Expression Of Interferon Induced Transmembrane Protein 3 In Lung Adenocarcinoma And Regulates The Growth And Invasion Of Lung Adenocarcinoma Cell

BackgroundCancer is the most common cause of deaths worldwide.Among all,lung cancer has the highest mortality,which could be attributed to the uncontrolled proliferation and early metastasis of cancer cells in the lung tissue.At present,long-term exposure to tobacco smoke,genetic factors,and air pollution are the main causes of lung cancer.Many studies have shown that lung cancer has obvious polymorphism and genetic heterogeneity exerted via different genetic pathways,molecular expression profiles,and therapeutic responses.Several genes and proteins have been identified to play a major role in the complex signaling pathways of carcinogenesis.However,the exact mechanism of lung cancer is yet to be elucidated.Interferon induced transmembrane protein(IFITM gene family was first discovered in the cDNA of IFN-induced neuroblastoma in 1984.To date,five IFITMs(Ifitm1,Iftm2,Ifitm3,Ifitm5,and Ifitm10)subgenes have been identified in humans,and all are located on chromosome 11.The members of the IFITM protein family are composed of about 130 amino acids.There are two transmembrane protein regions intercalated in a conserved cytoplasmic region.Previous studies have shown that these regions belong to a family of mouse genes,consisting of two short transmembrane domain proteins with high similarity but different core sequences of N-terminal and C-terminal.Human homologous genes(Ifitml,Ifitm2,and Iiftm3)are clustered in the 18 kbp genome sequence of chromosome 11.The main functions of the IFITM family are regulation of immune function,inhibition of virus synthesis or replication,and regulation of osteoblast bone mineralization.IFITM protein is also considered to play a role in cell cycle control and apoptosis.IFITM3,also known as 1-8U,was first isolated from colon cancer and ulcerative colitis patients.Previous studies have confirmed that IFITM3 can be used as the preferred marker of ulcerative colitis-related colon cancer.The expression of IFITM3 is positively correlated with the metastasis and prognosis of colorectal cancer.The overexpression of IFITM3 in head and neck squamous cell carcinoma indicates poor prognosis of patients.On the other hand,the expression level of IFITM3 in astrocytoma cells is higher than that in normal mouse astrocytes,and the upregulation of IFITM3 affects the proliferation and invasion of glioma,which is considered as a key factor in the occurrence and invasion of glioma.In addition,IFITM3 plays a critical role in the invasion and bone metastasis of prostate cancer patients.Ifitm3 gene knockout inhibits tumor cell proliferation,induces cell cycle arrest,senescence,and apoptosis.IFITM3 regulates the migration,invasion,proliferation,and apoptosis of HCC cells and plays a key role in the occurrence and progression of HCC,deeming it to be a new potential target for the treatment of HCC.The overexpression of IFITM3 indicates poor prognosis in patients with stage IIA esophageal squamous cell carcinoma.Ifitm3 gene is a predictor of postoperative lymph node metastasis and recurrence in esophageal squamous cell carcinoma.The overexpression of IFITM3 was related to the degree of tumor differentiation,lymph node metastasis,and distant metastasis,while the downregulation of the expression significantly inhibited the migration,invasion,and proliferation of tumor cells.IFITM3 protein was highly expressed in invasive breast cancer and significantly related to estrogen receptor and progesterone receptor status.Silencing the Ifitm3 gene significantly reduced the mRNA and protein expression of IFITM3 in breast cancer cells.The expression level of the molecule inhibited the growth and invasion of tumor cells.IFITM3 may be a key factor in tumorigenesis and crucial in regulating tumor cell migration andinvasion.Interestingly,IFITM3 regulates the invasion and development of tumor cells in solid tumors.However,the specific function and potential mechanism of IFITM3 in the pathogenesis of lung cancer are yet unclear.In this study,Ifitm3 gene was used as the breakthrough point,and the level of IFITM3 protein in lung adenocarcinoma tissues was detected to analyze the correlation between the expression of IFITM3 in lung adenocarcinoma tissues and the clinicopathological characteristics of lung adenocarcinoma patients.Thus,we silenced and overexpressed the Ifitm3 gene in lung adenocarcinoma cells by lentivirus-mediated gene silencing and overexpression techniques,and observed the effects of IFITM3 protein on the growth,proliferation,and metastasis of lung adenocarcinoma cells.Hence,we aimed to investigate the regulation of migration and invasion and the tumorigenicity of nude mice in vitro.ObjectiveTo determine the role of IFITM3 in the carcinogenesis of lung adenocarcinoma,analyze the expression of IFITM3 protein in lung adenocarcinoma,observe the effects of silencing and overexpression of IFITM3 on the growth,migration,invasion,apoptosis,and the cell cycle distribution of lung adenocarcinoma cells,and the tumorigenesis of nude mice,explore the mechanism of IFITM3 on the proliferation and invasion of lung adenocarcinoma,and lay a foundation for finding new biological markers and treatments.Methods1.Expression of IFITM3 in lung adenocarcinomaLung adenocarcinoma tissues were collected,and the expression of IFITM3 in lung adenocarcinoma was detected by immunohistochemical staining.Also,the expression of IFITM3 in lung adenocarcinoma and its clinical significance were analyzed.2.mRNA and protein expression levels of IFITM3 in 16HBE,H1299,H1395,H1437,H1975,and A549 cells were detected by qRT-PCRThe highly expressed cell lines were selected,and the IFITM3-specific shRNA expression vector was constructed by lentivirus-mediated gene silencing technique.The Ifitm2-specific over expression vector was constructed by lentivirus-mediated gene overexpression and transfected into lung adenocarcinoma cell line with low expression of IFITM3.The silencing efficiency of the gene after shRNA and oeRNA lentivirus infection was detected by qRT-PCR and Western blot,respectively.3.Effect of silencing and overexpression of Ifitm3 gene on cell proliferationThe clone formation ability of lung cancer cells with silencing and overexpression of Ifitm3 gene was detected by soft agar culture clone formation test.4.Flow cytometry was used to detect the effects of silencing and overexpression of Ifitm3 gene on the distribution of lung cancer cell cycleWestern blot was used to detect the effect of silencing and overexpression of Ifitm3 gene on the cyclin of lung cancer cells.Flow cytometry was used to analyze the early apoptosis of lung cancer cells after silencing and overexpression of the gene.5.Effects of silencing and overexpression of Ifitm3 gene on the invasion and migration of lung cancer cellsThe effects of silencing and overexpression of Ifitm3 gene on the invasion and migration of lung cancer cells were detected by Transwell invasion and migration assays.6.Effects of silencing and overexpressing Ifitm3 gene on tumorigenesis in nude miceH1299 cells with silenced Ifitm3,A549 cells with overexpressed IFITM3 and control cells were inoculated into the left axilla of nude mice to observe the tumor,measure the tumor volume,and kill the nude mice on the 30th day after inoculation.7.Effects of silencing and overexpressing IFITM3 gene on the expression of matrix metalloproteinases(MMPs)and epithelial-stromal transformation in lung cancer cells were detected by Western blotThe expression of MMP-2 and MMP-9 after silencing and overexpressing Ifitm3 gene was detected by Western blot,and the effects of silencing and overexpressing Ifitm3 gene on the expression of epithelial mesenchymal transition(EMT)markers Vimentin,Snail,and E-cadherin were detected by Western blot.8.Western blot was used to detect the role of the p38-MAPK signaling pathway in IFITM3-induced EMTWestern blot was used to detect the expression of phosphorylated p38,Vimentin,Snail,and E-cadherin in silenced IFITM3 genome after adding p38-MAPK pathway activator TNF-? and in overexpressed IFITM3 genome after adding SB203580,a specific inhibitor of the p38-MAPK signaling pathway.Results1.Expression of IFITM3 protein in human lung adenocarcinoma and its clinicalsignificance.The results showed that the expression of Ifitm3 gene was positive in 41/50(82.00%)cases of lung adenocarcinoma.The expression of IFITM3 in patients with TNM stage(?+? stage)and with lymph node metastasis was higher than that in patients with TNM stage(?+? stage)and without lymph node metastasis,respectively.Thus,the expression of IFITM3 in lung cancer was related to lymph node metastasis and TNM stage.2.Expression and transfection efficiency of IFITM3 in lung adenocarcinoma cells.The mRNA expression of Ifitmi in 16HBE,H1299,H1395,H1437,H1975,and A549 cells was detected by RT-qPCR,and the protein expression of IFITM3 in each cell line was detected by Western blot.The results showed that only a small amount of IFITM was expressed in human bronchial epithelioid cells 16HBE,and the expression was higher in H1299 and H1975 cells as compared to that in H1395 and A549 cells.Therefore,H1299 and H1975 cells were selected for gene silencing,and H1395 and A549 cells were selected for gene overexpression for subsequent studies.The transfection efficiency was detected by RT-qPCR and Western blot.The expression of IFITM3 mRNA and protein was significantly inhibited after lv-shIFITM3 transfection into H1299 and H1975 cells.The expression of Ifitm3 mRNA and protein increased significantly after lv-oeIFITM3 transfection into H1395 and A549 cells.3.Effects of silencing and overexpression of Ifitm3 gene on cell proliferation.The soft agar culture showed that the clone formation ability of H1299 and H1 975 cells decreased significantly after silencing the Ifirm3 gene,indicating that the growth of tumor cells was inhibited.On the other hand,the clone formation ability of H1 395 and A549 cells was significantly enhanced after overexpression of the gene,suggesting that overexpression of IFITM3 promotes the growth of tumor cells.4.Effects of silencing and overexpression of Ifitm3 gene on cell cycle distribution,cyclin,and early apoptosis of lung cancer cells.The results of flow cytometry showed that the number of cells in the G0/G1 phase increased significantly with the downregulation of IFITM3,while that decreased in the G2,M,and S phases.Furthermore,the number of cells in the G0/G1 phase decreased with the downregulation of G0/G1 phase.Silencing the expression of IFITM3 arrested the H1299 and H1975 cells in G0/G1 phase,thereby inhibiting the proliferation of tumor cells,while the overexpression of IFITM3,decreased the number of cells in G0/G1 phase significantly,and the number of cells in the G2,M,and S phases increased.The overexpression of IFITM3 promoted the proliferation of H1 395 and A549 cells.The results of Western blot assay showed that silencing the expression of IFITM3 decreased the expression of CDK4 and cyclinDl and increased the expression of P21 in H1299 and H1975 cells,while the overexpression of IFITM3 increased the expression of CDK4 and cyclinDl and decreased the expression of P21 in H1395 and A549 cells.Early apoptosis of lung cancer cells was detected by flow cytometry using Annexin V-FITC and PI staining.The results showed that silencing IFITM3 promoted early apoptosis in H1299 and H1975 cells,while the overexpression of the protein inhibited early apoptosis in H1395 and A549 cells.5.Effects of silencing and overexpression of Ifitm3 gene on the invasion and migration of lung cancer cells.The results of Transwell invasion assay showed that the invasive ability of H1299 and H1975 cells decreased significantly after silencing the Ifitm3 gene,while that of HI 395 and A549 cells was significantly enhanced after overexpression of the gene.The results of Transwell migration assay showed that the migration ability of H1299 and H1975 cells decreased significantly after silencing the gene,and that of H1395 and A549 cells was significantly enhanced after the overexpression of the gene.6.Effects of silencing and overexpression of Ifitm3 gene on tumorigenesis in nude mice.Silencing the Ifitm3 gene inhibited the tumorigenesis ability and the overexpression enhanced the tumorigenesis ability of H1299 cells and A549 cells,respectively,in nude mice.7.Effect of silencing Ifitm3 gene on the expression of MMPs and the interstitial transformation of epithelial cells.Western blot detected the expression of MMP-2 and MMP-9 and showed that silencing the expression of Ifitm3 reduced the expression of both MMPs in H1299 and H1975 cells,while the overexpression elevated the expression of the MMPs in H1395 and A549 cells.Furthermore,Western blot revealed that silencing IFITM3 reduced the expression of Vimentin and Snail and increased the expression of E-cadherin in H1395 and H1975 cells,while the overexpression of IFITM3 increased the expression of Vimentin and Snail and decreased the expression of E-cadherin in H1395 and A549 cells.8.Role of p38-MAPK signaling pathway detected by Western blot in EMT induced by IFITM3.The results of Western blot assay showed that the expression of non-phosphorylated p38 altered significantly after silencing the Ifitm3 gene,and the expression of phosphorylated p38 decreased significantly.When TNF-? was added to H1299 and H1975 cells,the expression of E-cadherin decreased and that of phosphorylated p38,Vimentin,and Snail increased after the activation of p38-MAPK pathway.The expression of non-phosphorylated p38 in overexpressed IFITM3 showed no significant change,but the expression of phosphorylated p38 decreased significantly The addition of SB203580 specifically inhibited the p38-MAPK signaling pathway in H1395 and A549 cells.The expression of E-cadherin increased,while the expression of phosphorylated p38,Vimentin,and Snail decreased significantly.These results suggested that IFITM3 can induce the occurrence of EMT through the p38-MAPK pathway.Conclusions1.Ifitm3 gene is highly expressed in lung adenocarcinoma,which is closely related to lymph node metastasis and TNM stage.2.After silencing the Ifitm3 gene,the proliferation ability of lung cancer cells decreased and apoptosis was promoted.After overexpression of Ifitm3 gene,the proliferation ability of lung cancer cells increased and apoptosis decreased.3.Silencing of Ifitm3 gene significantly reduces the invasion and metastasis ability of lung cancer cells,while overexpression enhances the invasion and metastasis ability of lung cancer cells.4.Silencing Ifitm3 gene inhibits the tumorigenicity of lung cancer cells in nude mice,and the overexpression of the gene enhances the tumorigenesis ability of lung cancer cells in nude mice.5.Silencing Ifitm3 gene decreases the expression of MMP-2 and MMP-9 proteins.IFITM3 induces interstitial epithelial transformation through p38-MAPK signaling pathway,thus promoting the invasion and metastasis of lung cancer cells.These results suggested that IFITM3 plays a critical role in the proliferation,invasion,and metastasis of lung adenocarcinoma cells.The expression of IFITM3 is also related to the development of lung adenocarcinoma.Thus,IFITM3 seems a promising target for the treatment of lung adenocarcinoma.