Clinical Significance And Functional Study Of CPA4 Expression In Lung Cancer

Objective:The incidence and mortality of lung cancer rank first in malignant tumors.Lymph node metastasis is one of the common metastases of lung cancer,and it is also an important indicator of lung cancer treatment strategy and prognosis.Some studies have found that carboxypeptidase plays an important role in tumor metastasis.The purpose of this study was to investigate the biological function of CPA4 in lung cancer cells,the expression of CPA4 in serum of lung cancer patients,lung cancer tissues and their paired tissues of lymph node metastasis and its clinical significance,as well as the mechanism of abnormally high expression of CPA4 in lung cancer.Contents:The expression of CPA4 gene in lung cancer tissues was analyzed in the Oncomine database.Furthermore,Immunohistochemistry and ELISA were used to detect the expression of CPA4 in lung cancer tissues and serum samples of lung cancer patients and and its clinical significance.We also studied the effects of CPA4 on invasion and migration in vivo and in vitro.The relationship between the abnormal high expression of CPA4 in lung cancer with DNA methylation,histone acetylation and EGF expression were also studied.Methods:The expression of CPA4 and EGF in lung cancer tissues and the correlation with lung cancer metastasis were analyzed by Oncomine database.Genecards database predicted the cell localization of CPA4.The subcellular localization of CPA4 was detected by cellular immunofluorescence.Expression level of CPA4 in lung cancer cells was detected by Real time PCR and Western blot.CCK-8,Transwell and serum-free balling test were used to detect the effects of CPA4 on cell proliferation,invasion,migration,and self-renewal ability.The nude mice were used to detect the effect of CPA4 on tumorigenesis and invasion in vivo.And we detected the effects of deacetylase inhibitor(TSA),DNA methylase inhibitor(DAC)and epidermal growth factor(EGF)on the expression of CPA4 in cells,detected the expression of CPA4 in tissues by immunohistochemistry;while ELISA was used to detect the levels of CPA4 in serum.The statistical analysis was used to analyze CPA4 expression in lung cancer tissues and serum of lung cancer patients,and the correlation between CPA4 expression and clinicopathological features of lung cancer patientsResults:Oncomine database showed that CPA4 was highly expressed in lung cancer tissues with lymph node metastasis or distal metastasis.Western blot analysis showed that CPA4 is an exocrine protein.The results of immunofluorescence showed that CPA4 is also localized in the cytoplasm and nucleus of H226 and SPCA-1 cells.Functional experiments showed CPA4 knockdown significantly inhibited the invasion of H226 and SK-MES-1 cells,with inhibition rates of 62.34%and 77.3%,respectively,and significantly inhibited migration,with inhibition rates of 48.09%and 42.4%,but had no effect on cell proliferation.Overexpression of CPA4 significantly promoted the invasion,migration and self-renewal of H1299 cell lines,increased by 50.50%,58.11%and 75.80%respectively,but had no effect on cell proliferation.Animal experiments showed that CPA4 could promote the invasive ability of H1299 cells to surrounding tissues in vivo,but had no effect on the size of tumors in vivo.The results showed that the higher TSA concentration,the higher CPA4 expression;the higher DAC concentration,the higher CPA4 expression;the higher EGF concentration,the lower CPA4 expression.Immunohistochemical results showed that the positive rates of CPA4 were 12.5%(1/8),50%(23/46)and 76.1%(35/46)in normal lung tissues,primary lung cancer tissues and matched lymph node metastasis tissues,and the expression of CPA4 was significantly increased in turn.Further analysis showed that the expression of CPA4 was closely related to the pathological type,differentiation degree and tumor stage of lung cancer,but not related to the patients gender,age and depth of invasion.Serum CPA4 levels in 90 patients with lung cancer showed that serum CPA4 level in patients with lymph node metastasis(2882.89±875.57pg/ml)was significantly higher than that in patients without lymph node metastasis(2166.47±845.88pg/ml).Conclusion:CPA4 can significantly promote the invasion,migration and self-renewal of human lung cancer cells in vitro,and the invasive ability in vivo.The levels of CPA4 in primary lung cancer tissues was significantly higher than that of lymph node metastatic tissues.Serum CPA4 levels in lung cancer patients with lymph node metastasis were significantly higher than those without lymph node metastasis.CPA4 might be a potential marker to detect lymph node metastasis and a novel target for inhibiting lymph node metastasis of lung cancer.