Preliminary Research Of NSUN2 Promoting Lung Cancer Cell Proliferation,Metastasis,Invasion And Drug Resistance

Objectives:Lung cancer is one of the malignant tumors with the highest morbidity and mortality in China.In the past 20 years,the emergence of various targeted therapies and the effective application of immunotherapy,in the population of patients with advanced lung cancer,has made the treatment of lung cancer get a tremendous progress.But the targets that have been discovered so far are limited,and some targeted therapies also have drug resistance by unknown mechanism.These are huge challenges we have to face.So,it is urgent to find more effective targets and strategies for the treatment of lung cancer.In the early stage of this study,it was found that the expression of the gene NSUN2(NOP2 RNA methyltransferase family member 2)in the tumor tissues of three patients with non-small cell lung cancer was significantly higher than that of adjacent normal tissues checking by the gene chip,and this found difference was confirmed by RT-qPCR in 57 clinical lung cancer samples this.NSUN2 is a kind of RNA m5C methyltransferase that can methylate a variety of RNAs.m5C methylation at different sites on mRNA has different regulatory effects on the translation or stability of the mRNA.Several reports have shown that this type of RNA modification can regulate the physiological function of cells in many ways,and some studies suggested that this RNA modification also plays a role in cancer.In order to explore the role of m5C methylation in lung cancer.We planned to reveal the role and underlying molecular mechanism of the RNA methyltransferase,NSUN2 in the tumorigenesis and development of lung cancer.Its functions in the proliferation,metastasis,invasion and drug resistance of lung cancer cells were detected and analized,and the possible target genes that NSUN2 methylates their mRNAs and changes their protein expression to alter tumor phenotype were checked,in order to evaluate the value of NSUN2 in clinical diagnosis and treatment of lung cancer.Methods:1.Collecting cancer tissues and adjacent tissues of patients diagnosed with non-small cell lung cancer in the Third Affiliated Hospital of Kunming Medical University,Kunming City,Yunnan Province,and extracting total RNA,then using RT-QPCR to detect the mRNA level of NSUN2 in cancer tissues and adjacent cancers.2.Designing and constructing shRNA expression vector(shNSUN2)that knock down NSUN2 and NSUN2 overexpression vector(pNSUN2),then preparing lentivirus to infect two lung cancer cell lines A549 and H1299 cells in vitro.Screening and obtaining stable cell lines of A549 and H1299 in which NSUN2 was knocked down or overexpressed of and their control groups.3.Performing cloning formation experiment,CCK8 detection,scratch healing experiment,Transwell experiment,Cell resistance experiment and nude mouse tumorigenicity experiment in vitro and in vivo By using the obtained A549 and H1299 stable cell lines and control groups.in oeder todetecting the effect of NSUN2 on the proliferation,metastasis,invasion and drug resistance of lung cancer cells.4.Extracting RNA and protein from A549 and H1299 stable cell lines with knockdown or overexpression of NSUN2 and their control groups,and performing RT-PCR and Western blot tocheck the expression of three target genes of NSUN2 and the key genes in their downstream pathways which play important roles in the tumorigenesis and development of lung cancer.Results:1.RT-qPCR results showed that the mRNA level of the gene NSUN2 in lung cancer tissues is significantly higher than that in adjacent tissues,it is necessary to further study its role in lung cancer.2.Under the in vitro A549 and H1299 culture,the proliferation,metastasis,invasion and drug resistance of lung cancer cells are reduced after NSUN2 knockdown,while overexpression of NSUN2 only enhances the invasion ability of lung cancer cells;in vivo environment,knocking down NSUN2 significantly reduce the proliferation ability of lung cancer cells in the nude mice.3.The mRNA levels of MUC1 in A549 and H1299 cells with knockdown or overexpression of NSUN2 did not change.But the protein level of MUC1 in A549 cells after NSUN2 knockdown was significantly increased,but the expression levels of MUC1 were extremely low in all H1299 cells.The expression level of MUC1 was increased afterthe NSUN2 knockdown,but its downstream PI3K/AKT pathway and glycolysis pathway were not activated,and the expression level of some key genes in these pathways decreases after NSUN2 knockdown.4.The mRNA levels of CDK1 in A549 and H1299 cells after knockdown and overexpression of NSUN2 did not change.But the level of CDK1 protein was positively correlated with the expression level of NSUN2 in A549 cells after knockdown or overexpression of NSUN2,but there was no significant correlation in H1299 cells.5.The mRNA levels of CDC25C in A549 and H1299 cells did not change after knockdown and overexpression of NSUN2,but the expression level of CDC25C protein was positively correlated with the expression level change of NSUN2.The rise of CDC25C level,which is raised by NSUN2 overexpression,failed to dephosphorylate CDK1 to activate it.Conclusions:Changes in the expression level of NSUN2 would affect the proliferation,metastasis,invasion and drug resistance of lung cancer cells.The expression change of NSUN2 could affect the expression of MUC1,CDC25C and CDK1 genes at the post-transcriptional level,but It seemed that their expression change resulted from NSUN2 change could not mediate the effect of NSUN2 on the proliferation,metastasis,invasion and drug resistance of lung cancer cells.