Expression And Molecular Mechanism Of Junction Adhesion Molecule Like Protein To Promote Tumor Progression In Lung Adenocarcinoma

BackgroundLung cancer mainly includes non small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Non small cell lung cancer accounts for 80%-85%of the total number of lung cancer.According to the pathological type,NSCLC can be divided into lung adenocarcinoma(LUAD),lung squamous cell carcinoma(LUSC)and large cell lung cancer,Among them,LUAD and LUSC are the two most important subtypes of NSCLC.It can be seen that lung adenocarcinoma is the type with the highest incidence rate in lung cancer.Although many progress has been made in early lung cancer screening,minimally invasive treatment technology,chemical therapy,molecular targeted therapy and immune checkpoint inhibitors,the 5-year survival rate of lung adenocarcinoma is still very low.This characteristic of high mortality and low survival rate is closely related to the continuous proliferation and anti-apoptosis of lung adenocarcinoma cells.Therefore,clarifying the important molecular mechanism of proliferation and antiapoptosis of lung adenocarcinoma,searching for new biological markers of lung adenocarcinoma and exploring related therapeutic targets play an extremely important role in the prevention and treatment of lung adenocarcinoma and improving the prognosis of patients.Junctional adhesion molecules(JAML)are a glycoprotein family,belonging to the immunoglobulin superfamily(IgSF).JAM-A,JAM-B,JAM-C,JAM-4 and JAML are the main family members of JAMs.JAMs proteins play an important role in many processes.In recent years,studies have found that JAMs also play an important role in tumorigenesis and development.JAM-A is the most widely studied protein.Several studies have shown that the increased expression of JAMA drives tumorigenesis and promotes metastasis by activating intracellular signaling pathways independent of adhesion.Similarly,there are also reports supporting the tumor inhibitory effect of JAM-A.JAM-A plays different functions in different tumors.Even in the same type of tumor,such as breast cancer,JAMA has also been reported to promote or inhibit cancer.Therefore,there is no clear consensus on the function of JAM-A in cancer.It can be seen that the functional mechanism of JAM-A is extremely complex.However,the research on JAML is not in-depth,especially the relationship between JAML and tumors,especially lung adenocarcinoma,is still unclear.JAML is a newly discovered member of the JAMs family.The important difference between JAML and other JAMs in the family is that its C-terminal lacks the traditional PDZ binding motif,which is crucial in the process of targeting intercellular connections.Compared with other members of JAMs,JAML has different possibilities in protein function and targeting mode due to the deletion of PDZ binding motif,that is,JAML may have diversified expression and may play a role in different diseases.For example,JAML was found to be highly expressed in human coronary atherosclerotic plaques and in aortic plaques of apolipoprotein E(ApoE)mouse models.Blocking JAML was found to reduce the degree of atherosclerosis in ApoE mice and enhance the stability of porridge like atherosclerotic plaques.In the case of acute inflammatory injury of intestinal epithelium,in the case of imbalance of neutrophil recruitment,neutrophils can continuously release JAML,further destroy the intestinal barrier and inhibit intestinal mucosal healing.In view of this situation,studies have found that targeting JAML can improve mucosal healing response.There are still many unknown functions that have not been discovered by people,which need more indepth experimental research to explore.The relationship between JAML and malignant tumors has attracted extensive attention of scholars.A recent study found that JAML is highly expressed in human gastric cancer tissue and promotes the development of gastric cancer,partly due to the activation of p38 mediated signaling pathway.In 2022,two bioinformatics analyses on the role of JAML in lung adenocarcinoma found that the low expression of JAML in lung adenocarcinoma was predicted from the database,but it was lack of specific experimental data support.In view of the existing research findings,JAMs,especially JAM-A,have different roles in different tumors,which may be tumor suppressive or cancer promoting.Moreover,JAML has more diversity in protein function and targeting mode due to the lack of PDZ binding motif.What is the expression level of JAML in patients with lung adenocarcinoma,the impact of JAML on cell proliferation and migration,whether JAML plays a role in the progression of lung adenocarcinoma,and what signal pathways may be involved,These problems have not been solved by research so far.1 Expression and clinical significance of JAML in lung adenocarcinomaObjective:To explore the expression of JAML in lung adenocarcinoma and the correlation between JAML expression and clinical characteristics of patients with lung adenocarcinoma.Methods:1.Collect the freshly resected cancer and the matched surgical specimens adjacent to the cancer in patients with lung adenocarcinoma,extract mRNA and protein respectively for RT-qPCR and Western blotting experiments,and analyze the expression difference of JAML in lung adenocarcinoma and adjacent tissues from the mRNA and protein levels.2.Collect the matched pathological sections of lung adenocarcinoma patients and adjacent tissues,detect the expression level of JAML protein by immunohistochemical staining,and locate the expression of JAML in lung cancer tissues at the same time;And collect the relevant clinical information of lung adenocarcinoma patients from the collected tissue sections,and analyze the correlation between the level of JAML protein expression and the clinicopathological characteristics of patients.3.In four lung adenocarcinoma cell lines,the expression location of JAML in lung adenocarcinoma cells was located again by cellular immunofluorescence technique.Results:1.In the freshly resected paired surgical specimens of 32 patients with lung adenocarcinoma,it was found that JAML expression was significantly increased in cancer tissues compared with adjacent tissues.2.Immunohistochemical staining in paired tissue sections of 126 patients with lung adenocarcinoma and adjacent tissues also confirmed that JAML expression was significantly higher in cancer tissues than in adjacent tissues,and JAML expression was localized in cytoplasm;High expression of JAML is associated with poor pTNM stages.3.Cell immunofluorescence assay also confirmed that the expression of JAML in lung adenocarcinoma cells was located in the cytoplasm.Conclusions:The expression of JAML in tumor tissues of patients with lung adenocarcinoma was significantly higher and related to the clinical characteristics of the progression of patients with lung adenocarcinoma;JAML expression was localized in the cytoplasm of lung adenocarcinoma tissues and lung adenocarcinoma cell lines.2 Effects of JAML on the proliferation,migration,invasion,cell cycle and apoptosis of four lung adenocarcinoma cell linesObjective:To explore the effects of JAML on the malignant biological behaviors of four LUAD cell lines in vitro.Methods:1.The expression level of endogenous JAML in A549,H1299,PC9 and H1975 lung adenocarcinoma cell lines was detected by Western blotting.2.Small interfering RNA was transiently transfected into A549 and H1299 cells for JAML knockdown,and plasmids were transfected into PC9 and H1975 cells for JAML overexpression.3.Using lentivirus to transfect lung adenocarcinoma cells,puromycin was screened,JAML knockdown and control group lentivirus(A549 shJAML,A549 shNC)were constructed against A549 cells,JAML overexpression and control group lentivirus(PC9 JAML OE,PC9 vector)were constructed against PC9 cells.4.At the cell level in vitro,the changes of cell proliferation after JAML knockdown or overexpression in four kinds of lung adenocarcinoma cells were detected by CCK8 assay,EdU assay as well as clone formation assay;cell migration and invasion ability were detected by cell scratch assay,transwell migration and invasion assay after JAML knockdown or overexpression in four kinds of lung adenocarcinoma cells.5.Flow cytometry was used to detect cell cycle and apoptosis after JAML knockdown or overexpression.Results:1.There are differences in the expression level of endogenous JAML in four lung adenocarcinoma cell lines.The expression of endogenous JAML is relatively high in A549 and H1299 cells,and relatively low in PC9 and H1975 cells.2.CCK8 assay and EdU assay confirmed that the application of small interfering RNA knockdown JAML in A549 and H1299 cells can inhibit the proliferation of cells,and the application of plasmid overexpression JAML in PC9 and H1975 cells can promote the proliferation of cells;Clone formation experiments using A549 cells transfected with lentivirus knockdown JAML and PC9 cells overexpressing JAML can also confirm that JAML knockdown can inhibit the proliferation of cells,and JAML overexpression can promote the proliferation of cells.3.Cell scratch assay,Transwell migration and invasion assay confirmed that the application of small interfering RNA knockdown JAML in A549 and H1299 cells can inhibit the migration and invasion of cells,and the application of plasmid overexpression JAML in PC9 and H1975 cells can promote the migration and invasion of cells.4.Flow cytometry showed that the application of small interfering RNA knockdown JAML in A549 and H1299 cells could cause cell cycle arrest in G0/G1 phase,and increase the proportion of apoptosis;The application of plasmid overexpression JAML in PC9 and H1975 cells can promote the proportion of S phase+G2/M phase cells,and reduce the proportion of apoptosis.Conclusions:JAML plays a role of oncogene in lung adenocarcinoma cell line.JAML knockdown can inhibit the malignant biological behavior in LUAD cells;JAML overexpression can promote the proliferation,migration and invasion of cells,increase S phase+G2/M phase cells,and inhibit apoptosis.3 Molecular mechanisms of JAML promoting lung adenocarcinoma progressionObjective:To explore the molecular mechanism of JAML in the biological processes of proliferation,invasion,metastasis and apoptosis of four lung adenocarcinoma cell lines.Methods:1.Western blotting was used to detect MMPs,apoptosis related proteins,EMT phenotype related proteins,Wnt/β-canetin and PI3K/AKT/mTOR signaling pathways after JAML knockdown or overexpression in four lung adenocarcinoma cells.2.Select Wnt/β-canetin activator or PI3K activator respectively to treat with A549 cells which was JAML knockdown.The changes of MMP2,MMP9 and EMT phenotype related proteins and related pathway proteins were detected by Western blotting.The changes of cell proliferation,migration and invasion after pathway function recovery were studied by EdU assay and Transwell migration and invasion assay.Results:1.Knockdown of JAML in A549 and H1299 cells can down-regulate the expression of MMP-2 and MMP-9,up-regulate of Bax,down-regulate of Bcl-2 and Survivin,and also cause the decrease of cyclin D1 expression closely related to cell cycle;Overexpression of JAML in PC9 and H1975 cells can up-regulate the expression of MMP-2 and MMP-9,inhibit Bax expression,promote Bcl-2 and Survivin expression,and also cause the increase of cyclin D1 expression.2.Knockdown of JAML in A549 and H1299 cells can promote EMT,and down-regulate expression of β-catenin in Wnt/β-catenin pathway and downstream cyclin D1 and Survivin;overexpression of JAML in PC9 and H1975 cells can inhibit EMT,and up-regulate expression of β-catenin in Wnt/β-catenin pathway and downstream cyclin D1 and survivin.3.In A549 and H1299 cells,knockdown of JAML could down-regulate the expression of PI3K,p-Akt and p-mTOR,while the expression of total Akt and total mTOR were not affected;In PC9 and H1975 cells.4.Wnt/β-catenin agonist CHIR99021(5μmol/ml,24h)was treatment with A549 cells when JAML knockdown,Western blotting confirmed that CHIR99021 could activate Wnt/β-Catenin signaling pathway can partially reverse the EMT phenotype changes caused by JAML knockdown,and partially reverse the decrease of MMP2 and MMP9 caused by JAML knockdown.EdU assay and Transwell migration and invasion assay found that CHIR99021 could partially restore the reduction of A549 cell proliferation,migration and invasion caused by JAML knockdown.5.PI3K agonist 740Y-P treatment(10μg/ml,24h)Western blotting confirmed that 740 Y-P could activate PI3K/AKT/mTOR signal transduction pathway and partially reverse the decrease of MMP2 and MMP9 caused by JAML knockdown.EdU assay and Transwell migration and invasion assay found that 740 Y-P could partially rescue JAML effects.Conclusions:JAML knockdown can down-regulate the expression of proliferation and apoptosis related proteins,inhibit EMT,and partial inactivated the Wnt/β-catenin and PI3K/AKT/mTOR pathways;JAML overexpression can up-regulate the expression of proliferation and apoptosis related proteins,promote EMT,and make Wnt/β-Catenin and PI3K/AKT/mTOR pathways are overactivated.The pathway recovery experiments confirmed that Wnt/β-Catenin pathways were partly involved in the effect of JAML on the proliferation,migration,invasion and EMT phenotype of lung adenocarcinoma cell lines,and the PI3K/AKT/mTOR pathway is partly involved in the effect of JAML on the proliferation,migration and invasion of lung adenocarcinoma cell lines.4 Effect of JAML on the growth of transplanted lung adenocarcinoma in nude miceObjective:To explore the effect of JAML on the proliferation and key proteins of Wnt/β-catenin pathway,PI3K/AKT/mTOR pathway in lung adenocarcinoma cells in vivo.Methods:1.BALB/c nude mice were randomly divided into four groups,with 6 in each group.Subcutaneously injected A549-shJAML,A549-shNC,PC9 JAML-OE,PC9-Vector cells to bliud tumor formation model.2.Use vernier caliper to measure the long diameter and short diameter of subcutaneous tumor twice a week,and draw the growth curve.3.On the 28th day,the nude mice were killed,the tumor tissue was stripped,and calculate the volume of the tumor.4.The expression of JAML and Ki-67 and key proteins of Wnt/β-catenin pathway,PI3K/AKT/mTOR pathway were evaluated by immunohistochemical staining.Results:1.After the down-regulation of JAML expression,the tumorigenesis time of transplanted tumor in nude mice was delayed,the growth was slow,suggesting that downregultion of JAML reduced the tumorigenicity of A549 cells in nude mice;JAML overexpression in nude mice resulted in shorter tumor formation time and rapid growth.The volume of the tumor was higher than that of the negative control group,suggesting that JAML overexpression enhanced the tumorigenicity of PC9 cells in nude mice.2.Further using immunohistochemical staining method,it was found that JAML in nude mouse tumor tissue decreased after JAML knockdown can cause positive rate of Ki67 decreased,suggesting that the proliferation of lung adenocarcinoma cells decreased after JAML knockdown;Compared with the negative control,the expression of JAML in nude mouse tumor tissue increased after JAML overexpression,and the positive rate of Ki67 increased,suggesting that the proliferation of JAML overexpressed lung adenocarcinoma cells was enhanced.Compared with the negative control,the expression of β-catenin、Cyclin D1 and Survivin in tumor tissue of nude mice decreased after JAML knockdown,and decreased expression of PI3K,p-AKT and p-mTOR suggested that the activity of Wnt/β-catenin pathway and PI3K/AKT/mTOR pathway were inhibited;JAML overexpression can activated Wnt/β-catenin pathway and PI3K/AKT/mTOR pathway.Conclusions:In vivo experiments confirmed that JAML knockdown could inhibit the growth of lung adenocarcinoma xenografts,and JAML overexpression could promote the growth of lung adenocarcinoma xenografts.It was also confirmed that JAML may play a cancer promoting role through Wnt/β-catenin pathway and PI3K/AKT/mTOR pathway in nude mice.Full Text Conclusion1.JAML is highly expressed in lung adenocarcinoma,and the highly expressed JAML is closely related to the clinical characteristics of lung adenocarcinoma patients.2.JAML knockdown can inhibit the malignant biological behavior of LUAD;JAML overexpression can promote the proliferation,migration and invasion of lung adenocarcinoma cells,affect the cell cycle,increase S phase+G2/M phase cells,and inhibit apoptosis.JAML plays an important role in regulating the malignant biological behavior of lung adenocarcinoma.3.JAML knockdown can down regulate the expression of proliferation and apoptosis related proteins,inhibit EMT,and partial inactivated Wnt/β-catenin and PI3K/AKT/mTOR pathways;JAML overexpression can up regulate the expression of proliferation and apoptosis related proteins,promote EMT,and overactivated Wnt/β-Catenin and PI3K/AKT/mTOR pathways.The pathway recovery experiments confirmed that Wnt/β-Catenin pathways were partly involved in the effect of JAML on the proliferation,migration,invasion and EMT phenotype of lung adenocarcinoma cell lines,and the PI3K/AKT/mTOR pathway is partly involved in the effect of JAML on the proliferation,migration and invasion of lung adenocarcinoma cell lines.4.In vivo experiments have confirmed that JAML knockdown can inhibit the growth of lung adenocarcinoma xenografts,and JAML overexpression can promote the growth of lung adenocarcinoma xenografts.It was also confirmed that JAML may play a cancer promoting role through Wnt/β-catenin pathway and PI3K/AKT/mTOR pathway in nude mice.5.JAML plays a role of oncogene in lung adenocarcinoma and is expected to become a new biomarker and effective therapeutic target of lung adenocarcinoma.