The Role And Mechanism Of NRK In Cancer-associated Fibroblasts Promoted Migration And Invasion Of Lung Adenocarcinoma

Objective:Cancer-associated fibroblasts(CAFs)are the major components of Tumor microenvironment(TME).Targeting CAFs is a promising therapeutic strategy for cancer treatment.According to RNA-seq,integrated bioinformatics analysis,cells and clinical model,we identified that NRK was highly expressed in CAFs.Therefore,we further explored the role and mechanism of NRK in CAFs promoted proliferation,migration and invasion of Lung adenocarcinoma(LUAD).Our study will provide a promising therapeutic target for cancer treatment.Methods:1.The primary of CAFs and Normal Fibroblasts(NFs)were isolated from LUAD tissue and adjacent normal tissue,respectively.Western Blot(WB)and Immunofluorescence(IF)were performed to verify the markers of fibroblasts.2.CAFs and NFs were cultured with serum-free DMEM medium for 72 h.Then the medium was collected and centrifuged to remove cell debris.The supernatant was CAFs conditional medium(CAF-CM)or NFs conditional medium(NF-CM).Explore the effects of CAF-CM and NF-CM on the proliferation,invasion and migration of LUAD cells(A549 and H1299).3.According to RNA-seq data,our previously reported Lnc RNA microarray and public datasets from Array Express.Differentially expressed genes(DEGs)with|fold change| > 2 and adjusted P-value < 0.05 were selected as the overlaying DEGs.q RT-PCR was used to detect the relative m RNA expression levels of the overlaying DEGs.Besides,q RT-PCR,WB and IF were used to detect the expression of NRK in CAFs.4.The expression levels of NRK in LUAD tissues and adjacent normal tissues were detected by Immunohistochemistry(IHC).And the correlation between NRK and clinical pathology of LUAD patients were further explored.5.Overexpression plasmid and interference lentivirus vector of NRK were constructed,separately.NFs were transfected with overexpression plasmid of NRK,while CAFs were transfected with interference lentivirus vector of NRK.The m RNA and protein levels of NRK-depleted CAFs or NRK-overexpressing NFs were detected by q RT-PCR and WB assay.6.Explore the effect of conditional medium from NRK-depleted CAFs or NRK-overexpressing NFs on migration and invasion of LUAD cell lines.The changes of CXCL5 in conditional medium were detected by ELISA.7.Wound healing,Transwell assays,WB and IF were used to detect the motility,the expression of phospho-Cofilin(p-Cofilin)and the changes of F-actin in NRK-depleted CAFs or NRK-overexpressing NFs.8.Coculture was used to detect the effect of NRK-depleted CAFs on leading LUAD cells migration.Results:1.CAFs displayed the typical spindle-shaped,fattened,and fibroblast-like morphology similar to NFs under the light microscope.WB and IF results showed both CAFs and NFs strongly expressed mesenchymal marker Vimentin,but did not express epithelial marker E-cadherin.CAFs strongly expressedα-SMA and FAP-α,while NFs weakly expressed these two biomarkers of activated fibroblasts(P < 0.05).2.Compared to NF-CM,CAF-CM promoted the proliferation,migration and invasion of LUAD cells(P < 0.05).3.According to RNA-seq and comprehensive bioinformatics analysis,we identified that NRK was the most significantly differentially up-regulated gene in CAFs.The results of q RT-PCR,WB,IF showed that NRK was strongly expressed in CAFs(P < 0.05).Besides,IF results showed that NRK was predominantly expressed in nuclear and cytoplasm of CAFs.4.Compared with normal lung tissues,NRK was strongly expressed in LUAD tissues.Besides,high expression level of NRK was associated with clinicopathologic features including smoking,T stage and N stage(P < 0.05).5.The m RNA and protein expression levels of NRK were decreased in NRK-depleted CAFs,while the m RNA and protein expression levels of NRK were increased in NRK-overexpressing NFs.6.Compared with CAF-CM,CXCL5 was increased in conditional medium from NRK-depleted CAFs(P < 0.05).However,compared with NF-CM,CXCL5 was decreased in conditional medium from NRK-overexpressing NFs(P < 0.05).7.The motility,the expression level of p-Cofilin and the polymerization of F-actin were decreased in NRK-depleted CAFs(P < 0.05).While the motility,the expression level of p-Cofilin and the polymerization of F-actin were increased in NRK-overexpressing NFs.8.Compared to CAFs,the ability of leading LUAD cells migration was decreased in NRK-depleted CAFs.Conclusion:1.The high expression of NRK in CAFs could promote proliferation,migration and invasion of lung adenocarcinoma cells.2.High expression level of NRK may inhibit the release of CXCL5 from CAFs,as well as increased the expression of p-Cofilin and the polymerization of F-actin to enhance the motility of CAFs,thereby inducing lung adenocarcinoma migration and invasion.