| Objective: PRC1(Protein regulator of cytokinesis1),which is located on chromosome 15 in human,encodes a protein that is present at high levels during the S and G2/M phases of mitosis and involved in cytokinesis.PRC1 was originally identified as a mitotic spindle-associated Cdk(cyclin-dependent kinase)substrate in vitro and was subsequently shown to be a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone.Microtubules play essential roles in cell cycle,trafficking,signaling and migration.Alterations in microtubule-associated proteins have been demonstrated in cancers.More and more findings show that PRC1 was associated with cell migration.Gene expression profiles obtained by cDNA microarray analysis can provide a considerable amount of information for characterizing the nature of individual cancers.Through genome-wide expression profile analysis,people have found that expression of protein regulator of cytokinesis1 was significantly upregulated in many clinical cancers such as cholangiocarcinoma,colon cancers,non-small cell lung cancers,pancreatic cancers and breast cancers.Accumulating evidences indicate that PRC1 is overexpressed and plays an important tumor promoting role in the progression of various malignant tumors,including cervical cancer,prostate cancer,hepatocellular carcinoma and breast cancer.Until now,PRC1 has been reported to be associated with poor prognosis in non-small cell lung cancer patients.However,the function of PRC1 in the carcinogenesis of non-small cell lung cancer patients have not been reported.Method: Gene expression pfofiles obtained by cDNA microarray can provide a considerable amount of information for characterizing the nature of individual cancers.we previously downloaded data of 285 non-small cell lung cancer cases with Stage I–III disease and distinct clinical outcomes from three datasets(GSE42127,GSE11969,GSE8894)of Gene Expression Omnibus(GEO).Then,we carried out a genome-wide expression profile analysis of these datas by means of a combination of c DNA microarray and a systems biology approach.According to the analysis results,we identified several survival-related key genes which were consistently co-expressed in NSCLC and associated with various aggressive clinicopathological characteristics and overall prognosis of NSCLC patients.Among these upregulated genes,we focused on the functional significance of protein regulator of cytokinesis 1(PRC1)in the development of non-small cell lung cancer.The expression of PRC1 in NSCLC tissues and matched adjacent normal tissues from 36 cases of NSCLC patients were examined by means of real time quantitative PCR.Furthermore,data mining of PRC1 gene expression from NSCLC and adjacent matched tissues that are publically available in The Cancer Genome Atlas(TCGA)RNA-seq database was analyzed.These results indicated that PRC1 might play important roles in the development and progression of NSCLC.In order to establish NSCLC cell lines of overexpression and silencing expression of PRC1,plasmids were previously designed to express the entire coding sequences of PRC1 for transfecting into human non-small cell lung cancer cells and lentiviral vector-mediated siRNA was constructed to interfer PRC1 expression of non-small cell lung cancer cells.To detect the expression of PRC1 in mRNA and protein level in the transfected NSCLC cells,RT-PCR and Western blot analysis were carried out 2 or 3 days after transfection.The effects of PRC1 on the cell proliferation and migration in NSCLC cell lines were examined by performing soft agar colony formation assays and transwell assays.Because of cell proliferation depends on cell cycle progression,so then we further explore the impact of PRC1 on cell cycle by means of flow cytometry analysis.Additionally,to demonstrate that PRC1 can promote the oncogenesis of NSCLC in vivo,the effects of PRC1 on tumour growth in a subcutaneous xegograft were investigated.Finally,to study the relationship between PRC1 and the clinicopathologic stage and survival time of patients with NSCLC,a total of 150 cases of NSCLC paraffin-embeded tissue were retrieved from the pathology archive of NingBo First Hospital and the First Affiliated Hospital of Soochow University and the expression of PRC1 was then assayed by immunohistochemical staining.The study has been approved by two hospitial ethical committee.Result: In this study,it was found that PRC1 was significantly upregulated in tumour tissues compared with matched normal lung tissues from NSCLC patients.Moreover,based on the transwell assay,soft agar colony formation assays and flow cytometry analysis of NSCLC cells in vitro,overexpression of PRC1 in NSCLC cells can promoted cell proliferation,migration and cell cycle progression,while PRC1 knockdown significantly inhibited the migration and proliferation abilities of NSCLC cells and led to cell cycle arrest at the G2/M phase.Similarly,according to the growth curve of xenografts in xenograft mouse model,high PRC1 expression excerted an oncogenic effect by promoting tumour growth.Moreover,clinical patients with NSCLC and higher PRC1 expression exhibited significantly shorter overall survival(p=0.027)and progression-free survival duration(p=0.011)than patients with lower PRC1 expression.Conclusion: These findings suggest that PRC1 was upregulated in NSCLC tissues and promote cell proliferation and cell cycle progression.Meanwhile,overexpression of PRC1 may predict shorter overall survival and progression-free survival duration in patients with NSCLC.All of these results show that PRC1 should play oncogene role in process of NSCLC,and might be a novel promising prognostic biomarker and therapeutic target for non-small cell lung cancer.Objective: The results of the experimental study in Part I showed that the high expression of PRC1 promotes the proliferation and migration of NSCLC cells and promotes the NSCLC cell cycle into the division stage.And the animal model showed that PRC1 promotes the growth of NSCLC transplanted tumor;clinically,the pathological staging and survival prognosis of NSCLC patients with highly-expressed PRC1 are poorer to those in patients with lowly-expressed PRC1.These results suggest that PRC1 plays an important role in the development and progression of NSCLC.However,the molecular mechanism of PRC1 of affecting cell cycle and proliferation of NSCLC remains unclear and needs further study.Method: The expression and phosphorylation of proliferation-related regulatory pathway molecules and downstream target molecules were detected by Western blot to investigate the potential mechanism of PRC1 of promoting the NSCLC cell proliferation.The cell cycle-related regulatory proteins were detected by immunofluorescence to reveal how PRC1 affects the cell division of NSCLC.Further detection of NSCLC cell cycle-related molecules via q-PCR showed the possible signal pathways affecting the cell cycle.Western Blot was used to detect the expression and phosphorylation of NSCLC signaling pathway molecules and downstream signaling molecules with different PRC1 expression levels,so as to investigate the potential mechanisms of PRC1 of affecting NSCLC cell cycle.Result: It was found in the study on molecular mechanism that the level of FAK phosphorylation of NSCLC cells in highly-expressed PRC1 group was up-regulated.After si RNA-FAK was added,it was found that the promoting effect of PRC1 on cell proliferation was inhibited.The expression and phosphorylation of downstream target molecule Paxillin of FAK were further detected,and the results showed that highly-expressed PRC1 could also promote the phosphorylation level of Paxillin,suggesting that PRC1 may promote the proliferation of NSCLC cells via regulating the phosphorylation level of FAK-Paxillin pathway molecules.In the study on molecular mechanism affecting cell cycle,it was found that PRC1 promoted NSCLC cell into the division stage via regulating the expression of F-actin protein;highly-expressed PRC1 significantly inhibited the expressions of cell cycle-related molecules Cip1/p21 and Kip1/p27.At the same time,it was found that the phosphorylation of downstream molecules of Cip1/p21 and Kip1/p27,p107 and p130,was inhibited,but the phosphorylation of p RB was not affected,suggesting that PRC1 may regulate the NSCLC cell cycle through the regulation of phosphorylation level of p21/p27-p RB family molecules.Conclusion: The experimental study in this part revealed the possible molecular mechanism of PRC1 of affecting the NSCLC cell cycle and proliferation,which lays a theoretical basis for searching new therapeutic targets for NSCLC and study on targeted drug therapy. |
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