Investigation of HDM4 and It’s Transcript Variants in Tumor Regulation
|School||Huazhong University of Science and Technology|
|Course||Clinical Laboratory Science|
|Keywords||HDM4 HDM4-S amplification alternative splicing CML AML HDM2 HepG2 HUVEC P53 MDM4 p21 HDM2|
Objective To study the expression of HDM4 and HDM4-S in patients with Chronic Myelogenous Leukemia and Acute Myelogenous Leukemia. And analyse the changes of the relative ratio of the two to understand the role of HDM4 in leukemia.Method 35 patients with chronic myelogenous leukemia (CML) and 36 cases of acute myelogenous leukemia (AML) and 15 health persons were recruited. Real-time quantitative RT-PCR was carried out to detect the expression of HDM4 and HDM4-S. Using 2-△△(?) method to analysis the data of HDM4, HDM4-S mRNA expression. Another pair of primers designed to distinguish the products from FL-HDM4 and HDM4-S cDNA. According to the bands by agarose gel electrophoresis, HDM4-S and FL-HDM4 ratio was detect in each group.Result (1) HDM4-S, HDM4 were expressed in all the peripheral blood mononuclear cells of samples. HDM4 expression were higher than in normal (p<0.05), while the HDM4-S shows no significant changes in CML and AML (p>0.05). (2) There was equivalent expression of both transcripts in normal samples, the ratio of HDM4-S/HDM4 close to 1.00 but never exceed. FL-HDM4 was the main style in majority of leukemia, and the ratio of HDM4-S/FL-HDM4 broader range than in normal. (3) In addition, We also found some of the leukemia samples shows higher ratio of HDM4-S/FL-HDM4 (above 1.00).The percentage was 8.57%(3/35) of CML and 16.7%(6/36) of AML.Conclusion we concluded that HDM4 amplification higher in myeloid leukemia than normal. Although the HDM4-S levels did not have significant changes,the higher ratio of HDM4-S/FL-HDM4 may be related to the occurrence of leukemia and may be help in the diagnosis of leukemia rather than a single gene level. Objective Reconstrcted eukaryotic expression recombinant plasmid of HDM2' dissertation">HDM2 and identify its DNA sequence. In addition, transfect the recombined plasmid pEGFP-C2-HDM2、pEGFP-C2-HDM4-S into eukaryotic cell lines--HepG2 cell、HUVEC cells to screen out the cells with overexpressed wt genes.Methods A eukaryotic expression recombinant plasmid of pEGFP-C2-HDM2 and pEGFP-C2-HDM4-S was reconstrcted by clone techology. Liposomal lipofectamine2000 mediates and G418 selected transfection of HepG2 and HUVEC cells. Report gene pEGFP emitted green fluorescence were observed by fluorescence microscope method, which was estimated the position of exotic HDM2 and HDM4-S and the expression efficiency in cells.Results (1) The genes was successfully inserted into pEGFP-C2 vector and identified by PCR, enzyme digestion and DNA sequencing.(2) After transfection and G418 treatment, the single clone cells with aim genes overexpression was got at last, meanwhile the expression fusion protein was found by fluorescence microscope detection.Conclusion The vector pEGFP-C2-HDM2 and pEGFP-C2-HDM4-S has been successfully constructed and it could efficiently express in HepG2 cell and HUVEC cell individually, it may thus provide experimental basis for further research. Objective:To investigate the role of HDM4 and HDM4-S gene in the regulation of overexpress HDM2 and HDM4-S.And detect the protein level changes in HDM4-p53 pathway.Method:Collect the HepG2 and HUVEC cells which have the stable transfection of HDM4 and HDM4-S. The gene expression level of HDM4 and HDM4-S was detected with real-time PCR. Primer HDM4-Mix for normal PCR is acted to evaluate the relative level of FL-HDM4 and HDM4-S. Western Blot shows the levels of p53、p21、mdmx in each group.Result:In the HUVEC cells which overexpress of HDM2 and HDM4-S, HDM4 was inhibited and the ratio of HDM4-S/FL-HDM4 elevated. In the HepG2 group, P21 increased, but no changes shows in P53 and MDM4. In the HUVEC cells which overexpress of HDM2 and HDM4-S, the truncated protein was detected, P53 protein decrease but P21 increase according to control.Conclusion:After extrinsic expression of HDM2 and HDM4-S, the ratio of HDM4-S/FL-HDM4 changes and showed significant higher level. And the effect on P53、P21、MDM4 protein was different between HepG2 and HUVEC. We can concluded that HDM4 participate in cell homeostasis and the changes of alternative splicing was one of the force.