Expression Of RGMB And Its Correlation With Prognosis In Lung Cancer

BackgroundTumor recurrence and metastasis is the basic feature of malignant tumor progression,and it is also an important indicator of the prognosis of patients..Lung cancer now is the leading cause of cancer related mobility and mortality all over the world.Surgical resection is the primary treatment for early stage lung cancer.However,for patients who receive surgery,there will be still of risks of recurrence and metastasis.Because there are few symptoms of early stage lung cancer,most of which are diagnosed at their advanced stage.As a result,radiation and chemotherapy are the only ways of treatment to them.Therefore,it is important to understand the molecular mechanism of lung cancer to further provide solid basis for relative treatment.RGMB is one of the members of the family repulsive guidance molecules(RGMs).According to the related research,bone morphogenetic protein(BMPs)and RGMB protein co receptor relationship,and the occurrence and development of tumor and RGMB protein has a certain relevance.Repellency orientation points of lung cancer is one of the world RGMs is BMPs cooperation receptors,and BMPs is the important regulatory factors in the process of bone metastases of cancer,so the RGMs likely by BMP signaling pathways that regulate the occurrence of lung cancer and bone metastases.Previous research has shown that RGMs can alter tumor cells malignant biological behavior,suppress the ability of invasion and metastasis.The present study aims to study its important role in lung cancer and its metastasis and recurrence.ObjectiveWe detected RGMB gene expression in 120 cases of lung cancer tissues and 30 cases of normal lung tissue,and analyzed the correlation betweent RGMB expression and clinical indicators to predict the prognosis.At the same time,the detection of different RGMB gene expression level in lung cancer cell lines,select high expression of cell lines and build RGMB gene knock-out model,to verify its influence on tumor cell function and prepare for future cell signal study.Methods1.To explore the relationship between RGMB expression and lung cancer in TCGA database.2.The expression of RGMB in normal lung tissue and lung cancer tissue was detected by fluorescence quantitative PCR method.The relationship between RGMB expression level and clinical pathology,stage and prognosis was analyzed.3.The high expression of RGMB gene screening of lung cancer cell lines,the QPCR method is adopted in the mRNA level of testing RGMB mRNA expression in lung cancer cell lines,and therefore screening RGMB high mRNA expression in lung cancer cell lines for subsequent experiments.4.PLVX-shRNA-Puro-RGMB,such as the construction of eukaryotic expression vector and identified using the public website design and screening related sequence,to the biological synthesis company.With pLVX-Puro plasmid after restructuring,transfection competent e.coli,For identification of positive clones of the results,and then extract plasmid;gene transfection of 293 T cells to detect the transfection efficiency by fluorescence quantitative PCR interference detection,high efficiency of interference fragment screened and after lentiviral packaging and subsequent experiments.5.Slow virus LV-RGMB-scramble and LV-RGMB preparation,infection of shRNA A549 cellLine in 293 t cells for lentivirus packaging,and the drop degree and collection,after using the virus infection in lung cancer cell line A549,stable screening and training through puro,obtain stable RGMB gene silence lung cancer cell lines,transfection efficiency was determined by mRNA assay.The expression of RGMB in transfected cell lines was detected by blot western and fluorescence quantitative PCR respectively.6.Silence RGMB gene A549 lung cancer cell functionUsing MTT experiment of cell survival rate to describe the influence of cell growth;Using cell adhesion experiment to observe the adhesion ability;The cell migration and invasion ability of the cells were observed by experiments,cell migration assay and Transwell invasion assay.7.Detection of BMPs Signaling Protein Expression in Cell Lines with Reducing RGMB Expression by Western blot.Results1.The RGMB expression in lung cancer was analyzed through TCGA,and the RGMB gene was down-regulated in tumor tissue and had a significant negative correlation with T and N staging.The expression in male patients was lower than female.2.Of the RGMB gene expression levels were detected by fluorescence quantitative PCR,the results showed that compared with normal lung tissue,RGMB in lung cancer gene expression level is low(P< 0.05).At the same time,found that female patients with RGMB expression level is relatively high.In advanced lung cancer RGMB expression level is low,but early in RGMB the high level of expression(P< 0.05).RGMB high expression indicated better clinical prognosis and survival of the state.3.We successfully screened for lung cancer cell line A549.High expression of RGMB gene was successfully constructed RNAi eukaryotic expression plasmid targeting RGMB gene,identified by enzyme digestion,the resulting plasmid was transfected into 293 T cells,the interference effect of fluorescent quantitative PCR results indicated that the plasmid can produce the ideal.4.Successful lentivirus packaging LV-RGMB scramble,LV-RGMB shRNA,its infection in lung cancer cells,after puro pressure filter,using QPCR and Western blot test,the results show that the experimental group of A549 cells RGMB mRNA expression than untreated cells fell by 65%(p < 0.05).It Shows that we successfully build stable RGMB gene silence lung cancer cell lines.5.RGMB A549 gene stably silenced lung cancer cell lines in vitro proliferation capacity,adhesion,migration and invasion ability was significantly enhanced compared with the control group(P < 0.05).6.In order to investigate whether RGMB is involved in BMP signaling pathways,we used RGMB to stabilize silencing and to study the A549 cell line in control group.Western blot was used to detect the protein molecules in BMPs signaling pathway.The phosphorylation level of Smad-1/5/8 was increased after RGMB silencing(p <0.05).However,there was no significant change in ERK and JNK protein activation between the two groups.The results suggested that RGMB might play a role in inhibiting the tumor by inhibiting the Smad1 / 5/8 pathway.Conclusion1.The slow virus vector targeting RGMB gene was successfully constructed,which could inhibit the expression of RGMB gene in lung cancer cell line A549 in a long time.2.Silent RGMB gene can promote the growth,invasion and metasitasis of lung cancer cells.3、The tumor-suppressing effect of RGMB might be exerted through inhibition of the Smad1/5/8 pathway.