| Lung cancer is one of the most prevalent malignancies worldwide,also the the leading cause of cancer-related death,with a high incidence.The mechanism of NSCLC has not been elucidated yet,which brings many challenges to the diagnosis and treatment.Yunnan province,China,has a high incidence of lung cancer,so it is urgent to explore the specific driving mechanism of NSCLC.RNA binding protein(RBP)is a key molecule responsible for post-transcriptional regulation in eukaryotes.When RBPs binds to mRNA,it regulates the splicing,output,localization,translation and degradation of mRNA,and thus participates in the occurrence of tumor.RBM proteins refer to a group of RNA binding proteins containing RNA binding notif.As an important part of RBPs,RBM protein has gradually become a potential target for the diagnosis and treatment of malignant tumors.As a novel RBP,RNA-binding motif protein 47(RBM47)located on chromosome 4p14,has a high affinity for poly-A,-C and-U RNAs.Previous studies have demonstrated that RBM47 plays differing roles in breast cancer and lung cancer.Breast cancer patients with high RBM47 expression tend to achieve better clinical outcomes.RBM47 binds to dickkopf-related protein 1 mRNA 3'UTRto increase its expression level,which inhibits breast cancer recurrence and growth by antagonizing Wnt.However,in lung cancer,down-regulation of RBM47 accelerated tumor formation and metastasis in a mouse xenograft model.Therefore,We focus on the role of RBM47 in NSCLC.This study tried to explore RBM47 malignant biological behaviors with NSCLC through the cell lines and tumor tissues,by exploring the activity of cell proliferation,cell migration,cell cycle process.We attempt to obtain the evidences that proved RBM47 participating in the malignant biologicar features of NSCLC,providing new targets and new ideas for the diagnosis and treatment of NSCLC in Yunnan.In this study,the expression of RBM47 in NSCLC tissues was firstly detected by IHC method.The results suggested that RBM47 may be closely related to the occurrence of NSCLC,and the analysis of clinical characteristics showed that RBM47 was closely related to lung adenocarcinoma,indicating poor prognosis of patients.In order to further explore the mechanism of RBM47 in NSCLC,we found that after RBM47 was reduced by shRNA,the proliferation of lung cancer cells A549 was inhibited,cell cycle was blocked,apoptosis was increased,and migration ability was weakened.Part 1.Increased expression of RNA-binding motif protein 47 predicts poor prognosis in non-small-cell lung cancerObjective:Lung cancer is the leading cause of cancer associated mortality worldwide.In China,in particular,lung cancer mortality has markedly increased and is likely to continue to rise.RNA binding proteins are pivotal to the development and progression of a variety of cancer types,including non small cell lung cancer(NSCLC).RNA binding motif protein 47(RBM47)has been found to act as a tumor suppressor in breast cancer and NSCLC.However,to the best of our knowledge,RBM47 expression in NSCLC tissues has yet to be investigated.Methods:Analysis via the online database,six datasets from Gene Expression Omnibus explored the correlation between RBM47 and NSCLC.A total of 175 pairs of NSCLC cancer tissues and adjacent non-tumor tissues from 175 patients who underwent complete surgical resection,including primary tumor plus mediastinal and bronchial lymph node dissection were included in the current study.RBM47 protein expression levels were detected in all tissue specimens by immunohistochemistry staining.The positive rate of RBM38 was compared between cancer tissue and normal tissue,and its association with the clinical pathological characteristics and prognosis was elucidated.Results:Analysis via the online database,Gene Expression Omnibus,revealed that RBM47 was upregulated in NSCLC and associated with pathological type,suggesting that RBM47 may play different roles in lung adenocarcinoma and lung squamous cell carcinoma.In the present study,the expression of RBM47 was examined by immunohistochemistry in 175 pairs of tumor and adjacent non cancerous tissues resected from patients with NSCLC.The results indicated that the expression of RBM47 was significantly increased in NSCLC samples compared with that in the matched non cancerous samples.Furthermore,RBM47 expression was higher in Xuanwei compared with that in non Xuanwei NSCLC,suggesting that RBM47 is a more sensitive biomarker in Xuanwei NSCLC,and that it may serve as a candidate therapeutic target.In addition,RBM47 expression was associated with the pathological type,however not with the age,sex,lymph node metastasis,pT stage or pathological Tumor Node Metastasis stage of the patients.The increased expression level of RBM47 may indicate a worse overall survival rate for patients with NSCLC.In addition,multivariate survival analysis showed that the Xuanwei area is associated with poor prognosis for patients with NSCLC.Conclusion:In conclusion,the present study revealed that the upregulation of RBM47 accelerated the malignant progression of NSCLC,indicating that RBM47 may be a potential biomarker for NSCLC progression and a therapeutic target for NSCLC.Part 2.Studies on the relationship between RBM47 expression ans NSCLC cell biological behavior in vitroObjective:Preliminary results showed that RBM47 was significantly up-regulated in NSCLC tissues and was a sensitive marker in xuanwei NSCLC.The increased expression level may indicate a poor prognosis in NSCLC patients,suggesting that RBM47 may be involved in the malignant progression of lung cancer.Tumor cells have 10 hallmarkers,such as abnormal proliferation,disordered cell cycle,decreased apoptosis,invasion and migration.RBPs was found to be involved in the malignant phenotypes of lung cancer,breast cancer and other tumors.This part has explored the role of RBM47 in NSCLC,and provided theoretical and experimental basis for targeted NSCLC treatment strategies based on RBM47 through MTS experiment,cell cycle detection and cell apoptosis detection.Methods:1.Expression of RBM47 in lung adenocarcinoma cell lines and lung epithelial cells was measured by Westernblotting.2.Construction of RBM47-shRNA lentiviral vector.3.A549 cell line was transfected by RBM47-shRNA lentiviral vector,and Westernblotting was performed to detect the expressions of RBM47.4.The effect of RBM47 in NSCLC cell proliferation was analysed using MTS assay and flow cytometry.5.Flow cytometry was used to analyse the relationship between RBM47 and NSCLC cell apoptosis6.Transwell assay was used to analyse the relationship between RBM47 and NSCLC cell migration.Results:1.Westernblotting detected that RBM47 was expressed in all NSCLC cell lines,and A549 cell line was chosen as experimental model.2.Westernblotting confirmed that RBM47-shRNA lentiviral vector could decrease the expression of RBM47.3.MTS assay and cell growth curve experiment showed that the proliferation of A549 cells were suppressed significantly,flow cytometry cell cycle analysis results revealed significant G0/G1 arrest in cell cycle in RBM47-knockdown A549 cell model.4.The apoptosis rate had statistically significant change in A549 cell line after RBM47 was knocked down.5.Transwell showed that cell migration was decreased in A549 after RBM47 downexpression.Conclusions:RBM47 was highly expressed in NSCLC cell lines.RBM47 knock-down may inhibit the NSCLC cell proliferation through cell cycle arrest,cell apoptosis and cell migration... |
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