| Objective:Lung cancers comprise the most common malignant tumor and are associated with the highest morbidity and mortality worldwide,of which 80%are non-small cell lung cancer?NSCLC?in the advanced stage at the time of diagnosis.Surgery is the best treatment for NSCLC,but even patients with curative outcomes might have local or systemic recurrence and eventually die of the disease.Therefore,it is important to identify the molecules that play a role in the development of NSCLC and understand their mechanisms of action.Identifying biomarkers and drug targets for NSCLC will contribute to early diagnosis and effective treatment and is a high priority in the fight against NSCLC.The Notch signaling pathway is involved in development,differentiation,cell proliferation,and apoptosis.Some studies have shown that it also plays a regulatory role in malignant tumors such as breast cancer and T cell leukemia.Activated Notch is over-expressed in the alveolar epithelium and synergizes with the myc pathway to promote the development of NSCLC.Notch1 inhibits p53-mediated apoptosis by regulating the stability of p53 and is required for lung adenocarcinoma growth.The Notch signaling pathway is activated by the ligand–receptor binding of five ligands?DLL1,3,4,and Jagged-1 and Jagged-2?and four receptors?Notch 1,2,3,4?.Subsequently,the Notch intracellular domain?NICD?is released and transferred to the nucleus,where it induces the expression of target oncogenes encoding c-myc,p21,p27,and HES family proteins,which promote carcinogenesis.GRWD1?Glutamate-rich WD repeat containing 1?is a 446-amino acid WD repeat protein that contains seven WD repeats and a glutamate-rich acidic domain at the N-terminus.WD repeats are minimally conserved domains of approximately 40–60amino acids,which serve as sites of protein–protein or protein–DNA interactions.WD repeat proteins are highly conserved in eukaryotes and closely associated with various biological processes including cell division,apoptosis,and signal transduction.GRWD1is over-expressed in various cancer cell lines.It induces the carcinogenic transformation of human normal cells and is associated with poor prognosis in patients with cancer.Nevertheless,the biological function of GRWD1 in NSCLC is still unclear.In this study,we aimed to elucidate the role of GRWD1 in this disease by examining GRWD1mRNA and protein expression in fresh NSCLC tissues and lung cancer cell lines.We also analyzed the effects of GRWD1 on the proliferation and migration of NSCLC cells at the cellular level and explored its mechanism of action to clarify its role in NSCLC.Methods:1.Immunohistochemistry:A total of 170 paraffin sections of tumor tissues obtained from patients in 2015–2016 at the First Affiliated Hospital of China Medical University were used for immunohistochemistry.All tissues were surgically removed,and the patients had not undergone chemotherapy or radiotherapy before surgery.Immunohistochemical staining was performed using the streptavidin peroxidase method.Two experienced clinical investigators examined the data and scored all slides semi-quantitatively by assessing the staining intensity and percentage of stained cells in each of the representative areas of the slides.Based on the staining intensity,the expression of GRWD1 was divided into four classes:0?no staining?,1?weak staining?,2?medium staining?,and 3?high staining?.The percentage of stained cells was scored as follows:0?0%?,1?1–30%?,2?31–70%?,and 3?71–100%?.The two scores of each tumor sample were multiplied to obtain a final score between 0 and 9.A final score greater than 3 was designated“GRWD1-high,”whereas a score of less than 3 was designated“GRWD1-low.”All statistical analyses were performed using SPSS 17.0,The correlation between GRWD1 expression and clinical parameters was analyzed by the chi-square test,P<0.05 was considered statistically significant.2.Cell culture:All cell lines were purchased from the Shanghai Cell Bank and cultured in medium with 10%qualified fetal bovine serum and 100 IU/ml penicillin.Human bronchial epithelial?BEAS-2B?cells were cultured in Dulbecco's modified Eagle medium with high glucose.A549,H1299,H460,H226 and H292 lung cancer cells were cultured in Roswell Park Memorial Institute 1640 medium.SK-MES-1 lung cancer cells were cultured in MEM medium.All cells were cultured in a 5%carbon dioxide incubator at 37°C.3.Quantitative real-time polymerase chain reaction?PCR?:Quantitative real-time PCR was performed using the SYBR Green PCR Master Mix in a QuantStudio 3 Real-Time PCR Instrument with the following cycling conditions:95°C for 2 min,95°C for 15 s,and 60°C for 1 min for 40 cycles.Relative gene expression was calculated using the2-??CTmethod with reference to the?-actin encoding gene.All experiments were repeated three times under the same conditions.4.Cell proliferation assay:Twenty-four hours after transfection,the cells were plated in96-well plates containing 10%FBS medium at a density of approximately 5000cells/well;five replicate wells were prepared for each sample.The cells were cultured continuously for 5 days and stained with MTT reagent at the same time each day.Briefly, 10?l of 5 mg/ml MTT was added to each well,the cells were incubated at 37°C for 4 h,the medium in each well was carefully removed,and 150?l of DMSO was added.The results were quantified spectrophotometrically using a microplate reader at a wave length of 570 nm.5.Colony formation assays:Twenty-four hours after transfection,the cells were counted and cultured in a 6-well plate supplemented with medium at a density of 500 cells/well for approximately 10 days until macroscopic colonies formed.The colonies were fixed with ice cold methanol,stained with hematoxylin,air-dried,and scored according to the number of colonies.6.Cell cycle assay:Forty-eight hours after transfection,cells were harvested from the6-well plates by centrifugation,washed in PBS,and fixed with 70%ethanol at 4°C overnight.The cells were then collected by centrifugation,washed in PBS,and incubated with 450?l propidium iodide?PI?and 50?l RNaseA for 30 min,which was followed by flow cytometry.7.Cell migration assay:Cell migration assays were conducted in 24-well Transwell chambers with an aperture of 8 microns.Twenty-four hours after transfection,all cells were trypsinized and 6×104 cells were transferred to the upper perforation chamber in 200?l medium and 2%FBS,whereas 600?l medium and 10%FBS were added to the lower chamber.After 24h of culture,the cells on the surface of the membrane were removed with a cotton tip,fixed with paraformaldehyde,and stained with hematoxylin.Ten regions were randomly selected under high power magnification to calculate the number of migrating cells.The experiment was carried out three times.8.Statistical analysis and datasets analysis:We downloaded the transcriptome datasets of NSCLC from TCGA.The difference genes were obtained by R language analysis,and the correlation between genes was analyzed by R language.Survival curves were compared using the Kaplan–Meier database and log-rank test.ONCOMINE gene expression array datasets is an online cancer microarray database,it was used to analyze the expressions of GRWD1 mRNA in clinical cancer tissues,and compared with the normal control groups.The P value was obtained by Students't-test analysis.The fold change and cut-off of P value were defined as 1.5 and 0.01,respectively.Results:1.The UALCAN and GEPIA dataset indicated that the mRNA expression of GRWD1 was higher in cancer tissues than in normal tissues whether in LUAD or LUSC.Quantitative Real-time PCR to analyze the expression of GRWD1 mRNA showed that it significantly higher in the cancer tissues.In addition,GEPIA and Kaplan–Meier datasets analysis indicated that the increased GRWD1 mRNA levels were significantly associated with the overall survival.Immunohistochemical analysis revealed that the expression of GRWD1 was negatively correlated with the degree of pathological differentiation of NSCLC and positively correlated with tumor size,lymph node metastasis,and P-TNM stage.Western blot analysis of GRWD1 protein expression in fresh NSCLC and corresponding adjacent tissues showed higher GRWD1 expression in cancer tissues than adjacent tissues.2.We performed cell colony formation,cell proliferation assays and Transwell cell migration assay in A549 and H1299 cell lines,and found that GRWD1 can promote the growth and migration of NSCLC cell lines in vitro,and the cell cycle was mainly affected in the G2/M phase.3.Western blot analysis showed that GRWD1 promotes the proliferation of NSCLC cells through its effects on CyclinB1,CDK1,and p27,and promotes the migration of NSCLC cells by affecting RhoA,RhoC,and CDC42 expression.4.Western blot analysis of EMT-related proteins showed that GRWD1 can promote EMT in NSCLC cells by increasing the expression of N-Cadherin,Vimentin,Snail,and Zeb1 and decreasing the expression of E-Cadherin and ZO-1.5.The difference genes of NSCLC and the signal pathway related to GRWD1 were obtained by R language analysis,the result showed that there was a correlation between Notch pathway related genes and GRWD1.Western blot analysis showed GRWD1 can active the Notch signaling pathway.6.After inhibition of Notch pathway activity by DAPT,a Notch pathway inhibitor,Western blot,cell clone formation and Transwell migration assay confirmed that GRWD1 promoted NSCLC cell proliferation and migration through Notch pathway.Conclusion:1.GRWD1 is highly expressed in NSCLC,and the protein expression of GRWD1 was positively correlated with tumor size,lymph node metastasis and P-TNM stage,negatively correlated with the degree of pathological differentiation of NSCLC and prognosis.2.GRWD1 promotes cell proliferation by affecting the G2/M phase through regulation of the Cyclin B1–CDK1 axis.3.GRWD1 promotes the migration of NSCLC cells by affecting RhoA,RhoC,and CDC42 expression.4.GRWD1 can active the Notch signaling pathway,and promotes cell proliferation and migration through the Notch pathway. |
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