Synergistic Inhibition Of PKM2 And CCND1 By SiRNA Of PKM2

Objective: To study the inhibitory effect of an PKM2 siRNA on the expression of CCND1 and its mechanism. Methods: Detect siM2PK-1's influence on the proliferation and cycle by the colony formation assay and flow cytometry. Discover siM2PK-1's effect on CCND1 via real-time quantitative PCR and Western blot. Verify the specification of siM2PK-1's inhibition on CCND1 by over-expressing PKM2 and other PKM2-interference experiment, Using bioinformatics analysis and reporter gene assays to explore the mechanism of siM2PK-1's suppression on the expression of CCND1. Results: By the use of colony formation, we found that siM2PK-1 could inhibit the proliferation of SK-Hep-1 cells. Further more detection by flow cytometry revealed that siM2PK-1 can induce cell cycle arrest at G0-G1 phase. The results of real-time Quantitative PCR and Western blot demonstrated that siM2PK-1 could affect the expression of CCND1 at both RNA and protein levels, while the over-expression of PKM2 could not up-regulate CCND1 at neither RNA nor protein levels. The PKM2 siRNA could not down-regulate CCND1.Thus, siM2PK-1 is dual-targeting. Moreover, bioinformatics analysis combined with a reporter gene assay suggested that the siM2PK-1 could bind to the CCND1 promoter region by imcomplementary maner to cause its promoter region CpG island methylation. Conclusion: siM2PK-1, which could inhibit PKM2, could also suppress the expression of CCND1.Objective:To investigate miR-210 expression in HepG2 cells under three kinds of hypoxia models. Methods:Hypoxia induced HepG2 cells under three different hypoxia models, including physical hypoxia and chemical hypoxia-mimicking agents, such as CoCl₂ and Na₂SO₃. qRT-PCR was used to assay the expression of miR-210. Results : miR-210 could be up-regulated in these three hypoxia models. The expression of miR-210 has a dose dependent manner with the concentration of CoCl₂ and Na₂SO₃. Conclusion:Hypoxia induced by physical hypoxia and chemical hypoxia-mimicking agents could upregulate the expression of miR-210 in HepG2 cells. All these three models could be applied to research the relationship among hypoxia, miRNA and cancer. These results further prove that the miR-210 expression really is caused by hypoxia.