The Biological Function And Mechanism Of KIF18A In Lung Adenocarcinoma

Background:KIF18A is a member of the Kinesin-8 subfamily,which is downregulated in most human normal tissues and abnormally upregulated in various human malignant tumor tissues.KIF18 A is significantly associated with malignant pathological features and poor prognosis of patients with cancers,and promotes cancer cell proliferation and metastasis.Therefore,it may act as a new molecular target for tumor genetic therapy.However,few reports have focused on KIF18 A in LUAD,and its role and mechanism in LUAD remain unclear.Methods:LUAD tissue samples were collected from clinical patients,and the expression of KIF18 A in LUAD tumor tissues and adjacent normal lung tissues were detected by q PCR,western blot and immunohistochemistry.The association of KIF18 A expression levels with clinicopathologic characteristics as well as the survival analysis of LUAD patients were analyzed.By mining TCGA and GEO databases of LUAD RNA-seq data,the m RNA expression levels of KIF18 A in LUAD tissues were analyzed.The correlation between KIF18 A m RNA expression level and clinical pathological features and prognosis of patients were analyzed.LUAD cell lines with stable overexpressing or silencing of KIF18 A were constructed by transfecting with the lentivirus vector.The roles of KIF18 A in the cell proliferation,apoptosis,cell cycle distribution and cell senescence of LUAD cells were examined by CCK-8,plate clone formation assay,flow cytometry and ?-galactosidase staining,respectively.A nude mouse subcutaneous xenograft model was established to evaluate the effect of KIF18 A on the proliferation capacity of LUAD cells in vivo.Cell scratches,Transwell migration and invasion assay were performed to explore the effect of KIF18 A expression on the migration and invasion ability of LUAD cells.Western blot assays were used to detect the effect of KIF18 A expression on the expression of MMP2/9 in LUAD cells.A tumor metastatic model in nude mice was established to evaluate the effect of KIF18 A expression on the metastatic ability of LUAD cells in vivo.Immunofluorescence assay was performed to evaluate the relationship between KIF18 A expression and EMT in LUAD.Western blot assays and data mining from TCGA database were used to analyze the correlation between KIF18 A expression and EMT-related biomarkers and MMP9 expression.Cancer SEA database was used to explore the biological function of KIF18 A gene in LUAD at the single cell level.Western blot assays were used to examine the effects of KIF18 A silencing on the expression of Smad2/3 proteins.Transwell cell migration and invasion assays were performed to explore the effect of KIF18 A silencing on TGF-?1 induced cell migration and invasion in LUAD.After treating A549 cells with TGF-?1 or SB431542,western blot assays were used to verify the role of KIF18 A in the TGF-?/Smad signaling pathway.To predict the upstream regulatory mi RNAs of KIF18 A gene by bioinformatics analysis.Dual-luciferase reporter gene assay was used to verify the direct target relationship between mi R-26b-5p and KIF18 A.q PCR assay and data mining from TCGA database were used to analyze the expression level of mi R-26b-5p in LUAD tissues,the correlation between mi R-26b-5p and KIF18 A gene expression and its role in patient's prognosis.Western blot assays were used to examine the effect of mi R-26b-5p on KIF18 A protein expression.Cell plate clone formation,migration and invasion assays were used to examine the effect of mi R-26b-5p/KIF18 A on the proliferation and metastasis capacity of LUAD cells.Results:At the level of m RNA transcription and protein expression,the expression of KIF18 A in cancer tissues was significantly higher than that in normal lung tissues.The expression level of KIF18 A was significantly associated with lymph node metastasis,TNM stage and vital status in patients with LUAD.KIF18 A expression was negatively associated with overall survival,and was an independent factor for predicting poor prognosis in patients with LUAD.The expression of KIF18 A was relatively low in A549 cells and relatively high in PC9 cells,which were selected to establish stably transfected cell lines with KIF18 A knockdown and overexpression for subsequent experiments.Overexpression of KIF18 A promoted the proliferation of LUAD cells in vivo and in vitro,while knockdown of KIF18 A inhibited cancer cell proliferation and induced cell apoptosis,senescence and G2/M phase arrest.Overexpression of KIF18 A enhanced the migration and invasion ability of LUAD cells in vivo and in vitro,while knockdown of KIF18 A inhibited cell migration and invasion.KIF18 A overexpression promoted the expression of MMP2/9,while knockdown of KIF18 A inhibited the expression of MMP2/9.Overexpression of KIF18 A promoted the formation of metastatic nodules on the surface of lungs in nude mice,while knockdown of KIF18 A inhibited lung metastasis in nude mice.Overexpression of KIF18 A promoted EMT of LUAD cells,while knockdown of KIF18 A inhibited the process of EMT.The function of KIF18 A gene was significantly associated with cell proliferation,invasion,metastasis and EMT at the single cell level.KIF18 A regulated the phosphorylation level of Smad2/3 proteins in LUAD.Knockdown of KIF18 A inhibited cell migration and invasion induced by TGF-?1 in A549 cells.Knockdown of KIF18 A significantly inhibited TGF-?1-induced phosphorylation of Smad2/3 proteins.SB431542 could inhibit the phosphorylation of Smad2/3 proteins caused by KIF18 A overexpression.KIF18 A regulated the expression of EMT-related biomarkers and MMP2/9 through activating TGF-?/Smad signaling pathway.miR-26b-5p was the upstream regulatory mi RNA of KIF18 A gene.The expression of mi R-26b-5p was down-regulated in LUAD tissues,which was negatively correlated with KIF18 A expression and patients' prognosis.Transfection of mi R-26b-5p mimics inhibited the proliferation and metastasis ability of A549 cells,while KIF18 A overexpression could reverse the inhibition of mi R-26b-5p on proliferation and metastasis of A549 cells.Conclusions:KIF18A was overexpressed in LUAD and was an independent prognostic biomarker for poor prognosis in LUAD.KIF18 A promoted the proliferation of LUAD cells in vivo and in vitro,while knockdown of KIF18 A promoted cancer cell apoptosis and senescence,leading to G2/M phase arrest in the cell cycle.KIF18 A regulated EMT and extracellular matrix remodeling via activating TGF-?/Smad signaling pathway.mi R-26b-5p could directly target the KIF18 A gene and regulate the proliferation and metastasis of cancer cells.KIF18 A was a novel promising molecular target for the treatment of LUAD.