Functions And Mechanism Of CADM4 In The Growth And Metastasis Of Non-small Cell Lung Cancer

PrefaceThe incidence rate of lung cancer is the highest in the world.And the incidence rate of non-small cell lung cancer(NSCLC)is about 85% of all lung cancer patients,it is the major type of lung cancer.Smoking,environmental pollution,diet,respiratory diseases,family inheritance,and female hormone levels are all listed as risk factors for lung cancer.The early symptoms of lung cancer are not obvious and easy to be ignored.About 70% of lung cancer patients are locally advanced or have metastasized at the point of diagnosis.At present,the early stage of clinical treatment of NSCLC is mainly radical surgery,supplemented by chemoradiotherapy and targeted therapy.And the late stage is mainly systemic therapy.Among which,the effect of traditional radiotherapy and chemotherapy is limited,and the clinical application of targeted therapy,anti-angiogenesis therapy and immune checkpoint inhibitors have greatly improved the prognosis of patients.According to the latest statistics in 2021,the 2-year relative survival rate of NSCLC patients is 42%.This is closely related to the exploration of clinical treatment and basic research of NSCLC.Therefore,it is very important to study the molecular mechanism of NSCLC.CADM4 is an immunoglobulin-like cell adhesion molecule.Several papers proved that CADM4 is down-regulated in various tumor tissues or tumor cells.What can be listed are prostate cancer,glioma,as well as colorectal cancer,and breast cancer.The overexpression of CADM4 can inhibit proliferation of colorectal cancer cells and promote apoptosis in vitro.The low expression of CADM4 in patients with renal carcinoma is closely related to vascular invasion,tumor size and metastasis,and it is also demonstrated the anti-tumor growth effect of CADM4 in vivo.CADM4 is also associated with poor prognosis in patients with colorectal adenocarcinoma.However,the effect of CADM4 in NSCLC's growth,metastasis and the mechanisms have not been reported.PI3K/Akt is confirmed to be involved in tumor cell growth,metastasis and invasion.Akt has been proved to be activated in many tumors,including NSCLC.However,whether CADM4 can regulate the Akt signaling pathway in NSCLC is not certain.Based on this situation,our subject first clarified whether CADM4 was abnormally expressed in NSCLC tissues.Subsequently,the level of CADM4 was changed in human NSCLC cell lines,its effect on growth and metastasis was observed.Akt signaling pathway agonists or inhibitors were used on cells.Whether the growth and metastasis of NSCLC mediated by CADM4 were reversed was studied.We clarified the mechanism of CADM4 regulating cell growth and metastasis in NSCLC.Finally,the expression of CADM4 in vivo was intervened to clear and definite the internal tumor growth and metastasis.This further verified the conclusion of the cell experiment.This study aims to identify a potentially valuable target for NSCLC.MethodsThis thesis includes three parts.First,the expression level of CADM4 was detected in 40 pairs of NSCLC and adjacent tissues collected clinically.Then,two NSCLC cells were transfected with CADM4 overexpression vector or si CADM4(we choose A549 and NCI-H1299).Each group proliferation ability of NSCLC was detected by CCK-8and the change of NSCLC cell cycle was observed through flow cytometry.CDK6,cyclin E1,cyclin D1 and p27 were then detected using western blot.Each NSCLC cell group migration ability was observed through wound healing assay.Each NSCLC cell group invasion ability was observed through transwell.The activity of each NSCLC cell group invasion-related proteins MMP-2 and MMP-9 were observed through gelatin zymography method.On the basis of overexpression or down-regulation of CADM4 in NSCLC,Akt agonist SC79 or inhibitor LY294002 was adding into the cells.Whether the effects of CADM4-mediated tumor cell behaviour were reversed after activating or blocking the AKT signaling pathway were detected though the above methods.Finally,the CADM4 stable overexpressing or knockout nude mice xenograft was established.Some related data such as tumor volume and weight were measured.The level of CADM4 in the tumor was detected by western blot,and the expression of ki67 was detected by immunohistochemistry.Tumor metastasis model in nude mice was established by tail vein injection of CADM4 stably overexpressed or knocked down cells.HE staining was used to observe the pathological changes in lung and liver.Results1.Expression of CADM4 in NSCLC clinical samples40 pairs tumor and corresponding adjacent tissues of NSCLC patients were collected to detect the expression of CADM4 at protein and m RNA levels.The results showed: compared with adjacent tissues,the level of CADM4 protein and m RNA expression in NSCLC tissues were significantly reduced,which was statistically significant.2.CADM4 regulates the growth and metastasis of NSCLC cells in vitroA549 and NCI-H1299 cells were transiently transfected with CADM4 overexpression vector or si CADM4.The experiment was divided into Vector group,CADM4 overexpression group,si NC group,si CADM4 group.Proliferation ability,cell cycle change,proliferation and cycle-related protein expression,migration and invasion ability of each group were detected.The results showed that compared with Vector group,the proliferation,migration and invasion ability,the proportion of cells in the S phase and G2 and expressions of CDK6,cyclin E1,cyclin D1,the activities of MMP-9and MMP-2 in the CADM4 overexpression group were significantly reduced,while the expression level of P27 was significantly increased.Compared with the si NC group,the various results of the si CADM4 group showed a completely opposite trend to the above changes.In addition,the same conclusions were obtained both in A549 and NCI-H1299 cell lines.3.The role of Akt signaling pathway in CADM4 regulating NSCLC cell growth and metastasisA549 and NCI-H1299 cells were transiently transfected with CADM4 overexpression vector or si CADM4.The activation state of the Akt signaling pathway,ie the ratio of p-Akt/Akt was detected.The experiment was divided into Vector group,CADM4 overexpression group,si NC group,si CADM4 group.Based on this result,it was determined that the Akt signaling pathway agonist SC79 and inhibitor act on the overexpression and knockdown of CADM4 cells,respectively.The experiment was conducted in two batches.First,the experiment was divided into CADM4+DMSO and CADM4+SC79 groups to detect the cell proliferation ability,cell cycle change,proliferation and cycle-related protein expression,migration and invasion ability of each group.Subsequently,the experiments were divided into si CADM4+DMSO and si CADM4+LY294002,and the same tests were carried out.The results showed:Overexpression of CADM4 inhibited the activation of Akt signaling pathway,and knockdown of CADM4 promoted the activation of Akt signaling pathway.Compared with CADM4+DMSO group,the proliferation,migration and invasion ability,the proportion of cells in the S phase and G2 and expressions of CDK6,cyclin E1,cyclin D1 in the CADM4+SC79 group were significantly increased,while the expression level of P27 was significantly reduced.Agonist SC79 blocks the inhibitory effect of CADM4 overexpression on cell proliferation,migration and invasion.Compared with si CADM4+DMSO group,the proliferation,migration and invasion ability,the proportion of cells in the S phase and G2 and expressions of CDK6,cyclin E1,cyclin D1 in the CADM4+SC79 group were significantly reduced,while the expression level of P27 was significantly increased.Inhibitor LY294002 reversed the promotion of cell proliferation,migration and invasion due to CADM4 knockdown.4.CADM4 regulates the growth and metastasis of NSCLC cells in vivoIn cell experiments,we constructed and screened A549 and NCI-H1299 cells that stably overexpress and knock down CADM4.The model of nude mice xenograft was established by subcutaneous injection of CADM4 stable overexpressing or knockout cells.Tumor volume and weight were measured and calculated.The level of CADM4 in the tumor and the expression of ki67 was detected.Tumor metastasis model in nude mice was established by tail vein injection of CADM4 stably overexpressed or knocked down cells.HE staining was used to observe the pathological changes in lung and liver.The experiment was divided into Vector group,CADM4 overexpression group,sh NC group,sh CADM4 group.The results showed that compared with the Vector group,the tumor volume and quality of the CADM4 group were significantly reduced.Ki67 was significantly reduced,and the number of metastatic nodules in liver and lung tissue was significantly reduced.While,the expression level of CADM4 was still significantly increased.Compared with the sh NC group,the tumor volume and quality of the sh CADM4 group were significantly increased.The expression of Ki67 and the number of metastatic nodules in liver and lung tissue was significantly increased.While,the expression level of CADM4 was still significantly reduced.Conclusions1?CADM4 expression is significantly reduced in human NSCLC clinical tissues.2?After overexpressing CADM4,the proliferation ability,migration and invasion ability of NSCLC cells were significantly reduced.And knocking down of CADM4,the proliferation ability,migration and invasion ability of cells were significantly improved.3?CADM4 can inhibit the activity of Akt signaling pathway.4 ? After using the Akt signaling pathway agonist SC79,the previous effects of overexpression of CADM4 on cell proliferation,migration,and invasion were all reversed.After using the Akt signaling pathway inhibitor LY294002,the effects of CADM4 knockdown on cell proliferation,migration and invasion were all reversed.5 ? CADM4 regulates the proliferation,migration and invasion of NSCLC cells through the Akt signaling pathway.6 ? Overexpression of CADM4 in vivo experiments significantly reduced the growth and metastatic ability of non-small cell carcinomas;after knocking down CADM4,the growth and metastatic ability of cells increased.