| Background:Lung cancer is the most commonly diagnosed and cancer-related death in men,and breast cancer is the most commonly diagnosed and cancer-related death in women.Although in recent years significant progress has been made in delineating the moleclular pathogenesis of lung cancer and breast cancer,and clinical treatment techniques,such as surgery and chemotherapy,have been imporoved,the prognosis of patients still remains unoptimistic.Homeo box genes are a family of genes that share highly conserved structure while maintaining a high degree of diversity.Its conserved sequences encode proteins containing homologous domains that are capable of binding DNA which endow them with the ability to envolve extensively in the process of embryos,tissues and organs development and human diseases including tumor.We have previously identified two down-regulated homeobox genes: LHX6 and ALX4 in lung cancer and breast cancer.However,the clinical significance and indepth mechnasim remains unclear in both lung cancer and breast cancer.In this study,we first analyzed the prognostic significance of LHX6 and ALX4 in lung cancer and breast cancer patients,and then the function and regulation mechanism of LHX6 and ALX4 were investigated in tumorigenesisContents:1.Analysis the prognostic significance of LHX6 and ALX4 in lung cancer and breast cancer patients.Using the Kaplan-Meier method,we analyzed the prognostic significance of LHX6 and ALX4 in lung cancer and breast cancer patients in the public database(www.kmplot.com/ analysis.and https://genome-cancer.soe.ucsc.edu.).Moreover we applied TMA methods to further confirm the prognostic results2.The expression and clinincal significance of LHX6 in lung adenocarcinoma and ALX4 in breast cancer.First,the expression pattern of LHX6 in lung adenocarcinoma and ALX4 in breast cancer in was examined by RT-PCR,RT-qPCR,WB,IHC and bioinformatics methods.Furthermore,the relation between gene expression and clinical parameters such as prognosis,histological grade,stage and lymph node status was evaluated using TMA data and public data base.3.Expression regulation,function and mechanism study of LHX6 in lung adenocarcinomaThe MSP,de-methylation treatment and bioinformatics methods were applied to analyze relation between LHX6 promoter methylation and expression in lung adenocarcinoma.The function of LHX6 in lung adenocarcinoma was investigated in vitro by MTS,colony formation,transwell assay and in vivo nude mice tumor formation.The effect of LHX6 on cell cycle was detected by PI single staining.The effects on Wnt/?-catenin pathway were detected by qPCR,WB and TOP/FOP Flash methods,and the luciferase reporter vector containing different CTNNB1 promoter regions were constructed to analyze the impact of LHX6 on their transcriptional activity.Finally,the above results were further verified using the TCGA database.4.Expression regulation,function and mechanism study of ALX4 in breast cancerThe MSP and BSP methods were used to detect the methylation status of ALX4 promoter region in 8 normal breast tissues,3 breast cancer cell lines and 108 breast cancer tissues.The relationship between ALX4 expression and promoter methylation was analyzed by using de-methylation treatment and bioinformatics method(www.cbioportal.com).The function of ALX4 in breast cancer cells was further investigated in vitro by MTS,colony formation,EDU assay,Transwell assay and in vivo nude mice tumor formation methods.The effects of ALX4 on cell apoptosis and cell cycle were detected by V-APC/7-AAD double staining and PI single staining.The alteration of Wnt/?-catenin signaling pathway was first analyzed by bioinformatics and further by TOP/FOP Flash,qPCR and WB.The regulation mechanism of ?-catenin by ALX4 was analyzed by luciferase reporter assay and WB methods.Results:1.LHX6 is an independent favorable prognostic factor for lung adenocarcinoma patients and ALX4 is an independent favorable prognostic factor for breast cancer patients respectively.Kaplan-Meier analysis showed that LHX6 was a favorable prognostic marker in lung adenocarcinoma patients(N = 720,P <0.01),but had no significant correlation with survival outcome in breast cancer patients(N = 1402,P = 0.24).ALX4 was a favorable prognostic marker marker in breast cancer patients(N = 1080,P = 0.00),but had no significant correlation with survival outcome in lung cancer patients(N = 997,P = 0.67).Furhter using the Cox-regression analysis we demonstrated that LHX6 was an independent favorable prognostic marker in lung adenocarcinoma patients(N = 88,P = 0.01 and ALX4 was an independent favorable prognostic marker in breast cancer patients(N = 142,P = 0.01).2.LHX6 and ALX4 were down regulated in lung adenocarcinoma and breast cancer,respectively,and were significantly associated with clinical progressionExpression profile analysis showed that LHX6 was down regulated in lung adenocarcinoma and ALX4 was down regulated in breast cancer.Further analysis showed that LHX6 expression was significantly associated with histological grade(N = 88,P = 0.00),tumor size(N = 88,P = 0.02)and lymph node status(N = 84,P = 0.03)in lung adenocarcinoma patients.ALX4 expression was significantly associated with clinincal stage(N = 140,P = 0.03),tumor size(N = 141,P = 0.01)and lymph node status(N = 138,P = 0.00)in breast cancer patients and its expression could be used as a specific and sensitive marker in distinction between noemal and breast cancer patients(Area under the curve = 0.874,P <0.01,95% Cl = 0.84-0.90).3.LHX6 was methylation silenced in lung adenocarcinoma and inhibited lung adenocarcinoma cell proliferation and metastasis by interfering Wnt/?-catenin signaling pathway through transcriptional silencing the expression of ?-cateninDNA methylation analysis revealed that LHX6 expression in lung adenocarcinoma was regulated by its promoter methylation.In vitro study showed that LHX6 suppressed lung adenocarcinoma cell line proliferation by G1/S blocking and metastasis.In vivo functional experiments further indicated that LHX6 inhibited the tumor formation and metastasis of LTEP-a-2 cells.The mechanism by which of LHX6 inhibited lung adenocarcinoma cell proliferation and metastasis was suppressing the Wnt/?-catenin pathway by transcriptional silencing of ?-catenin,and its promoter region(-1161 bp to + 27bp)was a key sequence for the inhibitory function of LHX6.4.ALX4 was methylation silenced in breast cancer and inhibited breast cancer cell proliferation and metastasis by interfering Wnt/?-catenin pathway through promoting the phosphorylation degradation of ?-cateninMethylation assay showed that the promoter region of ALX4 was hypermethylated in three breast cancer cell lines and 108 breast cancer tissues,while no methylation was detected in 8 normal breast tissues.The expression of ALX4 was negatively correlated with the degree of methylation of the promoter(P <0.01).In vitro and in vivo functional analysis showed that ALX4 can inhibit the proliferation of breast cancer cells by inducing cell apoptosis and G1/S blocking.Further study found that ALX4 can significantly inhibit the invasion and metastasis of breast cancer cells.Mechanism study showed that ALX4 can not regulate the expression of ?-catenin at the transcriptional level,but can induce GSK3? to promote the phosphorylation degradation of ?-catenin protein thus exerted its anti-cancer function by interfering with the Wnt/?-catenin signaling pathway.Conclusions:In summary,our study revealed that LHX6 and ALX4 are two important tumor suppressor genes with significant clinical values,and play a vital role in tumor suppression by interfering with the Wnt/?-catenin signaling through transcriptional silencing or protein degardation of ?-catenin.This study not only enriches our understanding of the molecular mechanism of lung adenocarcinoma and breast cancer,but also provides a new clue for future study of the mechanisms of LHX6 and ALX4 in other cancers. |
PDF Full Text Request