Function And Mechanism Study Of PRKD1 In Invasion And Migration Of Non-Small Cell Lung Cancer

Objective:Lung cancer is the most widespread malignancy in the world.As the most common subtype of lung cancer,non-small cell lung cancer(NSCLC)accounts for approximately 85%of all lung cancers.Traditional treatments against cancer(surgery,radiotherapy and chemotherapy)and emerging targeted therapy and immunotherapy have made great achievements in inhibiting cancer progression,but the five-year survival rate of NSCLC has not been significantly improved,which is closely related to the prone of NSCLC to invasion and distant migration.With this reason,investigating the molecular mechanisms involved in the invasion and migration of NSCLC cells has important theoretical significance and clinical value for improving prognosis.Protein Kinase D(PRKD)family is a conserved silk/threonine kinase family.PRKD1,a member of this family,is down-regulated in a range of cancers,including progressive gastric,prostate and breast cancers.PRKD1 is involved in the formation of precancerous lesions and the invasion and migration of cancer cells and is considered as a new target for the prevention of precancerous lesions and tumor development.Studies have shown that PRKD1 is downregulated in NSCLC,but its exact functional mechanism is unclear.In this study,we aimed to explore the role and underlying mechanism of PRKD1 in invasion and migration,which is expected to provide a new theoretical basis for the treatment of NSCLC.Methods:1.The expression of PRKD1 in 33 tumors was analyzed using The Cancer Genome Atlas(TCGA)pan-cancer data.The expression of PRKD1 in NSCLC tissues and normal lung tissues was explored by TCGA、Gene Expression Omnibus(GEO)and Gene Expression Profiling Interactive Analysis(GEPIA).Analyze the association of the expression of PRKD1 and clinicopathological features and prognosis of patients.2.Real-time quantitative reverse transcription PCR(qRT-PCR)was used to detect the expression of PRKD1 in normal lung epithelial cells(BEAS-2B)and NSCLC cells(H1299,A549,H1975 and PC9),from which the NSCLC cells with the highest and lowest PRKD1 expression were selected for the next experiments.3.Small interfering RNA(siRNA)knocked down the expression or plasmid overexpression of PRKD1 in NSCLC cells A549 and H1299.CCK8,EDU cell proliferation experiments,healing and Transwell experiments were performed to observe the effect of PRKD1 on cell proliferation,migration and invasion ability.4.Stable knockdown and overexpression of PRKD1 in A549 and H1299 cells were constructed by lentiviral transfection technique,respectively.The lentiviral stably transfected cells were divided into 4 groups:null group(LV-NC)and PRKD1 stable overexpression group(LV-PRKD1);control group(sh-NC)and PRKD1 stable knockdown group(shPRKD1).These 4 groups of lentiviral-stabilized A549 cells were injected into nude mice via subcutaneous and tail vein,respectively.The subcutaneous tumor model and metastasis model in nude mice were built to study the impacts of PRXD1 on proliferation and metastasis of NSCLC in vivo.5.Total RNA was extracted from PRKD1 stable overexpression group(LV-PRKD1)A549 cells and null group(LV-NC)A549 cells.Transcriptome sequencing was performed to analyze the changes of relevant signaling pathways and mRNA transcription levels after PRKD1 overexpression.According to the sequencing results,the signaling pathway with the most significant enrichment genes was selected as the downstream signaling pathway of PRKD1 for further study.The effect of PRKD1 on the expression of key proteins in downstream signaling pathway was examined using Western blot.6.Co-immunoprecipitation(CO-IP)and mass spectrometry techniques were used to identify genes that bind to PRKD1.The gene with the highest peptide coverage was selected and its expression and prognostic significance in NSCLC were verified by GEO,TCGA and GEPIA databases.The correlation between this gene and PRKD1 expression was also analyzed.7.Expression of downstream target gene in normal lung epithelial cells(BEAS-2B)and NSCLC cells(H1299,A549,H1975 and PC9)was detected by qRT-PCR.A549 cells were transfected with target gene siRNA,and the effect of its knockdown on cell proliferation,migration and invasion ability were observed by CCK8,EDU cell proliferation experiment,healing and Transwell experiment.The effect of target gene knockdown on the expression of key proteins in downstream signaling pathway was displayed by Western blot.8.In PRKD1 stable knockdown group(shPRKD1)A549 cells,siRNA was applied to reduce the expression of the target gene.Rescue experiments further confirmed that target gene is involved in the undesirable biological phenotype of NSCLC mediated by PRKD1.Results:1.The expression level of PRKD1 in NSCLC is decreased and correlates with poor prognosisTCGA pan-cancer data showed that PRKD1 was lowly expressed in 13 tumors.TCGA、GEO and GEPIA databases showed lower expression of PRKD1 in NSCLC tissues compared to normal lung tissue.NSCLC patients were divided into two groups according to the median expression level of PRKD1.Both in TCGA database and Kaplan-Meier Plotter website,patients in the low PRKD1 expression group were found to have shorter survival time.2.PRKD1 inhibits proliferation,migration and invasion of NSCLC cells in vitroqRT-PCR showed that the expression of PRKD1 was significantly reduced in NSCLC cells compared with normal lung epithelial cells,among which PRKD1 expression being the lowest in H1299 cells and relatively high in A549 cells,so these two cells were selected for subsequent experiments.qRT-PCR and Western blot were used to verify the transfection efficiency,and it was found that PRKD1 mRNA and protein levels increased significantly in A549 and H1299 cells transfected with overexpression plasmid,while the mRNA and protein expression of PRKD1 was decreased after transfection with siRNACCK8 and EDU cell proliferation experiments showed that in A549 and H1299 cells.overexpression of PRKD1 significantly inhibited cells proliferation,while knockdown of PRKD1 expression significantly enhanced cells proliferation.Healing and Tran swell experiments showed that overexpression of PRKD1 significantly inhibited the migration and invasion ability of A549 and H1299 cells,while knockdown of PRKD1 expression promoted migration and invasion.3.PRKD1 inhibits proliferation and metastasis of NSCLC cells in vivoThe nude mice subcutaneous tumor model showed that tumors in the LV-PRKD1 group grew slowly and had significantly smaller volume and weight,while tumors in the shPRKD1 group grew faster and had larger volume and weight.Immunohistochemistry(IHC)staining again confirmed the decreased expression of Ki67,a proliferation indicator,in the LV-PRKD1 group.The metastasis model showed that lung metastasis nodules were found in both LVPRKD1 and LV-NC groups of nude mice,but the number of lung metastasis nodules in the LVPRKD1 group was much lower than that in the LV-NC group.4.PRKD1 regulates Wnt/β-catenin signaling pathwayAs shown by transcriptome sequencing,the differential genes(log FC>2,P<0.05)were significantly enriched in Wnt signaling pathway after PRKD1 overexpression.Western blot showed that in A549 and H1299 cells.PRKD1 overexpression inhibited the expression of βcatenin.c-myc and c-Jun,which are key proteins in the Wnt/β-catenin signaling pathway,while PRKD1 knockdown increased the expression of β-catenin.c-myc and c-Jun.These changes are consistent with the results of proliferation and migration experiments,and we hypothesized that PRKD1 is involved in regulating Wnt/β-catenin signaling pathway in NSCLC to exert tumor suppressive effects.5.The expression level of EIF5A in NSCLC is increased and correlates with poor prognosisCO-IP and mass spectrometry showed that EIF5A could combine with PRKD1.GEO and TCGA databases showed that EIF5A was significantly adversely correlated with PRKD1 expression in NSCLC,and EIF5A was highly expressed in NSCLC tissues.NSCLC patients were divided into two groups according to the median expression level of EIF5A.Both in TCGA database and Kaplan-Meier Plotter website,patients in the high EIF5A expression group were found to have shorter survival time.6.Down-regulation of EIF5A inhibits proliferation,migration and invasion of NSCLC cellsThe qRT-PCR results showed that EIF5A was highly expressed in NSCLC cells versus normal lung epithelial cells.qRT-PCR and Western blot were used to verify the transfection efficiency,and it was found that siRNA transfection reduced mRNA and protein expression of EIF5A in A549 cells.CCK8 and EDU assays proved that the proliferation competence of A549 cells was significantly inhibited after the down-regulation of EIF5A expression.Healing and Transwell experiments showed that the migration and invasion capacity of A549 cells were decreased after the down-regulation of EIF5A expression.Western blot results showed that the expression of key proteins β-catenin,c-myc and c-Jun in the Wnt/β-catenin signaling pathway was down-regulated after knocking down the expression of EIF5A.7.EIF5A participates in the malignant biological behavior of NSCLC mediated by PRKD1The expression of EIF5A was knocked down by transfecting siRNA in A549 cells with stable knockdown of PRXD1.The results showed that EIF5A could rescue,to some extent,the enhanced cell proliferation and invasion ability caused by knockdown of PRKD1 and the activation of Wnt/β-catenin signaling pathway caused by knockdown of PRKD1.Conclusion:1.PRKD1 is lowly expressed in NSCLC and is correlated with poor prognosis.2.Overexpression and knockdown of PRKD1 can effectively inhibit and promote NSCLC cells proliferation.migration,invasion.tumorigenesis,lung metastasis and Wnt/β-catenin signaling pathway.3.EIF5A is highly expressed in NSCLC and negatively correlated with PRKD1 expression.EIF5 A knockdown can inhibit proliferation,migration and invasion of NSCLC cells and Wnt/βcatenin signaling pathway.4.PRKD1 inhibits proliferation,migration and invasion ability as well as Wnt/β-catenin signaling pathway by regulating EIF5A expression in NSCLC cells.