The Effect Of Hypoxia Induced Factor-1α On Multidrug Resistance And Inhibition Of Expression Of MDR1 With Specific SiRNA Targeting HIF-1α In BEL-7402 Cell Line Under Hypoxia

Being one of the most common malignant tumors across China, the prevailing treatment approaches on hepatocellular carcinoma (HCC) include operation, chemotherapy and radiotherapy. However, because HCC is not sensitive to the chemotherapy, the effects of which are overall not satisfying enough. In such circumstances, either applying one chemotherapeutic drug alone or combining more than one does nothing significantly different. When the obstacles causing this difficulty are concerned, multi-drug resistance (MDR) to anti-neoplastic drugs is one of the major contributors. In spite of this, MDR is one of the reasons responsible for the cancer reoccurrence and metastasis as well. Taking these into account, the generation mechanisms and reversion method involved into MDR have increasingly attracted scientists' researchmphasis so far.HIF-1 is a hypoxia response regulating factor generally residing in mammalian cells. As an oxygen-dependent regulation transcription cell factor and regulates less than 40 putative target genes, is a master regulator of the transcription of many different factors involved in the biological progress of hypoxia malignant tumors. HIF-1a is a vital important biomarker of malignant tumor prognosis and helps reduce tumor chemosensitivity. Traditional reversion drugs have great side-effects to patients at its reversal dose and are limited in clinical therapy. So it is urgent and pivotal to find new means and drugs to reverse MDR. In recent years, gene transduct and gene therapy have become very valuable and important method to conquer tumor or MDR. The breakthrough in gene therapy relies on finding more efficient new targets. It thus requires finding out new pertinence target genes to treat human cancer.RNAi is a powerful tool to inhibit the target gene expression by post-transcriptional gene silencing. When 21bp small interference RNA (siRNA) is introduced into mammalian cells, sequence-specific destruction of endogenous target mRNAs occurs and gene expression is suppressed effectively.Partâ… Objectives:Study the expression of HIF-1α,PTEN,P-gp proteins and their relationships in patients of hepatocellular carcinoma (HCC) and analyze their clinical significance.Methods:HIF-1α, PTEN,P-gp proteins were detected by immunohistochemical technique with SABC or SP in 43 cases of HCC. Hepatic tissues from 7 normal patients were selected as controls.Results:The positive expression rates of HIF-1α,PTEN,and P-gp proteins in HCC tissues were 60.5%(26/432), 46.56%(20/43) and 51.2%(21/43), respectively. Whereas, their positive expression rates in normal hepatic tissues were 14.2%(1/7), 100%(7/7), 0 %(0/7) accordingly. Given so, the expression level of HIF-1α,PTEN,and P-gp proteins were significantly higher in HCC tissues than in normal hepatic tissues (p＜0.05). Among them, HIF-1αis in proportion to the size of the tumor and differentiation extent; except PTEN is more relate with the carcinomatous differentiation, while P-gp being more dependent on the tumor size, both of these two proteins have relevance with the existence of the portal vein or carcinomatous embolism in the biliary tract. The found relationship between the expression of HIF-1αand PTEN was negative with statistical significance, the same between the pair PTEN and P-gp, in comparison, HIF-1αand P-gp decrease or increase with the same trend.Conclusion:1. Immunohistochemistry demonstrated that HIF-1αand P-gp were highly expressed in HCC, oppositely, the expression of PTEN in HCC was deficient.2. The expression of HIF-1αwas in high relevance with differentiation and division of HCC. When HIF-1αwas overexpressed, the anti-cancer gene PTEN was deficient.3. MDR in HCC was triggered at least partially by up-regulating the expression of P-gp via PI(3)k/PTEN / Akt / HIF-1αsignaling transduction pathway.Partâ…¡Objectives:To examining the expression change of H/F-1αand mdrl in mRNA and protein levels in BEL-7402 cell line under chemical hypoxia induced by CoCL₂,Methods:1,MTT assay was employed to determine the sensitivity of CoCL₂ treated BEL-7402 cell line to anticancer drugs; 2,RT-PCR and Western Blot were used to examine the change of HIF- 1αand mdrl in mRNA and protein level in normoxia and hypoxia.Results:1,IC₅₀ of chemotherapy adriamycin (ADM) in BEL-7402 was emarkably higher than those treated by CoCL₂;2,RT-PCR demonstrated that the expression of HIF-1αand MDRl's mRNA increased continuously with the growing of the time. The increasing folds of HIF-1α's mRNA in hypoxia groups over blank group were1.49 (P＜0.05),1.97, 2.94 2.14 (P＜0.05), accordingly treated after 12h,24h,48h。The MDRls mRNA in hypoxia were 1.64, 2.01, 3.82, 5.18(P＜0.05), accordingly.3,Western Blot demonstrated that the expression of HIF-1α's and P-gp protein also increased continuously with the growing of the time. The increasing folds of HIF-1α's protein in hypoxia groups over blank group were 1.17,2.56,3.33,5.33(P＜0.05), accordingly treated after 6h,12h,24h,48h. The increasing folds of P-gp protein in every groups were1.44, 1.84, 5.96,6.84(P＜0.05),accordingly.4,Analyze the expression of HIF-1αin different groups on mRNA and protein level, we can get the equation of Y=-0.91+0.873X, P＜0.01 ; Analyze the expression of P-gp in different groups on mRNA and protein level, we can get the equation of Y=-0.214+1.776X, P＜0.01 ; Analyze the correlation of HIF-1α's and P-gp's Mrna expression: =0.758, P＜0.01; Analyze the correlation of HIF-1α's and P-gp's protein expression, r=0.906, P＜0.01。Conclusion:1,100μmol/L CoCL₂ can induce the expression of HIF-1α; 2,CoCL₂ can decrease BEL-7402cells sensitivity to chemotherapyagents ;3,In BEL-7402 cell line of hypoxia, HIF-1αtakes the role of regulating the expression of MDR1, and HIF-1αregulates the expression of MDR1 mainly in translation level.Partâ…¢Objectives:To construct a recombinant plasmid generating short hairpin RNA, and to explore the effect of recombinant vectorp shRNA transfection on the mRNA and the protein expression of HIF-1α,MDR1 in transfected BEL-7402 cell lines.Methods:Firstly three plasmids expressing hairpin siRNA were constructed and used to transfect BEL-7402 cell lines. Then RT-PCR and Western Blot were used to examine the change of HIF-1αand mdrl in mRNA and protein level in hypoxia.Results:The HIF-1αsequence-specific siRNA was designed by the authorsaccording to the principle of siRNA. No 100% homologous mRNA of othergenes was found in Blast. The result of computer-assistant analysis for the siRNA showed the HIF-1αspecific siRNA designed by the authors might be useful for RNAi.RT-PCR and Western Blot demonstrated that the expression of HIF-1αand MDRl's mRNA and protein decreased continuously after transfectde by the recombinant plasmid with lipofectmine 2000. Compared with the BEL-7402 cell lines untransfected, The HIF-1αmRNA level degradated and the protein expression was inhibited in the BEL-7402 cell lines transfected shRNA.Conclusion:The mRNA and protein expression of HIF-1αand MDRl can be down expressed obviously by transfection with the recombinant plasmid.