Effect Of MicroRNA-34a Targeting CSF-1R On Immune Microenvironment Of Non-Small Cell Lung Cancer And Its Mechanism

Objective: To clarify the effect of microRNA-34 a targeting CSF-1R on the immune microenvironment of NSCLC and to explore its possible mechanism,so as to provide a theoretical basis for looking for a new target of NSCLC immunotherapy.Methods: 1.MicroRNAs with matched binding sites to the mRNA3 ’-UTR of CSF-1R were predicted using the microRNA target gene prediction databases(Target Scan,Pic Tar,mi RDB,etc.).2.Collect NSCLC and normal lung tissues paraffin-embedded specimens,and culture human non-small cell lung cancer cells and human normal lungepith elial cells BEAS-2B.The expressions of CSF-1R,TAMs surface markers CD206 and microRNA-34 a in NSCLC were detected by immunohistochemistry,q RT-PCR and Western-blot.3.The constructed microRNA-34 a overexpressing lentivirus and negative control lentivirus were transfected into H1975 cells and divided into three groups:experimental group(transfection),negative control group(NC)and blank control group(Blank).After evaluating the transfection efficiency,the cells were co-cultured with PMA-induced macrophages for 48 hours.The morphological changes of macrophages in each group were observed under microscope.The expression of CD206 in macrophages after co-culture was detected by q RT-PCR.Then,the three groups of cells were respectively implanted subcutanically in nude mice to construct the xenograft tumor model in nude mice.Tumor growth was recorded and the growth curve was drawn.After tumor stripping,the volume and mass of the three groups of tumors were compared.The expression of CD206 and CSF-1R was detected by immunohistochemistry.Finally,dual luciferase reporter gene detection,q RT-PCR and Western-blot were used to verify the targeting relationship between microRNA-34 a and CSF-1R.4.Enrichment analysis of signaling pathways was performed through the KEGG Pathway public database to predict the downstream signaling pathway of CSF-1R,and Western-blot was used to detect the protein expression changes of this signaling pathway in each group of cells and xenografts.Results: 1.Bioinformation analysis and prediction showed that microRNA-34 a and CSF-1R m RNA 3’-UTR had matching binding sites,suggesting that CSF-1R might be the target gene of microRNA-34 a.2.Immunohistochemical results showed that the expression of CSF-1R in lung adenocarcinoma and squamous cell carcinoma was significantly higher than that in normal lung tissues,with significant differences between the two groups(P=0.000),while the expression of TAMs surface marker CD206 in NSCLC was also significantly higher than that in normal lung tissues,with significant differences between the two groups(P=0.000).Compared with normal lung epithelial cell BEAS-2B,CSF-1R was up-regulated in H1975,PC-9 and A549 cell lines,and down-regulated in H460 cell lines,and the expression difference between H1975 and BEAS-2B cells was the most significant,while microRNA-34 a was down-regulated in both NSCLC cell lines,with statistical significance(P < 0.05).Therefore,human lung adenocarcinoma cell line H1975 with low expression of microRNA-34 a and high expression of CSF-1R was selected for subsequent experiments.3.The overexpression of microRNA-34 a and negative control lentivirus were successfully transfected into H1975 cells;After the cells of the transfection group,NC group and Blank group were co-cultured with macrophages for 48 hours respectively,the morphology of macrophages in the three groups showed many obvious protrusion under the microscope,indicating that macrophages had gradually polarized to TAMs.The q RT-PCR results showed that the expression of CD206 in the transfection group was significantly lower than that in the NC group and the Blank group,with statistical significance(P < 0.05).Tumor growth curve of three groups of nude mice transplantation tumor model showed that Blank and NC group of nude mice transplantation tumor growth rate significantly faster than the transfection group,the difference is statistically significant(P < 0.05),after stripping tumors had found that transfection group of the size and the weight of tumors were significantly lower than Blank and NC groups,statistically significant difference(P < 0.05).Therefore,overexpression of microRNA-34 a inhibits tumor cell growth.Immunohistochemical staining results showed that the expression of CSF-1R and CD206 was strongly positive in the NC and Blank groups,and weakly positive in the transfection group.The results of dual luciferase reporter gene assay showed that compared with mi R-NC group,the relative luciferase activity of CSF-1R wild-type vector was significantly inhibited in mi R-34 a mimic group,and the difference was statistically significant(P=0.000),while there was no statistically significant difference in the co-transfection group with CSF-1R plasmid and mutant vector(P > 0.05).q RT-PCR and Western-blot results showed that the expression of CSF-1R in the transfection group was significantly lower than that in the NC and Blank groups,with statistical significance(P < 0.05).Therefore,overexpression of microRNA-34 a can inhibit CSF-1R and reduce TAMs expression in NSCLC immune microenvironment.4.Based on KEGG Pathway enrichment analysis and our previous studies,PI3K/AKT /m TOR may be the downstream signaling Pathway of CSF-1R;Western-blot analysis of the protein expression levels of PI3K/ AKT /m TOR signaling pathway in cells and transplanted tumors of the three groups showed that compared with the NC group and the Blank group,the protein expressions of PI3 K,AKT and m TOR in the transfection group were down-regulated,with statistical significance(P<0.05).Conclusion:1.MicroRNA-34 a can inhibit the expression of CSF-1R,reduce the amount of TAMs in the immune microenvironment of NSCLC,and thus delay tumor progression;2.The mechanism by which microRNA-34 a influences the immune microenvironment of NSCLC may be related to the PI3K/ AKT /m TOR signaling pathway.