The Mechanism Of Arecoline Regulating TPM1 In Epithelial Cell Apoptosis And EMT

BackgroundOral submucous fibrosis（OSF）is a chronic inflammatory progressive disease that occurs in the oral mucosa.The main pathological changes are the atrophy of the oral mucosal epithelial layer and the accumulation of collagen in the lamina propria.The main clinical manifestations include burning pain and progressive limitation mouth opening.OSF seriously affects the patient’s quality of life.Because its canceration rate is 7%to 13%,the World Health Organization lists it as precancerous state.The etiology of OSF is unclear,but is thought to be multifactorial.Betel nut chewing,pepper intake,nutritional factors,genetic factors,and immune factors are all incorporated^[1,2].Chewing betel nut is the main cause of OSF,and betel nut has been identified as a Class I carcinogen by the International Cancer Research Center.Arecoline which is the most important alkaloid in betel nut,plays an important role in the pathogenesis of OSF.The role of arecoline in OSF has became a hot research topic.Studies have found that arecoline is cytotoxic to epithelial cells which inhibited epithelial cell proliferation.The abnormal expression of cell cycle and apoptosis-related proteins were discoved in OSF tissues.But its pathogenic mechanism is still unclear.Arecoline induces epithelial-mesenchymal transformation（EMT）,which promoting the expression ofα-smooth muscle actin（α-SMA）and the transformation of epithelial cells into the myofibroblast phenotype,which is an important step in the process of tissue fibrosis and tumor formation.Previous study revealed that chewing betel nut could activate the EMT-related factors to promote OSF,but the mechanism is unclear.The mechanism of arecoline in epithelial atrophy and EMT need to be further studied.This study may provide experiment evidence for prevention and treatment of OSF.ObjectiveTo investigate the effect and mechanism of TPM1 up-regulation in OSF.It can provide a theoretical basis for the pathogenesis of arecoline-induced epithelial tissue lesions in OSF and a new clue for the diagnosis and treatment of OSF.1.Bioinformatics was used to find the differentially expressed gene Tropomyosin 1（TPM1）,and to explore the expression of TPM1 in human and mouse OSF.2.To investigate the effect of TPM1 on cell proliferation,apoptosis and EMT in HaCaT cells stimulated by arecoline.3.To explore the mechanism of TPM1 on apotosis and EMT in arecoline-induced HaCaT cells.Methods1.The GEO database was used to screen differentially expressed gene and the expression of the gene in human and mouse OSF tissues were detected.（1）Download the gene chip data GSE64216 from the GEO database,and analyze the data through the GEO database analysis tool GE02R,and screen the differentially expressed genes.Then use the Cytoscape software to process the PPI network map obtained by STRING,and use the Cytohubba plug-in to analyze the hubgene and screen key gene.DAVID online database was used for GO enrichment analysis.（2）Masson stain was used to detect the expression of collagen in different OSF periods.Electron microscopy was used to observe the ultrastructure of OSF tissues.（3）Immunohistochemistry was used to detect the expression of TPM1 in different OSF periods.（4）The OSF mice model was constructed.Masson staining was used to detect the expression of collagen and immunohistochemistry was used to detect the expression of TPM1 in mice OSF tissues.2.To explore the effect of arecoline on the apoptosis and EMT of HaCaT cells.（1）After the treatment of arecoline in HaCaT cells,the expression of TPM1 m RNA was detected by q RT-PCR,and the expression of TPM1protein was detected by Western blot.（2）CCK8 experiment and flow cytometry were used to observe the effects of arecoline on the proliferation,cell cycle and apoptosis of HaCaT cells.（3）Western blot was used to detect the protein expression of E-cadherin and Vimentin in HaCaT cells stimulated by arecoline.（4）Immunohistochemistry was used to detect the expression of E-cadherin and Vimentin in OSF of patients and mice.3.To explore the role of TPM1 in the mechanism of arecoline affecting HaCaT cell apoptosis and EMT.（1）After transfection of TPM1-si RNA,q RT-PCR was used to detect the expression of m RNA and Western blot was used to detect the expression of TPM1 protein.（2）After knocking down the expression of TPM1,the CCK8experiment and flow cytometry were used to detect the effects on cell proliferation,cell cycle and apoptosis of HaCaT cells stimulated by arecoline.（3）After knocking down the expression of TPM1,Western blot was used to detect the expression of TPM1,E-cadherin and Vimentin.q RT-PCR was used to detect the TPM1 m RNA expression.（4）After knocking down the expression of TPM1,q RT-PCR was used to detect the COL3A1 andα-SMA m RNA expression.（5）Western blot was used to detect the protein expression of ROCK1,Rho A,Smad 2/3 and p-Smad 2/3 in HaCaT cells stimulated by arecoline.After treatment with TGF-β/Smad signaling inhibitor SB431542 and Rho A/ROCK signaling inhibitor Y27632 respectively,Western blot was used to detect the protein expression of TPM1expression.q RT-PCR was used to detect the TPM1 m RNA expression.（6）After treatment with TGF-β/Smad signaling inhibitor SB431542in HaCaT cells stimulated by arecoline,the CCK8 experiment and flow cytometry were used to detect the effects on cell proliferation,cell cycle and apoptosis.Western blot was used to detect the expression of E-cadherin and Vimentin.Result1.The GEO database was used to screen differentially expressed genes and the expression of the gene in human and mouse OSF tissues were detected.（1）A total of 1088 differentially expressed genes were found through the GEO database,in which 634 genes were up-regulated and454 genes were down-regulated.Cytoscape software and Cytohubba were used to process the differentially expressed genes PPI network map,and20 hubgenes and key gene TPM1 were obtained.In GO enrichment analysis,cell function analysis,cellular components analysis and biological process analysis showed that TPM1 may be involved in the occurrence and development of OSF.（2）The protein expression of TPM1 in patients and mice OSF tissues was elevated.It was correlated with the severity of OSF patients.2.Arecoline promotes cell apoptosis and EMT in HaCaT cells.（1）The m RNA and protein expression of TPM1 were increased after arecoline stimulated HaCaT cells.It was most significant when arecoline was 0.16m M.（2）Arecoline could inhibit the proliferation of HaCaT cells,arrest HaCaT cell cycle at G1 and promote HaCaT cell apoptosis.（3）The protein expression of E-cadherin was decreased,while the protein expression of Vimentin was elevated in arecoline stimulated HaCaT cells.（4）Compared with normal tissues,the expression of E-cadherin was decreased in patients and mice OSF tissues.E-cadherin protein was negatively correlated with TPM1 protein expression in patients and mice OSF tissues（r_s=-0.374,P<0.05;r_s=-1.00,P<0.05）.The expression of Vimentin was elevated in OSF patient tissues.Vimentin protein was positively correlated with TPM1 protein expression（r_s=0.346,P<0.05）.The expression of Vimentin was elevated,but the difference was not statistically significant in mice OSF tissues（P>0.05）.3.Arecoline promoted the expression of TPM1 by inducing the activation of TGF-β/Smad signaling pathway,and then participated in regulating the apotosis and EMT of HaCaT epithelial cells.（1）After knocking down the expression of TPM1,the proliferation of HaCaT cells was weakened,cell cycle arrest in G1 phase was reduced and cell apoptosis was weakened.（2）After knocking down the expression of TPM1,the protein expression of E-cadherin was elevated,while the protein expression of Vimentin was decreased in arecoline stimulated HaCaT cells.（3）After knocking down the expression of TPM1,the m RNA expression of COL3A1,α-SMA were decreased in arecoline stimulated HaCaT cells.（4）Arecoline induced activation of TGF-β/Smad pathway and Rho A/ROCK pathway in HaCaT cells.TGF-β/Smad pathway mediated the up-regulation of TPM1 by arecoline in HaCaT cells.（5）After treatment with TGF-βinhibitor SB431542,the protein expression of E-cadherin was elevated,while the protein expression of Vimentin showed no differences in arecoline stimulated HaCaT cells.The proliferation of HaCaT cells was weakened,cell cycle arrest in G1 phase was reduced and cell apoptosis was weakened.Conclusion1.The expression of TPM1 were elevated in patients and mice OSF tissues and arecoline stimulated HaCaT cells.It showed that arecoline could affect the expression of TPM1.2.Arecoline could inhibit the proliferation of HaCaT cells,arrest HaCaT cell cycle at G1 and promote HaCaT cell apoptosis.Knocking down the expression of TPM1 could attenuate the effect of arecoline on the apotosis and EMT of HaCaT cells.3.Arecoline promoted the expression of TPM1 by promoting the activation of TGF-β/Smad signaling pathway in HaCaT cells,and participating in regulating the apoptosis and EMT of HaCaT epithelial cells.