The Effect Of LncRNA Hotair On OATP1B1 Expression Regulated By Sponging MiR-206/miR-613 And Its Mechanism

Background:miR-206/miR-613 regulated the expression and function of OATP1B1 at the post transcriptional level.LncRNA HOTAIR can act on miR-206/miR-613 to regulate the expression of its target genes,thus affecting the corresponding functional changes.However,whether LncRNA HOTAIR plays any role in regulating OATP1B1 of miR-206/miR-613 is still unknown.Objectives:The purpose of this article is to reveal the regulatory effect of LncRNA HOTAIR on the expression of OATP1B1 through miR-206/miR-613 and its molecular mechanism,to add new content for elucidating the regulatory mechanism of OATP1B1,and to provide theoretical and scientific basis for further clinical study of individual differences in drug response.Methods:1.miR-206/miR-613 mimics and miR-206/miR-613 inhibitors,were transiently transferred into HepG2 cells to construct miR-206/miR-613 over-expression and underexpression cell models.RT-qPCR,Western-blot technique was used to study the effect of miR-206/miR-613 on expression of OATP1B1 mRNA and protein.2.pcDNA3.1-HOTAIR,LncRNA HOTAIR Smart Silencer and pcDNA3.1 ?HOTAIR Smart Silencer NC were transiently transferred into HepG2 cells to construct LncRNA HOTAIR over-expression and under-expression cell models.RT-qPCR,Western-blot technique was used to study the effect of LncRNA HOTAIR on miR-206/miR-613 expression and OATP1B1 mRNA,protein expression.3.In HepG2 cells,transient co-transfection of LncRNA HOTAIR+ miR-206/miR-613 to further confirm the role of miR-206/miR-613 in the regulation of OATP1B1 by LncRNA HOTAIR.4.In HEK-293 T cells,miR-206/miR-613 mimics and LncRNA HOTAIR reporter plasmids were transiently co-transfected to study the specific binding of LncRNA HOTAIR and miR-206/miR-613 by Luciferase reporter gene assay;pcDNA3.1-HOTAIR or HOTAIR Smart Silencer and OATP1B1 reporter gene plasmids were transiently co-transfected to study the molecular mechanism of LncRNA HOTAIR competitive binding to miR-206/miR-613 and regulating OATP1B1 expression.Results:1.Compared with the control group,transient transfection of miR-206/miR-613 mimics,OATP1B1 protein decreased by 38.6% and 61.1% respectively.Transient transfection of miR-206/miR-613 inhibitors,OATP1B1 protein increased by 67.4% and 72.8%.However,there was no change in mRNA expression of OATP1B1 during transient transfection of miR-206/miR-613 mimics or inhibitors.The above results suggested that miR-206/miR-613 significantly decreased the expression of OATP1B1 protein at the post-transcriptional level.2.Transient transfection of pcDNA3.1-HOTAIR,the expression of miR-206/miR-613 mRNA were decreased to 0.45/0.34 times of the control group.Compared with the control group,the expression level of OATP1B1 protein increased by 103.5%.Transient transfection of HOTAIR Smart Silencer,the expression of miR-206/miR-613 mRNA were increased to 1.95/1.87 times of the control group.Compared with the control group,the expression level of OATP1B1 protein decreased by 43.1%,while there was no change of OATP1B1 mRNA expression after the transient transfection of pcDNA3.1-HOTAIR or HOTAIR Smart silencer.It has been shown that overexpression or inhibition of LncRNA HOTAIR also regulated OATP1B1 at post transcriptional level.In addition,LncRNA HOTAIR negatively regulated the expression of miR-206/miR-613 mRNA.The above results suggested that LncRNA HOTAIR may further regulate the expression of OATP1B1 by acting on miR-206/miR-613.3.Transient co-transfection with pcDNA3.1-HOTAIR + miR-206/miR-613 mimics,compared with the control group,the expression level of OATP1B1 protein decreased by 13.3% and 18.6% respectively.Transient co-transfection with HOTAIR Smart Silencer+ miR-206/miR-613 inhibitors,compared with the control group,the expression level of OATP1B1 protein increased by 46.9% and 30.9%.Thus,the activation or inhibition of overexpression or inhibition of LncRNA HOTAIR on the expression of OATP1B1 protein can be partially reversed by miR-206/miR-613 mimics or miR-206/miR-613 inhibitors,which effectively proves that the regulatory effect of LncRNA HOTAIR on OATP1B1 is realized by targeting miR-206/miR-613.4.Transient co-transfection of miR-206/miR-613 mimics and pmir/HOTAIR-WT reporter genes reduced luciferase activity by 50% or 57%,while the mutation contained 23 bases of the binding site 2186bp-2207bp/2186bp-2205 bp of miR-206/miR-613 and LncRNA HOTAIR 3'-UTR,and then co-transfected miR-206/miR-613 mimics and pmir/HOTAIR-Mut reporter genes,the luciferase activity had no significant change.It showed that there is a specific binding between LncRNA HOTAIR and miR-206/miR-613.5.Transient co-transfection of pcDNA3.1-HOTAIR or HOTAIR Smart silencer and pGL/OATP1B1-WT reporter gene,the luciferase activity was up-regulated by 100% or down-regulated by 54.3%.However,there was no significant change in the luciferase activity when transient co-transfected with pcDNA3.1-HOTAIR or HOTAIR Smart silencer and pGL/OATP1B1-Mut reporter gene after breaking the exact site of miR-206/miR-613 binding with OATP1B1 3'-UTR in 590-597 nt.It was further determined that LncRNA HOTAIR repress miR-206/miR-613 at the 3'-UTR binding site of OATP1B1,and the expression of OATP1B1 protain at post-transcriptional level.Conclusion:LncRNA HOTAIR could sponge miR-206/miR-613 to prevent its action on specific sites,thereby affecting the expression level of OATP1B1 protein.