Study On The Expression And Function Of HIF-1α And HIF-2α, Hypoxia-inducible Factor In Renal Carcinoma On Human

Renal carcinoma is a common malignant tumor in renal parenchyma withincreasing incidence rate. The biological behavior of renal carcinoma is verycomplicated and insensitive to the radiotherapy and chemical therapy. The postoperation and metastatic renal carcinoma patients are mainly treated withimmunotherapy in clinic at present. However, the antineoplasmic activity of treatmentusing interleukin (IL-2) and interferon-α(IFN-α) alone is common. The relieving ofdisease can not be lasted. In addition, IL-2 in large dose has greater toxicity, co manysubjects are not suitable for this treatment. Hypoxia-inducible factor HIF canstrengthen the expression of vascular endothelial growth factor (VEGF), glucosetranscript factor 1 (GLUT-1) and glycolytic enzyme in the down stream target genes,promote the generation of blood vessels and energy metabolism of cells, and possiblyplay an important role in the developing progress in the excessive expression ofmalignant tumor.In this project, firstly, we check the expression of HIFI-αand HIF2-α, Hypoxia-induciblefactor in human renal carcinoma strain 786-0 and OS-RC-2 on the level of gene transcription andprotein via using RT-PCR and Western-Blot, explore the correlation of HIFl-α, HIF2-αand thegeneration and development of human renal carcinoma. Constructed the eucaryon plasmid whichcan express HIF1-αand HIF2-αgenes stably further and transfect into the OS-RC-2 and 786-0.Designing and combining the small interferon RNA (siRNA) by chemical synthesis, RNAinterference the HIF gene transfect into the cellular strain, identify that synthetic siRNA gene andprotein level can inhibit the expression of HIF effectively. On this basis, checking the influence onthe proliferation of tumor cells by MTT, study the influence on the VEGF etc, HIF downstreamgenes by immonohistochemistry methods. Inoculating the renal carcinoma cells and interferencerenal carcinoma cells to the athymic mice, we learn the influence of HIF genes on theoncogenicity. In this project, we adopted the RNA interference method to study the influence ofHIF1-α和HIF2-α, Hypoxia-inducible factors on the proliferation of renal carcinoma cells and thedevelopment of tumor, providing the experimental evidences to the gene treatment of renal cellcarcinoma. Part IExpression and significance of HIF-1αand HIF-2αin renal carcinomaObjective: Exploring the expression and clinical significance of HIF-1αand HIF-2α,Hypoxia-inducible factor in suprarenal epithelioma and human renal carcinoma 786-0and OS-RC-2.Methods: Checking the expression of HIF-1αmRNA and HIF-2αmRNA in 56 casesprimarily diverged renal carcinoma and 52 cases normal renal tissues on transcriptionlevel by RT-PCR, checking the expression of HIF-1αand HIF-2αon the protein levelby Western-Blot. And also the OS-RC-2 and human renal carcinoma strain 786-0 ontranscription and protein level.Results: The expression rate of HIF-1αmRNA in tissue specimen of suprarenalepithelioma is 87.5%, increased than the control group with significant difference(P＜0.001); expression rate of HIF-2αmRNA in tissue specimen of suprarenalepithelioma is 94.6%, increased than the control group with significant difference(P＜0.001). in the 56 RCC tissue specimens, 52 cases express HIF-1αprotein, positiverate is 92.9%, increased than the control group with significant difference(P＜0.001).in 56 case RCC tissue specimens, 54 cases express HIF-2α, the positive rate is 96.4%,increased than the control group with significant difference(X~2=88.59,P＜0.001).HIF-1αmRNA, HIF-2αmRNA and proteins in OS-RC-2 and 786-0 are all positivelyexpressed.Conclusion: The expression of HIF-1αand HIF-2αon the mRNA and protein levelpossibly play an important role in the genesis and development of suprarenalepithelioma.PartⅡConstruction and identification of psilencer3.0-HIF-αsiRNA, HIFinterference expression vectorObjective: Construct psilencer3.0-HIF-αsiRNA recombination plasmid, to settle thebase for discussing the function of HIF-1 and HIF-2. Methods: Designing and synthesizing the siRNA which can inhibit the HIF-1αandHIF-2α, constructed in RNAi expression vector by cellular transfect method.Checking the inhibition of RNAi expression vector successfully constructed on themRNA and protein by real time quantitative PCR and Western blot.Results: The relative expressive quantum of HIF-1αmRNA in 4 transfectrecombination plasmid is less than 1, especially psilencer3.0-HIF-1αsiRNA2 andpsilencer3.0-HIF-1αsiRNA3 are obvious. In 786-0 cells after transfect 48h, theinhibit rate is 75.4% and 82.1% respectively. In the OS-RC-2 cells, the inhibit rate is82.3% and 91.2%. The relative expressive quantum of HIF-2αmRNA in 4 transfectrecombination plasmid is less than 1, especially psilencer3.0-HIF-2αsiRNA1 andpsilencer3.0-HIF-2αsiRNA3 are obvious. In 786-0 cells after transfect 48h, theinhibit rate is 76.1% and 87.4%respectively. In the OS-RC-2 cells, the inhibit rate is72.3% and 81.2%.In transfect 786-0 cells, protein expression quantum of 4 HIF-1αinterference plasmidexperimental groups is decreased in different level compared with the blank group.Among them, the HIF-1αprotein expression level of psilencer3.0-HIF-1αsiRNA2and psilencer3.0-HIF-1αsiRNA3 is relatively lower. In transfect 786-0 cells, proteinexpression quantum of 4 HIF-2αinterference plasmid experimental groups isdecreased in different level compared with the blank group. Among them, the HIF-1αprotein expression level of psilencer3.0-HIF-2αsiRNA1 and psilencer3.0-HIF-1αsiRNA3 is relatively lower.Conclusion: The RNAi expression vector constructed successfully can inhibit theexpression of HIF-1 and HIF-2 on mRNA and protein level effectively.PartⅢInfluence of HIF-1αand HIF-2αon the proliferation of human renalcarcinomaObjective: Studying the influence of psilencer3.0-HIF-1αsiRNA andpsilencer3.0-HIF-2αsiRNA, successfully constructed HIF-αRNAi expression vectors,and the down stream genes VEGF, etc. Methods: Studying the influence of psilencer3.0-HIF-1αsiRNA andpsilencer3.0-HIF-2αsiRNA on the proliferation of tumor cells by MTT and that onHIF downstream genes VEGF etc. by immunohistochemistry.Results: Psilencer3.0-HIF-αsiRNA can inhibit the proliferation in vitro of 789-0 cells.The relative inhibiting rate of psilencer3.0-HIF-1αsiRNA plasmid group is47.4±4.3%, that of psilencer3.0-HIF-2αsiRNA plasmid group is 52.7±5.8%, havesignificant difference when compared with blank group(P＜0.05) and also whencompared with no-load vector transfect group(P＜0.05). Psilencer3.0-HIF-αsiRNAcan inhibit the proliferation in vitro of OS-RC-2 cells. The relative inhibiting rate ofpsilencer3.0-HIF-1αsiRNA plasmid group is 55.3±5.6%, that of psilencer3.0-HIF-2αsiRNA plasmid group is 44.3±4.1%, have significant difference when compared withblank group(P＜0.05) and also when compared with no-load vector transfectgroup(P＜0.05).After interference the HIF-1αor HIF-2α, the expression of downstream gene VEGF inrenal carcinoma is obviously lower than the non interference group.Conclusion: RNAi expression vector of psilencer3.0-HIF-1αsiRNA andpsilencer3.0-HIF-2αsiRNA can inhibit the proliferation of human renal carcinomaand down regulate the expression of downstream genes, VEGF.PartⅣInfluence of HIF-1αand HIF-2αon the oncogenicity of human renalcarcinomaObjective: Exploring the influence of HIF-1αand HIF-2αon oncogenicity of humanrenal carcinomaMethods: 40 athymic mice are randomly divided into 4 groups, blank group,psilencer3.0-HIF-1αsiRNA, psilencer3.0-HIF-2αsiRNA and no load vector group.Observing the tumor in each 3 days after the inoculation, measuring the longestdiameter(a), shortest diameter(b) by using sliding caliper, calculating the volume oftumor according to the formula. All mice are executed in 6 weeks after the inoculation,stripped and put out the tumor which to be weighed and then calculate the inhibit rate of tumor weight.Results: The volume of tumor in psilencer3.0-HIF-1αsiRNA and psilencer3.0-HIF-2αsiRNA is smaller than that in blank and no load vector group in the samestage(P＜0.05); the volume of tumor in psilencer3.0-HIF-2αsiRNA is a bit smallerthan that in psilencer3.0-HIF-1αsiRNA group in the same stage with nosignificance(P＞0.05).The weight of tumor in psilencer3.0-HIF-1αsiRNA and psilencer3.0-HIF-2αsiRNAgroup is a bit lighter than control group(P＜0.05);the weight of tumor inpsilencer3.0-HIF-2αsiRNA is a bit lighter than that in psilencer3.0-HIF-1αsiRNAwith no significance(P＞0.05)。Conclusion: After transfect psilencer3.0-HIF-1αsiRNA and psilencer3.0-HIF-2αsiRNA, the genesis and development of renal carcinoma can be obviously inhibitedand possibly play an important role in the genesis and developing progress.