Regulatory Mechanism Of APRIL And MRNA In Non-small Cell Lung Cancer(NSCLC)

Lung cancer is a common clinical malignancy.According to statistical data,the number of deaths due to lung cancer in China amounts to hundreds of thousands each year.The increasing incidence and high mortality rate make lung cancer get more and more attention.There are many ways to classify lung cancer,which are divided into two categories according to pathology.One is small cell lung cancer(SCLC),with neuroendocrine differentiation,whose treatment and prognosis are significantly different;The other type is non-small cell lung cancer(NSCLC),including adenocarcinoma,squamous cell carcinoma,and large cell carcinoma.Studies have confirmed that the mortality of non-small cell lung cancer is much higher than SCLC,more than 80%.In recent years,although NSCLC has made great progress in diagnosis and treatment,the 5-year survival rate of patients still does not exceed 15%.APRIL is a type II transmembrane protein transcribed from the chromosomal 17p13.1 gene.A new member of the tumor necrosis factor superfamily(TNFSF)ligand was first discovered and cloned by Hahne et al.in the late 1990 s.Except for some normal immune cells(such as B lymphocytes,dendritic cells,and macrophages,etc.),almost no APRIL expression is detected in human normal tissues,but many cancers such as liver cancer,colon cancer,leukemia,and lymphoma are present.Its expression in the organization is quite high.Now,it is clear that APRIL has two specific receptors,BCMA and TACI.APRIL binds to receptors in two forms: soluble and membrane-bound.After ligand and receptor binding,it regulates the body’s immune response and stimulates lymphocyte maturation and activation.In recent years,with the in-depth study of APRIL-BCMA-TACI,it has been found that this system can also promote the proliferation,migration,and proliferation of cancer cells and is highly expressed in NSCLC.At the same time,in the lung cancer cell line H1299 cells in vitro,APRIL can affect the transmembrane transduction of some signal pathways and the activation of some enzyme systems,resulting in obvious biological effects.Various studies have suggested that APRIL-BCMA-TACI system has a certain position in the overall layout of lung cancer,especially non-small cell lung cancer.ObjectivesThe main purpose of this study was to detect different expressions of APRIL and its receptor BCMA-TACI in different tissues and material locations(NSCLC cancer tissue,adjacent normal tissues,and peripheral blood),together with statistics and analysis of APRIL-BCMA-TACI.The correlation between the content of the system in different tissues and various clinicopathological parameters of NSCLC patients;In vitro experiments were performed to observe the effects of APRIL-BCMA-TACI system on the proliferation and metastasis of lung cancer cells and the transmembrane signal transduction pathways.To explore the specific mechanisms of APRIL and its receptor BCMA-TACI in NSCLC,in order to guide clinical treatment,And provide a theoretical basis for the development of new treatments for lung cancer.Methods(1)Immunohistochemical staining was used to detect the expression of APRIL and its related receptors TACI and BCMA in cancer tissues and paracancerous tissues in 80 NSCLC patients.(2)The H1299 and A549 lung cancer cell lines were knocked out of BCMA and TACI gene expression by shRNA interference technology to detect whether there was any change in the cell proliferation of the lung cancer cells in the absence of BCMA and TACI receptors during in vitro culture.The method used was CCK-8;(3)H1299 and A549 lung cancer cell lines were transfected with empty plasmids and BCMA-TACI-shRNA plasmids,respectively,and the changes in the growth and migration of these cells after transfection were detected by APRIL.The cell scratch method was used.(4)Real-time fluorescence quantitative PCR was used to detect the expression of APRIL,BCMA and TACI mRNA in peripheral blood of normal people and NSCLC patients.(5)H1299 and A549 lung cancer cell lines were selected and shRNA technology was used to inhibit the expression of BCMA and TACI genes.It was examined whether APRIL acted on A549 cells without receptors BCMA and TACI and control cells,whether it affected the phosphorylation of MAP kinase ERK1/2 signal.Methods using Western blot.Results(1)The results of immunostaining showed that APRIL protein was mainly stained in the cytoplasm of cells.The positive rate of 72.50%(58/80)of APRIL protein in 80 NSCLC tissues was significantly higher than that in adjacent normal tissues(11.25%,9/80).Statistical analysis of the two sets of data yielded P<0.05,was considered statistically significant.(2)There was a correlation between the expression of APRIL by immunohistochemical staining and the clinicopathological features of NSCLC: the gender,age and histological classification of NSCLC patients did not affect the expression of APRIL protein.Statistical results showed that P>0.05;but the tumor size,differentiation and Lymph node metastasis significantly affected the content of APRIL in different tissues(P<0.05).(3)The positive rate of TACI protein expression in NSCLC patients was significantly higher than that in adjacent normal tissues(P<0.05),and there was a statistically significant difference.As for the correlation analysis with clinicopathol ogical features,the age,gender,tumor size,and differentiation of NSCLC patients were found.The degree and lymph node metastasis did not affect the expression of TACI in different tissues(P>0.05).However,the histological classification of glands and squamous cell carcinoma signifycantly affected the expression of TACI.The statistical result was P<0.05.(4)The positive rate of BCMA protein expression in NSCLC patients was significa ntly higher than that in normal tissues adjacent to cancer(P<0.05).There was a statistically significant difference.The correlation analysis between clinical characteristics and clinical pathological features showed that the age,sex,tumor size,and histology of NSCLC patients.Classification and differentiation did not affect the expression of BCMA protein(P>0.05),but lymph node metastasis significantly affected the positive expression of BCMA protein(P<0.05).(5)In vitro experiments confirmed that the H1299 and A549 lung cancer cells treated with APRIL had scratches on the edges and scratches.The cell density increased and the clustering tendency increased significantly,but H1299 and A549 cells affected by shRNA transfection(BCMA and TACI genes.The expression was disturbed.The experimental results showed that the edge and center of the scratch were small in cell density,and the clustering and migration tendency was weak.(6)After the human lung cancer cell line H1299 and A549 cell chromosome receptor genes were artificially inhibited transcription and translation,the BCMA and TACI were no longer expressed in the cell structure;at this time,after APRIL cells were stained,real-time dynamic observation showed that The number and density of stained cells increased significantly.After 24 hours,the A549 cells first showed the above-mentioned conditions.After 48 hours,the number of stained cells in the H1299 cells was also decreased.After statistical analysis,the phenomenon of slower amplification of the two cells was statistically significant(P<0.05).(7)The results of real-time fluorescence quantitative PCR showed that the expression levels of APRIL mRNA in different groups were compared with each other,and there was a statistically significant increase in the value of peripheral blood test group in lung cancer patients(P<0.05).The relationship between different clinical features and pathological parameters and the level of APRIL mRNA in peripheral blood was analyzed.The results showed that the age,sex,pathological type,and pathological stage of NSCLC did not interfere with the change of APRIL mRNA content in blood(P>0.05).However,lymph node metastasis and different degree of tumor differentiation significantly increased APRIL mRNA content(P<0.05).The difference of BCMA mRNA content was not affected by the clinical features and pathological parameters of any NSCLC patients,but the content of TACI mRNA in the peripheral blood was significantly higher in the presence of lymph node metastasis and poorly differentiated tumors.This difference was statistically significant(P<0.05).(8)The transmembrane signal transduction of A549 cells mediated by MAPK and ERK1/2 was significantly increased after receiving APRIL.Compared with the negative control group,the statistical result obtained was P<0.05,indicating statistical significance.However,APRIL did not increase the phosphorylation of MAPK and ERK1/2 in the A549 cell line where BCMA and TACI gene expression was disturbed,resulting in P>0.05.Conclusions(1)The occurrence,development,invasion and metastasis of NSCLC tumors are related to the high expression of APRIL and its related receptors TACI and BCMA.(2)APRIL mRNA is highly expressed in the peripheral blood of patients with NSCLC,and is associated with some of the clinicopathological parameters of the tumor,suggesting that this index can be used to guide clinical diagnosis and prognosis.(3)Soluble APRIL and BCMA-TACI proteins can promote the growth,differentiation,and metastasis of lung cancer cells in vitro,suggesting that APRIL-TACIBCMA mechanism is involved in the development and progression of lung cancer,while activating MAP kinase and enhancing ERK1/2 Phosphorylation pathway may be one of the mechanisms for its molecular biological effects.(4)This experimental study provides a theoretical basis for the diagnosis and treatment of NSCLC for late multifactorial combined detection.Significance:The expression of APRIL and its receptors(TACI and BCMA)in non-small cell lung cancer tissue,peripheral blood and the cells in vitro is high,and the signaling pathway was involved in the occurrence and development of human lung cancer in some clinical pathological features;The expression levels of APRIL,BCMA and TACI and NSCLC in patients with significant correlation,suggesting that APRIL and its receptors BCMA and TACI may be involved in the occurrence,development and transfer process of NSCLC,which may become a new therapeutic target for NSCLC.