SAHA Suppresses Cell Proliferation And Induces Apoptosis Via Mir-769-5p/3p In Gastric Cancer

Background and ObjectiveHistone deacetylases inhibitors(HDACi)are a class of anticancer agents targeting HDACs,which have been increasingly developed and gradually applied in clinical practice.Vorinostat/suberoylanilide hydroxamic acid(SAHA)is a broad-spectrum HDACi,which can inhibit the activity of certain HDACs and increase the histone acetylation levels,thereby inducing apoptosis and playing an important role in inhibiting the cancer progression.More and more researches reported the anticancer mechanisms of SAHA in lymphoma,prostate cancer,gastric cancer(GC),pancreatic cancer and other tumors.HDACi can exert antitumor effects through miRNAs.However,the relationship between SAHA and miRNAs in GC has not been reported yet.By miRNA-seq,we find that SAHA can upregulate the expression of miR-769-5p/3p in GC.Bioinformatic analyses indicate that IGF1R may be a target of miR-769-5p/3p,and STAT3 may be a transcription factor that regulates miR-769.In this study,we aim to explore the effect of miR-769-5p/3p on the malignant biological behavior of GC,clarify the molecular mechanism of SAHA regulating miR-769-5p/3p at the transcriptional level through the STAT3-IGF1R-HDAC3 complex in GC,in order to find potential therapeutic targets and provide a theoretical basis for exploring the anticancer effects of SAHA in GC.Methods1.miRNA-seq analysis was used to investigate the miRNAs induced by SAHA in GC cells.Survival analysis was applied to observe the associations between the miRNAs expression and prognosis of GC patients.qPCR was used to evaluate the miR-769-5p/3p expression in GC cells after SAHA treatment.We detected the miR-769-5p/3p expression in GC cell lines and tissues by qPCR and ISH and analyzed the miR-769-5p/3p expression in different clinical groups stratified according to differentiation,T,lymph node metastasis,TNM stage and other clinical parameters.2.We transfected mimics or inhibitor into GC cells or normal gastric mucosal cell line and detected the cell proliferation capacity by colony formation assay and EdU assay.Invasion or migration ability was observed by transwell assay or wound healing assay.3.We performed the screening of the target of miR-769-5p/3p by bioinformatics,information retrieval and qPCR.Western blot assay and dual-luciferase assay were applied to explore the target relationship between miR-769-5p/3p and IGF1R.Western blot assay was used to evaluate the association between SAHA and IGF1R.Colony formation assay,EdU assay,transwell assay and subcutaneous tumor model in mice were carried out to observe whether miR-769-5p/3p regulated the GC cell malignant abilities though IGF1R.Hoechst 33258 staining was used to detect the apoptosis in GC cells after miR-769-5p/3p overexpression or/and IGF 1R interference.4.We selected STAT3 as the upstream regulator of miR-769 by bioinformatics,information retrieval and qPCR.Dual-luciferase assay and ChIP assay were used to investigate the binding of STAT3 to the miR-769 promoter.Colony formation assay,EdU assay,transwell assay and subcutaneous tumor model in mice were performed to evaluate the role of miR-769-5p/3p in STAT3 regulating GC cell proliferation and invasion.Hoechst 33258 staining was used to detect the GC cells apoptosis cells after STAT3 interference or/and miR-769-5p/3p overexpression.5.Co-IP assay was used to detect the interactions between STAT3,IGF1R and HDAC3.QPCR,dual-luciferase assay,ChIP and re-ChIP were performed to detect the binding of STAT3-IGF1R-HDAC3 complex to the miR-769 promoter.Subcutaneous tumor model in mice was used to detect STAT3-IGF1R-HDAC3 complex inducing regulation for miR-769-5p/3p in vivo.Western blot,qPCR,ISH and IHC were applied to detect the STAT3,IGF1R,HDAC3 and miR-769-5p/3p expression in GC tissues.Pearson correlation was used to analyze the correlation between STAT3,IGF1R,HDAC3 and miR-769-5p/3p.We analyzed the STAT3,IGF1R and HDAC3 expression in different clinical groups.6.We evaluated whether SAHA could synergize with miR-769-5p/3p to promote GC cells apoptosis by flow cytometry,TUNEL assay and subcutaneous tumor model in mice.Results1.miR-769-5p/3p was upregulated in GC cells after SAHA treatment.Low miR-769-5p/3p expression was observed in GC cell lines and GC tissues and associated with poor prognosis in GC.Low miR-769-5p/3p expression could be found in GC patients with poor differentiation,T3-T4,lymph node metastasis or stageⅢ-Ⅳ.2.miR-769-5p/3p suppressed the proliferation and invasion abilities of GC cells by targeting IGF1R.3.STAT3 could bind to the promoter of miR-769,and downregulated the miR-769-5p/3p expression.4.STAT3,IGF1R and HDAC3 interacted with each other and formed a multiprotein complex,regulating the activity of the miR-769 promoter and the miR-769-5p/3p expression.5.SAHA could synergize with miR-769-5p/3p to promote GC cells apoptosis.ConclusionSAHA upregulates miR-769-5p/3p in GC cells.miR-769-5p/3p blocks the malignant abilities of GC cells by targeting IGF 1R.STAT3-IGF 1R-HDAC3 complex modulates the transcriptional activity of miR-769 promoter to regulate miR-769-5p/3p expression.SAHA synergizes with miR-769-5p/3p to promote cells apoptosis in GC.