The Influence Of NLRP6 On HCT116 Cells’ Proliferation And Migration And Exprseeion On Colorectal Cancer

Objective: This study interference the NLRP6 expression of HCT116 cells, and observations it influences the proliferation and migration in mice with HCT116 cells. Discussion on the role of NLRP6 in the occurrence and development of colorectal cancer, and lay the foundation for targeted gene therapy for colorectal cancer.Methods: HCT116 cells groups: the control group is Si-CON(NCsh RNA cell suspensions, NLRP6 normal expression), the experimental group is Si-NLRP6(NLRP6-sh RNA cell suspensions, interference and reduced expression of NLRP6). 1) the study first cultured colorectal carcinoma cell lines Lo Vo, SW620, SW480, HCT116, HT29 and SW1116, and using quantitative real-time PCR(q RT-PCR) method to detect the expression of NLRP6 m RNA and detection of NLRP6 protein expression by Western blotting in cell lines, then filter out the high expression of NLRP6 cell line, preparation of follow-up experiments. 2) then transfected with lentiviral packaging plasmids NLRP6-sh RNA into HCT116 cells, interfering with NLRP6 gene expression, to detect the influence of NLRP6 on migration and proliferation of HCT116 cells by cell wound scratch assay and soft agar colony formation assay. 3) detected the expression of P38 MAPK, p-P38 MAPK, AKT, p-AKT, EGFR, MMP-2 and MMP-9 proteins in HCT116 cells by Western blotting. 4) using nude mouse tumorigenicity experiments to detection of interference NLRP6 expressive impact on HCT116 cells in vivo tumorigenicity. The nude mice were randomly divided into two groups: Si-CON group, Si-NLRP6 group, each group of nine mice. Injection of the cells at a concentration of 2×106/ml, each portion inoculation 0.1ml. Feeding mice were sacrificed after four weeks. The tumor weighed after stripping, with Vernier caliper measure maximum tumor diameter a and minimum diameter b, according to the formula V = ab2/2 to calculate the volume of tumor nodules. Meanwhile, anatomy of nude mice with lung, observation of lung metastasis and measured the transfer area, making fresh lung pathological specimens, HE stained microscopic pathologic conditions of the lung. 5) collected 71 cases of colorectal cancer resection specimens and 45 adjacent normal tissue specimens were detected the NLRP6 protein expression by immunohistochemical SP method.Results: 1) q RT-PCR and Western blotting: NLRP6 m RNA and protein are expressed at different levels in six species of colorectal cancer cell lines, the expression of the highest levels in HCT116 cells. 2) cell wound scratch assay: Si-NLRP6 group scratch repair rate is(37.30±1.90)%, Si-CON group is(10.00±1.92)%, Si-NLRP6 group cell migration increased capacity, there was a statistically significant difference(P<0.05). Soft agar colony formation assay: Si-NLRP6 group of cells to form the number of colonies than Si-CON group increased significantly, Si-NLRP6 group of colony formation rate is(63.1±14.2)%, Si-CON group of colony formation rate is(20.1±9.5)%, and that the NLRP6-sh RNA promoting HCT116 cells proliferation, the difference was statistically significant(P<0.05). 3) Western blotting: P38 MAPK, pP38 MAPK, AKT, p-AKT, EGFR, MMP-2, and MMP-9 proteins all have varying degrees of expression in HCT116 cells, and expression level in comparison with the Si-CON group, Si-NLRP6 group of protein expression level was significantly increased, the difference was statistically significant(P<0.05). 4) four weeks after inoculation of HCT116 cells, subcutaneous implanted tumor formation in nude mice. SiNLRP6 group tumor volume was significantly greater than the control group, HE staining more number of cancer cells in lungs, lung metastasis area larger, and more number of lung metastases, the difference was statistically significant(P<0.05). 5) positive expression of NLRP6 protein in the cytoplasm, as brown-yellow or brown granules. NLRP6 protein in adjacent normal tissues positive rate is 68.9%, and the positive rate of colorectal cancer is 42.3%, the difference was statistically significant(?2=7.837, P=0.005). NLRP6 expression in colorectal cancer is not related to the patients’ age, gender, tumor location, histological grades, and depth of invasion(P>0.05). NLRP6 in no lymph node metastasis positive rate is 52.4%, significantly higher than the lymph node metastasis of 27.6%, the difference was statistically significant(?2=4.322, P=0.038). NLRP6 in no distant metastasis positive rate is 47.5%, significantly higher than the distant metastasis of 16.7%, the difference was statistically significant(?2=3.875, P=0.049). In stage TNM I/II NLRP6 positive rate is 55.0%, higher than stage III/IV positive rate of 25.8%, a statistically significant difference(?2=6.100, P=0.014).Conclusions: 1) NLRP6 high expression in colorectal cancer HCT116 cell lines. 2) NLRP6-sh RNA interfere with NLRP6 gene can promote HCT116 colorectal cell proliferation and migration, and raised P38 MAPK, p-P38 MAPK, AKT, p-AKT, EGFR, MMP-2 and MMP-9 protein expression, while promoting the subcutaneous tumor formation in nude mice and pulmonary metastasis occurs. 3) NLRP6 protein is low expression in colorectal cancer tissue and related to lymph node metastasis and tumor metastasis and TNM stage. NLRP6 expression may be associated with the development of colorectal cancer, have a correlation between the invasion and metastasis.