Effect And Mechanism Of Metformin On Papillary Thyroid Carcinoma Cells Via MiR-21/PTEN/AKT Signaling Pathway

Background and purposeThyroid cancer is the most common thyroid malignant tumor,and showed a trend of increased morbidity year by year,which seriously impacts on human health.Metformin is one of the oral biguanides.Due to its safe and effective hypoglycemic effect,metformin has been recognized as the first-line drugs for treatment of type 2 diabetes by experts at home and abroad.In recent years,several studies have reported that metformin can inhibit the development of tumor,not only reduce the incidence of tumor in diabetic patients,but also inhibit tumor growth in diabetic patients with thyroid cancer,as well as improve the prognosis of patients with thyroid cancer.But the specific anti-cancer mechanism of metformin is not yet clear.Our team pilot study found that expression of miR-21 in thyroid cancer cells was increased.After treated thyroid papillary carcinoma IHH4 cells with metformin,we found that:(1)Metformin decreased the proliferation of IHH4 cells in a time-and dose-dependent manner,increased the proportion of cells in G0/G1 phase and decreased that in S and G2/M stage.And also,the apoptosis rate of IHH4 cells was increased.(2)Microarray chip technology showed that a total of 114 miRNAs in IHH4 cells differential expressed significantly after intervened with metformin.We selected the miRNAs which expressed difference and was of great research significance,including the decreased miR-125-a and miR-21,as well as the increased miR-29b-3p.The biology function analysis of the significantly differential expressed miRNAs' target genes significantly enriched in transduction and transcriptional regulation;Pathway items were significantly enriched in tumor related signaling pathway by KEGG analysis.Therefore,basic on part of our previous studies,we will further explore whether metformin can inhibit thyroid papillary cancer cells proliferation by regulating the expression of miR-21 and controlling its downstream signaling pathways,thereby explore the anti-cancer molecular mechanism of metformin on thyroid carcinoma.Methods1.In order to validation the role of metformin on miR-21,PTEN and AKT genes,human papillary thyroid carcinoma IHH4 cells were incubated in vitro and intervened with metformin for 48h,and the expression levels of miR-21,PTEN(cell proliferation related genes)and AKT were detected by quantitative real-time PCR.2.Cells were cultivated in vitro and transfected with miR-21 mimics or miR-21inhibitor,resulting in the over-or lower-expression of miR-21.Then,we detected the expression levels of PTEN and AKT genes by qRT-PCR to verify the impact of miR-21 on PTEN and AKT.3.Cells were transfected with miR-21 mimics and miR-21 inhibitor respectively followed by metformin treatments for 48h.Next,we detected the expression of PTEN and AKT by qRT-PCR and western blotting to confirm the role of miR-21 to PTEN and AKT in the case of metformin intervention.4.Cells were transfected with miR-21 mimics and miR-21 inhibitor respectively followed by metformin treatments for 48h.We detected the cell proliferation by CCK8 assay to clear the effect of miR-21 on cell proliferation under the influence of metformin.5.We treated the cells with metformin after transfected miR-21 mimics or miR-21 inhibitor into IHH4 cells.Cell apoptosis was detected using flow cytometry to explore the effect of miR-21 on IHH4 cells apoptosis under the influence of metformin.Results1.Compared with the control group,metformin inhibited the expression of miR-21,up-regulated that of PTEN gene and down-regulated AKT gene markedly in IHH4 cells.2.MiR-21 mimics up-regulated miR-21 expression significantly,down-regulated PTEN gene and increased the expression of AKT gene in IHH4 cells.On the contrary,miR-21 inhibitor decreased the miR-21 expression obviously,up-regulated PTEN and down-regulated AKT.3.MiR-21 mimics can partially counteract the effect of metformin on promoting the expression of PTEN and inhibiting the downstream gene AKT.On the other hand,after transfected miR-21 inhibitor into IHH4 cells,the effect of metformin on PTEN and AKT was enhanced.4.After over expressed miR-21,the antiproliferative effect of metformin on IHH4 cells was weakened.Inversely,there was a synergistic effect between miR-21 inhibitor and metformin on cell proliferation.5.Over-expression of miR-21 partly eliminated the effect of metformin induced apoptosis in IHH4 cells,and down-regulated expression of miR-21 mildly collaborated metformin induced apoptosis in IHH4 cells.ConclusionsMetformin may inhibit the papillary carcinoma thyroid IHH4 cells proliferation by control the expression of miR-21 and up-regulate the expression of tumor suppressor gene PTEN,as well as down-regulate the AKT signaling pathway.