Expression Of P125in Gastric Intratumoral Tissues And Peritumoral Tissues, And The Study Of POLD1Expression Regulation By P21

Purpose:Polymerase δ (Pol δ) is one of the most important DNA polymerases in eukaryotic DNA replication. Pol δ consists of four subunits. p125, encoded by POLD1, is the catalytic subunit of Pol8, and it includes both polymerase and exonuclease activity. POLD1is regulated and controlled by many kinds of proteins. Studies show that abnormalities of Pol8have certain relations with tumors. The abnormal expression or abnormal regulation of Pol8may lead to tumorigenesis. To explore the correlation between tumorigenesis and abnormal expression of p125, protein levels of p125in gastric carcinoma tissues were investigated. Then POLD1gene was silenced in gastric cancer cells, and proliferation rate of POLD1silenced cells was detected, so as to analyze the correlation between POLD1and malignant proliferation of gastric cancer cells. As known, p21is the first discovered cell cycle inhibitor, it can block cell cycle progression via p53-dependent or independent pathway. And POLD1is regulated by cell cycle. Therefore we speculated that p21participated in the regulation of POLD1. And we explored this preliminary regulation model. It is helpful for providing a more effective target for specific drug design for tumor therapy.Mathods:1. Protein levels of p125in peritumoral and intratumoural areas from surgery excised tissues of62gastric carcinoma patients were detected by immunohistochemistry assay, and the correlation between p125levels and clinic pathological feature of these patients were analyzed. Recombinant plasmid pGPU6/GFP/neo-POLD1i was constructed with POLD1specific interference sequence and pGPU6/GFP/neo-shNC vector. Plasmid pGPU6/GFP/neo-POLD1i was transfected into gastric carcinoma cells MGC-803, and Cell Proliferation Rate was detected by MTT assay. Subcellular locations of Protein P21and p125were also detected by cell immunohistochemistry assay.2. To acquire p21overexpressed cells803-p21or p21silenced cells803-p21i, recombinant plasmids pXJ41-p21and pGPU6/GFP/neo-p21i were transfected into MGC-803cells respectively. Proliferation rate of803-p21cells was detected by MTT assay, and apoptosis level of803-p21was detected by Flow cytometry (FCM).3. To investigate the expression regulation of POLD1by p21, mRNA levels and protein levels of POLD1and other relative factors (including p53, CDK4, CDK2and PCNA) in803-p21and803-p21i cells were detected by real-time fluorescence quantitative PCR (qPCR) and Western blot respectively. Interaction of P21and p125was detected by immunoprecipitation assay. To make clear the detailed correlation of p53or CDK4with the regulation of POLD1by p21, p53interference plasmid pGPU6/GFP/neo-p53i or CDK4expression plasmid pEGFP-CDK4was transfected into803-p21cells, then relative mRNA level of POLD1was detected by qPCR. And the subcellular locations of P53and CDK4were investigated by immunofluorescence assay.Results:1. Mean p125level of intratumoural areas was much higher than that of peritumoral areas. And p125level was unrelated with age, sex, differenciation or size of tumor, lymph node metastasis, etc. MTT assay showed that803-POLDli cells proliferated much slower than negative control cells803-NC or blank cells803.2. Gastric carcinoma cells803-p21proliferated much slower than that of negative control cells803-pXJ or blank control cells803. Apoptosis percentage was much higher in803-p21cells than in negative control cells803-pXJ or blank control cells803detected by FCM.3. Results of qPCR and western blot showed that negative correlations were found between POLD1, CDK2, CDK4, PCNA and p21, positive correlation was found between p53and p21. Immunoprecipitation assay showed that there wss no detectable P21in p125antibody precipitated protein complex, or no detectable p125in P21antidody precipitated protein complex. Relative mRNA level of POLD1was increased in p53silenced or CDK4increased803-p21cells.Conclusions:1. Mean protein level of p125is much higher in intratumoural areas than in peritumoral areas, and proliferation of gastric carcinoma cells MGC-803was inhibited when POLD1gene was silenced. These imply that malignant proliferation of gastric carcinoma cells may be related to high expression level of POLD1.2. p21can increase POLD1expression level, and p21inhibits the proliferation of gastric carcinoma cells, so it is suggested that p21might inhibit gastric carcinoma cells proliferation via affecting the expression level of POLD1.3. p21may indirectly regulate POLD1expression via CDK4, and the expression regulation of POLD1by p21is also related to p53, CDK2and PCNA.