| Background: Lung cancer has the highest incidence rate and mortality rate in China,and 80% of them are non-small cell lung cancer.FMO3(flavin containing monoxygenase 3)is an important liver microsomal enzyme that catalyzes the nitrogen oxidation of nucleophilic heteroatomic compounds such as nitrogen,sulfur,phosphorus and selenium.Its abnormal expression is involved in tumor related biological processes such as drug / toxicology metabolism,endoplasmic reticulum stress,and plays an important role in the development,and drug resistance of many kinds of tumors,such as liver cancer,colorectal cancer and breast cancer.However,there is no report about its expression and function in lung cancer.Objective: This study aims to study the expression level,function,clinical significance and signal pathway mechanism of FMO3 in non-small cell lung cancer in order to reveal the development mechanism of non small cell lung cancer and provide guidance for screening diagnostic and therapeutic markers.Methods: 1 10 pairs of tissue samples of non-small cell lung cancer(including lung adenocarcinoma and lung squamous cell carcinoma)and corresponding adjacent tissue samples(20 in total)were collected.Immunohistochemical experiments were carried out to verify the expression level of FMO1-5.Then 115 cases of non-small cell lung cancer tissues and their corresponding adjacent tissues were collected to make tissue chips.And then we carried out immunohistochemical experiments to test the expression of FMO3 and Ki67.The tissue chips were scanned to obtain high-definition pictures,and then the average cumulative optical density(AOD)was calculated via Image J software as the score of immunohistochemistry.SPSS 24.0 statistical analysis software was used to analyze and compare the expression difference of FMO3 and Ki67 in nonsmall cell lung cancer and adjacent tissues,and its relationship with clinical characteristics,and overall survival is calculated as well.2.The up-regulated cell line of non-small cell lung cancer cell A549 and the down-regulated cell line of the lung squamous cell carcinoma H1703 were constructed by genetic engineering technology.CCK-8,plate cell clone formation,Ed U proliferation staining and flow cytometry were used to explore the cellular function of FMO3.3.The expression of proteins of endoplasmic reticulum stress related pathway were detected by Western blot to explore the relationship between FMO3 and apoptosis of non-small cell lung cancer cells.Results: 1.Immunohistochemical results showed that five flavin monooxygenases of FMO1-5 were all expressed in non-small cell lung cancer tissues and adjacent tissues,but compared with adjacent tissues,there was no significant difference in the expression of other flavin monooxygenases(P > 0.05)except that FMO3 was significantly higher in non-small cell lung cancer tissues and significantly different from paired adjacent tissues(P = 0.0003).2.Immunohistochemical results showed that the expression of Ki67 in non-small cell lung cancer was significantly higher than that in adjacent tissues(P = 0.0002),and its expression level was significantly positively correlated with the expression of FMO3(P < 0.0001).3.We analyzed the correlation between the expression of FMO3 in non-small cell lung cancer and the clinicopathological features of patients with non-small cell lung cancer.The results showed that the expression level of FMO3 was significantly correlated with tumor size(P = 0.0035)and T stage(P = 0.0224)of non-small cell lung cancer.The 5-year survival rate of high FMO3 expression group was significantly lower than that of low FMO3 expression group(P = 0.0007).Univariate and multivariate analysis showed that high FMO3 expression(HR = 3.32,95% CI: 1.59-6.93,P = 0.01),smoking(HR = 2.47,95% CI: 1.11-5.51,P = 0.02),tumor size(> 5cm)(HR = 2.54,95% CI = 1.26-5.13,P = 0.01)T stage(T3T4)(HR = 1.47,95% CI: 0.71-3.05,P = 0.02)and TNM stage(HR = 1.78,95%CI: 0.88-3.58,P = 0.02)were risk factors affecting the overall survival of patients with non-small cell lung cancer,while only high expression of FMO3 and TNM stage were independent risk factors.4.The results of CCK-8 experiment,plate cell clone formation experiment and Ed U staining proliferation experiment showed that the proliferation ability of A549-FMO3 cells was significantly stronger than that of the control group(P < 0.05),while the proliferation ability of H1703-sh2 cells was significantly weaker than that of the control group(P< 0.05).5.Flow cytometry showed that compared with the control group,A549-FMO3 group had less apoptosis,while H1703-sh2 group had more apoptosis than the control group.6.WB test showed that the expression of IRE1α(P<0.05),pIRE1α-S724(P<0.001),cleaved caspase12(P<0.001),cleaved caspase3(P<0.001)and cleaved PARP(P<0.01)was significantly lower in A549-FMO3 group compared to the control group,and there was no significant difference in other related proteins.In the H1703-sh2 group,the phosphorylation level of p-IRE1α-S724 was significantly higher(P <0.05),and the expression levels of cleaved caspase 12(P < 0.01),cleaved caspase 3(P < 0.05)and cleaved PARP(P < 0.05)were significantly lower compared to the control group,and there was no significant difference in other related proteins.7.We used immunohistochemical staining to verify the expression of p-IRE1α-S724 in non-small cell lung cancer and its relationship with FMO3 expression.The results showed that the expression of p-IRE1α-S724 in non-small cell lung cancer was significantly higher than that in adjacent tissues(P < 0.0001),and it was negatively correlated with the expression of FMO3(P < 0.001).Conclusion: 1 The expression level of FMO3 in non-small cell lung cancer was significantly higher than that in adjacent tissues.2.The expression of FMO3 expression had a significantly positive correlation with tumor size and T-stage in patients with non-small cell lung cancer,and had a significantly negative correlation with the 5-year survival rate in patients with non-small cell lung cancer.Its high expression predicts poor prognosis in patients with non-small cell lung cancer.3.High expression of FMO3 and TNM stage are independent risk factors of the prognosis of non-small cell lung cancer.4.Overexpression of FMO3 enhanced the proliferation of A549 and inhibited its apoptosis.Knockdown of FMO3 inhibited the proliferation of H1703 and promoted its apoptosis.5.FMO3 downregulates IRE1 α/ Caspase12 / Caspase3 / PARP signal transduction pathway to inhibit endoplasmic reticulum stress and thus inhibits apoptosis of A549 cells. |
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