| Background:The latest cancer epidemiology data shows that lung cancer has become the highest mortality and incidence of malignant tumors.More than 85%of lung cancer belongs to non-small cell lung cancer(NSCLC).NSCLC is mainly divided into two types of lung adenocarcinoma and lung squamous cell carcinoma,lung adenocarcinoma accounts for about 40%of all lung cancer,and lung squamous cell carcinoma accounts for about 25%of all lung cancer.Recently,the continuous development of molecular tageting drugs for multiple driving genes,the overall survival time of patients with advanced NSCLC with specific gene mutation has been prolonged.However,the overall five-year survival rate of lung cancer is still hovering at about 15%.One of the important reasons is the abnormal proliferation and metastasis of lung cancer cells.Protein regulator of cytokinesis 1(PRC1)gene is a key protein molecule of cytokinesis.Previous studies have shown that PRC1 promoted the proliferation of breast cancer,liver cancer and gastric cancer,and was determined as as predictors of poor prognosis.However,the expression of PRC1 gene in non-small cell lung cancer,especially in lung adenocarcinoma,and the correlation between clinicopathological features,and its effect on invasion and metastasis of lung adenocarcinoma are still unclear.This study mainly focuses on the following five aspects:1.Expression of PRC1 in NSCLC and its correlation with clinicopathological factors and prognosis.2.The effect of PRC1 on the growth,proliferation,migration and invasion of lung adenocarcinoma cells.3.Animal experiments study PRC1 regulates the proliferation and metastasis of lung adenocarcinoma;4.The effect of PRC1 on cell cycle and apoptosis in lung cancer;5.Study on the mechanism of regulating Wnt/?-catenin signaling pathway.Materials and Methods:1.Analysis of PRC1 mRNA expression and prognostic significance using Oncomine(www.oncomine.org)and KMplot(http://kmplot.com)public database.Real-time PCR,Western blot and immunohistochemistry(IHC)were used to detect mRNA and protein expression of PRC1 in NSCLC and matched paracancerous tissues.The expression of PRC1 was detected by IHC method in 90 lung tissues and adjacent tissues.The clinicopathological features and prognostic value were analyzed.2.In the three lung adenocarcinoma cell lines(A549,SPC-A1 and H1299)with high expression of PRC1,the transfection efficiency was verified by real-time PCR and Western blotting by lentivirus infection.MTT assay,clone formation assay and Transwell migration assay were used to detect the changes of cell growth,proliferation and migration ability.3.The A549 cell line stably transfected with lentivirus infection was divided into two groups(A549 empty plasmid group and A549-shRNA-PRC1-1 cell group).The model of subcutaneous tumor of nude mice and experimental model of pulmonary metastasis of tail vein were established.The rate of tumor growth,tumor tumor and the number of lung metastatic nodules were observed.4.The variants of cell cycle and apoptosis were detected by flow cytometry,and the related cell cycle and apoptotic protein were detected by Western blot.5.In the A549 cells knocked down PRC1,the difference gene of Wnt signal pathway was found to be the most significant by Next Generation Sequencing(NGS)and bioinformatics method.The gene expression of Wnt/?-catenin pathway was validated by quantitative PCR and Western blot.The protein expression of PRC1 and Wnt/?-catenin signaling molecules(c-Jun,?-catenin,cyclin D2 and cMyc)was detected by immunohistochemistry in 20 cases of lung adenocarcinoma tissues,and the correlation analysis was performed.Experimental results:1.In this study,we found that PRC1 showed high expression in four NSCLC data cohorts of the Oncomine(www.oncomine.org)public database.In NSCLC,the overexpressed mRNA of PRC 1 is a poor prognostic factor,subgroup analysis found that PRC 1 showed poor prognostic significance only in lung adenocarcinoma in KMplot(http://kmplot.com).Real-time PCR,Western blot and immunohistochemistry(IHC)were used to detect the expression of mRNA and protein in cancer tissues.The results showed that PRC1 was highly expressed in lung adenocarcinoma,and the expression of PRC1 was correlated with lymph node metastasis and TNM stage in patients with lung adenocarcinoma.Survival analysis showed that the 5years overall survival time of patients with high expression of PRC1 was significantly lower than that of PRC1 low expression group.Further Cox multivariate analysis showed that PRC1 overexpression could also be classified as an independent factor in the prognosis of lung adenocarcinoma.2.The expression of PRC1 was detected by Western blot and quantitative PCR in three lung adenocarcinoma cell lines(A549,SPC-A1,H1299)and lung squamous cell carcinoma cell lines(H1703,SK-MES).The results showed that PRC1 showed high expression in the three lung adenocarcinoma cell lines(A549,SPC-A1,H1299).The transfection efficiency was down-regulated by A549,SPC-A1 and H1299 cell lines by lentivirus infection using Real-time PCR and Western blot.The results showed that the expression of PRC 1 was significantly decreased.Cell proliferation and invasion function showed that the cell growth ability of A549,SPC-A1 and H1299 cell lines was significantly decreased by MTT assay.The clone formation assay showed that the proliferation ability of cells was significantly decreased after knockdown PRC1;Transwell migration experiments showed that the migration ability of cells was significantly decreased after knocking down PRC1.3.The growth of subcutaneous tumor in nude mice began to observe the growth of subcutaneous tumor in nude mice after 6 days of subcutaneous injection of A549 cells.After 21 days,the subcutaneous tumor volume of shRNA-PRC1 group was significantly decreased compared with the control group.And with the extension of time,the difference between the two groups gradually increased.The lentivirus-infected A549 cells were injected into the nude mice through the tail vein.The shRNA-PRC1 group could significantly inhibit the in vivo metastases of lung adenocarcinoma cells compared with the control group.H&E staining showed that the number of lung metastases after PRC1 knockdown was significantly lower than that of the control group.4.Cell cycle test showed that in A549,SPC-A1 and H1299 cell lines,the G2 phase cells were significantly increased and the G1 phase cells were decreased,and the cell cycle was mainly arrested in G2 phase.The protein level of the cell cycle negative control factor(P21,P27)was significantly up-regulated,(Cdc25c,CyclinBl,cdc2)protein levels were significantly reduced.In A549 and SPC-A1 cell lines,Annexin-V PI staining was used to detect the number of apoptotic cells in PRC1 knockdown.Western blot was used to detect the expression of apoptotic cells in A549 and SPC-A1 cell lines after PRC1 knockdown.PARP,Bcl-xl and Bcl2 were down-regulated,while the level of apoptosis inhibitor Bax increased.5.In the A549 cells knocked down PRC1,the differential gene was mainly enriched in the Wnt/?-catenin signaling pathway by Next Generation Sequencing(NGS)and the bioinformatics method.The transcriptional level of Wnt/?-catenin pathway-related genes such as WNT8B,CCND2,SFRP4,TCF7L1 and FZD3 in A549 cells was down-regulated by quantitative PCR.The results of Western blot showed that WNT8B,CCND2,TCF7L1,FZD3,c-Jun,c-Myc protein levels were also down-regulated.The protein expression of PRC1 and Wnt/?-catenin signaling molecules(c-Jun,?-catenin,cyclin D2 and cMyc)was detected by immunohistochemistry in 20 cases of lung adenocarcinoma tissues.The results of Spearman correlation analysis showed that there was a positive correlation between the expression of PRC 1 and the expression of c-Jun,?-catenin,cyclin D2 and cMyc.Conclusion:1.The mRNAexpression in PRC1 was highly expressed in NSCLC specimens of the public database,and mRNA overexpression could consider as a predictor of poor prognosis in patients with lung adenocarcinoma.Our validated results showed that the mRNA and protein level of PRC1 were significantly higher in NSCLC tissues,and the protein expression was significantly correlated with the hippocampal lymph node metastasis and TNM stage in lung adenocarcinoma.The high expression of PRC1 protein could be classified as an independent of the poor prognostic factors in lung adenocarcinoma.2.In vitro experiments showed that PRC1 could be used as an oncogene to promote lung cell proliferation and invasion.3.In vivo,PRC1 can also promote the proliferation and metastasis of lung adenocarcinoma cells.4.PRC1 inhibition can lead to G2 phase arrest and promote apoptosis in lung adenocarcinoma cell lines.One of the mechanisms of promoting tumor invasion and invasion is through the promotion of cell cycle cytoplasmic progress and apoptosis inhibition.5.In this study,using Next Generation Sequencing(NGS)and bioinformatics analysis,we foundd that the difference genes of PRC1 knockdown were mainly enriched with Wnt/?-catenin signaling pathway,and further cultured in the cell line and human lung adenocarcinoma tissues by quantitative-PCR method and Western The results showed that there was a positive correlation between PRC1 and Wnt/?-catenin signaling pathway. |
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