BMP9 Inhibits The Migration And Invasion Of Breast Cancer Cell Through Down-Regulating LncRNA SNHG3

Objective: To investigate the mechanism of BMP9 inhibiting the migration and invasion of breast cancer cells from the perspective of lncRNA SNHG3 and its target gene ZEB1.Methods: LncRNA expression profiling microarray was used to detect the expression of lncRNA in breast cancer cells MDA-MB-231 with or without BMP9 treatment.The results were screened and validated by qRT-PCR.To observe the correlation between SNHG3 and breast cancer,bioinformatics analyse based on breast cancer clinical samples was performed.Fluorescence in situ hybridization was applied to observe the cellular localization of SNHG3.siRNA and SNHG3 plasmid were transfected into breast cancer cells respectively to reduce the endogenous expression of SNHG3,and construct overexpressing SNHG3 cell lines.The proliferation ability of breast cancer cells was measured by colony formation assay.The migration and invasion of breast cancer cells were detected by wound healing assay and transwell chamber assay.The direct binding site of miR-186-5p on SNHG3 was predicted by StarBasev2.0 database.Combined with literature and database analysis,which showed that miR-186-5p could directly regulate the expression of target gene ZEB1.The dual luciferase reporter gene assay and Western blot were performed to verify the regulation of miR-186-5p and ZEB1 by SNHG3;Moreover,EMT pathway related indicators were detected by qRT-PCR and Western blot.The effect of ZEB1 on breast cancer was further explored by above biological function experiments through overexpression ZEB1.Ad-BMP9 and overexpression SNHG3 plasmid were co-transfected into breast cancer cells.Transwell assay was carried out to evaluate the effect of SNHG3 on inhibition of BMP9 on breast cancer migration and invasion.Finally,the effect of SNHG3 on breast cancer was explored by subcutaneous tumor formation in nude mice.MCF-7 breast cancer cells of control group and knocking down with SNHG3 group were subcutaneously transplanted into the female nude mice respectively.The tumors were collected after 35 days.The mRNA expression of SNHG3 was detected by qRT-PCR.The protein expression of ZEB1,MMP2 and Snail was measured by Western blot.Immunohistochemical staining was performed to observe the expression of PCNA、E-cadherin and Vimentin.Results: Combined with lncRNA expression profiling chip and qRT-PCR,which showed that the expression of SNHG3 significantly decreased after BMP9 treatment of breast cancer cells.Using Oncomine and TCGA database analysis,it was found that compared with the normalpopulation,the expression of SNHG3 in breast cancer patients was highly expressed.At the same time,compared with the immortalized normal mammary epithelial cells MCF-10 A,SNHG3 was also highly expressed in breast cancer cells.FISH confirmed SNHG3 was mainly localized to the cytoplasm in the breast cancer cells.The qRT-PCR confirmed that the overexpression SNHG3 was successfully constructed.Through the wound healing experiment and transwell assay,it showd that knockdown of endogenous expression of SNHG3 could inhibit the migration and invasion of breast cancer cells;Overexpression of SNHG3 could promote the proliferation,migration and invasion of breast cancer cells.qRT-PCR and Western blot were used to detect the mRNA and protein levels of EMT markers,and it was found that SNHG3 can promote the transformation of breast cancer cells into EMT.The dual-luciferase reporter assay confirmed that SNHG3 had a direct binding site to miR-186-5p,and that miR-186-5p also directly binded to ZEB1.We found that the expression of ZEB1 increased after overexpression of SNHG3 and vice versa.In the rescue experiment,transfection of pcDNA3.1+/SNHG3-WT in breast cancer cells reversed the decrease of ZEB1 expression by si-SNHG3,whereas the expression of ZEB1 was unchanged after transfection of pcDNA3.1+/SNHG3-MUT.Therefore,SNHG3 promoted the transformation of breast cancer cells into EMT by competitively binding of miR-186-5p to up-regulate of ZEB1,andincreased the ability of migration and invasion.In addition,co-transfection of BMP9 adenovirus and SNHG3 overexpression plasmid partially reversed the inhibitory effect of BMP9 on migration and invasion of breast cancer cells.In vivo,the results showed that the tumorigenic ability of breast cancer cells was inhibited after knockdown of SNHG3 expression in MCF-7 cells;The expression of SNHG3 in experimental group was significantly reduced by qRT-PCR.Western blot showed that SNHG3 knockdown inhibited the expression of ZEB1,Snail and MMP2.The results of immunohistochemistry showed that SNHG3 knockdown promoted the expression of E-cadherin,inhibited the expression of PCNA and Vimentin.Conclusion: BMP9 could inhibit the migration and invasion of breast cancer cells through down-regulating LncRNA SNHG3,and its underlying mechanism may be related to EMT signaling pathway.