| Objective: To predict LKB1-related protein,and to verify the expression correlation between LKB1 and its related protein in non-small cell lung cancer(NSCLC)cell lines and their effects on radiosensitivity.Furthermore,to explore the regulatory mechanism between them and to demonstrate the pathway regulating radiosensitivity.Additionally,to verify the influence of their expressions on radiotherapy prognosis in NSCLC patients.To provide new molecular markers and targets for predicting and regulating the radiosensitivity and radiotherapy prognosis of NSCLC.Methods: 1.SRMAP database and RMBase v2.0 database were applied to predict m6 A modification sites of LKB1 mRNA.2.The mRNA levels of m6 A core regulatory proteins were detected by RT-qPCR at 0,2,4,6,8,10,12 and 24 h after X-ray irradiation in NSCLC cell line(H1299).3.The mRNA and protein expression levels of METTL3 and LKB1 in human bronchial epithelioid cell lines(HBE)and NSCLC cell lines(H1299,A549 and SK-MES-1)were detected by RT-qPCR and Western Blot.4.In NSCLC cell lines(H1299 and SK-MES-1),RT-qPCR and Western Blot were used to detect the expression levels of LKB1 mRNA and protein after siRNA silencing METTL3,and the expression levels of METTL3 mRNA and protein after siRNA silencing LKB1.5.After siRNA interfered with the expressions of METTL3 and LKB1 in NSCLC cell lines(siNC,siMETTL3,siLKB1 and siMETTL3+siLKB1 in the response verification group),the effects of METTL3 and LKB1 expressions on proliferation,activity and DNA damage of irradiated cells were detected by plate cloning,CCK8 and γH2AX immunofluorescence techniques.6.The m6 A level of LKB1 mRNA was detected by Me RIP-qPCR after METTL3 silencing.7.YTHDF2 was interfered by siRNA and overexpressed plasmid in NSCLC cell lines,respectively.Following this,the mRNA and protein levels of LKB1 were detected by RT-qPCR and Western Blot.Response experiment: METTL3 siRNA and YTHDF2 overexpression plasmid were co-transfected,METTL3 was silenced and YTHDF2 overexpressed at the same time,and the expressions of LKB1 mRNA and protein were detected by RT-qPCR and Western Blot.8.Si RNA was applied to interfere with the expressions of METTL3 and LKB1(siNC group,siMETTL3 group,siLKB1 group and siMETTL3+siLKB1 group),which were divided into two groups: X-ray irradiation and non-irradiation.The changes of p AMPK and γH2AX proteins were detected by Western Blot.9.Clinical and radiotherapy-related information and formalin paraffin-embedded pathological specimens were collected from 122 NSCLC patients who underwent postoperative radiotherapy in the First Affiliated Hospital of China Medical University.Immunohistochemistry was used to verify the expressions of METTL3 and LKB1,and to analyze the correlation between them.The effects of METTL3 and LKB1 expressions on locoregional recurrence-free survival(LRRFS),distant metastasis survival(DMFS),disease-free survival(DFS)and overall survival(OS)of NSCLC patients after radiotherapy were statistically analyzed.Results: 1.A total of 77 m6 A modification sites of LKB1 mRNA were predicted in SRMAP database,among which 34 m6 A modification sites own more than 95%confident,and the m6 A sites with high confidence accounted for 44.2%.Meanwhile,a total of 52 m6 A modification sites of LKB1 mRNA were predicted in RMBase v2.0database,among which 37 m6 A modification sites(71.2%)were found in Motif score>300,and 34 m6 A modification sites(65.4%)were found in Support Num≥3.2.Among the m6 A core regulatory proteins with high radiobiological correlation,METTL3 is the only m6 A methylation catalytic subunit.3.In NSCLC cell lines(H1299,A549 and SK-MES-1),the expression levels of METTL3 mRNA and protein were significantly higher than those of HBE,while the expression levels of LKB1 mRNA and protein were significantly lower than those of HBE.Among them,the expression of LKB1 was deficient in A549 cell line.4.In NSCLC cell lines(H1299 and SK-MES-1),when METTL3 was silenced,the expression of LKB1 was increased and the radiosensitivity of NSCLC cells was enhanced,when LKB1 was silenced,the radiosensitivity was decreased,when METTL3 and LKB1 were both silenced,the radiosensitivity of NSCLC cells was lesser than that only silenced METTL3.5.The m6 A modification level of LKB1 mRNA was decreased after METTL3 silencing.6.The expression levels of LKB1 mRNA and protein were increased after YTHDF2 silencing,the expression levels of LKB1 mRNA and protein were decreased after YTHDF2 overexpression.When silencing METTL3 and overexpressing YTHDF2 at the same time,the expression levels of LKB1 mRNA and protein were lower than that only silenced METTL3.7.Both irradiated and non-irradiated NSCLC cell lines showed the same effects: the expression levels of LKB1 and p AMPK were increased after METTL3 silencing,while the expression level of METTL3 had no change and the expression level of p AMPK was decreased after LKB1 silencing.When METTL3 and LKB1 were both silenced,the expression level of p AMPK was lower than that only silenced METTL3.At the same time,we also observed that in the irradiation group,when METTL3 was silenced,the expression level ofγH2AX protein was significantly higher than that in the control group,while,when LKB1 was silenced,the expression level of γH2AX protein was significantly lower than that in the control group.When METTL3 and LKB1 were both silenced,the expression of γH2AX was lower than that only silenced METTL3.8.In the pathological tissues of NSCLC patients,METTL3 was mainly expressed in the nucleus,LKB1 was mainly expressed in the cytoplasm.The high expression rate of METTL3 was 68.03%,and the low expression rate of LKB1 was 61.48%.The expressions of METTL3 and LKB1 protein were not correlated with age,sex,tumor location,pathological type,tumor size and number of positive lymph nodes.In NSCLC tissues,lung adenocarcinoma(LUAD)tissues,and lung squamous cell carcinoma(LUSC)tissues,the expression of METTL3 was negatively correlated with LKB1,respectively.9.In NSCLC patients,patients with low METTL3 expression tended to be better in LRRFS,DMFS,and DFS after radiotherapy,while the expression of METTL3 had no effect on OS.Patients with high LKB1 expression tended to be better in LRRFS,and with marginal statistical difference in DFS after radiotherapy,while the expression of LKB1 had no effect on DMFS and OS.In addition,a lower number of positive lymph nodes also tended to lead to better DMFS and DFS,and gender,pathological type,and tumor size were correlated with OS.Multivariate COX regression analysis confirmed that METTL3 is an independent prognostic factor in LRRFS,DMFS,and DFS of NSCLC patients after radiotherapy,LKB1 is an independent prognostic factor in LRRFS,the number of positive lymph nodes was an independent prognostic factor in DMFS and DFS,and no independent prognostic factor affecting OS were detected.Concluisons: 1.In NSCLC cell lines(H1299 and SK-MES-1),METTL3 plays as an upstream protein to negatively regulate the expression of LKB1,and low expression of METTL3 could increase the expression of LKB1 and improves the radiosensitivity.2.METTL3 regulates the m6 A modification level of LKB1 mRNA and depends on YTHDF2 recognition to affect the attenuation of LKB1 mRNA,thereby regulating the expression level of LKB1 mRNA and protein.METTL3-LKB1 regulates the expression of their downstream protein,p AMPK,to affect the degree of DNA damage of NSCLC cells after irradiation,so as to regulate the radiosensitivity of NSCLC cell line.3.In NSCLC patients,the expression of METTL3 is negatively correlated with that of LKB1,meanwhile,those patients who with low expression of METTL3 tend to have better LRRFS,DMFS,and DFS after radiotherapy,and those with high expression of LKB1 tend to have better LRRFS.However,METTL3 and LKB1 both had no significant effect on overall survival.In addition,fewer positive lymph nodes also tended to be associated with better DMFS and DFS.4.METTL3 and LKB1 could be potential radiosensitization targets and prognostic indicators of radiotherapy in NSCLC. |
PDF Full Text Request