanism of CENP-O regulating gastric cancer cell proliferation and apoptosis.6.Western-blot assay showed that down-regulated CENP-O expression,ATM protein expression was down-regulated,and CCND1 and c-Jun proteins were down-regulated simultaneously.Suggesting that CENP-O is possible to regulate the proliferation and apoptosis of gastric cancer cells through ATM signaling pathway.Conclusion:1.CENP-O is highly expressed in gastric cancer cells and gastric cancer tissues,which is closely related to the occurrence and development of gastric cancer and prognosis.Low expression of CENP-O can weaken the proliferation of gastric cancer cells in vitro,increase apoptosis.In vivo experiments revealed that the tumor-forming volume of gastric cancer cells with low expression of CENP-O was reduced and the weight was also reduced.2.The results of gene chip and IPA analysis suggest that ATM may be one of the downstream pathways regulated by CENP-O.CENP-O may regulate the expressionof CCND1 and c-Jun protein by regulating ATM signaling pathway,thereby affecting gastric cell proliferation and apoptosis.