Regulatory Mechanisms Of SIRT6 On The Malignant Transformation Of Bronchial Epithelial Cells Induced By Cigarette Smoke Extract

Background:Lung cancer incidence is closely linked to a history of smoking.Hence,smoking is an identified risk factor for lung cancer.Although smoking cessation is advocated in China,the lung cancer incidence and mortality induced by active or passive smoking remain high.Most studies on smoking-related lung cancer have an epidemiological or clinical scope,whereas those investigating molecular mechanisms are limited.Therefore,in this project we will conduct a preliminary exploration of lung cancer genesis by smoking.Sirtuin 6(SIRT6)is a member of the histone deacetylase III family,which plays an important role in cell proliferation,aging,DNA repair,and tumor development through its NAD~+-dependent deacetylase activity on histone H3.Studies analyzing tumor samples have shown that SIRT6 expression is tissue-specific.The expression of SIRT6 is low in liver cancer,renal cell carcinoma,gastric cancer,and colon cancer and is upregulated in osteosarcoma,prostate cancer,and papillary thyroid cancer.In breast cancer,SIRT6 has been characterized both as a tumor suppressor and activator.Most lung cancer and cell line studies have shown that SIRT6 expression is low,while its overexpression can inhibit tumor proliferation and invasion.The JAK2/STAT3signaling pathway is critical for intercellular signaling and its uncontrolled activation can drive tumorigenesis.In our previous study,we continuously induced BEAS-2B cells with cigarette smoke extract(CSE)using a microfluidic technology,finding that STAT3 plays an important role in CSE-induced chronic inflammation and malignant transformation.Studies have shown that SIRT6 can inhibit tumor cell proliferation and induce apoptosis by inhibiting JAK2/STAT3 pathway in colon cancer,gastric cancer,and glioma cells.However,the expression and regulation of SIRT6 activity in smoking-related lung cancer remains unclear.In this study,human bronchial epithelial cells(BEAS-2B)induced by CSE exposure were employed.We aimed to elucidate the role and molecular mechanisms of SIRT6 in malignant transformation of BEAS-2B cells and in the regulation of the JAK2/STAT3 signaling pathway.Our findings will enrich current knowledge on the molecular mechanisms of lung cancer and reveal new therapeutic targets for smoking-related lung cancer.Methods:1.Malignant transformation of CSE-induced BEAS-2B cells and SIRT6expression levels(1)The cell counting kit-8(CCK8)method was used to screen for the most suitable concentration and duration of CSE induction in BEAS-2B cells.(2)The expression levels of oncogenes,SIRT6,and related proteins in malignant cells were detected by RT-PCR and western blotting.(3)A subcutaneous transplantation model of CSE-induced malignant transformation in nude mice was established.2.Regulating the impact of SIRT6 on CSE-induced BEAS-2B malignant cells(1)CSE successfully induced the malignant transformation of BEAS-2B cells,referred to as�T3�cells herein.Lentivirus-transfected LV-SIRT6,LV-SIRT6-RNAi,and corresponding empty vector-transfected LV-GV358 and LV-GV248 T3 cell lines with stable SIRT6 overexpression and silenced expression were established.The transfected cells were divided into five groups:?.T3-con group(non-transfected group);?.T3-LV-GV358 group(SIRT6-overexpressing empty vector control group);?.T3-LV-SIRT6 group(SIRT6-overexpressing group);?.T3-LV-GV248 group(SIRT6-silenced empty vector control group);?.T3-LV-SIRT6-RNAi group(SIRT6-silenced group).(2)The CCK8 assay was used to detect the proliferation ability of the transfected cells.(3)m RNA and protein expression of genes involved in invasion,cell cycle,and apoptosis in each group were detected by RT-PCR and western blotting.(4)Transwell assays and scratch tests were conducted to evaluate the invasion and migration extent of each group after transfection.(5)Following transfection,the cell cycle of each group was analyzed by flow cytometry.(6)Flow cytometry and Mito Tracker staining were used to detect apoptosis in each group after transfection.(7)Western Blot was used to detect the overexpression and silencing regulation of SIRT6 on the JAK2/STAT3 signaling pathway in T3 cells.3.The regulatory role of SIRT6 on subcutaneous transplanted tumor growth in nude mouse T3 cells(1)Overexpression and silencing of SIRT6 established subcutaneous transplantation tumor models of T3 cells.Subcutaneous tumor formation in each group of nude mice was observed,and the tumor growth curves were drawn accordingly.(2)The m RNA and protein expression of genes participating in invasion,cell cycle,and apoptosis were detected in tumor tissues of each group by RT-PCR and western blotting.(3)Immunohistochemistry was used to detect the expression of invasion-,cell cycle-,and apoptosis-related proteins in each group.(4)Western blotting and immunohistochemistry were performed to detect the expression of JAK2/STAT3 signaling pathway-related proteins in subcutaneous transplanted tumor tissues of T3 cells after overexpression and silencing of SIRT6.Results:1.The T3 cell line was generated by continuously inducing BEAS-2B cells with50%CSE for seven days and facilitating their recovery for two weeks.Compared with the control group(BEAS-2B cells without CSE exposure),the expression of related oncogenes(K-ras,C-Myc,Cyclin A1,Cyclin D1,CDK4 and CDK6)was increased in T3 cells,while the expression of SIRT6 was decreased.T3 cells potentiated the formation of subcutaneous transplanted tumors in nude mice.BEAS-2B cells chronically exposed to CSE underwent malignant transformation and were oncogenic.2.Genetic modifications on the expression potency of SIRT6 can significantly affect the proliferation,apoptosis,invasion,and migration abilities of T3 cells.When SIRT6 was overexpressed,the expression levels of invasion-and migration-related genes and proteins in T3 cells were decreased,thus inhibiting their invasion and migration.The expression levels of cell cycle-related genes and proteins were significantly decreased,and the cells were arrested in the G1 phase.The expression levels of apoptosis-related genes and proteins were significantly increased,thereby promoting apoptosis.Compared with the overexpression of SIRT6,the silencing of SIRT6 had opposite effects on the invasion,migration,proliferation,and apoptosis of T3 cells.Overall,SIRT6 overexpression can inhibit the activation of the JAK2/STAT3signaling pathway,whereas silencing can promote the activation of the JAK2/STAT3signaling pathway.3.In T3 cells,the overexpression of SIRT6 can inhibit the growth of subcutaneous transplanted tumors,resulting in cell cycle arrest,increased apoptosis,downregulation of proteins involved in invasion and metastasis,and inhibition of the JAK2/STAT3signaling pathway activation.Compared with the overexpression of SIRT6,silencing the SIRT6 expression had opposite outcomes on the cell cycle,apoptosis,invasion,and metastasis of transplanted tumor cells and the JAK2/STAT3 signaling pathway.Conclusion:1.CSE can induce malignant transformation of bronchial epithelial cells and has tumorigenic potency in nude mice.It may be related to the low expression of SIRT6.2.Overexpression of SIRT6 can inhibit the invasion and migration of CSE-induced malignant transformation cells.Additionally,it can induce cell cycle arrest and apoptosis and inhibit the tumorigenic potential of nude mice,which may be a promising therapeutic target for smoking-related lung cancer.3.SIRT6 is involved in the regulation of the JAK2/STAT3 signaling pathway in T3 cells,which may decipher the underlying molecular mechanisms of smoking-related lung cancer.