Effect And Mechanism Of P55PIK On Invasion And Metastasis In Colon Cancer

Objective:To change the p55PIK content of the colorectal cancer cell lines and detected the change of mobility in vitro and in vivo.Method:Firstly, we validated the colon cancer cell lines'p55PIK content by western blot, then construct vector which containing p55PIK gene sequence:pcDNA3-p55PIK vector, through transfection method then transfected it into HT29and SW480cell lines, and screened two stable cell lines which overexpression p55PIK by using drugs neomycin. And we got lentivirus pLKO1.0-sh-p55PIK by using lentiviral packaging technology, then infected LoVo cells to got a stable cell lines with p55PIK silence. The alteration of cell lines in movement ability was derived by a scratch test and Transwell test, and at the same time, we used single cell colony formation assays and BrdU corporation assay to detect the changes of colony formation ability in the new stable cell lines. In the last, we use a colon cancer liver metastasis model in nude mouse to validate the role of p55PIK play on colon cancer metastasis ability.Results:the results showed that the three cell lines stem from primary clone cancer, HT29, HCT116and SW480, the p55PIK content of which is extremely low, and the p55PIK of SW20which comes from abdominal metastasis lymph node was slightly higher, the LoVo cell lines, which origin from supraclavicular lymph node metastasis clone, was with obvious endogenous p55PIK. And we successfully got p55PIK overexpression cell lines, HT29-p55PIK and SW480-p55PIK, and the p55PIK LoVo-sip55PIK cell lines which with p55PIK silence by gene cloning technology and lentiviral technology. At the same time we got controls cell lines which include the empty vector. The scratch test revealed that the mobile ability of SW480cell with p55PIK overexpression enhancement, the mean migration distance of overexpression group in the12hour compared with controlled group was1.7times (p=0.03), the24h time pointwas1.46times (p=0.046). In Transwell experiment, the p55PIK overexpression SW480, which group cell number of moved to the down chamber in48hours in the migration experiment was2.29times compared with the control group (p=0.009) and1.63times in invasion experiment(p=0.003); the24hours mean migration number of p55PIK knockdown LoVo cells were (30+14), which is significantly lower than control group (58+7),about51.7%of the control cells (p=0.004),the corresponding number was58%in invasion assay(p=0.004). Colony forming experiment showed that the average diameter of SW480control group clone was945.8+156.8m, and the corresponding data were1348.3+181um in overexpression group, increased significantly (p=0.0067); while in the HT29cells, the average diameter of control group clone were381.7+37u m, and overexpression group were644.3+148.5m, also with significantly enhancement (p=0.009). In colon cancer liver metastases model, SW480cells with p55PIK overexpressing group were found two mouse with liver metastasis, the control group showed no visible liver metastases.Conclusion:we got two p55PIK overexpression and one p55PIK konchdown stable colon cancer cell lines, and p55PIK overexpression could enhance the HT29and SW480cells' motility and invasion ability, and it could also enhance the SW480cells'ability of clone formation; and the motility of p55PIK knockdown LoVo cells diminished, also with clone forming ability reduced. p55PIK could enhance the metastasis ability of SW480in nude mice. Objective:To elucidate the mechanisms of p55PIK's effect on migration and metastasis and confirm the treatment role of drug direct at p55PIK.Method:we firstly used RealtimePCR technology to compared the difference of cytokines' mRNA associated with metastasis between of overexpression of p55PIK cells and their control cells, and the difference between of p55PIK knockdown cells and their control cells. Then used the Elisa assay to verify whether there were a corresponding factor protein changes. At the same time we analysis the change of NF-κB activation which related with the effect of p55PIK on migration and invasion through luciferace fluorescent reporter assay and Western blotting related to p55PIK. Finally, we use a competitive inhibitors of p55PIK, N-terminal peptide, treated the SW480cells, and detected the difference of mobility through Transwell chamber assay and scratch test.Results:RealtimePCR confirmed that the cytokine PDGF-A mRNA level in p55PIK overexpression SW480group was3.9±2times than control group (P=0.042), HT29was1.7±0.2times (P=0.04), TNFa mRNA change was3.2±0.5times in SW480group(P=0.004) and1.9±0.45times in HT29cells group(P=0.02), the Elisa assay found that, after p55PIK overexpression, the protein level of TNFa was inreased1.8times (SW480, P=0.0002)and1.75times (HT-29, P=0.003).The luciferace fluorescent reporter gene assay reminded us that the NF-κB activity was increased1.9±0.25times(P=0.0002) after p55PIK overexpression transient, and the corresponding data was1.5±0.2times (P=0.009) in HT-29cells. We made the p55PIK of LoVo cells knockdown by siRNA, NF-κB activity was inhibited to47±5%(P=0.002) of the control group, and p65536phosphorylation levels also decreased. Finally, SW480cell treated with N24was reduced in migration (p=0.02) and invasion(p=0.002).Conclusion:p55PIK affected the metastasis related cytokines such as, PDGF-A and TNF a though affecting the NF-κB signal pathway, resulted in the change of migration and invasion. The N-teminal of p55PIK peptide could inhibit the mobility of tumor cells.