MicroRNA-101 Contributes To HIF-1α-mediated Cell Cycle Arrest And Apoptosis Through Targeting Von Hippel-Lindau Tumor Suppressor(VHL)in Tumor Cells

Hypoxia may induce tumor cells to express HIF-1α.The activation/inactivation of HIF-1α is precisely regulated in an oxygen-dependent manner.When oxygen is adequate,HIF-1α is hydroxylated by prolyl hydroxylase(PHDs)and in turn recognized by the von Hippel-Lindau tumor suppressor(VHL),an E3 ubiquitin protein ligase,for unbiquitin-mediated degradation.Under hypoxia,the hydroxylase activity of PHDs is dramatically reduced;thus HIF-1α is stabilized,dimerized with HIF-1β to form HIF-1 complex and translocated to the nucleus,regulating multiple genes expressions such as vascular endotherlial growth factor A(VEGFA)and so on.HIF-1α is essential for hypoxia-induced apoptosis and cell cycle arrest.Several recent studies have indicated that the expression of mi RNAs can be modulated by hypoxia,termed “hypoxamirs”.However,the involvement of mi RNAs in the regulation of HIF-1α induction remains elusive.In present study,we demonstrated that mi R-101 was rapidly and transiently induced in breast cancer cells under hypoxia.The mi R-101 is recognized as a new hypoxamirs.Over-expression of mi R-101 significantly inhibited cell proliferation in breast cancer cells through promoting apoptosis and cell cycle arrest in normoxia condition.This cell proliferation inhibitory phenomenon seems due to mi R-101-mediated induction of HIF-1α,because we have identified that VHL,a negative regulator of HIF-1α,is a novel target of mi R-101.We have for the first time identified that VHL is a direct target of mi R-101 and demonstrated that mi R-101 could decrease VHL protein levels.Two binding sites on VHL-3′UTR can be recognized and regulated by mi R-101.Over-expression of mi R-101 decreased VHL levels and subsequently stabilized HIF-1α to induce its downstream target VEGFA.Overexpression of mi R-101 could increase HIF-1α protein levels by repressing VHL in normoxia condition.Furthermore,increase of HIF-1α levels by either direct HIF-1α overexpression or indirect knockdown of VHLcan induce apoptosis and cell cycle arrest.Moreover,HIF-1α downregulation by VHL over-expression,anti-mi R-101 oligo administration or 2-Me OE2 treatment,could rescue cells from such inhibition.These results reveal a novel regulatory mechanism for induction of HIF-1αin hypoxia and suggest that mi R-101 may inhibit tumor cell proliferation through HIF-1α mediated cell cycle arrest and apoptosis.Finally,under the hypoxia environment,overexpression of exogenous mi R-101 did not significantly affect apoptosis and cell cycle of tumor cells.However,under normoxic environment,overexpression of exogenous mi R-101 could significantly promote apoptosis of tumor cells,even reversing the anti-apoptosis mechanism caused by p53-dificiency in tumor cells.Our finding suggests that the mi R-101/VHL/HIF1α axis may play an important role in suppression of tumor growth.Overexpression of mi R-101 in tumor cells under normoxia or up-regulation of endogenous mi R-101 of tumor cells in hypoxia may be a potential therapeutic approach to cancers.