TMEM184A Promotes The Malignant Phenotypes Of Non-small Cell Lung Cancer Cells By Interacting With PI3Kp110α

Objective: Globally,the number of new cases and deaths from lung cancer is on the rise.Although we have learned more in recent years about the risk of malignant progression and the choice of immunotherapy,lung cancer is still one of the most lethal cancers.Non-small cell lung cancer(NSCLC)is the most common pathological type of lung cancer,accounting for about 80% of lung cancer.Some patients have metastatic tumors when diagnosed,resulting in a relatively low 5-year survival rate of lung cancer.Therefore,early diagnosis and early treatment is one of the most effective means to save the lives of patients with NSCLC.Effectively identify the potential prognosis of NSCLC biomarkers and targets for drug treatment,will provide effective in the treatment of patients with early diagnosis and accurate way.Transmembrane protein(TMEM)is a kind of proteins that exist across a membrane structure,studies have shown that its progress in malignant tumor play an important role in the process.Many transmembrane proteins were identified as potential prognostic biomarkers,prompting transmembrane protein family is an important group of tumor research.Transmembrane protein 184A(TMEM184A),also known as SDMG1,is located on human chromosome 7p22.3 with a length of 18587 bp and a molecular weight of45.8k Da.It is composed of 413 amino acids,including 7 transmembrane structures,a solute-trans-α domain and three phosphorylation sites at the C-terminal.TMEM184 A,which belongs to the TMEM184 family.In mammals,the TMEM184 family is highly conserved.Although it has been reported that TMEM184 B and TMEM184 C may be involved in tumor regulation,the specific molecular mechanism of their physiological functions remains unclear.Current studies have shown that TMEM184 A is involved in vesicle transport,and in the regulation of sex differentiation of germ cells and pancreatic development.TMEM184 A recently was identified as heparin receptor in vascular endothelial cells and vascular smooth muscle cells.Studies in vascular smooth muscle cell have shown that exogenous heparin specifically binds to TMEM184 A in a concentration-dependent manner,inducing the expression of dual specificity phosphatase 1(DUSP1).DUSP1 inactivates mitogen-activated protein kinase(MAPK)ERK1/2 and its downstream target Elk-1,eventually lead to a reduction in cell proliferation induced by growth factors.In heparin-treated endothelial cells,TMEM184 A acts as a heparin receptor,which can reduce the activity of JNK and p38 induced by TNFα and the phosphorylation of downstream targets,mediated the antiinflammatory response process of vascular endothelial cells.In vivo studies have shown that TMEM184 A is expressed in the vascular system of zebrafish and is necessary for the normal regeneration of blood vessels.Although TMEM184 A plays an important role in the process of vascular cell proliferation as a heparin receptor,the relationship between TMEM184 A and tumor development remains unclear.This study aims to determine the relationship between TMEM184 A and clinicopathological factors of NSCLC by detecting the protein expression level of TMEM184 A in fresh lung cancer tissues and NSCLC cell lines,and to clarify the effect of TMEM184 A on the proliferation and migration of NSCLC cells by biaxially regulating the expression of TMEM184 A,and to explore the possible mechanism of TMEM184A’s influence on the proliferation and migration of NSCLC cells.Methods: UALCAN database,immunohistochemistry and western blotting were used to predict and detect the expression level of TMEM184 A in NSCLC tissues,and to analyze the relationship between TMEM184 A and clinicopathological factors of patients.The expression of TMEM184 A protein was regulated by transfection of TMEM184 A overexpressed plasmid and specific small interfering RNA.Then,the effect of TMEM184 A on the proliferation ability of NSCLC cells was evaluated by MTS assay and cell clone formation assay,and the changes of cell cycle and apoptosis were analyzed by flow cytometry.Transwell migration assay and cell scratch assay were used to detect the altered migration ability of NSCLC cells.Immunofluorescence co-localization assay and immunoprecipitation assay were used to detect proteins that could interact with TMEM184 A.Proteins that related to cell proliferation,apoptosis and migration and the key proteins of PI3K/Akt pathway were detected by western blotting assay,and recovery experiments were performed by using pathway inhibitors and small interfering RNA.Results:1.TMEM184 A is highly expressed in NSCLC and is associated with adverse clinicopathological factors.UALCAN database analysis showed that the expression of TMEM184 A was higher in lung adenocarcinoma and lung squamous cell carcinoma than in normal lung tissue.The results of CCLE database search showed that in 1457 cell lines of 40 tumor types,the expression level of TMEM184 A m RNA in lung cancer cell lines was higher than that in other tumor cell lines.Real-Time quantitative PCR results confirmed that the expression level of TMEM184 A m RNA in six NSCLC cell lines was higher than that in normal bronchial epithelial cells.Western blotting analysis showed that the expression of TMEM184 A protein in 14 pairs of fresh NSCLC was higher than that in the corresponding paracancerous normal tissues.And the expression level of TMEM184 A protein in six NSCLC cell lines was higher than that in normal bronchial epithelial cells.Immunohistochemical staining analysis of 207 NSCLC specimens showed that TMEM184 A was mostly negative in normal bronchial epithelial and alveolar epithelial cells(the positive rate is only 15.5%,31/200),but the positive expression rate was higher in lung adenocarcinoma(66.2%)and lung squamous cell carcinoma(68.38%).The expression of TMEM184 A was positively correlated with P-TNM stage(P=0.029),tumor size(P=0.014)and positive lymph node metastasis(P= 0.039)of NSCLC.2.TMEM184 A promotes the proliferation of NSCLC cells by affecting cell cycle and inhibiting apoptosis.The expression of TMEM184 A was bidirectional regulated in A549 and H1299 cells.Flow cytometry results showed that TMEM184 A promoted the cell cycle progression of NSCLC by regulating the G2/M phase of cell cycle.Western blotting results showed that TMEM184 A promote cell proliferation by adjusting the expression of cycle-related protein Cyclin B1,CDC2,p27.Flow cytometry and western blotting confirmed that TMEM184 A inhibits apoptosis of NSCLC cells by regulating expression of Bcl2,Bax,Cleaved caspase-3 and Cleaved caspase-9.3.TMEM184 A induces the occurrence of EMT and promotes NSCLC cells migration.In A549 and H1299 cells that biaxially regulated the expression of TMEM184 A,Transwell migration assay and western blotting assay showed that TMEM184 A could regulate the expression of EMT key proteins E-cadherin,N-cadherin,ZO1,Vimentin and transcription factor Snail,Slug,ZEB1 and promote the migration of NSCLC cells by regulating Rho A and Rho C.4.TMEM184 A affects the proliferation and migration of NSCLC cells by promoting PI3K/Akt signaling pathway.In A549 cells transfected with TMEM184 A plasmid,the key proteins PI3Kp110α and p-Akt(ser473)of PI3K/Akt signaling pathway changed significantly.By adding LY294002(an inhibitor of the PI3K/Akt signaling pathway)into TMEM184 A transfected cells,the promotion effect of TMEM184 A on cell proliferation and migration was reversed and consistent results were observed in H1299 cells.5.TMEM184 A interacts with PI3Kp110α to regulate the PI3K/Akt pathway.Immunoprecipitation assay and immunofluorescence co-localization assay confirmed the interaction between TMEM184 A and PI3Kp110α and their co-localization in the cytoplasm of NSCLC cells.Transfection of specific si-PI3Kp110α can reverse the changes of PI3K/Akt pathway key proteins and downstream proliferation and migration related proteins induced by up-regulation of TMEM184 A,thus reversing the promotion effect of TMEM184 A on proliferation and migration of NSCLC cells.Conclusion:1.TMEM184 A is highly expressed in NSCLC and is positively correlated with adverse clinicopathological features in NSCLC patients.2.TMEM184 A promotes the proliferation of NSCLC cells by affecting cell cycle,inhibiting apoptosis and inducing EMT of NSCLC cells.3.TMEM184 A activates PI3K/Akt pathway via PI3Kp110α thereby promoting the migration and proliferation potential of NSCLC cells.