Expression Of IL-35 In Non-small Cell Lung Cancer And Its Effect On The Biological Function Of Lung Cancer Cells

Objective:.Interleukin-35(IL-35)is a newly discovered member of the IL-12(Interleukin-12)family,a heterodimer composed of EBI3 and P35.IL-35 is highly expressed in various tumors such as liver cancer and pancreatic cancer and participates in the growth and metastasis of tumors,but studies in non-small cell lung cancer(NSCLC)are rarely reported.In this study,the biological function and molecular mechanism of IL-35 in lung cancer cells were studied by detecting the expression level of IL-35 in NSCLC tissues and cells.Methods: 1.Seventy-four cases of NSCLC tissue paraffin specimens and 25 paraffin specimens of benign lung lesions were collected for serial sectioning.The two subunits of IL-35(EBI3 and P35)were stained by immunohistochemistry to analyze the expression levels and the relevance of the two subunits in NSCLC.Case datas were collected to analyze the correlation between the expression of IL-35 and clinicopathological features of NSCLC.2.Peripheral blood of 21 patients with non-small cell lung cancer and 11 healthy subjects were collected.The expression of IL-35 in venous serum was detected by ELISA.3.Real-time quantitative PCR(q PCR)and ELISA were used to detect the secretion levels of IL-35 in lung cancer cell lines,such as A549,NCI-H1975,PC-9 and HEK293 who is the negtive control.4.The CCK-8 assay,flow cytometry,scratch test and transwell assay were used to detect the proliferation,apoptosis,migration and invasion of lung cancer cells after the stimulation of human recombinant protein IL-35 in A549 and NCI-H1975 cells.The expression of proliferation-related protein and apoptosis-related protein was examined by q PCR.Results: 1.The results of immunohistochemical serial section staining showed that the expression levels of EBI3 and P35 in NSCLC tissues were significantly higher than those in benign lung lesions.Furthermore,EBI3 and P35 were interrelated to a great extent in spatial distribution and expression level(P < 0.001).Then we defined the high expressions both EBI3 and P35 as IL-35 positive.In 74 cases of NSCLC,there were 42 cases with the expression of IL-35,accounting for 56.8%.The expression of IL-35 was positively correlated with tumor T stage,lymph node metastasis(P<0.05).There was no correlation with gender,age,histological type,differentiation degree and distant metastasis(P>0.05).These were indicated that IL-35 may be a potential marker of NSCLC.2.The results of ELISA showed that IL-35 in serum of patients with NSCLC was(464.64±352.88)pg/ml,which was significantly higher than that of healthy subjects who was(27.15±16.50)pg/ml(P<0.001).3.Q-PCR results showed that IL-35 subunits and its receptors were expressed in different degrees in three lung cancer cell lines.The ELISA results showed that the secretion levels of IL-35 in lung cancer cell lines were significantly higher than that in normal epithelial cells(P<0.05).4.IL-35 can significantly promote the proliferation and inhibit the apoptosis of lung cancer cells,as well as enhance the migration and invasion of lung cancer cells.Q-PCR confirmed that the expression levels of Bcl-2 and Cyclin D1 were up-regulated,and the expression level of Fas m RNA was down-regulated(P<0.05,Fig.7),while the expression of P53 who is a tumor suppressor gene was not observed with significant differences.Conclusion: IL-35 is highly expressed in NSCLC tissues,peripheral blood and cells,and it can promote the proliferation and migration of lung cancer cells and inhibit its apoptosis.IL-35 may play a important role in promoting cancer by regulating the expression levels of proliferation-related protein and apoptosis-related protein in cells.