The Expression And Role Of SM22? In Colorectal Cancer

Part one The expression of SM22? in colorectal cancerObjective: To explore the differences in mRNA and protein levels of SM22? which were expressed between colorectal cancer tissue and normal tissue adjacent to cancer,as well as the correlation between mRNA and protein expression.Meanwhile,the correlation between expression level of SM22? protein and the clinicopathologic features of patients with colorectal cancer were analyzed.Methods: The expression level of SM22? mRNA was detected by RT-q PCR,and the expression level of SM22? protein was detected by Western blot.The Correlation between mRNA and protein expression of SM22? was analyzed by Pearson Correlation analysis.Results: The RT-qPCR results showed that the mRNA expression level of SM22? was significantly reduced in colorectal cancer tissues compared with the normal tissues adjacent to cancer,and the difference was statistically significant(P<0.001).The Western blot results showed that the expression level of SM22? protein was significantly decreased in colorectal cancer tissues compared with the normal tissues adjacent to cancer,and the difference was statistically significant(P<0.001).Pearson Correlation Coefficients test analysis showed that colorectal cancer tissues SM22? mRNA and protein expression were positively correlated(r = 0.718,P<0.01).In 78 patients with colorectal cancer,there were 50 cases(68.5%)with the characteristic of that the expression of SM22? protein in cancer tissues was lower than that of adjacent normal tissues.The 50 patients with the down-regulated expression of SM22? protein in cancer tissues were defined as low expression groups.In 28 patients(31.5%),the protein expression of SM22? in the cancer tissues was not significantly different or up-regulated compared to the normal tissues adjacent to the cancer and were defined as high expression groups.The expression level of SM22? protein were not related to the tumor differentiation degree,gender,age,clinical stage,tumor site,tumor site and lymph node metastasis of patients with colorectal cancer(P>0.05).Conclusion: The mRNA and protein expression of SM22? in colorectal cancer tissues are down-regulated,and the mRNA expression of SM22? is positively correlated with protein expression level.The down-regulation of SM22? protein expression is not related to the clinicopathological parameters of patients with colorectal cancer.Part two The methylation state of the promoter region of Sm22? gene in colorectal cancer tissueObjective: To detect the methylation status of Sm22? gene promoter region in colorectal cancer tissues and corresponding normal tissues adjacent to cancer.To explore the relationship between the methylation status of Sm22? gene promoter region and the clinicopathological parameters and prognosis of the colorectal cancer patients.Methods: With the application of Methylation specific polymerase chain reaction(Methylation specific PCR)technique,the Sm22? gene promoter region methylation status was detected in colorectal cancer tissues and corresponding normal tissues adjacent to cancer of 78 colorectal cancer patients.The ?2 test was used to analyze the relationship between the methylation of the promoter region of Sm22? gene and clinicopathological parameters between the two groups.Kaplan-Meier method was used to analyze the relationship between the Sm22? gene promoter region methylation status and the overall survival of colorectal cancer patients.Results: The Methylation specific PCR results showed: In colorectal cancer tissues,there were 43 patients(59.0%)in the Sm22? gene promoter region methylated status,and 35 patients in the non-methylated states.In corresponding normal tissues adjacent to cancer,there were 19 cases(21.9%)with methylation in the promoter region of Sm22? gene,and 59 patients(78.1%)in the non-methylated state.The methylation rate of Sm22? gene in colorectal cancer tissues was higher than that in the adjacent normal tissues(?2=15.418,P<0.001).Western blot and Methylation-specific PCR results showed that in the 78 cases of colorectal cancer,there were 40 cases in which the expression of SM22? protein expression was down-regulated and the gene promoter region of Sm22? was methylated.There were 25 cases in which the protein expression of SM22? was increased and the promoter region of Sm22? was not methylated.There were 10 cases in which SM22? protein expression was down-regulated and the promoter region of Sm22? gene was un-methylated.There were 3 cases in which the expression of SM22? protein was increased and the promoter region of Sm22? gene was methylated.The correlation analysis showed that the methylation status of Sm22? promoter region was negatively correlated with the expression of Sm22 alpha protein(r=-0.668,P<0.001).Follow-up for 3 years,the overall survival rates of Sm22? gene promoter unmethylated patiens and methylated patiens were 88.57% and 67.44% respectively.After 3 years of follow-up,the overall survival rate of unmethylated patients was higher than that of methylated patients(P<0.05).Follow-up for 3 years,the relapse-free survival rates of Sm22? gene promoter unmethylated patiens and methylated patiens were 80% and 67.44% respectively.The relapse-free survival rate of unmethylated patients was higher than that of methylated patients(P<0.05).The stratified analysis of the relationship between the methylation status of Sm22? and the clinical pathological features of patients with colorectal cancer showed that the Sm22? gene promoter region methylation level was not related to the patient's gender,age,differentiation,staging,lymph node metastasis,infiltration depth and tumor site(P> 0.05).Conclusion: The methylation rate of Sm22? gene promoter region is elevated in colorectal cancer tissues and negatively correlated with protein expression.The high methylation rate in the promoter region of Sm22? is related to poor prognosis in patients with colorectal cancer,but it is not related to the clinicopathological features.Part Three Effect of silencing SM22? expression on the biological behavior of human colon cancer cell line HCT116Objective: To observe the effect of silencing SM22? expression on human colon cancer cell line HCT116 proliferation,clone formation,cell cycle and MMP-9 expression.To investigate the mRNA and protein expression of SM22? after demethylation by 5-Aza-2-Deoxycytidine.Methods: After the human colon cancer cell line HCT116 was transfected with siRNA-Sm22?,RT-qPCR was used to detect the mRNA expression of Sm22? and Western blot was used to detect the expression of SM22? protein.After silencing SM22? expression,the proliferation activity of HCT116 cells was detected by MTS test,the cloning ability of HCT116 cells was detected by plate cloning experiment,flow cytometry was used to detect the effect of HCT116 cell cycle.After silencing SM22? expression,RT-qPCR was used to detect the mRNA expression of MMP-9 and Western blot was used to detect the protein expression of MMP-9.After demethylation by 5-Aza-2-Deoxycytidine,The mRNA expression of Sm22? was detected by RT-qPCR and the protein expression of SM22? was detected by Western blot.Results: The results of RT-qPCR showed that the Sm22? gene mRNA expression level of the siRNA group was lower than that of the siRNA-NC and untransfected group after siRNA-SM22? being transfected 48 hours later(P<0.05).Western blot results showed that the SM22? protien expression level of the siRNA group was lower than that of the siRNA-NC and untransfected group(P<0.05).The results of MTS showed that the OD value of human colon cancer cell line HCT116 in siRNA group was higher than that in si RNA-NC group and untransfected group after cultivating 48 hours,72 hours and 96 hours(P<0.05).The results of plate cloning experiment showed that the number of clones in the siRNA group was significantly increased compared with the siRNA-NC and non-transfection group(P<0.05),while the number of clones in the siRNA-NC group was not significantly different from that in the non-transfection group(P>0.05).Flow cytometry detection results showed that the Proliferation index in siRNA group was significantly increased compared with non-transfection group(P<0.05),while the Proliferation index in si RNA-NC group was not significantly different from that in the non-transfection group(P>0.05).RT-qPCR test showed that after silencing SM22? expression,compared with the non-transfection group,the mRNA expression level of MMP-9 in the siRNA group was significantly increased(P < 0.05).While there was no significant difference between the siRNA-NC group and the non-transfection group(P>0.05).Western blot showed that after silencing SM22? expression,compared with the non-transfection group,the protein expression level of MMP-9 in the siRNA group was increased(P < 0.05).While there was no significant difference between the siRNA-NC group and the non-transfection group(P>0.05).RT-qPCR results showed that after Sm22? demethylation by 5-Aza-2-Deoxycytidine,the mRNA expression levels of SM22? in treated group was higher than that in untreated group(P<0.05).Western blot tests showed that the protien expression levels of SM22? in treated group was higher than that in untreated group(P < 0.05).Conclusion: Silencing SM22? expression could promote the proliferation activity,clone formation capacity and MMP-9 expression of human colon cancer cell line HCT116.The mRNA and protein expression level of SM22? is increased after SM22? demethylation by 5-Aza-2-Deoxycytidine.Conclusion:1.The mRNA and protein expression of SM22? in colorectal cancer tissues are down-regulated,and the mRNA expression level of SM22? is positively correlated with protein expression level.The down-regulation of SM22? is not related to the clinicopathological parameters of patients with colorectal cancer.2.The methylation rate of Sm22? gene promoter region is elevated in colorectal cancer tissues and negatively correlated with protein expression.The high methylation rate in the promoter region of Sm22? is related to poor prognosis in patients with colorectal cancer,but it is not related to clinicopathological features.3.Silencing SM22? expression could promote the proliferation activity,clone formation capacity and MMP-9 expression of human colon cancer cell line HCT116.The mRNA and protein expression level of SM22? is increased after SM22? demethylation by 5-Aza-2-Deoxycytidine.