| Background:Hepatocellular carcinoma (HCC) is a common malignant tumor, it is also one of the death cause among the three global cancer. POLD1encodes the125-kDa catalytic subunit of DNA polymerase δ. In the S phase, the complex of P125and proliferation cell nuclear antigen (PCNA) and replication factor C (RFC), positioning in the replication fork, that the DNA gets completely duplicated with leading chain and subsequent chains of two different forms of DNA replication. In recent years, the study found that a high expression of POLD1gene in a variety of tumors promote the cell proliferation and enhanced the cell invasiveness.Studies have shown that cell proliferation of dependent on POLD1coding P125involved in the synthesis of DNA, and this process is tied to cell cycle control. The cyclin-dependent kinasRegulation formed a variety of compounds play an important role in different phases of cell cycle, and determine the cell cycle progression. CDK4/cyclinD complex is the key to controlling G1/S protein complex, which allows the Rb protein serine or threonine residue phosphorylation occurs.Under normal circumstances, phosphorylation of Rb protein binding with the transcription factor E2F was in low status. Phosphorylation Rb protein can led to E2F release, and induce the expression of cyclin E, at the same time the complex of cyclin E and CDK2protein make Rb protein phosphorylation, and increase the activity of E2F and cyclin E in G1/S period. Which lead to a series of G1/S-related target molecules expression, and complete quickly DNA replication.Recent research findings show that CDK4genes express abnormally in various tumor tissues and cells, such as the absence of nucleotide sequence and (or) insert, replace, over-expression changes of gene. CDK4is a key factor of cell cycle, it control the pathway and mechanism of POLD1on the occurrence, development and metastasis of tumor. CDK4regulation POLD1pathway and mechanism study on mechanism of the occurrence, development and metastasis of the tumor. It will be very meaningful on the depth of research in cancer diagnosis, treatment and prognosis judgement. Purpose:To detecte the expression of P125in HCC and the correlation between CDK4and POLD1in SMMC-7721; To explore the CDK4on regulation of POLD1/P125expression and its significance.Methods:1. Immunohistochemistry (IHC):47cases cases of HCC had been collected and all were diagnosed by the Department of Pathology of the GuangXi Medical University during2011,3-2011,9. The HCC tissues and surrounding non-cancerous tissues (2cm and above from the tumor) were collected as experimental group, and15cases of intrahepatic bile duct stones surrounding liver tissue as normal control group. The expression of P125protein in hepatocellular carcinoma, surrounding non-cancerous tissues and normal tissues were tested by using IHC and, the data will be analysed by SPSS software to demonatrate the relationship between protein and Clinical pathological features of HCC.2. Plasmid construction:CDK4CDS sequence were amplificated from total cDNA from SMMC-7721by PCR, connected with pEGFP-C1after digested. And the recombinant plasmid of GFP-CDK4was constructed.3.Cell transfection:A:transient transfection, transfection reagent lipo2000mixed with plasmid at proportion of1:2(plasmid GFP-CDK4was transfected as experimental group, plasmid pEGFP-C1as negative control group, untreated SMMC-7721as blank control group) for15min at room temperature and droped softly to cells, after6h change to complete medium.48h later collect cells. B:stable transfection,5days after transiently transfected, added final concentration of500μg/ml of G418to each hole, three weeks later the stably transfected cells keep alive.4. MTT:The stable transfection of GFP-CDK4plasmid of SMMC-7702was cultivated in96-well plates, The MTT assay of cell experimental group and the control group can be performed after24,48,72h, and drew the growth curve..5. Real-Time Quantitative PCR (qRT-PCR):The total RNA extraction from SMMC-7702trandfected GFP-CDK4by using Trizol and reversed into cDNA. Real-time PCR reactions are performed Real Master Mix (SYBR Green), while set the beta-actin for internal reference by ΔΔCt method. Testing the expression of CDK4and POLD1, as well as other relevant factors, and analysed the results data by using ABI7500Software v2.0.5.6. Western Blotting (WB):The expression of cdk4and P125proteins were tested after stable transfection of GFP-CDK4in SMMC-7702, while set the beta-actin for internal reference, scanned results by LI-COR Odyssey infrared fluorescence imaging system, and analysed data using Odyssey V3.0image software.7. Analysis of POLD1promoter binding sites with bioinformatic:using the online software of mainstream authoritative prediction of transcription factor binding sites.www.gene-regulation.com/pub/databases.html and www.cbrc.jp/research/db/TFSEARCH.html forecast on the promoter binding sites in POLD1gene find and analysis the factors related to the target factor of CDK4/POLD1.Results:1. The detection of P125protein positive expression IHC rate for (93.6%) in HCC, significantly higher than the surrounding (10.6%) and normal liver tissue (6.6%). There are significant differences in three comparison groups (P<0.01), of which, the expression of P125protein in HCC significantly higher than normal liver tissue and paracancerous tissues (P<0.01), para-cancer tissue and normal tissue, the difference was not statistically significant (P>0.05). There are no obvious correlation on the positive expression rate of P125protein and Ki67expression,tumor size, the level of HBV infection and AFP, extrahepatic or ntrahepatic metastasis, and associated with cirrhosis (P>0.05). But the degree of differentiation of HCC is significantly related (r=0.560, P<0.01).2. The eukaryotic expression plasmid GFP-CDK4was successfully constructed, and mutations and deletions were found in GFP-CDK4from DNA blast. After transfection, both transiently transfected and stably transfected SMMC-7702can be observed the green fluorescence within the cell. 3. MTT assay shown that, compare to control groups, cell proliferation of the experimental group was significantly proliferated in96h(P<0.05), but without difference between24-72h.4. qRT-PCR assay had proved CDK4gene expression level in experimental group was increased significantly than in control groups (P<0.01), while the relative gene expression of POLD1was increased in the experimental group (P<0.01).5. Western blot assay are correspondent with qRT-PCR assay. CDK4protein expression level in experimental group was promoted, so as P125, compare to control group and blank group the difference is statistically significant(P<0.05), while control group have no statistically difference from blank group(P>0.05).6. The bioinformatics forecast software indicated there is no direct binding site of CDK4in POLD1’s promoter. However, some other factors’ binding sites were found in POLD1’s promoter, and the cdk4binding site was found in those factors’promoter. We speculated CDK4may indrectly promote the expression of POLD1by regulate other factor.Conclusion:1. P125was in high expression in HCC, and hepatocellular carcinoma grade are significantly related to P125, but not with Ki67. It is meaning that P125played a role in DNA synthetics also responsible for hepatocellular carcinoma is related to other known or unknown function. 2. The cell line test confirmed that the CDK4high expression promote the expression of mRNA and protein levels of POLD1/P125, thereby promoting cell proliferation verified POLD1as CDK4downstream genes is regulated by CDK4... |
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