Study On The Molecular Mechanism Of NiCl₂ Regulating P62 Expression And Inducing Autophagy In BEAS-2B Cells By Upregulating P65

Background and PurposePrimary bronchial lung cancer is abbreviated as lung cancer,which is a kind of malignant tumor that often occurs in human bronchial mucosa epithelial tissue in the early stage.At present,the morbidity and mortality of lung cancer rank first in cancer worldwide.The environmental factors of nickel metal also play an important role in the early occurrence and prevention of human lung cancer.Nickel metal and its compounds are widely used in many large industrial production processes,and their presence will have various adverse effects on human health.Including skin allergies,pulmonary bronchial fibrosis and respiratory cancer.Although some studies have found that nickel and its compounds affect the autophagy of lung cancer cells,the regulation mechanism of nickel on autophagy during the formation of lung cancer is unknown.Therefore,this topic intends to investigate the role of NiCl₂ in autophagy pathway induced by human bronchial epithelial cells malignant transformation of BEAS-2B in the process of autophagy,in-depth discussion of the role of NiCl₂in the formation of lung cancer and the specific molecular mechanism.MethodsBEAS-2B cells were treated with different concentrations of NiCl₂,the proliferation of BEAS-2B cells was detected by RTCA method,the colony forming ability of BEAS-2B cells was detected by soft agar method,and the expression of autophagy marker protein LC3 and autophagy related protein in BEAS-2B cells treated with the same concentration at different times were detected by Westernblot method In addition,we used short hairpin RNA（sh RNA）to knock down p62 and Sesn2 in BEAS-2B cells,and further established stable cell lines of shp62 and sh Sesn2.We used Westernblot method to detect the expression level of autophagy related proteins in each group.We also used QRT PCR to analyze the m RNA level of p62 exposed to NiCl₂.We also used double luciferase reporter gene test to analyze whether p62 transcription is involved in malignancy The potential transcription factors that can be combined in p62 promoter region were analyzed by molecular bioinformatics software.Westernblot,fluorescence quantitative PCR,chip and other experiments were used to elucidate the specific molecular mechanism of malignant transformation of human bronchial epithelial cells BEAS-2B induced by NiCl₂ exposure through autophagy.ResultsNiCl₂promotes the proliferation and malignant transformation of BEAS-2B cells;The LC3A/B-II expression of protein showed a time-dependent effect,and the expression levels of Sesn2 and p62 increased with the time of NiCl₂ induction,showing a time-dependent effect;p65 combined with p62 promoter region under NiCl₂exposure,thus up regulating the expression of p62 at the transcription level,and then inducing autophagy of BEAS-2B cells;and then inducing malignant transformation of human bronchial epithelial cells BEAS-2B.ConclusionsThe proliferation and malignant transformation of BEAS-2B cells were enhanced by continuous NiCl₂ exposure.Subsequently,it was found that p62,an autophagy related protein,could regulate the autophagy of BEAS-2B cells and affect their malignant transformation ability.Further studies have shown that p65 protein can interact with p62 promoter region,indicating that NiCl₂ may affect the malignant transformation ability of BEAS-2B cells by up regulating p65 to induce autophagy.