| Objective：To investigate the mechanism and the possible approaches of solving drugresistance by silencing hypoxia-inducible factor-1alpha of human pancreatic cancercell line Patu8988/5-Fu cultured in hypoxia mimetic cobalt chloride.Methods：The effective jamming fragment screened by RT-PCR for silencing HIF-1αgene was transfected into pancreatic cancer cells Patu8988/5-Fu through lentivirus.Cells were divided into three groups:①Blank control group (Blank),②negativeshRNA transfection control group (Lenti-NC-shRNA) and③stable transfectiongroup group (Lenti-shRNA). The20μmol/L cobalt chloride was used to mimicchemical hypoxia model, MTT assay was to determine the Patu8988/5-Fuproliferation activity and rate of drug resistance compared to normoxia, JC-1fluorescence staining flow cytometric analysis was to analyse the mitochondrialmembrane potential changes. RT-PCR was to test HIF-1α and drug resistance genesMDR1expression before and after transfection; Western blot was to test proteinexpression level of HIF-1α and drug resistance genes MDR1. SPSS19.0softwareanalysis was used to analyse the experimental data.Results：The screening and lentivirus transfection of effective jamming fragments forsilencing HIF-1α gene of human pancreatic cancer cell line Patu8988/5-Fu, cobaltchloride mimic chemical hypoxia model and relative inspections were successfully done. RT-PCR results showed that the effective jamming fragments for HIF-1α inlentivirus transfection was Wtl-mus-1202:5’GGAGCTACCTTAAAGGGAATG-3’,5’CATTCCCTTTAAGGTAGCTCC-3’, its inhibition rate of HIF-1α gene in72h was82%, which met the needs of the experimental requirements. MTT assay suggestedthat under the cultivation of200μmol/L cobalt chloride for8h, the inhibition rates ofPatu8988/5-Fu cells were①2.03%②3.72%③5.48%separately,there was nosignificant differences between groups (P>0.05). JC-1fluorescence staining flowcytometric analysis showed that under the cultivation of200μmol/L cobalt chloridefor8h, the mitochondrial membrane potential in three groups were decreaseddramatically, which were①15.7%,②22.9%,③41.7%respectively, there werestatistical differences in group (P <0.05), but no significant differences betweengroups (P>0.05); Combined with MTT assay, the concentration of200μmol/LCoCl2for8h was chosen to mimic hypoxia cell environment. MTT assay showed thatin hypoxia the drug resistance rate of the three group compared to normoxiarespectively were①Blank1.95②Lenti-NC-shRNA1.72③Lenti-shRNA1.55，which meaned hypoxia could upgrade the drug resistance of the human pancreaticcancer cells Patu8988/5-Fu (P<0.01), while in the same condition hypoxia ornormoxia, compared to group①and②,group③showed lower drug resistanceto5-Fu,which meaned scilencing Hif-1α could reverse the multidrug resistance.RT-PCR detection showed the mRNA expressions of HIF-1α and MDR1: there is nostatistical differences between the groups①and②whether in normoxia orhypoxia (P>0.05), while group③was lower than group①and②（P<0.01）.And they all increased in hypoxia compared to normoxia in the three groups,(P <0.01). Western blot test showed the protein expressions of HIF-1α and MDR1: thereis no statistical differences between the groups①and②whether in normoxia orhypoxia (P>0.05), while group③was lower than group①and②（P<0.01）. And they all increased in hypoxia compared to normoxia in the three groups,(P <0.01), which is coincidence with the results of RT-PCR. The expression of HIF-1αand MDR1in Patu8988/5-Fu positive correlation (γ＝0.562,P<0.01).Conclusion：The upregulate of the HIF-1α and MDR1gene expression caused by cobaltchloride mimic hypoxia was related with the generation of multi-drug resistance ofPatu8988/5-Fu, targeted silencing HIF-1α may be a kind of way to reverse thechemotherapy drug resistance... |
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