Long Non Coding RNA OIP5-AS1 Promotes Metastasis Of Breast Cancer Via MiR-340-5p/ZEB2 Axis

Currently,breast cancer remains a leading health problem and constitutes one of the most severe burdensome diseases in females around the world despite understanding of underlying molecular mechanisms.Tumor metastasis is diagnosed in approximately 30% of breast cancer patients and is the major cause of cancer-related deaths.The prognosis for most patients with metastatic breast cancer is unfavorable with a median overall survival range from 2 to 3 years.Generally,breast cancer can be categorized into four subtypes[luminal A,luminal B,human epidermal growth factor receptor 2(HER2)positive and triple negative],which are defined using immunohistochemical breast tumor markers.These four subtypes have the potential risk of distant metastasis but with differential site?specific metastatic patterns.Currently,breast cancer metastasis from primary tumor to distant organs occurs through a sequential molecular cascade including local angiogenesis for tumor growth,invasion of the surrounding tissue,intravasation of the carcinoma cells into the blood or lymphatic vessels,dissemination and proliferation at secondary neoplastic foci.These carcinoma cells obtain mesenchymal features and suppress their epithelial features through the epithelial-to-mesenchymal transition(EMT)process to promote an invasive and metastatic phenotype.Multiple transcription factors coordinate EMT programs.Among them,zinc?finger E?box?binding(ZEB)transcription factors,ZEB1 and ZEB2,are two EMT regulators that either repress or activate transcription in various types of cancer.Furthermore,ZEB2 was reported to negatively correlate with the epithelial marker E-cadherin in breast cancer cells involved in breast cancer progression.Long non-coding RNAs(lncRNAs)are a class of transcripts containing more than 200 nucleotides in length with limited protein?coding capacity.Recent findings have shown that dysregulation of lncRNAs is involved in cell proliferation,tumor progression and metastasis in cancers.Functionally,lncRNAs interact with proteins and other RNAs to regulate their activities and cellular location.Furthermore,lncRNAs act as molecular sponges for miRNAs that block the binding activity for target transcripts.In breast cancer,several lncRNAs have been identified as either oncogenic or tumor suppressive factors,such as X-inactive-specifictranscript(XIST),HOX antisense intergenicRNA(HOTAIR),growth arrest specific 5(GAS5)and metastasis?associated lung adenocarcinoma transcript 1(MALAT1).A systematic analysis of the correlation has been carried out between these dysregulated lncRNAs and breast cancer clinicopathology and survival suggesting a pivotal role in cancer development.Increasing lncRNAs have been shown to participate in specific cancer types but more often exert general function in a broad spectrum of cancer.OPA-interacting protein 5 antisense transcript 1(OIP5-AS1)is an evolutionarily conserved long non-coding RNA that is transcribed from opposite direction to the OIP5 gene.It was first shown to be expressed in the nervous system and was essential for neurogenesis during embryonic development.The functions of OIP5-AS1 in multiple human cancers have been reported to be associated with oncogenesis.In breast cancer,OIP5-AS1 levels are upregulated in breast tumor tissue and correlated with tumor size,metastatic status of lymph nodes,pathological grading and TNM stage.Currently,the precise function and molecular mechanisms of OIP5-AS1 in breast cancer are not fully elucidated.Thus,our research focused on the metastasis properties and potential signalling of OIP5-AS1 in in vitro and in vivo models.We found that the high expression of OIP5-AS1 in the breast cancer cells was associated with migration and invasion.Further studies revealed that OIP5-AS1 regulates the expression of ZEB2,a key transcription factor in EMT,in breast cancer cells.One of the functions of long noncoding RNA is acting as ce RNAs in the cell.Thus,we tested the binding of OIP5-AS1 and miR?340?5p according to the complementary sequences.Besides,we also tested the binding of miR-340-5p and ZEB2 which was reported in other cancer types.In the present study,we investigated the role of OIP5-AS1 in breast cancer metastasis using the in vitro and in vivo models showing that OIP5-AS1 regulates ZEB2 expression by acting as ce RNA for miR?340?5p.Methods:Six cell lines(MCF-10A?MCF-7?MDA-MB-231?MDA-MB-468?ZR-75?SK-BR-3)were used in the In Vitro study.MCF-7 and MDA-MB-231 were mainly used to investigate the molecular mechanisms.The BALB/c nude mice were used in In Vivo metastasis assay.(1)To investigate the expression and the metastasis properties of Lnc OIP5-AS1 in breast cancer cell lines,the RT-PCR was used to detect the expression of OIP5-AS1 and to evaluate the expression of OIP5-AS1 with siRNA transfection.The wound healing assay and transwell invasion assay were used to detect the migration and invasion properties.(2)To investigate the expression and the metastasis properties of Lnc OIP5-AS1 in vivo,we packed OIP5-AS1 expression vector using lentivirus and infected MCF-7 cells,which injected into tail vein of nude mice.After eight weeks,the number of metastatic lung nodules were recorded.H&E staining and Ki-67 staining were performed to detect metastatic lung tissue in the LV-OIP5-AS1 group.(3)To investigate the molecular mechanism of metastasis of OIP5-AS1 in breast cancer,first we used Western Blot to detect the protein levels of E-cadherin,N-cadherin,and Vimentin with OIP5-AS1 siRNA knockdown.Then,the protein expression of several vital transcription factors(ZEB1,ZEB2,Snail,Slug,Twist)were also compared between negative control group and OIP5-AS1 siRNA knockdown group.(4)To investigate the role of OIP5-AS1 as a miRNA sponge,we initially used fluorescence in situ hybridization(FISH)to assess the subcellular location of OIP5-AS1.According to the prediction by Starbase and DIANA-Lnc Base software,we used anti-Ago2 RIP assay and dual luciferase reporter assay to verify the binding of OIP5-AS1 and miR-340-5p.Besides,the regulatory function of OIP5-AS1 on miR-340-5p was confirmed by RT-PCR which comparing the expression of miR-340-5p in control group vs OIP5-AS1 siRNA group.(5)To investigate the binding of miR-340-5p with ZEB2,we examined the expression level of miR-340-5p in breast cancer cell lines by RT-PCR method.Using mimics or inhibitors of miR-340-5p,we tested the expression of ZEB2 at mRNA level to confirm the direct regulation of miR-340-5p on ZEB2.In the meanwhile,the protein level of ZEB2 under each condition was tested by Western Blot.The direct binding of miR-340-5p and ZEB2 was verified by dual luciferase reporter assay.(6)To investigate the role of OIP5-AS1 on promoting migration and invasion was through miR-340-5p/ZEB2 axis,we used RT-PCR and Western Blot to confirm the expression of ZEB2 in the following groups: NC,si-NC,siOIP5-AS1,NC mimic,miR-340-5p mimic,siOIP5-AS1+ miR-340-5p inhibitors.The invasion properties of breast cancer cells in each group were detect using Transwell assay.Results:1.In vivo and in vitro studies of LncRNA OIP5-AS1 on breast cancer metastasis(1)OIP5-AS1 is up-regulated in breast cancer cell lines.(2)After knocking down OIP5-AS1 in MCF-7 and MDA-MB-231 cells,the wound healing test confirmed that the decreased expression of OIP5-AS1 can significantly reduce the migration ability of breast cancer cells;Transwell test confirmed that invasive cells of the siOIP5-AS1 knockdown group were significantly reduced.Through the above experiments,we believe that the reduction of OIP5-AS1 expression in breast cancer cells can reduce the migration ability and invasiveness of the cells.(3)MCF-7 cells overexpressing OIP5-AS1 can produce an average of about 3 lung metastases,while in the control group,there are about 0-1 lung metastases.At the same time,HE staining confirmed the observed phenomenon of lung metastases,and Ki-67 staining proved that these lung tumors have the characteristics of proliferation and differentiation.2.Study on the mechanism of LncRNA OIP5-AS1 on breast cancer metastasis(1)Knockdown of the expression of OIP5-AS1 leads to up-regulation of E-cadherin expression in cells,while down-regulation of the expression of N-cadherin and Vimentin,indicating that OIP5-AS1 can promote EMT in cells.(2)Knockdown of OIP5-AS1 in breast cancer cell lines can significantly reduce the protein expression levels of ZEB1 and ZEB2,indicating that the effect of OIP5-AS1 on EMT of breast cancer cells is accomplished by regulating the ZEB protein family.(3)RNA fluorescence in situ hybridization probe technology confirmed that lnc OIP5-AS1 is mainly located in the cytoplasm.(4)Bioinformatics analysis and anti-Ago2 RNA co-immunoprecipitation experiments and dual luciferase report analysis experiments indicate that OIP5-AS1 can combine with miR-340-5p and down-regulate the expression of miR-340-5p.(5)Bioinformatics analysis and dual-reporter luciferase experiments show that miR-340-5p binds to the non-coding region at the 3'end of ZEB2 and down-regulates the mRNA and protein levels of ZEB2.(6)OIP5-AS1 regulates the expression of ZEB2 mRNA and protein levels through miR-340-5p,and ultimately affects the invasiveness of breast cancer cells.Conclusions:In conclusion,we identified the OIP5?AS1/miR?340? 5p/ZEB2 axis in breast cancer cell metastasis.OIP5?AS1 facilitated breast cancer metastasis by sponging miR?340?5p to upregulate ZEB2 mRNA transcripts.The current results provide a new direction for the further investigation of molecular mechanism of breast cancer metastasis.Defining the underlying mechanisms of differentially expressed lncRNA in cancers may be useful in developing novel strategies for cancer diagnosis and treatment.