| Objective:This study aimed to investigate whether the LASS2/TMSG-1 gene promotes apoptosis in human lung cancer cells through mitochondrial apoptosis pathway and its specific mechanism.Methods:Immunohistochemistry S-P method was used to examine the expressions of LASS2/TMSG-1?ATP6V0C?Bcl-2 and Caspase-3 in pathological specimens of 87 cases of lung cancer tissue and 25 cases of normal lung tissue,and the correlation between the four indicators and the clinicopathological parameters and prognosis of patients with lung cancer was analyzed.LV-CERS2-RNAi(8649-2)(overexpressing the LASS2/TMSG-1 gene)was transfected into A549 cells by lentiviral transfection.The protein and mRNA level of LASS2/TMSG-1 were evaluated in lung cancer cell lines using western-blot and realtime fluorescence quantitative PCR.The changes of mitochondrial membrane potential and the apoptosis of lung cancer cells were observed by TMRM and flow cytometry.The expression levels of V-ATPase?Bcl-2?Caspase-3 and Caspase-9 in cells were detected by ELISA.Data were analyzed using SPSS 22.0 software,and P<0.05(as an analytical standard)was considered significant.Results:(1)Immunohistochemistry results show that the expression of LASS-2/TMSG-1 protein in lung cancer tissues(21/87,24.13%)was significantly lower than that in normal lung tissues(15/25,60.00%),the positive expression of ATP6V0C protein in lung cancer tissues(63/87,72.41%)was significantly higher than that in normal lung tissues(7/25,28.00%),the expression of Bcl-2 in lung cancer tissues(55/87,63.21)%)was significantly higher than in normal lung tissues(3/25,12%),and the expression of Caspase-3 protein in lung cancer tissues(27/87,31%)was significantly lower than in normal lung tissues(23/25,92%);the differences were significant(P<0.05).(2)The expression of LASS2/TMSG-1 and Caspase-3 in lung cancer tissues was significantly negatively correlated with the degree of differentiation and lymph node metastasis(P<0.05).The expression of ATP6V0C and Bcl-2 in lung cancer tissues was positively correlated with the degree of differentiation and lymph node metastasis(P<0.05).The expression of LASS2/TMSG-1 and V-ATPase in lung cancer tissues was related to the tissue type of lung cancer.The expression rate of LASS2/TMSG-1 in adenocarcinoma was higher than that in squamous cell carcinoma,while the expression rate of V-ATPase in squamous cellcarcinoma was higher.(3)The expression of LASS2/TMSG-1 was negatively correlated to the expression of ATP6V0C in Lung cancer tissues(r=-0.613,P=0.000);The expression of ATP6V0C was positively correlated to the expression of Bcl-2 in Lung cancer tissues(r=-0.482,P=0.000).(4)The expression levels of LASS2/TMSG-1?ATP6V0C?Bcl-2?Caspase-3 and the lymph node metastasis were independent risk factors in patients with Lung cancer(P<0.05).(5)The expression of mRNA and protein levels in the LASS2/TMSG-1 overexpression group was increased by Western-blot and real-time PCR,which was statistically significant compared with the other two groups(P<0.05).(6)Flow cytometry combined with TMRM probe showed that overexpression of LASS2/TMSG-1 gene decreased mitochondrial membrane potential and increased tumor cell apoptosis rate.(7)After overexpression of LASS2/TMSG-1 gene,ELISA analysis showed that the expression of V-ATPase?Bcl-2 was down-regulated in tumor cells,and the expression of proapoptotic proteins Caspase-3 and Caspase-9 was up-regulated,which was statistically significant compared with the other two groups(P<0.05).Conclusions:As a tumor metastasis suppressor gene,LASS2/TMSG-1 gene could not only inhibit tumor invasion and metastasis,but also bind to ATP6V0C subunit through HOX domain to inhibit V-ATPase activity and increase intracellular H~+concentration.Then,the downstream-cascade reactions led to the change of the ultrastructure of mitochondria,the decrease of mitochondrial membrane potential,the release of promote mitochondrial apoptosis pathway related proteins,and promote the apoptosis of A549 cells. |
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