BAG3 Inhibits The Proliferation And Induces Apoptosis Of Human Esophageal Cancer Cells By Inhibiting MiR-let-7g/i

As one member of BAG gene family,BAG3 can be induced by calcium influx inhibitor CAI,so it is also known as CAIR-1.Expression level of BAG3 in human normal myocardial tissue and skeletal muscle tissue is high,whereas very low in other tissues.In addition,BAG3 was highly expressed in hematological malignancies and solid tumors [Takayama et al.,2012].Previous studies have shown that BAG3 has a series of biological functions,in which BAG3 can specifically bind to anti-apoptotic proteins Bcl-2 and heat shock proteins HSP70 to form complex,and promote cell growth and signal transduction.BAG also participates in cell migration,apoptosis,adhesion,movement and other biological processes [Rosati et al.,2011].BAG3 can be induced by heavy metals,high temperature,protease inhibitors,HIV-1 infection and other stress stimuli,it is also the only member of BAG protein family found so far that can be induced by stress[Homma et al.,2006].Previous studies have shown that BAG3 was highly expressed in tumor tissues,knock-down of BAG can inhibit malignant phenotype of tumor cells,and participate in the process of human tumor resistance to chemotherapy[Mani et al.,2016].A recent study has indicated that knockdown of BAG3 in ovarian cancer cells caused a variety of miRNA expression level change,including miR-let-7g/i,BAG3 may be related to the regulation of miRNA expression in cells[Sugio et al.,2014].microRNAs(miRNAs)are small,non-coding RNAs that range in size from 18 to 25 nucleotides,miRNA itself does not have an open reading frame,nor can it encode proteins.miRNA is obviously different from most oligonucleotides and functional RNA degradation fragments,such as the 5' terminal of miRNA has a phosphate group,and the 3 ' terminal contains one hydroxyl group.miRNA has many biological functions and widely exists in human tissues and it can inhibit the translation or induce the degradation of target mRNA through the binding of target mRNA to 3'UTR [6-8].miRNAs including miR-let-7g/i differentially express in human cancers and are crucial for carcinogenesis [Winter et al.,2005].For instance,let-7 family members have been found to be downregulated in a number of human cancers,including lung,esophageal,colon,ovarian,uterine leiomyoma and breast cancers [Yanaihara et al.,2006],indicating that miR-let-7 may present as potential tumor suppressors.Moreover,miR-let-7g/i play an important role in drug resistance in tumor cells.Esophageal carcinoma(EC)is one of the highly malignant neoplasms,of which disease progression is frequently observed,even if at initial examination.Cisplatin is widely used as the preferred chemotherapeutic agent for different cancers,such as human non-small cell lung cancer and colon cancer [11-12].However,the development of drug resistance as a result of continuous infusion or multiple administration of DDP and the lethal regulatory pathway elicited by the drug transporter-mediated self-defense remains poorly understood.A previous study showed that ABCB9 confers resistance to cisplatin in lung cancer cells via miR-31 [Dong et al.,2014],which has also been shown to confer resistance to chemotherapy-induced apoptosis in prostate and colon cancer [14-15].Multidrug resistance protein 7(MRP7,gene symbols ABCC10)is a recently identified member of the C family of ATP binding cassette proteins,ABCC10 has been identified as a lipophilic anion transporter,which participates in the natural antitumor drug resistance process.However,ABCC10 has not been studied in esophageal cancer to date.By using a bioinformatic approach,we predicted that ABCC10 is a target gene of miR-let-7g and miR-let-7i.Although miRNAs may act as critical regulators in the development of drug resistance,the mechanisms by which miRNA regulates the ABCC10 and modulates the DDP-induced apoptosis in EC cells are still poorly understood.The purpose of this investigation is to determine whether BAG3 regulates miR-let-7g/i expression and miR-let-7g/i can affect drug sensitivity by monitor drug resistance gene ABCC10 in esophageal cell lines.Part? Correlation between expression of BAG3 and miR-let-7g and let-7iSurgically resected esophageal carcinoma tissues and matched adjacent normal esophageal tissues were collected from 24 primary esophageal squamous cell carcinoma patients at the First Affiliated Hospital of Zhengzhou University.All patients recruited in the study did not receive preoperative treatment.Written informed consent for the studies was obtained from all participants and their guardians.This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.Human esophageal epithelial cells HEEpiC,human esophageal carcinoma cells EC109 and TE10 cells were cultured in RPMI 1640 medium,which containing 10% fetal calf serum.Cells were passaged every 2-3 days and the log phase cells were used for experiments.Cells were counted using a hemocytometer.The expression levels of Bcl-2 related anti-apoptosis related gene 3(BAG3)and miR-let-7g/i in esophageal carcinoma tissues and esophageal cell lines were detected by qPCR and Western blot,and the correlation between BAG3 and miR-let-7g/i was analyzed.ResultsThe level of BAG3 mRNA in clinical esophageal squamous cell carcinoma tissues was increased compared to that in adjacent normal esophageal tissues.However,the levels of miR-let-7g and miR-let-7i were reduced in carcinoma tissues.The level of BAG3 protein expression in EC109 and TE10 cells was higher than that in HEEpiCs.We also observed that the levels of miRNA let-7g and let-7i expression were significantly lower in EC109 and TE10 cells compared with that in the HEEpiCs.Further analysis showed that the expression of BAG3 was negatively correlated with the expression of miR-let-7g(r=-0.842)in esophageal cancer patients,and was also negatively correlated with the expression of miR-let-7i(r=-0.815).ConclusionsThe expression of BAG3 in esophageal cancer tissues and cell lines were significantly up-regulated,and the expression of let-7g/i significantly decreased.In addition,the expression of BAG3 was negatively correlated with the expression of let-7g/i,which BAG3 can negatively regulate the expression of microRNA-let-7g/i,suggesting that BAG3 could regulate the expression of miR-let-7g/i in human esophageal carcinoma.Part? Effect of BAG3 inhibited miR-let-7g and let-7i on chemosensitivity of esophageal cancer cells MethodsOn the one hand,we deal with the EC109 and TE10 cells with DDP at different concentrations to detect the cell viability.miR-let-7g/i mimics or miR-let-7 mimic NC were transfected into EC109 and TE10 cells.After cellular adhesion,freshly prepared anticancer drugs DDP were added at 60 ?M concentration.The proliferation of cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry.In order to further explore the effect of let-7g/i induced by BAG3 on cisplatin induced proliferation and apoptosis of esophageal cancer cells.Esophageal cancer cells were transfected with shRNA BAG3 and observe cell proliferation.The effects of over-expression and down-regulation of BAG3 on the expression of miR-let-7g/i were detected by qPCR and Western blot.ResultsWith the increase of DDP concentration,the proliferation ability of EC109 and TE10 cells decreased significantly.Compared with control group,over-expression of let-7g and let-7i significantly inhibited the proliferation and induced apoptosis of EC109 and TE10 cells,thereby enhancing the cell sensitivity to cisplatin.Down regulation of BAG3 significantly inhibited the proliferation of EC109 and TE10 cells.Western blot and qPCR showed that down-regulation of BAG3 significantly increased the let-7g/i level,and up-regulation of BAG3 was decreased significantly let-7g/i level.ConclusionsDDP was inhibited significantly the proliferation of EC109 and TE10 cells.Let-7g and let-7i could reduce the proliferation of EC109 and TE10 cells,accelerate apoptosis,and enhance the sensitivity of cells to chemotherapy.Reduction of BAG3 and overexpression of let-7g and let-7i play a similar role in regulating the proliferation of esophageal cancer cells,suggesting that BAG3 may participate in cell proliferation and apoptosis by regulating the expression of miR-let-7g/i.Part? Molecular mechanism of miR-let-7g and let-7i inhibiting proliferation and enhancing apoptosis of human esophageal cancer cells MethodsThree bioinformatic algorithms(RNA22-HSA,MICRORNA.ORG and TARGETSCAN)prediction and dual luciferase reporter gene validation of the targeted regulation of ABCC10 by miR-let-7g/i.Western blot was used to detect the expression of ABCC10 protein in esophageal cancer cells by over-expression or inhibition of miR-let-7g/i,and to analyze the correlation between the expression levels.MTT assay and flow cytometry were used to examine the proliferation and apoptosis rate of EC109 and TE10 cells by down-regulation of ABCC10;the level of ABCC10 protein was detected by Western blot of down-regulation or over-expression of BAG3.ResultsBioinformatics predictions showed that the 3,UTR of ABCC10 have consequential pairing of miR-let-7g/i with 7-mer seed match.The dual-luciferase reporter assay showed that the relative luciferase activity was significantly decreased in the EC109 cells co-transfected with ABCC10 3,UTR and miR-let-7g/i mimics,whereas no significant changes were found in the cells co-transfected with MUT 3,UTR of ABCC10 and miR-let-7g/i mimics or miR-control.Overexpression of miR-let-7g/i significantly decreased the expression of ABCC10,while down-regulation of miR-let-7g/i was increased ABCC10 level significantly.miR-let-7g and ABCC10 mRNA had a significant inverse correlation(r =-0.865)and miR-let-7i and ABCC10 mRNA also had a significant inverse correlation(r =-0.842)in esophageal squamous cell carcinoma patients.Our results suggested the ABCC10 is a genuine target for miR-let-7g/i and we deduced that over-expression of miR-let-7g/i might inhibit cell proliferation and enhance apoptosis of esophagus cancer cells via down-regulating the protein expression of ABCC10.BAG3 significantly decreased the level of ABCC10 protein,but over-expressed BAG3 significantly increased the level of ABCC10 protein.ConclusionsmiR-let-7g/i can specifically regulate the expression of ABCC10;BAG3-induced miR-let-7g/i can inhibit cell proliferation and promote apoptosis of esophageal cancer cells by down-regulating the expression of ABCC10 protein,thereby enhancing the chemosensitivity of cells.