Effects Of Metformin On The Migration And Invasion Of Breast Cancer Cells And The Regulation Mechanism Of MiR-625

Aims:1. To investigate the effects of metformin on invasion and migration of breast cancercells MDA-MB-231and MCF-7.2. To investigate the effects of metformin on the expression of miR-625in breast cancercells MDA-MB-231and MCF-7.3. To investigate the effects of miR-625on invasion and migration of breast cancer cellsMDA-MB-231with miR-625mimics and MCF-7with miR-625inhibitors4. To investigate the expression of migtation and invasion relative proteins and targetprotein after transfected with miR-625mimics and miR-625inhibitors.Methods:1. In the background of MDA-MB-231and MCF-7,cells were treated with differentconcentrations of metformin for48h and72h, then the proliferation was measured byMTT assay. According the results, we use lower concentrate metformin to measure theeffects of metformin on invasion and migration of breast cancer cells.2. Investigate the effects on the migration and invasion of lower concentrate metforminof breast cancer cells MDA-MB-231and MCF-7by Transwell assay, then investigatethe effects of metformin on the expression of miR-625in breast cancer cellsMDA-MB-231and MCF-7by qRT-PCR.3. We examined the expression of miR-625in breast cancer cells MDA-MB-231andMCF-7by qRT-PCR,Total RNA was extracted using TRIzol (Invitrogen，USA) fromMDA-MB-231and MCF-7.4. Use target gene prediction software TargetScan、PicTar、miRanda and miRbase topredict the target gene through entering the search terms of miR-625,then Western blotwas performed and detect the expression of SCAI.5. After detecting the expression difference between MDA-MB-231and MCF-7, theMDA-MB-231was transfected with miR-625mimics while the MCF-7was transfected with miR-625inhibitors. Then the transwell assay and wound-healing assay were usedto detect the effects on cell migration and invasion.6. Western blot assay was used to detect the proteins related to migration and invasionafter transfected with miR-625.Results:1. The effects of the metformin on proliferation, invasion and migration of breastcancer cells MDA-MB-231and MCF-7MTT assay showed that compared to the NC group, the proliferation of breastcancer cells were significantly inhibited by metformin treatment in a dose-dependentmanner, so we used lower concentrate metformin to measure the effects on invasion andmigration of metformin,and the assay results showed that the invasion and migration ofMDA-MB-231andMCF-7cells were markedly decreased.2. Metformin influence the expression of miR-625of breast cancer cellsMDA-MB-231and MCF-7The breast cancer cells MDA-MB-231and MCF-7were treated withmetformin after48h, Total RNA was extracted using TRIzol (Invitrogen,USA)from MDA-MB-231and MCF-7. and Real-time PCR results showed that theexpression of miR-625was increased significantly compared to NC group(P<0.05).3. The expression of miR-625in breast cancer cells MDA-MB-231and MCF-7We examined the expression levels of miR-625in breast cancer cellsMDA-MB-231and MCF-7. The result showed that the expression of miR-625wassignificantly different in the two cells. The miR-625levels were crucially higher inMCF-7than in MDA-MB-231.4. The prediction and verification of the miR-625targetBy using online miRNA target gene prediction databases,we found that SCAI wasone of the target genes which we were looking for.5. The effects on migration and invasion of breast cancer cells MDA-MB-231andMCF-7after transfected with miR-625mimics and inhibitorsqRT-PCR results showed that expression of miR-625was crucially higher inMCF-7than MDA-MB-231.Therefore, the MDA-MB-231was transfected withmiR-625mimics while the MCF-7was transfected with the miR-625inhibitors. transfection of miR-625mimics into MDA-MB-231cells did not result in substantiallya reduction in distance migrated compared with negative control (NC) transfected cellsby using a Transwell assay. But in MCF-7cells, transfection with miR-625inhibitorsshowed a increase in distance migrated compared with transfected with negative control(NC) transfected cells by using a Transwell assay. Wound healing assay was used todetect the mobility of breast cells. Results showed that miR-625in MDA-MB-231cellusing mimics did not delay closure of the wound gap while transfected with miR-625inhibitors in MCF-7cell promoted closure of the wound gap in a time-dependentmanner(P<0.05).6. Western blot assay was used to detected the proteins related to migration andinvasion after transfected with miR-625.From the results we found that miR-625may inhibit the migration and invasion inbreast cell lines. Western blot results showed that the protein level of SCAI and TIMP1was significantly down-regulated in MCF-7cell after transfection with miR-625inhibitors,The result of western blot detecting the EMT relating proteins E-cadherin wasalso down-regulated,prompt that miR-625may induce EMT.Conclusion:1. Metformin can decrease the invasion and migration of breast cancer cellsMDA-MB-231and MCF-7, then the expression of miR-625was higher.2. The expression of miR-625was low expression in breast cancer cell MDA-MB-231with high invasion ability. On the contrary, The expression of miR-625was highexpression in breast MCF-7with low invasion ability.3. After transfected with miR-625inhibitors in MCF-7, we found that miR-625couldinhibit cell migration and invasion in vitro and the miR-625play a role in cancersuppressor gene.4. miR-625inhibit the cell migtarion and invasion through regulating SCAI in breastcancer MCF-7.