The Mechanism Of Histone Deacetlase 6 In The Development Of Esophageal Cancer

Section 1:The expression and mechanism of histone deacetylase 6 in esophageal cancerObjective:The aim of this study was to investigate the expression of histone deacetylase 6(HDAC6)in esophageal cancer tissue and the relationship with prognosis.Then,we further explored the mechanism of HDAC6 in the development of esophageal cancer in cell.Methods:First,we used fluorescence quantitative polymerase chain reaction(qRT-PCR)observed the expression of HDAC6 in 46 human esophageal cancer specimens and individually matched adjacent normal tissues from our hospital,and combined with the follow-up data to analyze the relationship between the expression of HDAC6 and prognosis of patients.Then,we used HDAC6 selective inhibitor Ricolinostat(ACY-1215)to inhibitor esophageal cancer cell proliferation and observe its effect by MTT colorimetric method.Alteration of cell cycle and apoptosis rate was detected by flow cytometry.Finally,we used western blot to detect the expression of cell cycle and apoptosis related proteins and nuclear histones.Furthermore,the related pathways in the upstream were explored and verified by the restore experiment to further clarify the mechanism of HDAC6 in the development of esophageal cancer.Results:The results of qRT-PCR showed that there was no significant difference in the expression of HDAC6 between tumor tissues and adjacent tissues(P=0.517).However,survival analysis revealed that the high expression of HDAC6 was associated with poor prognosis than low expression of HDAC6(P=0.001).In esophageal carcinoma cells,cell survival rate decreased gradually with the increase of ACY-1215 concentration,and G2/M arrest and apoptosis increased gradually.The reason of this phenomenon is that the HDAC6 inhibitor act on the PI3K/PDK1/AKT/mTOR pathway and ERK pathway proteins and H3K9 related proteins,thus affecting the expression of cell cycle and apoptosis related cyclinA2,CDK1,P21 P53,Bax,Bim,Bcl2,Cleaved caspse3,Cleaved caspase9 and Cleaved cleaved PARP protein,etc.Conclusion:Our study confirmed that the pathogenesis of HDAC6 through PI3K/AKT and ERK pathway and H3K9 related protein to promote the development of esophageal cancer.Our analysis may provide a theoretical basis for related targets in the application of diagnosis and treatment of esophageal cancer.Section 2:The epigenetic regulation mechanism of histone deacetylase 6 in esophageal cancer cellsObjective:We found that the high expression of HDAC6 was associated with the poor prognosis of esophageal cancer patients and HDAC6 through histone and PI3K/AKT,ERK pathway to affect cell cycle and apoptosis in section 1.In this section we aimed to investigate the epigenetic regulation mechanism of HD AC 6 in esophageal cancer cells.Methods:We used miRNA microarray to research the change of miRNA expression in esophageal cancer cells after HDAC6 inhibitor treatment.The expression increased and the target for PI3K miRNA was selected by bioinformatics analysis.QRT-PCR was used to validate the expression of selected miRNA and its correlation with PI3K.Then,we observed the effect of miRNA on cell cycle and apoptosis after HDAC6 inhibitor treatment.Finally,we used western blot to detect the expression of PI3K pathway proteins,cell cycle and apoptosis-associated proteins which affected by transfected miRNA inhibitor.Results:MiRNA microarray technology and bioinformatics analysis showed that the expression of miR-30d was increased after HDAC6 inhibition,and its target gene was PI3K,which was further confirmed by qRT-PCR.In vitro transfection of miR-30d inhibitor,G2/M phase arrest and apoptosis ratio was significantly decreased;the expression of PI3K/PDK1/AKT/mTOR pathway and cell cycle and apoptosis related protein also appears in the corresponding change.Conclusion:Our study confirmed that HDAC6 can promote the development of esophageal cancer by targeting PI3K with miR-30d,which provides a theoretical basis for further study of the related targets in the diagnosis and treatment of esophageal cancer.