Effect Of Limb Bud And Heart Development (LBH) On Proliferation And Metastasis Of Gastric Cancer Cell And Its Correlation With Clinical Significance

Objective:Gastric cancer(GC)is the most common and lethal type of malignancy worldwide.In 2012,approximately 989,600 new cases of GC and 738,000 cases of GCrelated deaths were recorded worldwide,accounting for 6.7% and 8.8% of new cancer cases and cancer deaths,respectively.Despite conventional postoperative adjuvant therapy,approximately 1/3 of patients still relapse.The poor prognosis is due to the lack of clear preventive measures,early detection methods,and effective treatment for GC.Therefore,identifying useful clinical biomarkers and molecular targets related to GC progression is necessary.The limb-bud and heart development(LBH)gene is a highly conserved,tissue-specific transcription cofactor in vertebrates that regulates multiple key genes in embryonic development.The gene is located on chromosome 2p23.1 and encodes a small nuclear protein with 105 amino acids.In addition to embryonic tissues,LBH is expressed in adult organs,including the spleen,gut,kidney,brain,and peripheral nervous system.Aberrant expression of LBH during heart development can lead to congenital heart diseases such as partial trisomy 2p syndrome and other growth defects.Increasing evidence indicates that embryonic development and tumorigenesis have similar molecular mechanisms.Rieger et al.first discovered the role of LBH in cancer in 2010.They observed that LBH acts as a target gene for the Wnt pathway to inhibit mammary epithelial differentiation and promote Wnt-induced tumorigenesis.In a study by Liu,overexpression of LBH was shown to lead to G1/S phase arrest in nasopharyngeal carcinoma and act as a transcriptional cofactor of NF-κB.Results from studies by Chen have confirmed that LBH predicted poor prognosis in hepatocellular carcinoma.Moreover,our team found that LBH is downregulated and predicts better overall survival(OS)outcome in lung adenocarcinoma.However,the expression and biological functions of LBH in GC remain unclear.Therefore,in this study,the expression pattern of LBH in GC was examined in public datasets and in our study samples.In addition,the effects of LBH on cell proliferation,migration,and invasion of GC cells was explored.The potential mechanism of LBH was also investigated using bioinformatics and Western Blot analyses.Methods: 1.TCGA-STAD and GSE29272 datasets was used to study the difference in the expression of LBH mRNA between GC and normal GC tissue.2.Overall survival between LBH high and low group of GC patients were examined by kaplan-meier analysis and logrank test in TCGA-STAD,GSE15459 and GSE29272 datasets.3.Cox univariate analysis and Cox multivariate proportional hazard model were used in the TCGA-STAD,GSE15459 and GSE62254 datasets to analyze whether LBH is an independent prognostic factor for GC.4.The protein expression level of LBH was detected by immunohistochemistry in the cancer and paracancerous tissues of 82 GC patients,and the expression difference of LBH was determined.5.The clinicopathological parameters and prognosis information of 82 patients with GC were collected and their correlation with LBH was analyzed.6.Establishment of a stable LBH knockdown HGC-27 cell line and a stable LBH overexpression BGC-823 cell line by transfection of lentivirus.7.Verify LBH knockdown and overexpression efficiency by qRT-PCR.8.The effect of LBH on cell proliferation was examined by MTT assay and colony formation assay in gastric cancer cell lines.9.The effects of LBH on cell migration and invasion were examined by wound healing assay and transwell assay in gastric cancer cell lines.10.The effect of LBH on peritoneal metastasis was determined by intraperitoneal injection of nude mice.11.Cell behavior and molecular pathways in which LBH may be involved were investigated by GSEA analysis in the TCGA-STAD,GSE15459 and GSE62254 datasets.12.The R software was used to calculate 1000 genes with the strongest LBH correlation(Spearman)in the TCGA-STAD dataset,and gene ontology(GO)and Kyoto Gene and Genomic Encyclopedia(KEGG)analysis were performed to study the biological processes involved in LBH.13.Western Blot was used to detect changes in molecular pathway after knockdown or overexpression of LBH.14.Using IP experiments to detect transcription factors that directly interact with LBH.15.RNA-seq and microarray related processing and analysis using R software.16.Statistical processing: Each experiment was repeated 3 times and the data was expressed as mean ± standard deviation(±s.d.).T test,spearman rank correlation test and Fisher exact calculation probability method were performed by SPSS 18.0 statistical software.P<0.05 was statistically significant.Results: 1.In the TCGA-STAD and GSE29272 datasets,the expression level of LBH mRNA in gastric cancer tissues was significantly higher than that in normal GC tissues.2.xIn the TCGA-STAD dataset,LBH expression was associated with T stage.In the GSE15459 dataset,LBH expression was associated with later stage,invasion Lauren classification and diffused subtype.In the GSE62254 dataset,LBH was associated with later T,N,and M stage.3.In the TCGA-STAD,GSE15459,and GSE62254 datasets,patients with LBH high expression group had a shorter overall survival.The COX multivariate proportional hazard model showed LBH is an independent risk factor for OS.4.Immunohistochemical analysis of 82 patients showed that the expression level of LBH in GC tissues was significantly higher than that in normal gastric tissues.According to the expression level of LBH protein,the GC patients were divided into LBH high expression group and LBH low expression group.The results showed that the prognosis of high LBH expression group was worse than that of LBH low expression group(P=0.012).5.The expression of LBH was detected in various gastric cancer cell lines.qRT-PCR results showed that LBH was the highest in MKN-45 cells,followed by HGC-27 cells,and lowest in BGC-823 cells.Western Blot results showed that LBH was highly expressed in HGC-27 cells and MKN-45 cells,and was not expressed in BGC-823 cells.6.Transfection of shRNA with lentivirus to establish a HGC-27 cell line stably knocking down LBH.The BGC-823 cell line stably transfected with LBH was established by transfecting the LBH overexpression plasmid with lentivirus.Both Western Blot and qRT-PCR showed good knockdown and overexpression efficacy of LBH.Compared with HGC27-NC-shRNA cells,the proliferation ability of HGC27-LBH-shRNA cells were reduce,and the migration and invasion ability were significantly weaken.8.Compared with BGC823-vector cells,the proliferation,migration and invasion ability of BGC823-LBH cells increased significantly.9.The intraperitoneal implantation of nude mice showed that the number of intraperitoneal nodules of BGC823-LBH increased significantly,and the intraperitoneal nodules of MKN-45-shRNA were reduce significantly,suggesting that LBH can promote peritoneal metastasis of gastric cancer.10.The GESA results in the TCGA-STAD,GSE15459,and GSE62254 datasets revealed high consistency of the three datasets,suggesting high expression of LBH related with "Focal adhesion","Extracellular matrixreceptor interactions" and " Cell-adhesion molecule cams” pathways.11.In the TCGASTAD dataset,GO and KEGG analysis of 1000 genes with the highest correlation with LBH indicated that high LBH expression is associated with PI3K-Akt pathway,focal adhesion and extracellular matrix-receptor interaction.12.Western Blot analysis showed that knockdown of LBH significantly inhibited the expression of integrin α1,integrin α5,integrin αV,integrin β1,integrin β3,while overexpression of LBH significantly increased integrin α1,integrin α5 Expression of integrin αV,integrin β1,integrin β3.13.Western Blot analysis showed that knockdown of LBH inhibited the expression of p-FAK and pAkt downstream of integrin,but did not alter the expression of FAK and Akt.Overexpression of LBH promoted the expression of p-FAK and p-Akt,but did not affect the expression of FAK and Akt.14.Western Blot analysis showed that knockdown of LBH inhibited the expression of NRP1,and overexpression of LBH promoted the expression of NRP1.15.Co-immunoprecipitation experiments showed that LBH can bind to the transcription factor c-jun,and inhibition of c-jun by SP600125 significantly inhibited the up-regulation of NRP1 caused by overexpression of LBH.Conclusion: 1.The expression of LBH in gastric cancer tissues is higher than that in normal tissues.2.LBH is associated with poor prognosis in gastric cancer.3.LBH promotes the proliferation of gastric cancer cells.4.LBH promotes migration and invasion of gastric cancer cells.5.LBH promotes peritoneal metastasis of gastric cancer.6.LBH is involved in the regulation of the integrin/FAK/Akt pathway.7.LBH is involved in the regulation of the c-jun/NRP1 pathway.