CKAP4 Positively Regulating Tumor Growth And Metastasis By The PI3K-AKT Signaling Pathway In Lung Adenocarcinoma Cell

Background and Purpose:Lung cancer is still the main cause of morbidity and cancer-related mortality worldwide.Part of advanced NSCLC(non-small cell lung cancer)were rapid drug resistance and lead to progressive disease by giving precise chemoradiotherapy,EGFR-TKIs or immunotherapy,and further treatment is needed.Therefore,the present investigation to find out the molecular mechanism of drug therapy resistance and explore new treatment schemes for advanced lung adenocarcinoma(LUAD)patients has become a hotspot in recent years.Cytoskeleton associated protein 4(CKAP4)is a reversible palmitoylated type II transmembrane protein,it was located in cell membrane and rough endoplasmic reticulum,which plays an important role in stabilizing protein structure.However,CKAP4 plays a role in oncogene or tumor suppressor is still controversial.Therefore,using the next-generation transcriptomic sequencing and bioinformatics tools,further explored the molecular mechanism of CKAP4 from the clinical level,cell level and animal level in LUAD.Moreover,this study will help to further study the abnormalities of LUAD at the multi omics level,clarify the up-to-date of molecular mechanism in pathogenesis from LUAD.Furthermore,the study will provide a better theoretical basis for new targeted drugs,and also have an extremely important medical transformation significance.Methods:(1)The differentially expressed genes were screened by tissue sequencing results after NSCLC treatment(three patients were progressive disease after accepted first-line immunotherapy,and three cases were partial response),and these related genes effects on diagnosis,survival and prognosis were observed in LUAD through the Cancer Genome Atlas(TCGA)and GEPIA2 dataset.(2)Fresh LUAD tumor tissues and paracancerous tissues were collected and properly stored in liquid nitrogen prior to further assay,the m RNA and protein expression levels of CKAP4 at the tissue and LUAD cell were further verified by RT-PCR and western blot assays.Moreover,paraffin embedded tissues of NSCLC were collected and analysed by immunohistochemistry(IHC),the correlation between CKAP4 expression and clinicopathological information of NSCLC was analyzed by univariate and multivariate logistic regression analyses.(3)The effects of small interfering RNA(si CKAP4)and overexpression plasmid(CKAP4-ox)were transfected into LUAD cells,and the expression efficiency of CKAP4 were verified by western blot and RT-PCR assays.(4)The effects of CKAP4 on the proliferation,invasion,migration and apoptosis of LUAD cells were detected by CCK-8,scratch,Ed U,chamber transwell,flow cytometry and western blot assays in vitro.(5)We downloaded and analysed the CKAP4-related differential expression genes in LUAD patients from TCGA database,the most relevant signaling pathways were screened by GSEA pathway enrichment analysis and webgestalt database,and the effect of CKAP4 expression on these signaling pathway related proteins was verified by western blot assays.(6)The upstream miRNAs having potential binding sites with CKAP4 were screened through bioinformatics database.(7)The miR-133 b inhibitor and overexpression mimics were transfectted into LUAD cells,and the expression efficiency of miR-133 b were verified by western blot and RT-PCR assays.(8)Dual-luciferase reporter assay was used to verify whether miR-133 b and CKAP4 have potential binding sites and interactions.(9)The effects of miR-133 b on the proliferation,invasion,migration,metastasis and apoptosis of LUAD cells were verified by cell function phenotype experiment.(10)The correlation between miR-133 b and CKAP4 and its effect on the biological behavior of LUAD cells were further clarified by cell recovery assays.(11)The effects of miR-133 b on downstream CKAP4 and signaling pathway related proteins were detected by western blot assays.(12)The effect of silenced CKAP4 expression on the tumorigenicity of subcutaneous transplanted tumors in nude mice by vivo assays.Results:Part Ⅰ.CKAP4 is an important oncogene in LUAD from clinical levels(1)The m RNA levels of expression was performed by next-generation transcriptomic sequencing,there are 83 differential expression of upregulated m RNAs and 280 downregulated m RNAs were identified in NSCLC patients.Moreover,according to TCGA dataset and relevant studies,there are six m RNA genes were screened,including,MAGED1,CKAP4,MDH2,FKBP4,MRPL12 and TUBG1.(2)The IHC staining showed that CKAP4 is mainly located in cell membrane and cytoplasm,it is higher expression in NSCLC tumor tissues and distant-metastasis tissues than paracancerous tissues.Multivariate logistic analysis identified serum NSE and PD-L1(22c3)as independent risk factors associated with CKAP4 expression by IHC.Part Ⅱ.CKAP4 suppresses cell apoptosis and promotes tumor cells growth and metastasis by PI3K-AKT-m TOR signaling pathway(1)The small interfering RNA(si CKAP4)and overexpression plasmid(CKAP4-ox)were transfected into LUAD cells with turbofect reagent,RT-PCR and western blot results showed that it could significantly affect the m RNA and protein expression level of CKAP4 in LUAD cells.(2)The cell function phenotype assays showed that suppressed CKAP4 expression could significantly inhibits ability of proliferation,migration,invasion,and promotes apoptosis in A549 and H1650 cells,while up-regulation of CKAP4 expression can significantly reverse above ability of LUAD cells.(3)We downloaded and analysed the related differential expression genes in LUAD patients from TCGA database,and the most relevant signaling pathways were screened by GSEA pathway enrichment analysis and webgestalt database,the PI3K/AKT signaling pathway maybe plays an important role in the regulation of LUAD.Moreover,western blot assays results showed that up-regulated CKAP4 was promoted the phosphorylation levels of P-PI3 K,P-AKT and P-m TOR,while no significantly change in non-phosphorylated PI3 K,AKT,and m TOR proteins.Part Ⅲ.miR-133 b suppresses tumor growth and metastasis and promotes apoptosis by CKAP4/PI3K-AKT-m TOR signal axis in LUAD cells(1)The bioinformatics database showed that miR-133 b is correlation and negatively regulated CKAP4 expression,and having a potential binding sites with CKAP4.(2)Inhibited and up-regulated miR-133 b expression can significantly affected CKAP4 expression at both m RNA and protein levels in LUAD cells.(3)The dual-luciferase reporter assay showed that up-regulated miR-133 b expression can significantly inhibited fluorescence activity in CKAP4 wild-type group,and miR-133 b can negatively regulate the expression of CKAP4 and affect the post-transcriptional level in LUAD cells.(4)Cell function assays showed that up-regulated miR-133 b could significantly inhibit the ability of proliferation,migration,invasion and metastasis,and promotes cells apoptosis in LUAD cells.While inhibited miR-133 b expression in LUAD cells,the above cell functions were opposite.(5)The cell recovery assays further showed that miR-133 b suppressed the tumor growth and promotes cell apoptosis by negatively regulate the CKAP4 expression.(6)miRWalk,KEGG database and western blot assays results showed that miR-133 b negatively regulates the expression level of CKAP4 protein in LUAD cells,and further inhibit the phosphorylated PI3K-AKT-m TOR signal axis related proteins.Part Ⅳ.CKAP4 depletion inhibits xenograft tumour growth in vivo(1)To further evaluate the effect of silenced CKAP4 expression on tumorigenesis in vivo assays,A549 cell was infected with lentiviral stable transfections and constructing xenograft mouse model.The results showed that the silencing of CKAP4 expression could significantly inhibit the growth of tumors.(2)The tumor tissue of nude mice were ground by using liquid nitrogen,RT-PCR and western blot showed that the CKAP4 expression was significantly decreased at both m RNA and protein levels.(3)The IHC staining showed that silencing of CKAP4,the expression protein levels of CKAP4,Ki-67,and Bcl2(anti-apoptotic protein)were decreased,however,Bax and caspase7(pro-apoptotic proteins)were increased.Conclusions:(1)CKAP4 could significantly promote tumor proliferation and metastasis of LUAD in vivo and in vitro.(2)The CKAP4 is related to regional lymph node metastasis,serum NSE and PD-L1(22c3)expression in NSCLC patients,higher expression with poor prognosis,and it is an important biomarker for the diagnosis and prognosis of LUAD.(3)miR-133 b could significantly inhibit the ability of migration,proliferation,invasion and metastasis,and promotes apoptosis by CKAP4/PI3K-AKT-m TOR signal axis in LUAD cells.