NIBP Expression In Colorectal Cancer And Its Impact On Colon Cancer Cell Proliferation

Background and PurposeColorectal cancer is one of the most common malignant tumors. In our country, with continuous improvement of the peoples living standards, the incidence of colorectal cancer is on the rise. The occurrence of colorectal cancer is the result of the interaction with the number of genetic and environmental factors lead to many genetic changes, which involves multiple signal transduction pathways, such as the NF-κB signal transduction pathways. By NF-κB transcription factor that regulates inflammation and tumor related vital few ways, the signaling pathways affect the survival of cancer cells and angiogenesis, tumor cell motility, and aggressive, thus promotes the proliferation of tumor cells, inhibiting cell apoptosis, and lead to tumor invasion and metastasis.NIBP (NIK and IKKβ binding protein) can links the NF-kB key enzyme-NIK that in the non-canonical way and-IKKβ that in the classic way to become a timer scaffold protein.It has a conserved sequence with Transport protein particles (TRAAP, Transport protein particle), and to TRAAP complex Bet3 role. Therefore, NIBP probably though regulating the NF-κB signaling pathway play a role in protein transport. Study found that the expression of NIBP increase can promote IKK beta and downstream p65 phosphorylation, in the classic NF-κB play a role of the activator. But inhibit the expression of NIBP can reduce the NF-κB pathway activation by IKK beta and NIK mediated, in addition, NIBP may improve the invasive ability of tumor cells caused by TNFastimulate. Recent studies have also found that in human colon cancer tissues NIBP rise obviously, may be associated with the NF-κB activation, and participate in the evolution process of adenoma to carcinoma. Therefore, in this study,to NIBP as the research object, to detect colon cancer tissues the expression of NIBP, p-p65 and c-myc, cyclinDl,to understand their relationships with the clinical staging and clinical pathology of colorectal cancer. Use of genetic engineering to build NIBP carrier, gene silencing in vitro experiment validation NIBP gene expression level changes on the influence of the growth of colon cancer cells, discusses their role in colorectal cancer occurrence, development invasion and metastasis.Methods1.Using immunohistochemical SP method respectively detection the expression of NIBP, p-p65 and c-myc, cyclinD1 in 16 cases of normal colon mucosa,21 cases of colonic tumor and 114 cases of colon cancer patients’colon tissue. To analysis NIBP, etc in colorectal cancer clinical and clinical pathologic stage, to speculation its role in development, invasion and metastasis of colorectal cancer.2.Using genetic engineering, HCT116 colorectal cancer cells as the research object, designing and building of NIBP gene slow virus expression vector pLenti6.3-EGFP-NIBP-miR-1/2/3, designing has nothing to do at the same time sequence as a negative control (negative control, NC). Use NIBP slow virus gene interference and the negative control slow viruses venom Lenti-EGFP infection of target cells and empty cells to be contrast. Extract protein of cells and WB detect target protein expression, screen the best jamming targets. Purpose gene interfere targets lentivirus Lenti-EGFP-NIBP-miR transfect HCT116 cells and then joined BSD (Blasticidin, BSD) to screen, to secure a stable transfect NIBP lower expression of monoclonal HCT116 cells. Using qPCR detection transfect efficiency, to select the high efficiency NIBP interference cell lines used in subsequent cell function test.3.to be Blank control group (Blank group, the infection of HCT116 cell lines), negative control group (NC group, Lenti-EGFP-miR transfect HCT116 cell lines), NIBP low expression group (NIBP group, Lenti-EGFP-NIBP-miR transfect HCT-116 cell lines) and NIBP cells low expression group plus NF-κB classic way activator TNFα group (NIBPT group) as the research object, apply CCK8 to test each group proliferation ability, flow cytometry instrument test each group cell apoptosis and cell cycle distribution, transmission electron microscope obser cell apoptosis, QRT-PCR and Western Blot test the expression of cell cycle related protein c-myc and cyclinD1.Result1.Immunohistochemistry showed that colon cancer tissues positive expression of NIBP, p-p65, c-myc and cyclinD1 were higher than colonic adenoma and normal colonic mucosa tissues, P<0.05. NIBP, pp65, c-myc and cyclinD1 expressed in colon cancer tissue were related to tumor differentiation degree, organization type, infiltration depth, TNM and Duke’s stage, lymph node metastasis and distant metastasis, P<0.05. NIBP, c-myc and cyclinD1 was positively correlated with p-p65.2.Establish NIBP silence carrier and stable transfection HCT116 cell lines. This part of the test using plasmid vector pLenti6.3-MCS/V5 DEST series slow virus expression vector successful build NIBP slow virus gene carrier Lenti-EGFP NIBP-miR (NIBP), Lentil-EGFP-miR (NC) carrier, confirmed by enzyme digestion, recombinant clone, identify vehicle building is successful. Green fluorescence was observed the vector transfect HCT116 cell lines under the fluorescence microscope to select stable transfect of monoclonal cell lines. QRT-PCR and Western Blot confirmed NIBP jamming effect is satisfied. After we design the NIBP gene 1#,2#,3# interference targets lentivirus packaging and infection HCT116 cells,the three targets on reducing efficiency are very obvious, NIBP protein were no obvious stripe. We select the#3 targets for subsequent steady turning strain screening. According to the testing results of QRT-PCR, NIBP disturbance stability strains of HCT116 cells compared to negative control stability strains, NIBP jamming efficiency is hight, NIBP gene interference stability strains of HCT116 cells building is successful.3.Research on the influence of the growth of colon cancer cells with NIBP: CCK-8 test showed that NIBP transfect cells grew slower than blank group, negative control group and NIBPT group, P<0.05. Apoptosis detection for four groups of cells apoptosis rate difference has no statistical significance, P>0.05. Cell cycle detection results show that the NIBP and NIBPT group in Go/G1 phase (respectively 73.500%±3.061%, and 70.133%±5.181%), significantly higher than the HCT116 cells group and NC group (55.933%±3.963% and 52.000%±2.193%), P<0.05. NIBP and NIBPT group in Sphase were 6.700%±1.249% and 9.467%±4.858%, lower than the HCT116 cells group and NC group (11.300%±3.751% and 13.567%±1.563%), but there was no statistically significant difference, P>0.05. NIBP and NIBPT group in G2/M phase were 14.967%±1.041% and 16.067%±1.380%, significantly lower than the HCT116 cells group and NC group (29.933%±2.309% and 31.200%±2.007%), P<0.05.The result show that NIBP transfect colon cancer cell apoptosis has no obvious increase, but their proliferation ability decrease. Transmission electron microscopy observed NIBP transfect cell apoptosis also no significant increase. And QRT-PCR and WB test results show that the four groups expression of c-myc and cyclinDl there was also no significant difference.Conclusion1. NIBP, p-p65, c-myc and cyclinDl high expression in colorectal cancer.2. The expression of NIBP, p-p65, c-myc and cyclinD1 are associated with tumor differentiation degree, infiltration depth, TNM and Duke’s stages and lymph node metastasis and/or distant metastasis.3. NIBP can promote HCT116 colon cancer cell lines proliferation, may be by activating the NF-kappa B classical way.4. NIBP had no obvious effect of apoptosis of HCT116 colon cancer cell lines.5. NIBP had no obvious effect of the expression of c-myc and cyclinDl in HCT116 colon cancer cell lines also.