NRSN2 Promotes Non-small Cell Lung Cancer Growth Through PI3K/AKT/mTOR Pathway

Background and Objective:Non-small cell lung cancer(NSCLC)remains the leading cause of cancer death in the world.Statistical results suggest that less than 15% of NSCLC patients surviving beyond 5 years.Newer molecularly targeted treatments have shown activity in advanced stages,but many patients do not respond to those treatments for NSCLC.So,newly predictive and prognostic biomarkers in NSCLC should be excavated for effective treatment.NRSN2,a newly isolated gene,encodes a small neuronal membrane protein.Previous study revealed that NRSN2 was mainly located in small vesicles and might be involved in organelle transport and conduction of nerve signals.To our knowledge,NRSN2 was mainly reported in nervous system.Recently,the inverse role of NRSN2 was investigated in hepatacellular carcinoma(HCC)and malignant melanoma.Factors in nerves have been found could exert some effects on cancer.NSCLC,especially lung adenocarcinoma,is related to the dysregulation of neuroendocrine.We retrieved Oncomine Database and found that NRSN2 is commonly highly expressed in NSCLC.So we proposted that NRSN2 might play a necessary role in NSCLC.PI3K/AKT/mTOR signal pathway is an important intracellular signal transduction pathway.It plays important roles in cell proliferation and survival by affecting the activity of downstream effector molecules,and it is closely associated with the development and progression of NSCLC.It is unclear whether NRSN2 can affect phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)signal pathway.In the current study,gene clone and RNA interference(RNAi)were employded to investigate the role of NRSN2 in the biological behavior and molecular mechanisms of NSCLC.It would result in finding new target for drugs and providing an experimental basis for targeted therapy of NSCLC.Methods:(1)The mRNA and protein level of NRSN2 in NSCLC tissues were detected by quantitative realtime PCR,immunohistochemistry staning and western blot.we analyzed the correlations between the NRSN2 expression status and clinicopathological characteristics of 48 NSCLC samples by statistic analysis.(2)Gene clone and RNA interference(RNAi)were employed to explore the relationship of NRSN2 expression and biological charateristics in A549 cell line.Western blot was used to test the expression of NRSN2 in A549 transfected with NRSN2 or in the absence of NRSN2.(3)The abilities of cell growth,migration,invasion were examined by CCK-8、colony formation and Transwell in vitro.(4)Western blot was used to examine the effect of NRSN2 silencing and over-expressing on the expression of phosphorylated AKT and phosphorylated mTOR in A549 cells.Results:(1)Elevated NRSN2 expression in NSCLC and closely related to tumor size and tumor differentiation.The mRNA and protein level of NRSN2 were increased in NSCLC tissues detected by quantitative real-time PCR,immunohistochemistry and western blot.Immunohistochemistry was performed to investigate the correlation between the clinicopathologic characteristics and expression of NRSN2 protein in NSCLC tissues.The expression level of NRSN2 in the NSCLC tissues was positively associated with the size of tumor(p<0.05)and tumor differentiation(p<0.05).(2)Silencing NRSN2 inhibited cell viability and proliferation。The CCK-8 cell viability assays showed that NRSN2 played a positive role in cell viability.Silencing NRSN2 significantly decreased the cell viability compared with control group.The results from cell clone formation assays suggested that knockdown NRSN2 inhibited the ability of cell proliferation.(3)High expression level of NRSN2 promoted cell viability and proliferation.A549 cells which stable over-express NRSN2 were subject to perform CCK-8 cell viability assays and cell viability remarkably elevated after NRSN2 heightened.Further tests showed that the ability of clone formation significantly increased in high level of NRSN2(p<0.01).(4)NRSN2did not affect cell invasion.A549 transwell invasion assays after over express or knock down NRSN2,statistical analysis showed that there was no difference between control and treatment groups.(5)NRSN2 affected cell viability or proliferation and activated PI3K/AKT/mTOR pathway.Protein was extracted from A549 after treatment and examined by western blot.Silencing NRSN2 reduced phosphorylation levels of AKT and mTOR,while high NRSN2 level upregulated phosphorylation levels of AKT and mTOR.Conclusions: NRSN2 is highly expressed in NSCLC tissues.NRSN2 promotes NSCLC growth through PI3K/AKT/mTOR pathway probably.Our study validates the carcinogenic role of NRSN2 in both clinicopathologic and biological aspects in NSCLC tumorigenesis.