The Molecular Regulatory Mechanism Of Regulation Of The Expression Of HIF-1α MRNA By Renal Clear Cell Carcinoma Von Hippel Lindau-(VHL) Protein

Background:Existing research think VHL tumor-suppressor genes play an important role inprimary sporadic renal clear cell carcinoma (RCC),the mechanism of tumor formationare still have more arguments.VHL genes exists in cytoplasm, by forming E3ligasecomplexes with some other proteins, which make the protein ubiquitindegradation,Hypoxia inducing factor (HIF) is factors related to the tumor formationresearched more popularly in recent years.Under constant oxygen condition, HIF αproline,when hydroxylated, combining with VHL protein, and trigger the ubiquitin-proteasome ubiquitin-proteasome proteolytic pathway, making itself rapiddegradation [15].Previous studies have found that in renal cancer cell lines andclinical renal clear cell carcinoma, VHL proteins facilitate HIF-1α mRNAexpression.Further study found that VHL protein has a promoting effect ontranscription of HIF-1α gene, but its molecular mechanism is unclear, and aboutwhich did not see the reports.Early studies suggest that VHL proteins can not onlyregulate the expression of HIF-1α from the level of protein, and can regulate theexpression of HIF-1α mRNAfrom the level of molecule.Objective:Based on the above theoretical background, the purpose of this study is topreliminarily verificate that VHL protein related to transcription regulatory regions ofHIF-1α genes and can promote expression of HIF-1α mRNA in renal cellcarcinoma, and further clarify the molecular mechanism and significance.Methods:Get the logarithmic phase of RCC4VHL (-) and RCC4T3-14VHL (+) cells,added into10%bovine serum and DMEM medium and then placed in cell culturebox to extend,80%adherent cells are used in the experiment.use Western blot todetect quantity and difference expression of two groups of VHL proteins in humankidney cells, through the real-time fluorescent quantitative PCR lab experimenttesting the expression and differences of HIF-1α mRNA in these two groups of cell lines to deduce whether the VHL protein can promote the expression of HIF-1αmRNA and in order to determine the interactions between VHL proteins and thetranscription regulation zone of HIF-1α gene.Results:The result of Western blot detection show that the quantity of expression of VHLprotein in RCC4VHL (-) cell lines is zero, statistically significant difference (P <0.05). Illustrate that the VHL gene of this cell lines were knock out successfully. Byreal-time fluorescent quantitative PCR detection, showing that the expression of HIF-1α mRNA is difference between kidney cancer cell lines of VHL negative and VHLpositive kidney cancer cell lines (RCC4T3-14),the expression of HIF-1α mRNA inVHL positive kidney cancer cell lines (RCC4T3-14) is significantly higher thannegative kidney cancer cell lines, statistically significant difference (P <0.05).Theexperiment at least repeat3times.Conclusion:VHL proteins can not only down regulate expression of HIF-1α protein from theprotein level, but also from the molecular level to promote the expression of HIF-1αmRNA, VHLprotein structure suggest that pVHL is not a direct with DNA-bindingtranscription factors, which may be through an intermediate material effedting on HIF-1α gene, and regulating the transcription and expression of it, the middle material isnot clear, needs to be studied further.