Linc-ROR Regulates The Mechanism Of Ovarian Cancer Stem Cells Function And Invasive Metastasis

Objective:The objective of this study was to explore the role of intergenic long chain non-coding RNA-recoding regulator(Linc-ROR)in the functional regulation of ovarian cancer stem cells(OCSCs),and further verify that Linc-ROR regulates stem cell-like characteristics,EMT activation and invasion and metastasis of ovarian cancer stem cells through miR-181c-5p/HIF-1α axis under hypoxia.Methods:(1)A2780 cells were induced to produce OCSCs by serum-free suspension culture method.The self-renewal and directional differentiation characteristics of the induced spheres were identified by sphere-forming experiments and adherent redifferentiation experiments.The positive expression rate of CD 133,a stem cell marker on the surface of OCSCs,was detected by flow cytometry.(2)Bioinformatic analysis predicted the targeting binding site between Linc-ROR RNA and miR-181c-5p RNA,and the relationship between Linc-ROR RNA and miR-181c-5p RNA was further validated by dual-luciferase assay.The mRNA expression levels of Linc-ROR and miR-181c-5p in A2780 cells and OCSCs were detected by qRT-PCR to investigate the relationship between Linc-ROR and miR-181c-5p at the mRNA level.(3)Lentiviral transfection was performed and cell lines with low as well as high expression of Linc-ROR were selected,and the mRNA expression level of Linc-ROR was measured by qRT-PCR to assess the transfection efficiency.Similarly,the corresponding ovarian cancer stem cells control-OCSCs,si-ROR-OCSCs,and p-ROR-OCSCs were induced by serum-free suspension cμlture;sphere-forming ability was assessed by sphere-forming rate,maximum sphere-forming diameter,and sphere-forming cycle;after 6 hours of hypoxia,the mRNA expression levels of control-ROR and miR-181c-5p in the three groups of OCSCs were detected by qRT-PCR;and the changes of core transcription factors(Oct-4 and Nanog),HIF-1α expression,and EMT-related proteins(vimentin and E-cadherin)in each group of Linc-OCSCs,si-ROR-OCSCs,and p-ROR-OCSCs at 6 hours of hypoxia were detected by Western blot to verify whether the changes in control-ROR expression could regulate the stemness characteristics of OCSCs,promote HIF-1α expression,and activate EMT.(4)Under hypoxia,miR-181c-5p was further transfected on the basis of ovarian cancer stem cells to verify whether miR-181c-5p was able to reverse the effect of Linc-ROR on OCSCs.They were divided into control-OCSCs,p-ROR-OCSCs,p-ROR+p-181c-OCSCs,and cultured under hypoxia,and the sphere-forming ability of the three groups of control-OCSCs,p-ROR-OCSCs,and p-ROR+p-181c-OCSCs was assessed by comparing the sphere-forming rate,sphere-forming cycle,and maximum sphere-forming diameter;after 6 hours of hypoxia,the mRNA expression levels of Linc-ROR and miR-181c-5p in the three groups of OCSCs were detected by qRT-PCR;and the relative protein expression levels of core transcription factors(Oct-4 and Nanog),EMT-related proteins(vimentin and E-cadherin),and HIF-1α in control-OCSCs,p-ROR-OCSCs,p-ROR+p-181 c-OCSCs,and p-ROR+p-181 c-OCSCs were detected by Western blot after 6 hours of hypoxia.(5)CCK-8/plate cloning assay and Transwell assay were used to detect the changes of proliferation,invasion and metastasis of ovarian cancer stem cells in each group after intervention.Resμlts:(1)Adherent A2780 cells were successfully induced to generate OCSCs.OCSCs were grown in sphere-forming suspension,and OCSCs could readhere and differentiate from serum-free culture to serum-containing culture.Flow cytometry revealed that the positive expression rate of a surface stem cell marker CD 133 was significantly increased(P<0.05),demonstrating that the induced OCSCs had stem-like characteristics.(2)Linc-ROR has a targeted binding site with miR-181c-5p.qRT-PCR revealed that the mRNA expression level of Linc-ROR was significantly increased and the mRNA expression level of miR-181c-5p was significantly decreased in induced OCSCs compared with A2780 cells.(P<0.05)(3)After lentiviral transfection,the intervention effect of Linc-ROR was significant.After lentiviral transfection,Linc-ROR intervention was effective.Compared with the control-OCSCs group,the relative mRNA expression of Linc-ROR was significantly higher,the mRNA expression level of miR-181c-5p was significantly decreased,the sphere-forming rate was increased,the sphere-forming cycle was shortened,the maximum spheroid diameter was longer,the relative expression of core transcription factors(Oct-4 and Nanog),HIF-1α,and vimentin protein was significantly increased,the relative expression of E-cadherin protein was decreased,and the proliferation,invasion and metastasis ability of OCSCs was significantly enhanced in the p-ROR-OCSCs group.The si-ROR-OCSCs group had the opposite experimental result.(P<0.05)(4)Compared with the p-ROR-OCSCs group,the p-ROR+p-181c-OCSCs group had restored sphere-forming ability,the expression of core transcription factors(Oct-4,Nanog),HIF-1α,and EMT-related protein(vimentin,E-cadherin),and no significant changes were observed compared with the control-OCSCs group.Overexpression of miR-181c-5p can reverse the effect of overexpressing Linc-ROR on OCSCs.(P<0.05)Conclusion:(1)Linc-ROR can promote the expression of core transcription factors Oct-4 and Nanog,improve the sphere-forming ability of OCSCs,shorten the sphere-forming cycle,and promote ovarian cancer proliferation,invasion and metastasis by regulating the stemness characteristics and function of OCSCs.(2)miR-181c-5p is an important downstream target of Linc-ROR in regulating the stemness characteristics as well as invasion and metastasis of OCSCs.Under hypoxia,Linc-ROR promotes the stem-like properties of OCSCs and activates EMT by negatively regulating miR-181c-5p and up-regulating the expression of HIF-1α,which may be one of the important mechanisms by which Linc-ROR promotes the proliferation,invasion and metastasis of tumor cells.