Study Of The Mechanism Of METTL3-Regulated TROAP In Non Small Cell Lung Cancer

Objective(s):Non small cell lung cancer is a malignant tumor originating from the bronchial mucosa or lung glands,accounting for about 80% of lung cancer patients.However,most lung cancer patients cannot be diagnosed in time,and they often have tumor metastasis at diagnosis.Although great progress has been made in many common treatments for lung cancer,such as surgical resection,chemotherapy,radiotherapy,and targeted drugs,considering the poor survival rate,it is still urgent to improve the prognosis and and treatment of lung cancer.Further studies of the molecular mechanisms of lung cancer development and identification of biomarkers and targets are crucial for early detection,intervention and treatment.TROAP is a class of protein located in the cytoplasm.Several studies have proved that this protein is associated with the prognosis of cancer.Currently,only one literature has reported the presence of m6 A modification in TROAP,which elaborated the regulatory mechanism of methyltransferase METTL14 in ovarian cancer.However,there is no relevant study in non-small cell lung cancer to associate methyltransferase METTL3 with TROAP,and the specific mechanism of action is still unclear.The purpose of this study was to examine the expression of TROAP in non-small cell lung cancer samples and lung cancer cell lines,explore the effects of TROAP on proliferation,migration and invasion of non-small cell lung cancer,and clarify the regulation mechanism of m6 A modification on TROAP expression and the mechanistic factors of TROAP.Methods:1.Cancer tissues from patients and their corresponding adjacent tissues were collected from xx Hospital from September 2018 to December 2019.2.The RNA expression amount of TROAP in non-small cell lung cancer tissues and adjacent tissues,non-small cell lung cancer cell lines A549 and H1299,human normal lung epithelial cell line KMB 17 was measured by quantitative real-time PCR(Q-PCR)technology.3 The protein expression level of TROAP in non-small cell lung cancer was determined using IHC technology.4.TROAP,METTL3 knockdown by sh RNA expression and TROAP expression after METTL3 by sh RNA knockdown by Q-PCR,and protein expression of TROAP,METTL3,TROAP expression after knockdown of METTL3,and PI3 K / AKT and EMT signaling protein expression after knockdown and overexpression.5 The proliferative capacity of the cells was determined by the CCK-8 assay.6.Cell migration ability was measured by scratch assay.7 The invasive ability of the cells was determined by the transwell assay.Results: 1.TROAP RNA expression was significantly higher in non-small cell lung cancer tissues than in adjacent tissues(P<0.001)。2.TROAP expression was higher in both A549 and H1299 than in KMB 17(P for all <0.001).3.IHC test results showed that TROAP expression was higher in non-small cell lung cancer tissues than in adjacent tissues(P<0.0001).4.In Progno Scan database for non-small cell lung cancer,TROAP expression was positively correlated with Prognostic value,The ROC curve drawn by the SPSS software showed that TROAP has some diagnostic value for lung cancer,but the clinical data analysis showed that the expression of TROAP was not related to the clinical information of the patients.5.Cell function experiments showed that the knockdown of TROAP could significantly inhibit the proliferation,migration and invasion of lung cancer cells(P<0.05),but the opposite was for overexpression.6.SRAMP database prediction showed the presence of m6 A modification sites in the m RNA of TROAP,and the knockdown increased the RNA and protein expression of TROAP after METTL3(P<0.05).7.After TROAP knockdown,P-AKT and P-PI3 K decreased in A549 and H1299 cells(P<0.05),opposite overexpression.8.After TROAP knockdown,N-cadherin expression decreased in A549 and H1299 cells(P <0.05),with the opposite overexpression.Conclusion(s): TROAP is highly expressed in clinical non-small cell lung cancer samples and lung cancer cell lines.The high expression of this gene can promote the proliferation,migration and invasion of non-small cell lung cancer cells.The expression of TROAP increased after the downregulation of M6A-related factors,METTL3.After TROAP knockdown,P-AKT and P-PI3 K and N cadherin expression were downregulated,and the opposite trend appeared after overexpression.As an oncogene,TROAP has the potential to be an effective target for clinical diagnosis and treatment of non-small cell lung cancer.