The Role And Molecular Mechanism Of PLEK2 In The Metastasis Of Non-small Cell Lung Cancer

Research Background:Lung cancer is the most malignant tumor with the highest morbidity and mortality in the world,of which,non-small cell lung cancer(NSCLC)accounts for about 85% of all lung cancers.Despite advances in the treatment of lung cancer,the prognosis of advanced NSCLC is still very poor,tumor metastasis is the main cause of death.However,a large number of studies have shown that Epithelial-to-mesenchymal transition(EMT)plays a crucial role in tumor metastasis.Transforming growth factor-?(TGF-?)is an important inducer of EMT.However,its specific mechanism of EMT has not yet been fully elucidated and remains to be further studied.Therefore,studying the molecular mechanism of TGF-?-mediated NSCLC metastasis and searching for relevant molecular targets and metastatic diagnostic markers has become a hot and difficult issue for researchers and clinicians at present.Objectives:In this study,bioinformatics analysis was used to screen differentially expressed genes in TGF-?-treated tumor cells.PLEK2,a gene highly expressed in NSCLC A549 cells,was screened for the association with TCGA database and NSCLC data in GEO database,Meta-analysis,and then clear the clinical significance of PLEK2 gene screening.Subsequently,the expression of PLEK2 in TGF-?-induced NSCLC cells and the expression of PLEK2 gene in clinical samples were verified.The biological functions of NSCLC cells and pulmonary microvascular endothelial cells(HMVEC-L)were studied after overexpression and silencing of PLEK2.Finally,the bioinformatics method was used to find the protein SHIP2 that may interact with PLEK2,and the corresponding cellular level of verification and molecular mechanisms were studied in order to provide a molecular basis for the personalized diagnosis and treatment of lung cancer.Methods:1.TGF-?-induced tumor cell differentiation of genes and bioinformatics analysisBased on the data in the GEO database,bioinformatics methods were used to screen the key genes of TGF-? induced tumor epithelial-mesenchymal transition(EMT).Combined with the data of TCGA database and NSCLC in GEO database,Meta analysis was performed to clarify the clinical significance of PLEK2 gene screening.2.Expression of PLEK2 in TGF-?-induced cells and its expression in clinical samplesThe mRNA and protein levels of PLEK2 in TGF-?-induced NSCLC cells A549,SPC-A1 and pulmonary vascular cell HMVEC-L were detected by qRT-PCR and western blot.To verify the expression of PLEK2 in TGF-?-induced cells;To immunohistochemical staining of lung adenocarcinoma tissue with the survival of the chip,statistical expression of PLEK2 in lung cancer patients,and PLEK2 expression levels in patients with The overall survival of the statistical analysis.3.PLEK2 promotes EMT,migration and invasion in non-small cell lung cancer cellsPLEK2 overexpression plasmid PCDNA3.1-PLEK2 and PLEK2 interference siRNA sequences were purchased,and the effective interference sequence was screened for the construction of PLEK2 silencing lentivirus shPLEK2.The expression of PLEK2 was detected by qRT-PCR and western blot,and the changes of the markers of EMT were detected by western blot and immunofluorescence(IF)in cells induced by TGF-?overexpressing PLEK2 and silencing PLEK2.The cell migration ability was detected by cell scratch assay and transwell chamber migration assay.The cell invasion assay was performed by transwell chamber invasion assay.The nude mice tail vein pulmonary metastasis model was constructed to detect the in vivo metastasis ability of PLEK2 silenced cells.4.PLEK2 promotes pulmonary vascular endothelium EndoMT and barrier function damageThe expression of PLEK2 was detected by qRT-PCR and western blot in HMVEC-L cells overexpressing PLEK2 and silencing PLEK2 induced by TGF-?,Western blotting and immunofluorescence(IF)were used to detect EndoMT markers Protein and tight junction protein changes,the use of vascular permeability test monolayer endothelial cell permeability.Vascular invasion assay was used to detect the invasion ability of A549 cells to monolayer HMVEC-L cells.5.The mechanism of PLEK2 in NSCLC cell metastasisThe protein SHIP2,which may interact with PLEK2,was predicted by on-line analysis software,and co-immunoprecipitation(Co-IP)and immunofluorescenceco-localization were used to verify the interaction.The phosphorylation and ubiquitination sites of SHIP2 were found by the predictive analysis.The effects of TGF-? treatment and PLEK2 overexpression and silencing on the phosphorylation and ubiquitination of SHIP2 were detected by Western blot.The related experimental methods of Methods 3 and 4 Effect of SHIP2 on EMT and Migration in NSCLC Cells.Western blot was used to detect the effect of TGF-? treatment and PLEK2 overexpression and silencing on SHIP2-related TGF-? / PI3 K / AKT signaling pathway.Result:1.TGF-?-induced tumor cell differentiation of genes and bioinformatics analysisThe data set of TGF-?-treated tumor cells in the GEO database was analyzed based on the R language,and the differential gene PLEK2 was obtained.Further meta-analysis showed that PLEK2 was highly expressed in NSCLC tissues,which was negatively correlated with disease-free survival and overall survival.2.Expression of PLEK2 in TGF-?-induced cells and its expression in clinical samplesThe m RNA and protein levels of PLEK2 in TGF-?-treated tumor cells A549,SPC-A1 and pulmonary vascular endothelial cells HMVEC-L were significantly increased.Immunohistochemical staining of lung adenocarcinoma with survival of the chip,PLEK2 expression in tumor tissue was significantly higher than adjacent tissues and was negatively correlated with overall survival.3.PLEK2 promotes EMT,migration and invasion in non-small cell lung cancer cellsThe expression of E-cadherin in epithelial cells of A549 and SPC-A1 cells was significantly decreased,while the expression of N-cadherin and Vimentin in interstitial cells was significantly increased.The migration and invasion of cells were significantly increased.After silencing the expression of PLEK2,the result of the cell experiment was exactly opposite to that of the overexpression,and the cell migration ability in vivo was significantly weakened.Simultaneous silencing of PLEK2 partially inhibited TGF-?-induced decrease in E-cadherin and increased N-cadherin,Vimentin and migration and invasion.4.PLEK2 promotes pulmonary vascular endothelium EndoMT and barrier function damageEndothelial marker VE-cadherin,Zo-1,and Occludin were significantly decreasedin HMVEC-L cells overexpressing PLEK2,while N-cadherin and Vimentin were significantly increased in HMVEC-L cells;the permeability of monolayer HMVEC-L cells The effect of silencing PLEK2 could inhibit the decrease of VE-cadherin and the expression of N-cadherin induced by TGF-?,Vimentin increased as well as increased cell permeability and the role of the barrier to the invasion of A549 cells weakened role.5.The mechanism of PLEK2 in NSCLC cell metastasisProtein interaction prediction interaction between PLEK2 and SHIP2,PLEK2 and SHIP2 interactions confirmed in A549 cells,and PLEK2 high expression can significantly reduce the expression of SHIP2 protein levels;TGF-? treatment and over-expression of PLEK2 SHIP2 Phosphorylation and ubiquitination levels were significantly increased.The overexpression of SHIP2 significantly inhibited the EMT,migration and invasion of A549 cells,and the phosphorylation of Akt in TGF-? / PI3 K /AKT signaling pathway was significantly inhibited.After further treatment of A549 cells with TGF-? and the phosphorylation of Akt after overexpression and silencing of PLEK2,we found that both PLEK2 overexpression and TGF-? treatment can significantly promote the phosphorylation of Akt,whereas the phosphorylation of Akt after silencing PLEK2 The level is significantly lower.Conclusion:We successfully screen out the differentially expressed gene PLEK2 by using TGF-?-induced tumor cell data set and carry out a meta-analysis using GEO and TCGA datasets.The results showed that the expression of PLEK2 in tumor tissues was significantly higher than that in adjacent normal tissues,and the expression of PLEK2 was significantly negatively correlated with disease-free survival and overall survival of cancer patients.At the cellular level,it was confirmed that TGF-? could obviously induce the expression of PLEK2 mRNA and protein.The clinical tissue chip was used to verify the expression of PLEK2 in lung cancer tissues,which was consistent with the meta-analysis.Cell function experiments show that PLEK2 can not only promote the EMT process and migration and invasion of NSCLC cells on the one hand,but also promote EndoMT of HMVEC-L cells and disrupt the tight junctions between the cells to increase cell permeability and finally destroy the vascular endothelium Cell barrier.TGF-? can significantly promote the high expression of PLEK2,and whether TGF-?treatment or overexpression of PLEK2 can cause SHIP2 ubiquitination degradation and activation of TGF-? / PI3 K / AKT signaling pathway.In summary,our study shows that TGF-? treatment leads to high expression of PLEK2,and then ubiquitination of SHIP2,making SHIP2 weakened the PI3K/AKT pathway and ultimately promote the metastasis and invasion of tumor cells,and therefore PLEK2 is expected to serve as Molecular targets for the treatment of NSCLC and metastasis diagnostic markers.