| Objective: It has been proved that BMP9 could inhibit the growth,migration and invasion of TNBC cells.Based on our former researches,we tried to explain the further mechanisms of BMP9 on restraining TNBC through TMEM100.Methods: Part 1: To predict the expression of TMEM100 in breast cancer by searching the UALCAN database,and the results were verified by qRT-PCR.Transfected the TMEM100 plasmid into MDA-MB-231 cells to construct TMEM100 / MDA-MB-231 recombinant cells,and shTMEM100 plasmid into BT-549 cells to construct shTMEM100 / BT-549 cells.The effect of TMEM100 on the proliferation in TNBC cells was detected by MTT experiment;transwell experiment was used to study the migration and invasion of TNBC cells;the protein expressions of Vimentin and Snail were measured by westernblot.Part 2: Applied GCBI and TargetScanHuman7.2 databases to predict miRNAs which regulated the expression of TMEM100.Transfected Ad-BMP9,Ad-siALK1 and miR-195-5p-mimic into MDA-MB-231 cells to construct BMP9/MDA-MB-231,siALK1 / MDA-MB-231 and mimic / MDA-MB-231 recombinant cells.qRT-PCR was performed to exam the effect of BMP9 on the expression of ALK1,miRNA and TMEM100.The expression changes of TMEM100 caused by siALK1 treatment was detected by qRT-PCR.The dual luciferase report experiment was applied to measure the luciferase activity.The expression of TMEM100 protein was detected by IHC in BMP9 / MDA-MB-231 transplanted tumor.Part 3: Co-transfected Ad-BMP9 and shTMEM100 into MDA-MB-231 cells to construct BMP9 / shTMEM100 / MDA-MB-231 recombinant cells.Flow cytometry experiments were used to detect the cell cycle.The expressions of E-Cadherin,N-Cadherin,Snail and Vimentin were examined by westernblot.Apoptotic bodies were stained and observed under microscope.mRNA array was used to analyze the gene expressions related with autophagy.The protein expression of LC3 and localization were detected by IF experiment.Applied westernblot assay to evaluate changes of LC3,ATG7,ATG5,AMPK-α and p-AMPK-α.Subcutaneous tumor formation experiment was used to detect changes in tumor growth.The expression of PCNA were examined by IHC in transplanted tumors.Results: Part 1: qRT-PCR results confirmed that the expression of TMEM100 decreased in breast cancer,which was consistent with the prediction of UALCAN database.In shTMEM100 / BT-549 recombinant cells,the expression of TMEM100 was significantly reduced(P <0.001);cell proliferation(P <0.05),migration and invasion(P <0.001)were enhanced;the expression of Vimentin and Snail increased.But the opposite results were observed in TMEM100/MDA-MB-231 cells.Part 2: Predictions of GCBI and TargetScanHuman7.2 databases showed that miR-195-5p might be related to TMEM100.And qRT-PCR results displayed that miR-195-5p could be adjusted by BMP9.In BMP9 / MDA-MB-231 cells,the mRNA expressions of ALK1,TMEM100 and miR-195-5p were up-regulated(P <0.05).But the expression of TMEM100 was significantly down-regulated in siALK1 / MDAMB-231 cells(P <0.05).After overexpression of miR-195-5p in MDA-MB-231 cells,the expression of TMEM100 increased(P <0.05).The dual luciferase report assay results indicated that TMEM100 could be targeted and up-regulated by miR-195-5p(P <0.05),while,the inhibitor of miR-195-5p reduced the expression of TMEM100(P <0.05).Silencing the binding site of miR-195-5p and TMEM100 3’-UTR region,no changes were observed.In vivo,overexpression of BMP9 could also promote the expression level of TMEM100.Part 3: In BMP9 / shTMEM100 / MDA-MB-231 cells,BMP9 failed to depress cell proliferation and the expressions of N-Cadherin,Snail and Vimentin.E-Cadherin remained the same.At the mean time,after analyzing the mRNA expression profile data,it was found that BMP9 might promote the expressions of autophagy-related genes LC3,ATG5,ATG7 and ATG12.After overexpressing of BMP9 and TMEM100,LC3 accumulated in the nucleus and the protein expression levels of LC3,ATG7,AMPK-α and p-AMPK-αtotally increased.But,in BMP9 / shTMEM100 / MDA-MB-231 cells,the protein expressions of LC3,ATG7,AMPK-α and p-AMPK-α decreased.The protein expression of LC3 and ATG5 greatly reduced in TMEM100 / WZ4003 / MDA-MB-231 cells exposing to AMPK pathway inhibitor.Conclusion: TMEM100 inhibits the proliferation,migration,invasion and EMT of TNBC cells,and its expression is regulated by BMP9.BMP9 can up-regulate the expression of TMEM100 through ALK1 and miR-195-5p.Furthermore,BMP9 inhibits TNBC cell proliferation and EMT via TMEM100.Additionally,BMP9 activates the TMEM100 / AMPK signaling pathway to promote cell autophagy.In summary,BMP9 up-regulates the expression of TMEM100 through miR-195-5p and ALK1 mediating,thereby inhibiting the proliferation and EMT of TNBC cells.Meanwhile,BMP9 promotes the occurrence of autophagy via TMEM100 / AMPK signaling pathway. |
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