| BackgroundIn China,esophageal cancer is the third leading cause of tumor-related death,and deep invasion and metastasis of esophageal cancer are the main causes of death in patients with esophageal cancer.Metformin is a biguanide oral hypoglycemic drugs commonly used in the treatment of type 2 diabetes.Recently,more and more epidemiological data and preclinical studies have demonstrated the anti-tumor effects of metformin in a wide range of cancers,including esophageal cancer.Many studies have found that the anti-tumor effect of metformin is related to many signaling pathways,but the specific mechanism is still unclear.LncRNAs are expressed widely in cancers and play a key role in the overall progression of cancer and radiochemotherapy resistance.It has been confirmed that lncRNAs are involved in metformin-mediated anti-tumor processes,and some studies have confirmed the importance of lncRNAs in esophageal squamous cell carcinoma(ESCC).However,in ESCC,the role and the mechanism of LncRNAs in anti-tumor process mediated the effect of metformin has not been reported.This study will explore the potential role and possible mechanism of LncRNAs in metformin-mediated anti-tumor process in ESCC.ObjectiveTo identify and clarify the role of lncRNAs in the proliferation and migration inhibition process mediated by Metformin and to explore the mechanism of lncRNAs in the proliferation and migration inhibition process mediated by Metformin,so as to provide a new target for clinical treatment of esophageal cancer.Methods(1)Esophageal cancer cells were treated with metformin at different concentrations,and ESCC cell viability was measured by MTT assay;ESCC cell migration ability was measured by the wound healing assay and Transwell in vitro migration test;(2)Opti-MEM I and Lipofectamine 2000 reagents(Invitrogen),CA,USA)were used to transfect the overexpression vectors pcDNA3.1-CCAT1(CCAT1),pcDNA3.1-SPRY4-IT1(SPRY4-IT1)and empty vector pcDNA3.1(as a control),and metformin was treated by administered.Real-time PCR was used to detect the expression of CCAT1 and SPRY4-IT1 RNA in ESCC cells,and Western blot was used to detect the expression of Vimentin and c-Myc protein in ESCC cells.(3)Statistical analysis was performed using SPSS 21.0.P < 0.05 was considered statistically significant.Results1.Metformin inhibited the proliferation and migration of ESCC cells in a dose-dependent manner: MTT results showed that metformin at different concentrations(2.5,5,10,20,40 mM)has inhibitory effects on the proliferation of esophageal cancer Eca-109 cells in vitro(P <0.05),and the inhibition increased with the increase of metformin concentration and treatment time(P<0.001).This result indicates that metformin exerts a time-dose-dependent inhibition of proliferation of Eca-109 cells in a certain concentration and time range,and the inhibition effect of metformin on the proliferation of ESCC cells was also observed in other ESCC cell lines(EC9706,TE1,TE7),with statistically significant differences(P < 0.05).The wound healing assay results showed that metformin at different concentrations(1.25,2.5,5,10,20 mM)had an inhibitory effect on the migration of esophageal cancer Eca-109 cells in vitro,and the inhibitory effect was gradually enhanced with the increase of metformin concentration and treatment time,and the differences are statistically significant(P < 0.05).Transwell in vitro migration experiment showed that metformin at differentconcentrations(5,10mM)had an inhibitory effect on the migration of Eca-109 and TE-1cells in vitro,and the difference was statistically significant(P < 0.001).2.CCAT1 and SPRY4-IT1 were the LncRNAs most significantly affected by metformin in ESCC cells: RT-PCR results showed that two of the seven LncRNA,CCAT1 and SPRY4-IT1,were significantly down-regulated in ECa-109 and TE1 cells after treatment with metformin(10mM)for 48 hours(P < 0.001).ECa-109 cells were treated with metformin at different concentrations(0,2.5,5,10 mM)for 48 h.The levels of CCAT1 and SPRY4-IT1 were down-regulated in a dose-dependent manner,and the differences were statistically significant(P<0.05).3.Overexpression of CCAT1 and SPRY4-IT1 reversed metformin-induced inhibition of proliferation and migration of ESCC cells: After 48 hours of transfection of Eca-109 cells with empty vector(pcDNA3.1),plasmid expressing CCAT1(CCAT1)or plasmid expressing SPRY4-IT1(SPRY4-IT1),real-time RT-PCR results showed that the proliferation of Eca-109 cells was significantly improved.Metformin inhibited the proliferation of Eca-109 cells,while the overexpression of CCAT1 or SPRY4-IT1 reversed the proliferation inhibition of Metformin on Eca-109 cells(P < 0.001).Similar phenomena were observed in cell migration.The results of cell scratch and Transwell test showed that the migration and invasion ability of Eca-109 cells increased.Metformin inhibited migration of Eca-109 cells,and overexpression of either of the two lncRNAs alleviated this inhibition.The difference was statistically significant(P < 0.001).4.Metformin inhibited the expression of E-cadherin,Vimentin and c-Myc in ESCC cells: ESCC cells(Eca-109,TE1)were treated with different concentrations of metformin(0,2.5,5,10 mM)for 48 h.Western blot analysis showed that metformin down-regulated the protein levels of Vimentin and c-Myc in a dose-dependent manner,and the difference was statistically significant(P<0.05).5.Metformin modulated c-Myc and Vimentin levels via CCAT1 and SPRY4-IT1: we overexpressed pcDNA3.1,CCAT1 or SPRY4-IT1 in Eca-109 cells.Then cells were treatedwith metformin(0 or 10mM)for 24 h.Real-time PCR revealed the RNA levels of CCAT1 and SPRY4-IT1 in four groups(P < 0.001).Western blotting showed that when CCAT1 was up-regulated,there was a concomitant increase in the protein level of c-Myc and Vimentin(P<0.01).The protein levels of c-Myc and vimentin were decreased by metformin(P<0.001),which were also rescued by over-expression of CCAT1 partially(P<0.01).The similar phenomena were observed in cells transfected with SPRY4-IT1.The similar phenomena were observed in cells transfected with SPRY4-IT1.Vimentin and c-Myc leves were increased by overexpression of SPRY4-IT1(P<0.01).The down-regulation of vimentin and c-Myc by metformin was also rescued by overexpression of SPRY4-IT1 at least partially(P < 0.05).6.There is a positive correlation between CCAT1 and c-Myc in human ESCC tissues:we measured the expression of c-Myc,CCAT1 and SPRY4-IT1 in the 15 pairs of ESC tissues and matched adjacent normal tissues by real-time RT-PCR.Our data indicated that the CCAT1 levels were remarkably up-regulated in ESC cancer tissues compared with normal tissues,and the CCAT1 up-regulation rate was 10/15(66.7%)(P<0.05).Meanwhile,significantly higher levels of SPRY4-IT1 and c-Myc were also found in ESC tissues compared with normal tissue.The up-regulation rate of SPRY4-IT1 and c-Myc was 11/15(73.3.7%)and 12/15(80%)(P<0.05).Furthermore,A positive correlation was observed between CCAT1 levels and c-Myc mRNA levels in vivo(r2 = 0.306,P? 0.05),while nocorrelation was observed between SPRY4-IT1 levels and c-Myc mRNA levels(r2 = 0.0748,P>0.05).These data suggested that RNA levels of CCAT1 and SPRY4-IT1 were significantly increased in ESC tissues,and there is a positive correlation between CCAT1 and c-Myc.Conclusion1.Metformin may inhibit the proliferation and migration of ESCC cells by regulating two lncRNAs(CCAT1 and SPRY4-IT1).2.There is a positive correlation between CCAT1 and c-Myc in human ESCC tissues.3.CCAT1 and SPRY4-IT1 are therapeutic targets for metformin in ESCC. |
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