Study On The Axial Inhibition Of NK Cell Activity By MiR-422a/DNAM-1 To Promote The Progression Of Non-Small Cell Lung Cancer

Objective: Lung cancer is the most common malignant tumor in the world.Long-term survival rate of patients is poor.Natural killer cell(NK)is one of the most important cells in the body that mainly play the role of natural anti-tumor immune function.Recent studies have shown that mirco RNA can affect the anti-tumor effect of NK cells by regulating the expression of NK cells’ surfactant receptors and ligands.The purpose of this study was to explore the effect of miR-422 a on the surface receptor molecule DNAM-1 of NK cells,as well as the related mechanisms and the ultimate role of miRNA-422 a in the development and progression of lung cancer.Methods: First,the screening conditions were set and miRNAs closely related to non-small cell lung cancer(NSCLC)were screened using the GEO database.Potential targets of miRNA were predicted by the miRNA target gene prediction database,and miRNAs related to NK cell surface receptors were selected to determine the target miRNA.Peripheral blood PBMC was collected from 117 patients with NSCLC(confirmed by preoperative puncture and postoperative pathology).NK cells were isolated by magnetic bead sorting and the serum was frozen storage.Through matching,excluding the influence of age,gender and smoking history,30 pairs of patients with NSCLC and lung benign tumor were successfully matched.Primary NK cells(h NK)and NK92 cell lines were cultured in vitro at the same time.The expression of h NK,NK92 activator receptor DNAM-1 and mi422 a in different culture conditions were detected by q RT-PCR,flow cytometry and Western blot.The effect of miR-422 a on the expression of DNAM-1 and its action sites were examined by cell transfection and dual-luciferase reporter assay.NK92 cells were co-cultured with K562,A549 and H520 cells(target to effect ratio 20:1),respectively.Exogenous TGF-1,Kyn and culture medium were added,and then the effect of TGF-1 and Kyn on NK cells was detected by lactic dehydrogenase(LDH)release method.Results: 1.Six miRNAs were selected from the GEO database: miR-106 a,miR-200 b,miR-210,miR-422 a,miR-155 and miR-183.Using the miRNA target gene prediction databases found that miR-422 a could match the gene of the activated receptor DNAM-1(CD226)on NK cells.2.miR-422 a was highly expressed in tumor tissues and cells.The expression of miR-422 a in NSCLC was significantly higher than that in benign lung tumors.3.Both in vivo and in vitro experiments showed that miR-422 a could significantly reduce the expression of NK cell surface receptor DNAM-1 and inhibit the cytotoxicity of NK cells.Further experiments showed that TGF-β1,rather than L-kynurenine,significantly increased the expression of miR-422 a in NK cells,which inhibited the killing effect of NK cells on lung cancer cells by inhibiting the expression of DNAM-1 on the surface of NK cells.On the contrary,reducing the expression level of miR-422 a in NK cells could significantly increase the expression of DNAM-1 on the surface of NK cells,thus enhancing the killing effect of NK cells on cancer cells mediated by DNAM-1.Conclusion: TGF-β1 can inhibit the expression of DNAM-1 on NK cells by up-regulating miR-422 a in NSCLC patients.miR-422 a is expected to be an important target for the regulation of immune surveillance of lung cancer.