Study On Signaling Pathway Mechanism Of P55PIK Regulation Of Alpha-fetoprotein (AFP) Gene Expression

Objective: P55 PIK is a regulatory subunit of PI3 K,plays an important role in cell cycle regulation and inflammation,and p55 PIK is expressed in a large number of clinical samples of hepatocellular carcinoma.It is found that adding p55 PIK specific inhibitor P15 to hepatocarcinoma cells can inhibit Liver cancer cell growth,Real time PCR test also confirmed that the diagnosis of liver cancer AFP is inhibited,so the focus of this experiment is to clarify the molecular mechanism of p55 PIK regulation of AFP expression.Methods: The expression of AFP mRNA and protein in cells were detected by real-time quantitative PCR and western blot.The effect of P55 PIK on AFP in hepatocellular carcinoma cells was detected by immunohistochemistry and immunoprecipitation;The activity of NF-kappaB was detected by P55PIK-specific inhibitor P15,and it was confirmed that P55 PIK could regulate the activity of NF-kappaB in HCC cells;The expression of AFP mRNA and protein in the cells were detected by real-time quantitative PCR and western blot.The expression of NF-kappaB-specific inhibitor PDTC and P55PIK-specific inhibitor P15 were determined by HepG2 cell line HepG2.The effect of NF-kappaB on AFP in cells;To investigate whether NF-kappaB can up-regulate the up-regulation of AFP up-regulation induced by p55 PIK by simultaneous up-regulation of p55 PIK and inhibition of NF-kappaB,and to confirm that P55 PIK cleaves AFP expression through NF-kappaB signaling pathway.The AFP gene was identified by luciferase reporter system The promoter was subjected to NF-kappaB-regulated fragments;The NF-kappaB-regulated fragment of the AFP gene promoter was determined by using the luciferase reporter system.The fragments of NF-kappaB regulated by AFP gene promoter were analyzed and predicted by bioinformatics.Of the locus;The effect of the above DNA sites on the transcriptional activity of AFP on PFPPK / NF-kappaB was observed by point mutation technique.It was clear which sites played an important role in P55 PIK / NF-kappaB regulation of AFP transcription.Resures: The mRNA level of AFP was observed in a concentration-dependent manner,and the expression of AFP mRNA was decreased gradually with the addition of P15.The protein expression of AFP was significantly decreased after silencing P55 PIK,and the expression of AFP The expression was significantly increased;AFP was significantly decreased at the protein level after PDT was added to HepG2 cells,and the change of AFP mRNA expression was in a concentration-dependent manner with the amount of PDTC added,that is,the amount of AFP m RNA expression increased with the amount of PDTC Much less;The levels of p65(ser536)pHospHorylation were significantly decreased,the expression of IKB? was significantly increased,and the pHospHorylation level of IKB?(ser36)was significantly decreased after P15 and PDTC were added to HepG2 cells respectively.The level of pHospHorylation of p65(ser536)was significantly decreased,the expression of IKB? was significantly increased and the pHospHorylation level of IKB?(ser36)was significantly decreased,and the control group(p55PIK,p55 PIK + PDTC)(-5229 /-16)-AFP,(-3375 /-16)-AFP(AFP),and the expression of AFP in the upstream regulatory region of AFP gene was significantly higher than that in the control group(P <0.05)(-1865 /-16)-AFP and(-225 /-16)-AFP,and the molecular weight sequences were identified by restriction enzyme digestion and sequencing.The changes of luciferase activity of each luciferase reporter gene vector were observed before and after P15 and PDTC,(-5184 / + 29)-AFP and(-3330 / + 29)-AFP were the most significant,that is,NF-kappaB was mainly in the(-5184,-1820)fragment;(-4717 /-4726),(-4372/4381),(-3171/3180)may be the NF-kappaB and AFP promoter(-5184,-1820),which were the most frequently reported fragments(-5229 / 16)-AFP was used as a template to analyze the above-mentioned sites,and the results were compared with those of P15 and PDTC before and after the addition of P15 and PDTC.(-4717 /-4726)and(-4372/4381)mutations,P15 and PDTC no longer regulate the transcriptional activity of AFP,that is,the site(-4717 /-4726),and the effect of gene mutation on the transcriptional activity of AFP gene.(-4372/4381)is a key site in P55 PIK / NF-kappaB regulation of AFP transcription activity.Conclusion: In parenchymal cells,p55PIK-mediated signaling pathway is involved in the expression of AFP,a commonly used hepatocellular biomarker.The results also show that p55 PIK regulates the expression of AFP by NF-kappB signaling pathway.In the AFP promoter,NF-kappB of the main role of the site(-4717 /-4726),(-4372/4381).