The Roles And Mechanisms Of MiR-301a In Hypoxia-induced Gemcitabine Resistance In Pancreatic Cancer

Background & Objetive: Hypoxia is a hallmark of pancreatic cancer and is associated with gemcitabine resistance.This study aimed to investigate the molecular mechanisms of hypoxia-induced gemcitabine resistance in pancreatic cancer,and to explore the role of miR-301 a in hypoxia-induced chemoresistance in pancreatic cancer.The result of our study will probably provide a promising therapeutic strategy to reverse the gemcitabine resistance in patients with pancreatic cancer.Methods: The effects of hypoxia on gemcitabine resistance and miR-301 a in pancreatic cancer cells were studied by cell viability assay?flow cytometry ?RealTime PCR and other biological technology.The roles of miR-301 a expression on gemcitabine resistance of pancreatic cancer cells was studied after overexpression and knockout of miR-301 a in pancreatic cancer cell lines.Si RNA interference technique and NF-?B p65 overexpression plasmid were used to study the regulation of miR-301 a by NF-?B p65.The effects of miR-301 a on TAp63?PTEN?HIF-1? and p-Akt was studied by Western blot.Results:(1)Compared with normoxia,hypoxia increased gemcitabine resistance in pancreatic cancer cells(P<0.05).Meanwhile,the expression of miR-301 a was up-regulated under hypoxia compared with normoxia(P<0.05).Compared with negative control group,overexpression of miR-301 a promoted gemcitabine resistance in pancreatic cancer cells under normoxia and hypoxia(P<0.05);Compared with negative control group,knockout of miR-301 a decreased gemcitabine resistance in pancreatic cancer cells under normoxia and hypoxia(P<0.05).(2)Knockdown of NF-?B p65 inhibited expression of miR-301 a under normoxia,and overexpression of NF-?B p65 promoted expression of miR-301 a under normoxia(P<0.05).However,no significant changes of miR-301 a were observed after knockdown and overexpression of NF-?B p65 under hypoxic conditions.(3)Compared with normoxia,the expression of TAp63 and PTEN protein were downregulated in pancreatic cancer under hypoxia.In addition,miR-301 a downregulated TAp63 and PTEN under normoxia and hypoxia.Moreover,miR-301 a promoted activation of p-Akt and accumulation of HIF-1?.Compared with negative control group,overexpression of TAp63 plasmid decreased gemcitabine resistance in pancreatic cancer cells under hypoxia(P<0.05).However,no significant changes were observed under normoxia.Further investigation indicated that TAp63 destabilized the HIF-1? protein in a proteasome-dependent manner,which generally accumulated under hypoxia.Our results also observed that the overexpression of TAp63 in hypoxic MIA Pa Ca-2 and Bx PC-3 cells leaded to upregulation of PTEN expression and inactivation of the Akt signaling pathway.Rescue assay showed that overexpression of TAp63 partially reversed gemcitabine resistance initiated by miR-301 a under hypoxic treatment(P<0.05).Conclusion: Hypoxia-induced miR-301 a inhibits the expression of TAp63 and PTEN under hypoxic microvironment and thus,promotes accumulation of HIF-1? and phosphorylation of Akt,which lead to promoted gemcitabine resistance in pancreatic cancer cells.