| Background:TTC9A,a newly identified chaperone protein of promyosin Tm5NM-1,is a member of the TTC9 family with functions in stabilizing the cellular microfilament backbone and participating in cell metastasis.Studies have shown that TTC9 A is up-regulated in malignant tumour tissues such as breast cancer,involved in metastasis and invasion of cancer cells,and is likely to play a role as a pro-oncogene.However,the expression,biological function and mechanism of action of TTC9 A in lung adenocarcinoma are still unclear,and its in-depth study will help provide a theoretical basis for early diagnosis,chemotherapeutic treatment and prognostic assessment of lung adenocarcinoma.Methods:1.Immunohistochemical assays combined with multiple bioinformatic database mining were used to analyze TTC9 A expression in tumor tissues and normal lung epithelial tissues of lung adenocarcinoma patients.To assess the correlation between TTC9 A m RNA expression levels and the clinicopathological characteristics and prognosis of patients.Bioinformatics analysis of the function and enrichment pathways of genes co-expressed with TTC9 A in lung adenocarcinoma.To predict the relationship between TTC9 A m RNA expression and the tumour immune microenvironment using tools such as x CELL and TIMER.2.The m RNA and protein expression of TTC9 A in four lung adenocarcinoma cell lines,A549,H1299,PC9 and NCI-H1975,were detected by q PCR and Western blot.Lentiviral vectors were constructed to silence TTC9 A expression and negative control(NC),and lung adenocarcinoma cell lines were transfected to screen and establish stable transfected cell lines.In in vitro assays,the effect of silencing TTC9 A expression on the proliferation ability of lung adenocarcinoma cells was examined using CCK-8 assay and plate clone formation assay.Flow cytometry was used to detect the effect of silencing TTC9 A expression on apoptosis and cycle distribution of lung adenocarcinoma cells.The effect of altered TTC9 A expression on the tumorigenic ability of lung adenocarcinoma cells in vivo was observed by establishing a subcutaneous transplantation tumor model in nude mice.3.Wound healing assay and Transwell assay were used to observe the effect of TTC9 A expression on the migration and invasion ability of lung adenocarcinoma cells.Western blot assay was used to detect the effect of TTC9 A expression on epithelial-mesenchymal transition(EMT)-related indexes and MMP-9 expression in lung adenocarcinoma cells.4.The effect of cisplatin on the activity of PC9 cells after silencing TTC9 A expression was examined by CCK-8 assay and plate clone formation assay.The expression levels of MRP1,a multidrug resistance-associated protein,in lung adenocarcinoma cells after silencing TTC9 A expression were detected by q PCR and Western blot.Flow cytometry was used to detect the effect of cisplatin on the apoptosis of PC9 cells after silencing TTC9 A expression.Western blot was used to detect changes in the expression of p38 MAPK and key molecules of the MDM2/p53 signalling pathway after silencing TTC9 A expression in lung adenocarcinoma cells.Result:1.The TCGA database analysis and immunohistochemical results showed that TTC9 A expression levels in lung adenocarcinoma tissues were significantly higher than those in normal lung tissues,and TTC9 A expression was strongly correlated with gender,lymph node metastasis and mortality status.TTC9 A expression is an independent biomarker for predicting overall survival(OS),progression-free interval(PFI)and disease-free interval(DFI)in lung adenocarcinoma.TTC9 A may serve as a novel biological indicator for the diagnosis and prognostic evaluation of lung adenocarcinoma.2.At the genetic and epigenetic levels,TTC9 A expression was regulated by DNA deletion and amplification and by hypomethylation levels.TTC9 A expression was strongly correlated with the level of tumour immune infiltration.Gene enrichment analysis identified the most significant genes co-expressed with TTC9 A as TSPAN15,SYTL2 and GPR110,which were positively correlated with TTC9 A m RNA expression.The results of gene enrichment analysis showed that the up-regulated genes co-expressed with TTC9 A in lung adenocarcinoma were mainly involved in protein binding transport,growth and developmental aspects,involving signalling pathways such as p53,extracellular matrix binding and cell adhesion.3.Both q RT-PCR and Western blot assays showed that TTC9 A was expressed in lung adenocarcinoma cell lines A549,PC9,NCI-H1975 and H1299,with the highest expression of PC9.Stable transduced lung adenocarcinoma cell lines with silent TTC9 A expression and negative control were successfully screened.4.Silencing TTC9 A expression inhibited proliferation,migration and invasion of PC9 cells in vitro and in vivo,promoted apoptosis,led to cell cycle G2 phase arrest and increased sensitivity to cisplatin.5.Western blot showed that silencing of TTC9 A expression significantly down-regulated the expression levels of N-cadherin,Vinmentin,Snai1,Snai2,p38,p-p38,MDM2 and MRP1,and up-regulated the expression levels of E-cadherin and p53.Conclusion:TTC9A is significantly highly expressed in lung adenocarcinoma tissues and correlates not only with patient gender,lymph node metastasis and survival status,but also with poor prognosis and immune infiltration of lung adenocarcinoma.Silencing TTC9 A expression inhibits the proliferative capacity of lung adenocarcinoma cells,promotes apoptosis and prevents the G2 phase of the cell cycle from proceeding smoothly.TTC9 A is likely to be a novel molecular target for diagnosis,prognosis and treatment of lung adenocarcinoma. |
