LINC02163 Regulates The Proliferation,apoptosis,migration And Invasion Of Breast Cancer Via MicroRNA-511-3P/HMGA2 Axis

BackgroundBreast cancer is a common malignant tumor,and it is also the second cause of death of female cancer globally.Considerable improvements in the anticancer treatments have notably increased the outcomes of patients with breast cancer;however,the clinical efficiency of patients with advanced stage remains unsatisfactory.Thus,it is essential to illustrate the molecular events responsible for the occurrence and development of breast cancer,which may be aid in the identification of effective targets for cancer diagnosis,prognosis and management.Long noncoding RNAs(lncRNAs)are identified as a new family of non-coding RNA molecules,with lengths over 200 nucleotides.Over 15779 lncRNAs have been identified in the human genome,which execute ctrictl regularoty activities in normal development and various human diseases.LncRNAs can regulate development and differentiation,gene imprinting and antiviral response.Thus,studying the involvement of lncRNAs in breast cancer progression and elucidating their working mechanism may provide potential targets for the treatment strategies of breast cancer.Previously,our data confirmed that LINC02163 and HMGA2 were overxpressed in breast cancer,while miR-511-3p was downregulated in breast cancer.Until now,there is no report concerning whether LINC02163 affects the biological functions of breast cancer and its underlying mechanisms.Therefore,in this study,we explored the effects of LINC02163 on the malignant behaviors of breast cancer in vivo and in vitro,and further clarified whether LINC02163 implementes its regulatory roles through miR-511-3p/HMGA2 axis.Part Ⅰ:Expression of LINC02163,miR-511-3p and HMGA2 in breast cancerObjective:Long intergenic non-protein-coding RNA 02163(LINC02163)is considered to be an important regulator of gastric cancer.It is unclear whether LINC02163 is associated with the malignancy of breast cancer,The purpose of this study was to determine the expression profile and clinical value of LINC02163,miR-511-3p and HMGA2 in breast cancer.Methods:Trizol was applied for extracting total RNA from breast cancer tissues and adjacent normal tissues.In addition,total RNA was exerated from breast cancer cell lines and MCF-10A.After RNA isolation,LINC02163,miR-511-3p and HMGA2 levels were detected by real-time quantitative PCR(RT-qPCR),and the correlation among them was determined by Spearman’s correlation analysis.Results:The data of RT-qPCR presented that LINC02163 was evidently highly expressed in breast cancer tissues and cell lines.Breast cancer patients with high LINC02163 level manifested shorter overall survival.LINC02163 expression manifested an obvious relation with tumor size(P=0.041),lymph node metastasis(P=0.002)and TNM stage(P=0.01).miR-511-3p was lowly expressed,while HMGA2 was overexpressed in breast cancer tissues.Furtheremore,an inverse expression relationship between miR-511-3p and LINC02163 was revealed by Spearman’s correlation analysis.Additionally,miR-511-3p level was negatively correlated with HMGA level in the breast cancer tissues.On the contrary,LINC02163 level was positively correlated with that of HMGA2 level in the same breast cancer tissues.Conclusion:LINC02163 and HMGA2 were highly expressed in breast cancer,while miR-511-3p was lowly expressed.Expression of LINC02163 exhibited a norbale correlation with aggressive clinicopathological parameters and poor prognosis.LINC02163,miR-511-3p and HMGA2 in breast cancer tissues showed an inverse or a positive relation in the breast cancer tissues.Part Ⅱ:Effects of LINC02163 and miR-511-3p on the proliferation,apoptosis,migration and invaison of breast cancer cellsObjective:LINC02163 was knocked down,while miR-511-3p was increased in breast cancer cells.Subsequently,the regulatory activities of LINC02163 depeltion or miR-511-3p overexpression on the malignant behaviors of breast cancer cells were explored.Methods:The siRNAs targeting LINC02163 or miR-511-3p mimic were transfected into MDA-MB-231 and MCF-7 cells.Cell Counting Kit-8 assay and flow cytometry analysis was performed to detect the proliferation and apoptosis of breast cancer cells,respectively.Transwell assays were conucted to assess the migration and invasion of breast cacner cells.Besides,xenogeneic tumor model in nude mice was applied by subcutaneous injection of LINC02163 shRNA(sh-LINC02163)cells,and then the effect of LINC02163 silencing on the growth of breast cancer cells in vivo was studied.Results:1.After si-LINC02163 or miR-511-3p mimic transfection,CCK-8 assay was implemented to measure the proliferative activity.Treatment of si-LINC02163 or miR-511-3p mimic clearly reduced the proliferation of MDA-MB-231 and MCF-7 cells.2.Flow cytometry analysis was performed to detect the effect of LINC02163 knockdown or miR-511-3p overexpression on the apoptosis of breast cancer cells.The results showed that the apoptosis rate of MDA-MB-231 and MCF-7 cells was evidently increased after si-LINC02163 or miR-511-3p mimic transfection.3.We also used Transwell cell migration and invasion assay to study the effect of LINC02163 downregulation or miR-511-3p overexpression on the migration and invasion of breast cancer cells.The results showed that the number of migrated or invaded MDA-MB-231 and MCF-7 cell was significantly lower than that in control group.4.The anti-growth effect of LINC02163 knockdown on breas cancer cells in vivo was proved using xenograft experiments.We established xenograft tumor model in nude mice by subcutaneous injection of MDA-MB-231 cells stably expressing sh-LINC02163 or sh-NC.The growth of sh-LINC02163-transfected xenografts was notably suppressed in comparison with sh-NC-transfected xenografts.The xenografts excised from sh-LINC02163-injected mice were striking smaller and lighter as compared with the sh-NC group.In addition,we detected the expression of LINC02163,miR-511-3p and HMGA2 in subcutaneous xenografts.The xenografts obtained from the sh-LINC02163-injected mice manifested downregulated LINC02163 compared with xenografts in the sh-NC group.Also,a significnat overexpression of miR-511-3p and downregulation of HMGA2 protein were detected in sh-LINC02163-transfected subcutaneous xenografts.These results indicated that the absence of LINC02163 decreased the growth of breast cancer cells in vivo.Conclusion:LINC02163 and miR-511-3p exert crucial regulatory actions on the proliferation,migration,invasion and apoptosis of breast cancer cells.Part III:LINC02163 regulates the proliferation,apoptosis,migration and invaison of breast cancer cells via miR-511-3p/HMGA2 axisObjective:The mechanisms underlying the oncogenic functions of LINC02163 in breast cancer cells were elucidated in detail.Also,whether LINC02163/mR-511-3p/HMGA2 pathway was involved in the control of breast cancer cell’s oncogenicity was unveiled too.Methods:The distribution of LINC02163 in MDA-MB-231 and MCF-7 cells was detected by Nuclear/Cytoplasmic fractionation.Bioinformatics tools were used to predict the miRNAs that can directly bind to LINC02163,which were further confirmed by luciferase reporter assay,RNA immunoprecipitation,western blot,RT-qPCR and rescue experiments.LINC02163 siRNA in parallel with miR-511-3p inhibitor or HMGA2 overexpression plasmids were cotransfected into MDA-MB-231 and MCF-7 cells,followed by a series of function experiments.Results:1.Linc02163 is mostly ocated in the cytoplasm of breast cancer cells,which indicates that LINC02163 may promote the progress of breast cancer by sponging certain miRNA.2.By utilizing bioinformatics tool,we found that miR-511-3p contains the complementary binding site within LINC02163.3.We used luciferase reporter gene to identify the binding between LINC02163 and miR-511-3p.In MDA-MB-231 and MCF-7 cells,exogenous of miR-511-3p lowered the luciferase activity of wt-LINC02163.But,the luciferase activity of mut-LINC02163 was unaffected by miR-511-3p overexpression.These results indicate that LINC02163 can bind to miR-511-3p.In addition,RIP results showed that,LINC02163 and miR-511-3p were clearly enriched in the Ago2 antibody contained-magnetic beads.It was also demonstrated the direct interaction between miR-511-3p and LINC02163 in breast cancer cells.4.We also detected whether LINC02163 can regulate the expression of miR-511-3p in breast cancer.The absence of LINC02163 raised miR-511-3p level in MDA-MB-231 and MCF-7 cells.Thus,LINC02163 could directly interact with miR-511-3p and inhibit its activity5.We used the miRNA target prediction database to predict the potential target genes of miR-511-3p.The results showed that the 3’-UTR of HMGA2 contained two potential miR-511-3p binding sites.After that,we confirmed the binding of miR-511-3p to 3’-UTR of HMGA2 with luciferase reporter assay.The results showed that upregulation of miR-511-3p could decrease the luciferase activity of wt-HMGA2 in MDA-MB-231 and MCF-7 cells;however,the luciferase activity of mut-HMGA2 was unchanged by miR-511-3p mimic.In addition,the expression of HMGA2 mRNA and protein in MDA-MB-231 and MCF-7 cells were significantly decreased after miR-511-3p mimic transfection.These results indicate that HMGA2 is a direct target of miR-511-3p in breast cancer cells.6.LINC02163 has been proved to be able to competitively bind and adsorb miR-511-3p,and HMGA2 is the target of the latter,we speculated that LINC02163 was involved in the regulation of HMGA2 expression in breast cancer cells.As expcted,HMGA2 expression in si-LINC02163-transfected MDA-MB-231 and MCF-7 cells was lower than that in si-NC group.Then,si-LINC02163 and miR-511-3p inhibitor or NC inhibitor were co-transfected into MDA-MB-231 and MCF-7 cells.The expression of HMGA2 in the transfected cells was analyzed by RT-qPCR and western blot.The results showed that low expression of LINC02163 significantly suppressed the expression of HMGA2 mRNA and protein.miR-511-3p inhibitor cotransfection significantly increased the expression of HMGA2 that was decreased by LINC02163 knockdown.Thus,LINC02163 could competitively bind miR-511-3p through ceRNA mechanism,and thus regulated the expression of HMGA2 in breast cancer cells.7.We further confirmed that the role of LINC02163 in breast cancer cells is realized by regulating miR-511-3p/HMGA2 signal axis.The results showed that inhibition of miR-511-3p or up regulation of HMGA2 could eliminate the anti-tumor activity induced by si-LINC02163 on breast cancer cells.Conclusion:miR-511-3p inhibition and HMGA2 overexpression can reverse the antitumor activities induced in breast cancer cells by LINC02163 depletion.LINC02163 promotes the malignancy of breast cancer cells by regulating miR-511-3p/HMGA2 axis.