The Effects And Mechanisms Of TWIST Up-regulating SYT7 In Non-small Cell Lung Cancer

Part I The screening of TWIST downstream genes associated NSCLC invasion and metastasisObjective:TWIST is highly expressed in a variety of tumors.It plays an important regulatory role in tumorigenesis,invasion and metastasis,angiogenesis and apoptosis resistance by transcriptionally regulating downstream target genes.This study aims to screen the downstream genes of TWIST related to the invasion and metastasis of NSCLC by whole gene expression profiling technology,and to explore the new mechanism of TWIST and its downstream regulatory network to promote NSCLC invasion and metastasis.Methods:The expression of TWIST in four lung cancer cells was detected by real-time quantitative PCR,and the cell line with the lowest expression of TWIST was selected for further study.The changes of metastasis ability of lung cancer cells after over-expressing TWIST were detected by scratch test and transwell migration test.The function genes associated with invasion and metastasis of NSCLC were obtained by whole genome expression profile microarray experiment,PCR and high throughput function screening method.Results:Real-time quantitative PCR showed that TWIST was highly expressed in A549 and H1299 cells,was moderately expressed in H358 cells and was low expressed in H1975 cells for four commonly used NSCLC cell lines(A549,H1299,H1975,H358).The scratch test and transwell migration test showed that the over-expression of TWIST significantly enhanced the metastasis ability of H1975 cell.The results of whole genome microarray experiment showed that 1297 genes were obtained as differentially expressed genes in H1975 cells after over-expressing TWIST according to the criteria:fold change more than 1.3 or less than-1.3 and a p-value less than 0.05,including 589 up-regulated genes and 708 down-regulated genes.Through bioinformatics analysis and validation of PCR,20 up-regulated genes were selected to perform high throughput function screening experiment,including DUSP5,TNIP1 TMEM154,CCBE1,CYB5R2,MYEOV,NUAK2,RPS6KL1,PPIF,RIMS2,SYT7,ABHD5,CEP85,LST1,NFKBIE,HYI,POLR3G,SNHG12,UPP1 and PXK.The results showed that the migration ability of p-TWIST-H1975 cells was significantly decreased after SYT7 and CEP85 were knocked out.The ratio of cells migration distance between experimental group and control group was 0.12 and 0.35(p<0.05),respectively,suggesting that the two genes were metastasis-related genes from high-throughput screening experiment.Conclusion:1.The over-expression of TWIST enhances the metastasis ability of NSCLC H1975 cells.2.SYT7 and CEP85 may serve as the functional genes associated with:NSCLC metastasis in TWIST downstream network.The study of the function of TWIST and its downstream genes will contribute to clarify the new mechanism of NSCLC invasion and metastasis.Part II The effects and mechanisms of SYT7 in non-small cell lung cancerObjective:Cell function experiments validate the possible function gene SYT7 obtained in part I,to clarify its role in NSCLC and explore its mechanism.Method:Real-time quantitative PCR was used to detect the expression level of SYT7 in A549,H1299,p-TWIST-H1975(TWIST over-expressed cell line)and H125 cells.shRNA technique was performed to inhibit SYT7’s expression in A549 cells(TWIST endogenous high-expression),H1299 cells(TWIST endogenous high-expression)and p-TWIST-H1975 cells(TWIST exogenous high-expression),respectively.The effects of SYT7 knockdown on invasion and metastasis of lung cancer cells were detected by transwell invasion and migration assays and tumor formation in nude mice.MTT proliferation and FACS apoptosis assays were used to detect the proliferation and apoptosis of A549 cells after SYT7 knockdown,and the changes of EMT related markers after SYT7 knockdown were detected by Western-blot.The TCGA data was applied to detect the expression of SYT7 in lung cancer tissues compared with normal lung tissues and the methylation level,and assess the association with clinical prognosis.The differently expressed genes after SYT7 knockdown were obtained by whole genome expression profile microarray experiment,and expressions of some genes were further validated by Western-blot.The potential mechanism of SYT7 in regulating the tumorgenesis and development of NSCLC was also explored preliminarily.Results:Real-time quantitative PCR showed that SYT7 was highly expressed in p-TWIST-H1975 cells,and moderately expressed in other three lung cancer cells.Transwell invasion and migration experiments and tumor formation in nude mice showed that the knockdown of SYT7 significantly inhibited the invasion and metastasis of p-TWIST-H1975,A549 and H1299 cells(p<0.05).The knockdown of SYT7 inhibited cell proliferation,promoted cell apoptosis,and up-regulated the expression of E-cadherin(the epithelial cell marker),downregulated the expression of N-cadherin and Vimentin(mesenchymal cell markers)in A549 cells.The results of TCGA data showed that SYT7 was high-expressed in lung cancer tissues and associated with the poor prognosis for lung squamous cell carcinoma patients.Compared with normal lung tissues,SYT7 gene’s CpG islands had lower methylation level(P<0.05)in lung adenocarcinoma cancer tissues.A total of 746 differentially expressed genes were obtained after SYT7 knockdown(|FC|>2,P<0.05),including 376 up-regulated and 370 down-regulated genes.The changes of protein level of DDX58、HMGB1、JUN、STAT1 validated by Western-blot were consistent with the results of microarray experiment.The results of protein interaction network showed that hnRNPs(SYNCRIP,HNRNPD,etc.)and EIFs(EIF4G1,EIF4E,etc.)had extensive and close relation.Conclusion:1.The knockdown of SYT7 inhibits the proliferation,invasion and metastasis of lung cancer cells and EMT,and promotes cell apoptosis.SYT7 can be regarded as a new oncogene for NSCLC.2.SYT7 is highly expressed in lung cancer tissues,and is associated with the prognosis of lung squamous cell carcinoma patients.DNA methylation may be one of the mechanisms of the high expression of S YT7 in lung adenocarcinoma.3.SYT7 may up-regulate the expression of EIFs(EIF4G1,EIF4E,etc.)by the interaction with SYNCRIP and/or its complexes,which may play an important role in the genesis and development of non-small cell lung cancer.