Studies Of NESG1Gene On Its Function And Molecular Basis In Lung Adenocarcinoma

Background and ObjectivesLung cancer is one of the most common carcinomas with a high degree of malignancy in the world. The incidence of lung cancer is a complex process of a multi-stage, multi-channel and multi-mechansim,which may be related to genetic susceptibility to environmental risk factors and other factors.It’s morbidity tends to have significantly higher occurrence in most countries. The development of lung cancer involves the joint participation of the structure and expression regulation changes of multiple associated genes and several signaling pathway. The imbalance of oncogene activation and the inactivation of tumor suppressor genes and their interaction play a critical role. Therefore, the study of lung caner related genes and signaling pathways will help further reveal the pathogenesis of lung cancer.NESG1gene （Genbank AF094758）, official name CCDC19（NM₀12337.1）, was found by Dr Zhongkui Li who is guided by Professor Kaitai Yao in1999.It was re-cloned,sequenced analysis and corrected sequence by Dr. Liu Zhen in2009, which upgrated its Genbank database from the initial version （NM₀12337.1） to （NM₀12337.2）.The study found that NESG1widely expressed in many human tissues and organs. NESG1was a candidate tumor suppressor gene in NPC,which involved in the MAPK and cell cycle mediated signaling pathyway.As a new tumor suppressor gene, The research about the function and mechanism of this gene in lung cancer haven’t been reported by now. MicroRNA （miRNA） is a non-coding small RNA which has a length of about18to24nucleotide, and is produced from a single stranded RNA precursor （pre-miRNA） with about70-90bases and a hairpin structure under the help of dicer enzyme. MicroRNA has an effect of post-transcriptional negative regulation on the gene expression by the way of complementary base-pairing reactions with target mRNA, leading to the degradation of mRNA or inhibition of translation. Studies show that more than50%miRNA positioned in the tumor related area of genome. Their expression levels changes in a variety of tumors, miRNA negative regulation of gene expression through the regulation of transcription and translation of the target gene involved in tumor cell proliferation, differentiation, migration, invasion and metastasis.Their disorders are closely related to tumor development is.It is worth going on for further study about the issues of whether NESG1regulated by miRNA in lung cancer, and which miRNA modulates NESG1in reverse in lung cancer,Phosphatidylinositol-3-kinase/protein kinase B （PI3K/Akt）,which participate in the regulation of various cellular processes such as growth, proliferation, differentiation, apoptosis and glucose transportation, is one of the most important intracellular signal transduction pathways. The available clinical evidence of PI3K-pathway deregulation in various cancers and the identification of downstream kinases that are involved in mediating the effects of PI3K （AKT,mTOR, PDK1and ILK） provide potential targets for the development of small-molecule therapies. Althouth other signalling pathways are also known to be regulated by PI3K activity and might be involved in PI3K-mediated tumorigenesis,Akt is the key of PI3K/Akt signalling pathwany which acts downstream of PI3K to regulate many biological processes, such as proliferation, apoptosis and growth.. The abnormal increase of PI3K and Akt activity was closely associated with angiogenesis, invasion, metastasis, anti-apoptosis, and abnormal proliferation of tumor cells.Based on the above vestigation, we identified the function of NESGl gene and the signal transduction pathway that it involved in,to explore the mechanism of this gene. This provide a reference for further elucidate the pathogenesis of lung cancer. Contents and methods 1. Detection of NESG1gene expression level in lung cancer cells and organization:to test NESG1expression level by immunohistochemisty and quantitative real-time PCR（qPCR）.2. Effects of NESG1gene over-expression on biological characteristiscs in lung cancer cellsConstruct enhanced green fluorescent protein fusion expression NESG1of slow virus carrier.An "empty" vector pGC-FU-GFP was utilized as a negative control. Lentiviral particles were used to infect A549and SPCA1cells. The abilities of cell growth, cell cycle, migration, invasion were examined by MTT, colony formation, FACS Caliber cytometry,Transwell, and boyden chamber in vitro. In vivo subcutaneous tumorigenestiy in nude mice was used to evaluate the effct of overexpressed NESG1on lung cancer cells.3. Effects of suppressing the expression of NESGlon biological behavior of lung cancer cellssynthetic siRNA was transfected into NESG1-overexpression lung cancer cells through lipofection. MTT,Transwell and Boyden chamber assay were utilized to detect the change of the ability of cell cycle, migration and invasion in NESG1silencing.4. Screening differentially expressed miRNA through Quantitative PCR. Select significantly different miRNA as further research objects5. NESG1gene mediated the molecular mechanisms of lung cancer（1）The alterations of PI3K/Akt signalling pathway associated genes,such as p-PI3K.pAkt,c-myc were examined by Western blot in NESG1-overexpression lung cancer cells.（2）Western blot was used to test the expression of cell cycle-related and EMT-associated genes in lung cancer cells transfected with NESG1or in the absence of NESG1.Results1. Measurement of NESG1gene expression levels in lung cancer cell and tissue. Immunohistochemistry staining was performed for26normal lungl tissues and83 lung cancer tissues. Immunohistochemistry showed that NESG1gene expression was down-regulated in most lung cancer tissues, but major in lower expression,the difference being statistically significant （P=0.038）. No significant associations were found between age, gender, histological type, tumor differentiation,T,N,M classification and clinical stage （P>0.05） of lung cancer patients.Interestingly, Prognostic analysis showed the patients with low expression of NESG1had markedly shorter overall survival than those with high NESG1of expression （P=0.012）.Multivariate analysis suggested that the level of NESG1expression was an independent prognostic indicator for the survival of patients with lung cancer （P=0.016）. Quantitative real-time PCR found that NESG1expressed differences in mRNA level with statistical significance in lung cancer cells compared with ARF.2. Effects of NESG1gene over-expression on biological characteristiscs in lung cancer cells.（1） Afer NESG1Lentiviral particles were infected in lung cancer cells,we established stable NESG1overexpression lung cancer cell lines.Empty vector was used as negative control.Protein expression level of NESGlwas dected by western blot. Positve overexpression polyclonal was named NE-A549,NE-SPCA1,negative control polyclonal NC-A549and NC-SPCA1,respectively.（2） Compared to negative control cells, both two polyclonal NESG1-overexpressed cells showed to be inhibited markedly in MTT cell proliferation assay. There were significant differences （A549F=46.788, P＜0.001; SPCA1F=306.681, P＜0.001）)（3）The colony formation assay showed that colony forming ability of NESG1overexpression cells wer significantly weakened, the difference being statistically significant.（A549t=46.568, P=0.003,SPCA1t=4.46P=0.01）（4）In transwell migration experiments, the number of over-expression of NESG1cells through the polycarbonate film was fewer than the control group, respectively. And the difference was statistically significant （A549t=16.841, P＜0.001, SPCA1t=11.732, P＜0.001）.Suggest that after overexpression NESGl, the ability of cells migrating weakened.（5） Boyden invasion assay, compared with no-load cells, the number of NE-A549and NE-SCPA1cells through the polycarbonate film significantly decreased, with significant difference（A549t=15.128, P＜0.001, SPCA1t=9.923, P＜0.001）.Suggest that the ability of cells invasion declined after NESG1overexpression.（6） Cell cycle flow cytometry analysis, compared to negative control cells,the S phase population decreased significantly in NESGl-overexpressed treatment groups Suggest that it slowed down cell into the S phase （A549t=3.254, P=0.031, SPCA1t=4.259, P=0.013）（7） In vivo subcutaneous tumorigenesity in nude mice showed that Cell growth speed of the treatment group was significantly slower comparing with the no-load control group（t=7.027, P=0.003）.3.Effects of NESG1expression suppressed on biological characteristics in lung cancer cells.（1） si-NESG1RNA was synthesized and transfected into human lung cancer cells. Western blot was used to test the efficiency of interference after72h.The result showed that02fragments had the better silence efficiency of the NESGl-overepressed cells among the three sequences design of synthesis siRNA,which was used for the next experiment.（2）MTT assay showed that,compared with the control group, si-NESG1cells grew faster,there were significant differences （A549F=19.922, P<0.001;SPCAl F=32.682, P＜0.001）（3）The transwell experiments foud that the number of si-NESG1cells through the polycarbonate film was markedly increased compared to control cells,with significant difference （A549t=13.016, P＜0.001, SPCA1t=7.067, P＜0.001）（4） Boyden invasion assay showed the number of si-NESG1cells through the polycarbonate film was more than the control group, and the difference was statisitically significant （A549t=6.4, P＜0.001, SPCA1t=8.264, P＜0.001）4. Search the function in the difference of miRNAs after overexpressed NESG1in lung cancer through QPCR.（1） QPCR showed that expression of miR-494, miR-206increased1.5-fold after expressing NESG1.（2） MTT assay showed that, compared with the control group, over-expression of miR-494cells grew slower, there were significant differences （P<0.001）. The transwell and boyden experiments showed that the number of over-expression of miR-494cells through the polycarbonate membrane was fewer than the control group, and the difference was statistically significant （P<0.001）.（3） Western blot assay showed that oncogenic cell cycle factor CCND1and mesenchymal-marked genes Vimentin and N-Ca expressions were markedly weakened in miR-494mimic treatment compared with miR-Ctr group.5.NESG1gene mediated the molecular mechanisms of lung cancer（1） Western blot assay found that the protein expression level of PI3K/Akt signalling pathway associated genes,such as p-PI3K.pAkt,c-myc was weakened in lung cancer cells after the overexpression of NESG1compared with contro groups,but there was no significant change in the protein level of PI3K.（2） Western blot assay showed that oncogenic cell cycle factor CCND1and mesenchymal-marked genes Vimentin and N-Ca expressions were markedly weakened in NESG1-overexpression treatment compared with control group, Meantime, the protein expression level of cell cycle factor p27,p15and EMT-associated gene E-cadherin was increased, while the protein expression level of CDK4had no significant difference after the overexpression of NESG1.（3） Furthermore,compared with si-control group,the protein level of CCND1increased after the inhibition of NESG1, accompanied with the decreased p27and p15,and increased N-Ca protein expression level. In summary,our studies suggest that NESG1is able to retard the progression of cell cycle and EMT occurrence.Conclusions1.Afer NESG1overexpression, the ability of proliferation in vivo and in vitro,migration and invasion were markedly reduced, and cell cycle progression was blocked from G1to S phase. In contrary, afer NESG1expression was inhibited, the ability of proliferation,migration and invasion was recovered, which suggested that NESG1should be a tumor-suppressive gene.2. NESG1may inhibit the signal pathway of PI3K/Akt. NESG1may suppress cell proliferation, migration, and invasion by reducing the expression of E2F1,CCND1, Vimentin,N-CA and upregulating the expression of P15, P27,E-Ca in A549and SPCA1lung cancer cells.3. miR-494may suppress cell proliferation, migration, and invasion by reducing the expression of CCND1, Vimentin and N-CA in A549and SPCA1lung cancer cells.