The Study About The NLRP6 Expression Level In Gastric Cancer And The Effect Of Proliferation In Gastric Cancer Cells

Objective:In this study,we investigated the expression of NLRP6 in gastric cancer tissues and gastric cancer cell lines,and researched the proliferation in Gastric Cancer Cells,when overexpression / knockdown of NLRP6,and to explore the relationship between NLRP6 and prognosis of gastric cancer patients.Methods:1.The mRNA and protein expression level of NLRP6 in gastric cancer tissues and normal mucosa tissues were detected respectively by the Realtime fluorescent quantitative polymerase chain reaction(qRT-PCR)、 Western-blot and immunohistochemistry.2.The expression level of NLRP6 in differernt gastric cancer cell lines and normal mucosa cell line were detected respectively by using qRT-PCR and Western-blot.3.Construction of recombinant plasmid carrying with the NLRP6 gene,infected MKN45 cell line and AGS cell line with lentivirus vector and obtained stable expressed NLRP6 gene gastric cancer cell lines.By the function experimental,such as MTT assay and tumor formation in nude mice,we detected the effect of biological functions when overpression NLRP6 gene in the gastric cancer cell lines--MKN45 cell line and AGS cell.4.The siRNAs were used to down-regulate the expression level of NLRP6 gene through transfecting AGS cell line.Then the knockdown cell line was used to detect the effect of biological functions.5.The relationship between the expression level of NLRP6 gene and either clinic pathological or overall survival of patients are also analyzed in this study by immunohistochemistry of tissue array.Results:1.The qRT-PCR and Western-blot detection showed that the expression of NLRP6 in the gastric carcinoma tissues is significantly lower than normal gastric mucosa tissues of 32 cases(P < 0.05)and 10 cases(P < 0.05)respectively.Also,Immunohistochemical of tissue array showed the same result.2.The expression level of NLRP6 in 4 gastric cancer cell lines(SGC7901、MKN45、AGS、MGC803)and normal gastric mucosa cell line(GES-1)was detectedby the qRT-PCR and Western-blot.The expression level of NLRP6 of all gastric cancer cell lines were lower than GES-1(P<0.05).3.The stable expressed NLRP6 gene in gastric cancer cell lines(MKN45-NLRP6 and AGS-NLRP6)were obtained.MTT assay showed that the ability of proliferation in MKN45-NLRP6 cell line was significantly weaker than MKN45-Ctrl group(P<0.05).The same result got from AGS-NLRP6 cell line.The number of colony formation in MKN45-NLRP6 and AGS-NLRP6 were both less than the respective control group(P<0.001).The weight and volume of tumor in the nude mice,which subcutaneous injected with MKN45-NLRP6 cell line was less than MKN45-Ctrl group(P<0.01).4.Compared to the AGS-NC group,the ability of proliferation in the AGS-siRNA was enhanced,after down-regulating NLRP6 gene in AGS(P<0.05).5.The analysis of the clinical pathological parameters in 72 gastric pateints suggested that the expression level of NLRP6 in gastric cancer was not relevant to the patient’s sex,depth of tumor infiltration and distant metastasis as there is no statistically significance(P>0.05),however,it striking related to patient’s age(P<0.05),local lymph node metastasis(P<0.05)and the clinical stage(P<0.05).The Kaplan-Meier survival analysis indicated that patients with high levels of NLRP6 had longer overall survival(P<0.05).Conclusion:1.The expression of NLRP6 in the gastric cancer tissue was lower than the gastric mucosa tissue,which showed that NLRP6 had the function of the tumor-suppressor genes on the tumorigenesis of gastric cancer.2.NLRP6 can suppress the proliferation of gastric cancer cells in vivo and in vitro.3.The high expression level of NLRP6 could improve the prognosis of gastric cancer patients,since the high expression level of NLRP6 was significantly related to patient’s age,local lymph node metastasis,the clinical stage and overall survival.