Effect And Mechanism Of Progestin And AdipoQ Receptor 3 On The Growth Inhibition Of Non-small Cell Lung Cancer

Objective: In this study,in order to explore novel therapeutic targets to improve the quality of life and long-term prognosis for NSCLC patients,we analyzed the expression level of PAQR3 in NSCLC and its potential clinical value,and constructed a stable NSCLC cell model in which the expression of PAQR3 was upregulated or downregulated to explore its function and possible regulatory mechanism in the progression of NSCLC.Methods: 1.Clinical value analysis: The expression level of PAQR3 mRNA in NSCLC was analyzed in UALCAN database to observe its correlation with the clinicopathological features of NSCLC patients.The cancer tissues and the adjacent normal lung tissues of 60 NSCLC patients were collected from the Department of Thoracic surgery,affiliated Hospital of Zunyi Medical University,and the mRNA and protein expression levels of PAQR3 in the cancer tissues and adjacent normal lung tissues collected were detected by Real-time fluorescent quantitative PCR(qRT-PCR)and Western-blot(WB).In addition,the clinical information of the 60 patients was sorted out to explore the relationship between PAQR3 mRNA expression and the clinicopathological features of 60 NSCLC patients.Moreover,the correlation between the level of PAQR3 mRNA and the prognosis of NSCLC patients was analyzed in PrognoScan and Kaplan-Meier Plotter database.2.Function analyses in vitro: A549 and H1299 cells of NSCLC were selected and used to construct stable NSCLC cell strains of elevated and interfered PAQR3 through lentivirus transfection.Groups: Lentivirus overexpression control group(Vector)vs PAQR3up-regulated group(PAQR3-OV);Interfered control group(Si-NC)vs PAQR3 interfered group(Si-PAQR3).The transfection efficiency was detected under fluorescencemicroscope at 24 hours after lentivirus transfection.And the complete culture medium of RPMI-1640 without virus and puromycin would be used to continue culture if the transfection was done.Complete culture medium containing puromycin(A549 2 ?mol/L and H1299 4 ?mol/L)were used during cell passage to screen stable transfected cells.The expression level of PAQR3 in the transfected cells was detected by qRT-PCR and Wester-Blot to determine whether the model was established successfully.Finally,CCK-8,colony formation assay and Flow cytometry analyzer were used to detect the effects of PAQR3 in the proliferation,colony formation,cycle and apoptosis of A549 and H1299 cells.3.Mechanism research: The data of gene expression of lung cancer cells in the Cancer Cell Line Encyclopedia(CCLE)was download,and the regulatory mechanism through which PAQR3 may be involved in the progression of lung cancer was analyzed by Gene Set Enrichment Analysis(GSEA).And in the established stable models of NSCLC A549 and H1299,the level of NF-?B(p65),p53,p-p53 and Bax proteins were detected by Western-Blot.Results: 1.The PAQR3 mRNA expression level of NSCLC tissues was significantly increased in UALCAN database and the level was correlated with the subtype and lymph node metastasis for NSCLC patients(P < 0.05).However,the mRNA and protein expression levels of PAQR3 were significantly decreased in the 60 collected clinical NSCLC tissues.The expression level of PAQR3 mRNA was correlated with the tumor size of NSCLC patients(P < 0.05).Furthermore,the analysis in PrognoScan and Kaplan-Meier Plotter databases showed that the patients of LUAD with increased level of PAQR3 tended to have a better prognosis.2.The function analyses in vitro showed that,compared with the lentivirus positive control group(Vector),the increased PAQR3 could significantly inhibit the proliferation and clone formation,hinder the transformation of cell cycle from G1 to S phase,and induced apoptosis of NSCLC cells.On the contrary,interfering the expression of PAQR3 would significantly promote the proliferation,clone formation,transformation of cellcycle of NSCLC cells and reduce its apoptosis(P < 0.05).3.It was found that PAQR3 may participate in regulating cell cycle,DNA replication,p53 signaling pathway and other signal pathways in lung cancer through GSEA.And increased PAQR3 expression in A549 cells inhibited NF-?B(P65)protein expression and increased p53 protein,p53 phosphorylation and Bax protein expression.Increased PAQR3 expression in H1299 cells inhibited NF-?B expression and promoted Bax protein expression,but p53 protein and p53 phosphorylation expression were not affected.Inversely,interference with PAQR3 expression in A549 cells promoted NF-?B expression and inhibited p53 protein,p53 phosphorylation,and Bax protein expression.Interference with PAQR3 expression enhanced NF-?B protein expression and inhibited Bax protein expression in H1299 cells.Conclusions: 1.PAQR3 mRNA and protein expression were decreased in NSCLC tissues,and their expression levels were significantly correlated with tumor size in NSCLC patients.The LUAD patients with increased PAQR3 expression tended to have a better prognosis.2.It was found that PAQR3 could inhibit the growth of NSCLC cells through NF-?B/p53/Bax signal pathway.