Effect Of Limb-Bud And Heart On Prognosis Of Lung Adenocarcinoma Cancer And Its Mechanism

Objective: Lung cancer is one of the leading causes of cancer-related death.Non-small cell lung cancer(NSCLC)accounts for 85% of lung cancers,of which lung adenocarcinoma(LUAD)is the most common subtype.The 5-year overall survival of patients remains low.Therefore,further exploration of prognostic markers for lung cancer and potential drug targets to provide new treatments is expected to greatly enhance patient survival.Limb-bud and heart(LBH)is an important transcription cofactor involved in embryonic development.In the early stage of vertebrate embryogenesis,LBH regulates the formation of the limbs and heart.The LBH-encoded protein(molecular weight,12.3 kDa)contains a conserved nuclear localization signal and a glutamate-rich transcriptional activation domain with transcriptional co-activation and co-suppression.Abnormalities in LBH function are closely related to a variety of congenital diseases and developmental defects.A number of studies have confirmed that abnormal expression of LBH is closely related to tumorigenesis.The researchers confirmed that LBH is overexpressed in basal breast cancer,and as a novel target of Wnt signaling pathway regulates tumor development.In addition,in nasopharyngeal carcinoma and prostate cancer,overexpression of LBH significantly inhibits the proliferation and migration of tumor cells and inhibits the malignant biological behavior of tumor cells.In a study of liver cancer,high expression of LBH indicates poor prognosis in patients with liver cancer,and overexpression of LBH may contribute to the progression of liver cancer.However,the role of LBH in the development and progression of lung cancer has not been elucidated.Therefore,our study will explore the potential functions of LBH in lung cancer through bioinformatics,clinical specimens,and molecular biology experiments.To provide a theoretical basis for LBH as a prognostic marker and drug target for patients Methods: 1.The Cancer Genome Atlas(TCGA),Gene Expression Omnibus(GEO),Kaplan-Meier plotter and tissue microarray(TMA)containing 41 lung cancer tissues and paired normal lung tissue were used to compare the differences in LBH expression and clinical characteristics of lung cancer patients.2.Another 70 LUAD tissues with follow-up records were conducted to explore the correlation with clinical characteristics and prognosis of LUAD patients by Kaplan-Meier and Cox-regression analysis 3.Cell viability assay,wound healing assay,colony formation assay,transwell assay and flow cytometry were used to explore the biological function of LBH on LUAD cells in vitro.4.Bioinformatics analysis and Western were used to assess signaling pathways that may be associated with LBH.Results: 1.LBH is significantly down regulated in NSCLC and correlates with clinical characteristics in public datasets.Data from TCGA and GSE19188 indicate that LBH is down regulated in lung cancer tissues.Kaplan-Meier plotter results showed that the LBH low expression group showed lower OS values relative to the LBH high expression group(HR = 0.62,p < 0.001).2.Decreased LBH protein expression in NSCLC patients.The expression level of LBH protein in lung cancer tissues was significantly down-regulated relative to normal lung tissue.Subtype analysis showed that the expression levels of LBH in LUAD and lung squamous cell carcinoma were lower than those in matched normal tissues.Further analysis showed that LBH expression levels were significantly associated with TNM phase(P = 0.014).3.LBH protein level can indicate prognosis in LUAD patients.Chi-square test showed that LBH expression was significantly correlated with invasion depth(p = 0.048),lymph node status(p = 0.031)and TNM stage(p = 0.007)in LUAD;COX univariate analysis showed: LBH expression level(HR)=0.095,95% CI = 0.013-0.698,p=0.021)is a risk factor for patient prognosis.Further COX multivariate analysis showed that LBH expression levels(HR = 0.120,95% CI = 0.016-0.894,p=0.039)were independent risk factors for prognosis.4.LBH knockdown increases LUAD cell proliferation,migration,and invasion.After LBH was knocked down in A549 cells using siRNA,the cell proliferation,migration and invasion ability were significantly enhanced.5.LBH influences key proteins to regulate cell adhesion.Bioinformatics analysis suggests that LBH is closely related to the cell adhesion pathway.Western blot confirmed that LBH can regulate the expression levels of integrin family proteins(such as integrin-?1,integrin-?2,integrin-?4,integrin-?v and integrin-?4),but does not affect epithelial-mesenchymal transition(EMT)-related proteins.Conclusion: LBH plays an important role in lung cancer,and as an independent prognostic factor for lung adenocarcinoma,inhibits the proliferation and metastasis of LUAD cells,and may serve as a potential prognostic biomarker and therapeutic target.