Study on P21Waf1/Cip1 Upregulation by Overexpression of hPOT1 in Hep-2 Cells
|School||Huazhong University of Science and Technology|
|Course||Clinical Laboratory Science|
|Keywords||Telomere hPOT1 pEGFP-C2 P21Waf1/Cip1 MDM2 P53 GFP-hPOT1 fusion protein|
Objective: To construct the eukaryotic expression plasmid pEGFP-C2-hPOT1.Method: Total RNA extracted from chronic myelogenous leukemia K562 cells with TRIzol were transcribed reversely into cDNA, then hPOT1 were amplified with high-fidelity Taq enzyme from the above cDNA. The amplified products were inserted into eukaryotic expression vector pEGFP-C2 to construct eukaryotic expression plasmid pEGFP-C2-hPOT1, following by transforming E. Coli, finally gene sequencing was adopted to identify pEGFP-C2-hPOT1.Result: Sequencing showed that mutation and frame shifting were not found in the insert hPOT1 of eukaryotic expression plasmid pEGFP-C2-hPOT1 and the sequence of insert was totally matched with the reference sequence of hPOT1( NM_ 015450.2 )in GeneBank.Conclusions: The eukaryotic expression plasmid pEGFP-C2-hPOT1 was constructed successfully.PartⅡStudy on P21Waf1/Cip1 upregulation by overexpression of hPOT1 in Hep-2 cellsObjective: To study influence and its possible mechanism of overexpression hPOT1 on P21Waf1/Cip1 in Hep-2 cells.Method:①Real-time quantitative PCR was used to examine hPOT1 mRNA levels and P21 mRNA levels in experimental groups(Hep-s cells transfected with pEGFP-C2-hPOT1) and control groups(Hep-s cells transfected with pEGFP-C2) respectively.②The location of GFP-hPOT1 fusion protein and MDM2 in Hep-2 cells were visualized with laser scanning confocal microscope.③The protein levels of GFP-hPOT1 fusion protein, MDM2, P53, P21Waf1/Cip1 in experimental groups and control groups were detected by Western blot.Result:①When Hep-2 cells were transfected with 4μg of plasmid for 24hr and 48hr respectively, hPOT1 mRNA levels in experimental groups was 92233.29 and 25870.14 times higher than that in corresponding control groups respectively, and moreover, only 98KD size of GFP-hPOT1 fusion protein was expressed in experimental groups, while there was 27KD size of GFP protein expressed in control groups;②GFP-hPOT1 fusion protein localizes mostly in the nucleus of HEp-2 cells, but there was also a small amount of the fusion protein exist in the cytoplasm;③There was no significant differences of P21 mRNA level between experimental groups and control groups, however, the protein level of P21Waf1/Cip1 in experimental groups was much higher than that in control groups, and moreover, with the addition of the amount of the plasmids that transfected into cells, the expression of GFP- hPOT1 fusion protein was increasing, and the expression of P21Waf1/Cip1 was unceasingly rises with increasing GFP- hPOT1 fusion protein;④The expression of MDM2 in experimental groups was much higher than that in control groups, while there was no significant differences of the expression of P53 between the two groups;⑤GFP- hPOT1 fusion protein and MDM2 in experimental groups co-locate in the same place.Conclusion: Overexpression of hPOT1 up-regulates the expression of P21Waf1/Cip1 in Hep-2 cells in dose-dependent manner, furthermore, GFP-hPOT1 fusion protein and MDM2 in experimental groups colocate in the same place. Overexpression of hPOT1 up-regulates possibly the expression of P21Waf1/Cip1 by inhibiting the activity of E3 ligase of MDM2 and the degradation of P21Waf1/Cip1 by proteasomes,not by increasing the P21 mRNA expression level.