Molecular Mechanism For PTBP1 Regulating Mena Alternative Splicing And Positive Effect On Migration And Invasion Of Lung Carcinoma Cells

Objective: The majority of genes have alternative splicing(AS)events,which mediates many physiological and pathological processes including tumor metastasis.For instance,one of Mena isoforms Mena INV,is a conserved regulator of actin dynamics that has important role in tumor metastasis.However,the exact mechanism mediated Mena AS has not been elucidated.To explore the molecular mechanism of PTBP1 in regulating Mena alternative splicing and role of PTBP1 in lung carcinoma cells metastasis.Thus,the aim of this study is to propose a new mechanism of PTBP1 regulating lung cancer cell metastasis by discussing that PTBP1 mediates lung carcinoma cells metastasis is partially dependent on Mena alternative splicing.Methods: 1.The expressions and survival probability of PTBP1 between the lung adenocarcinoma(LUAD)tissues and normal tissues were analyzed from Cancer Genome Atlas(TCGA)Database.q PCR and Western blot analysis were conducted in metastatic 95-D cell line and non-metastatic A549 cell line.The regular reverse transcription(RT-PCR)was used to detect the levels of Mena INV and Mena11 a.2.TGF? was used to induce EMT in A549 cells,and the changes of PTBP1 and EMT markers(E-cadherin,N-cadherin and Vimentin)during EMT were detected using Western blot.Moreover,the changes of the ratio of Mena11 a and Mena INV after TGF? induction were detected.3.The retrovirus was used to overexpress or knockdown PTBP1,wound healing assay and Transwell assay were used to analyze migrated ability and invasive ability after PTBP1 overexpression or knockdown in lung cancer cells,RT-PCR was used to detect Mena isoforms ratio changes.4.The wild and mutant Mena minigene vectors were constructed and transfected into PTBP1 overexpressed cells as well as PTBP1 knockdown cells.RT-PCR was used to detect the percentage of Mena isoforms in minigene level.5.According to the consensus sequences of the PTBP1,the binding sequences of PTBP1 in Mena precursor m RNA were predicted by Human Splicing Finder,RBP map and RBPDB.Then,the biotin-labeled RNAs were synthesised in vitro and was used to verify binding sequences of PTBP1 using RNA-pulldown technology.6.The retroviral overexpression vector of Mena INV and retroviral knockdown vectors of Mena isoforms were constructed,wound healing assay and Transwell assay were used to detect the metastasis and invasion of lung cancer cells after changes of PTBP1 and Mena isoforms.Phalloidin fluoresced the cells and the laser confocal system was used to observe the changes in the number of filopodia in lung cancer cells treated PTBP1 and Mena isoforms.Results: 1.The expression of PTBP1 was significantly increased in lung adenocarcinoma tissues and metastatic lung cancer cells.Patients with high PTBP1 expression had smaller survival probability than those with low PTBP1 expression(p<0.01).Western blot and q PCR results showed that the PTBP1 level of 95-D was higher than A549.RT-PCR results showed that the level of Mena INV was 3.8-fold in 95-D cells compared with A549 cells.2.Overexpression of PTBP1 increased the levels of N-cadherin and vimentin while reduced the levels of E-cadherin.The results of wound healing and transwell invasion assays showed that 95-D,A549 and TGF?-induced A549 cells displayed increase in migratory abilities compared with control treatment.The percentage of Mena INV was significantly increased after PTBP1 overexpression.Furthermore,the opposite effect was detected when PTBP1 was knockdown.3.After overexpression of PTBP1,the expression levels of N-cadherin and Vimentin increased and E-cadherin levels decreased.The metastasis and invasiveness of cells were significantly increased.RT-PCR results showed that the radio of Mena INV increased by 27% after PTBP1 overexpression;N-cadherin and Vimentin expression levels decreased,E-cadherin level was after knockdown of PTBP1,cell migration and invasion ability were significantly decreased,RT-PCR results showed that the proportion of Mena INV significantly reduced by 37%..4.Results of RNA-pulldown indicated that promotion of exon11 a skipping was mediated by PTBP1 binding sites on both upstream and downstream intron,which were chracteriatced by polypyrimidine sequences.5.The Mena was knockdown in PTBP1 overexpressed 95-D cells,the formation of filopodia was significantly reduced before the knockdown of Mena,and metastasis capacity was also significantly reduced.There was no significant change in specifically knockdown Mena11 a cells.After knockdown of PTBP1,overexpressed Mena INV significantly increased the formation of filopodia in cells and enhanced the ability of cell metastasis.Therefore,PTBP1 might partially depend on the expression of Mena INV to promote lung cancer cell metastasis.Conclusions:1.The expression of PTBP1 is related to the metastasis of lung cancer cells and EMT process;overexpression of PTBP1 promotes the metastasis of lung cancer cells and knockdown of PTBP1 inhibits the metastasis of lung cancer cells.2.PTBP1 binds to the polypyrimidine sequences(TTTTCCCCTT and TTTTTTTTTCTTT)in Mena precursor m RNA and regulates the alternative splicing of exon 11a;the alternative splicing form is exon skipping.3.Overexpression of Mena INV can partially rescue the effect of PTBP1 knockdown,resulting in increased formation of filopodia and promotion of lung cancer cell metastasis and invasion.Overall,this study proposes a model for PTBP1 to enhance the exon 11 a skipping in Mena precursor m RNA to promote lung cancer cell metastasis.