The Mechanism Of MicroRNA-506-5p Targeting Opioid Growth Factor Receptor(OGFR) To Regulate Cell Cycle In Non-small Cell Lung Cancer(NSCLC)

Objective Lung cancer is the first malignant tumor with morbidity and mortality in China,accounting for 22.7% of all malignant tumor deaths,and it is still rising.Operation has been used to treat NSCLC at home and abroad,but the effect is still not ideal.At present,some research achievements have been made on the biological characteristics of the occurrence and development of Non-small cell lung cancer,but the specific mechanism remains unknow.miRNA-506-5p encoding gene is located on human chromosome Xq27.3,and it can bind to the 3’ untranslated region of downstream gene messenger RNA and regulate gene expression by changing the stability of messenger RNA,thereby affecting cell proliferation,apoptosis and other biological processes.OGFR pathway regulates cell proliferation mainly by delaying G1/S interface of cell cycle.This paper will explore whether miRNA-506-5p can target OGFR downregulation to affect tumor cell proliferation and lead to tumor metastasis.This study will be carried out from three aspects: in vitro cell experiment,in vivo animal experiment and clinical specimen detection.Methods 1.In vitro,dual luciferase reporter gene assay was used to verify whether miRNA-506-5p targeted OGFR,and the effect of simultaneous inhibition of miRNA-506-5p and OGFR expression on proliferation of NSCLC cells was analyzed.Human lung cancer cell line A549 was cultured,and the expression levels of miRNA-506-5p,OGFR,OGFR,P16,CDK4,CDK6,P-Rb and PCNA in each group were detected by q TR-PCR and Western blot.Cell cycle changes were detected by flow cytometry.2.In vivo experimental results showed that compared with the blank group,tumor growth was inhibited rapidly subcutaneously in nude mice with miRNA-506-5p overexpression group and miRNA-506-5p overexpression control group.HE staining results showed that miRNA-506-5p could promote the proliferation of lung cancer cells in the transplanted tumor tissue.Immunohistochemistry(IHC)was used to detect the high expression of PCNA in A549 cells transfected with Mir-506-5p mimic.Qrt-pcr and Western-blot results showed that the expression levels of miRNA-506-5p,CDK4,CDK6 and PCNA genes were increased,while OGFR and P16 were low.3.Clinical detection The expressions of miRNA-506-5p,OGFR and P16 in cancer tissues and adjacent tissues of patients undergoing radical resection of NSCLC in our department were detected by q PCR and Western blot.The pathological factors of specimens were analyzed to observe the correlation between Mir-506-5P and OGFR in non-small cell lung cancer tissues.Results In vitro experiment results showed that mir-506-5P negatively regulated OGFR expression in non-small cell lung cancer cells(P<0.001).Qrt-pcr and WB detection results showed that the expression levels of Mir-506-5P,CDK4,CDK6 and PCNA were increased in A549 cells,while OGFR,P16 and Rb were low.Cell cycle detection results in each group showed that mir-506-5p inhibitor group and Mir-506-5p +OGFR overexpression group had reduced cells in G0/G1 phase,G2/M phase and increased cells in S phase(P<0.001).2.In vivo experimental results showed that compared with the control group,tumor growth was inhibited rapidly subcutaneously in nude mice with Mir-506-5p overexpression group and mir-506-5p overexpression control group.HE staining results showed that miRNA-506-5p could promote the proliferation of lung cancer cells in the transplanted tumor tissue.Immunohistochemistry(IHC)was used to detect the high expression of PCNA in A549 cells transfected with miRNA-506-5p mimic.qRT-pcr and Western-blot results showed that the expression levels of miRNA-506-5p,CDK4,CDK6 and PCNA genes were increased,while OGFR and P16 were low.3.qRT-PCR and Western-blot detection of non-small cell lung cancer tissues and adjacent tissues showed that miRNA-506-5P expression was higher in the cancer tissues than that in the adjacent tissues,and OGFR expression was higher in the adjacent tissues,with statistically significant differences.There was no significant difference in the expression levels of miRNA-506-5p and OGFR in clinical pathological factors,and they were negatively correlated in NSCLC cancer tissues.Conclusions 1.In vivo and in vitro experimental results confirm that miRNA-506-5P can promote the proliferation of tumor cells in NSCLC tissues.2.miRNA-506-5p can down-regulate the expression level of OGFR in NSCLC,promote the expression level of proliferating nuclear antigen(PCNA)and cyclin-dependent kinase(CDK)and their mediated Rb protein products,and promote the progression of NSCLC.3.miRNA-506-5p was highly expressed in lung cancer tissues,while OGFR was low expressed in lung cancer tissues,and the two expressions were negatively correlated.Early detection of miRNA-506-5P and OGFR expression in lung cancer tissues may be helpful to judge the prognosis of patients,and is expected to become a new therapeutic target for non-small cell lung cancer.