The Mechanism Of LRP5 Promoting The Progression Of Esophageal Cancer

BackgroundEsophageal cancer(EC)ranks second in esophageal cancer and is the sixth leading cause of cancer death globally.Esophageal cancer(EC)is an aggressive malignancy that usually presents in local advanced stages and has a poor prognosis.Low density lipoprotein receptor-related protein 5(LRP5)is one of the coreceptors of Wnt ligand molecules in WNT signaling pathway.It plays an essential role in the signal transduction process of Wnt/β-catenin(classic Wnt pathway).The classical Wnt pathway controls cell proliferation and differentiation and is a key regulatory factor in promoting embryonic development and maintaining tissue stability.Therefore,abnormal Wnt pathway signal transduction may lead to many diseases,including cancer.Abnormal activation of classical Wnt signaling pathway caused by abnormal LRP5 expression is closely associated with the occurrence of gastric cancer,colorectal cancer and other digestive tract related cancers.However,its role and molecular mechanism in the process of EC lesions have not been reported in the literature.Therefore,this study intends to clarify the relationship between LRP5 and EC and its molecular mechanism,which is expected to reveal the molecular mechanism of EC lesions from a new perspective,enrich the theoretical basis of EC pathogenesis,and provide new drug action targets for its treatmentObjectiveTo explore the expression changes of LRP5 in clinical EC tissue samples;To explore the influence of LRP5 expression level change on EC progression and its molecular mechanism;To explore the influence of the change of LRP5 expression level on macrophage polarization.Method1.The relationship between LRP5 gene expression level and EC occurrence was analyzed(1)Subjects: clinical EC tissues and paracancer tissues of different tumor grades.The expression of LRP5 gene was detected by Western blot.(2)Bioinformatics means were used to mine EC-related data in the TCGA database,analyze the difference in the mRNA level of LRP5 gene in EC tissues and normal esophageal tissues,and determine the correlation between the mRNA level of LRP5 gene and the age,clinical grade,stage,survival time and other clinicopathologic data of EC patients.2.Construct EC cell lines with stable overexpression or knockout of LRP5 geneTwo EC cell lines,EC9706 and Eca-109,were used to transfect LRP5 gene overexpression plasmid or CRISPR/Cas9 KO plasmid targeting LRP5 gene knockout and its negative control plasmid.Subsequently,G418 drug or purinomycin was used to screen positive cells,and monoclonal cells were selected for expanded culture.qRT-PCR and Western blot were used to identify the changes in mRNA and protein levels of LRP5 gene.The cells with the most obvious up-regulation or down-regulation of LRP5 gene were used as EC cell lines with stable overexpression or knockout of LRP5 gene for follow-up studies.3.The influence of overexpression of LRP5 gene on EC progression was identified by cell and animal experimentsThe effects of overexpression or knockout of LRP5 gene on the proliferation,invasion and migration of EC cells were investigated by CCK-8 assay,colony cloning assay,cell scratch assay and Transwell assay.The mRNA levels of genes related to cell proliferation and migration were detected by qRT-PCR.EC9706 cells with stable overexpression of LRP5 gene were inoculated subcutaneously into nude mice to explore the effect of EC9706 cells on tumor formation ability in nude mice.4.Explore whether the overexpression of LRP5 gene affects the sensitivity of EC cells to chemotherapy drugsThe EC cells overexpressing LRP5 gene and its control cells were treated with cisplatin.The survival rate of EC cells was detected by CCK-8 assay,and the mRNA and protein levels of apoptosis-related genes were detected by qRT-PCR and Western blot.5.To explore the influence of overexpression of LRP5 gene on classical Wnt signaling pathway(1)qRT-PCR and Western The mRNA and protein levels of the key genes CTNNB1(encodingβ-catenin protein),CCND1(encoding Cyclin D1)and Myc(encoding Myc protein)in the classical Wnt pathway were detected by blot technique after overexpression of LRP5 gene.(2)The classical Wnt pathway inhibitor ICRT3 was added into the EC cells overexpressing LRP5 gene and its control cells.Western blot was used to detect the changes of the active form of β-catenin protein in the classical Wnt pathway and the downstream target genes at the protein level.After ICRT3 was added,nuclear proteins and cytoplasmic proteins were separated,and Western blot was used to detect the changes of classical Wnt path-related proteins in the nucleus and cytoplasm.pGL4.49 [luc2P/TCF-LEF RE/Hygro]plasmid and LRP5 gene overexpressed plasmid were co-transfected into EC9706 and Eca-109 cells,and ICRT3 was added.The effect of overexpression of LRP5 gene on LEF/TCF transcriptional activity under normal and ICRT3 conditions was detected by single fluorescein reporting assay6.To explore the influence of overexpression of LRP5 gene on macrophage polarizationCulture medium of EC9706 and Eca-109 cells overexpressing LRP5 gene and control cells were collected to culture macrophages induced by differentiation of human monocyte THP-1 cells.qRT-PCR and Western blot were used to detect changes in mRNA and protein levels of polarization related markers in M1 and M2 macrophages.7.The effect of knockout of endogenous LRP5 gene on EC progression was identified through cell and animal experiments(1)EC cell lines with stable LRP5 gene knockout and its negative control cells were selected to explore the effects of endogenous LRP5 gene knockout on the malignant phenotype of EC cells and tumor formation ability in nude mice.(2)Cisplatin was given to detect the effect of knockout of endogenous LRP5 gene on EC cell survival rate and apoptosis related genes.(3)To detect the effect of knockout of endogenous LRP5 gene on key genes of classical Wnt pathway in EC cells;The intracellular proteins were separated and the changes of key genes of classical Wnt pathway at the protein level were detected by Western blot.The effect of LRP5 gene knockout on LEF/TCF transcriptional activity in normal or LiCl activated classical Wnt pathway was detected by single fluorescein reporting assay.(4)After knockout of endogenous LRP5 gene,changes in mRNA and protein levels of polarization related markers in M1 and M2 macrophages were detected.Results1.The expression level of LRP5 gene in EC tissue was increased(1)Compared with paracancer tissue,LRP5 gene protein expression level in EC tissue samples was significantly increased.(2)The results of TCGA data analysis showed that the mRNA level of LRP5 gene in EC tissue was significantly positively correlated with the malignant degree of patients,such as clinical grade and T stage,but had no significant correlation with N and M stage,age of onset and other factors.Overall survival was lower in EC patients with higher LRP5 mRNA levels.2.EC cell lines with stable overexpression or knockout of LRP5 gene were successfully constructedThe mRNA and protein levels of LRP5 gene were significantly increased or decreased in EC9706 and Eca-109 cell lines with stable overexpression or knockout of LRP5 gene.3.Overexpression or knockout of LRP5 gene can enhance or inhibit malignant progression of EC(1)The proliferation rate of EC cells was accelerated after overexpression of LRP5 gene.Knockout of endogenous LRP5 gene inhibited the proliferation rate of EC cells in vitro.(2)The number of EC cells cloned after overexpression of LRP5 gene increased;Knockout of LRP5 gene inhibited the formation of EC cell clones.(3)The migration rate of EC cells was accelerated after overexpression of LRP5 gene.Knocking out endogenous LRP5 gene inhibits EC cell migration.(4)The invasion ability of EC cells after overexpression of LRP5 gene was enhanced in vitro;Knockout endogenous LRP5 gene inhibited the invasion ability of EC cells.(5)Overexpression of LRP5 gene can accelerate the tumor formation rate of EC cells in nude mice;Knockout of LRP5 gene inhibits EC cell tumorigenesis in vivo.4.After overexpression of LRP5 gene,the survival rate of EC cells under the same concentration of cisplatin was higher than that of control cells.The results of qRT-PCR and Western blot showed that the mRNA and protein levels of apoptosis-related genes were decreased to varying degrees.Therefore,overexpression of LRP5 gene could reduce the sensitivity of EC cells to chemotherapy drugs.In contrast,knockout of LRP5 gene enhances EC cell sensitivity to chemotherapy drugs.5.Overexpression or knockout of LRP5 gene affects EC progression by activating or inhibiting classical Wnt signaling pathways(1)qRT-PCR results showed that mRNA levels of key genes CTNNB1,CCND1,Myc and CD44 in the classical Wnt pathway in EC cells were significantly up-regulated after overexpression of LRP5 gene.The protein expression levels of ABC,CCND1 and Myc were also significantly increased.After knockout of LRP5 gene,the mRNA and protein levels of these genes showed opposite changes.(2)The addition of the classical Wnt pathway inhibitor ICRT3 inhibited the expression levels of the Wnt pathway key proteins ABC,Cyclin D1 and Myc in overexpressed LRP5 gene and control EC cells.The expression levels of these proteins in nuclear proteins were also significantly decreased after the separation of cell proteins.Single fluorescein reporting assay results showed that overexpression of LRP5 gene enhanced LEF/TCF transcriptional activity in normal and ICRT3-inhibited states.In contrast,knockout of endogenous LRP5 gene down-regulates the expression of these proteins and reduces the transcriptional activity of LEF/TCF,but the addition of LiCl,the classical Wnt pathway activator,reverses this effect.6.Overexpression of LRP5 down-regulates the mRNA and protein levels of polarization markers in M1-type macrophages,and up-regulates the mRNA and protein levels of polarization markers in M2-type macrophages.Knocking out endogenous LRP5 has the opposite effect.Conclusion1.The expression level of LRP5 gene in EC tissues was significantly up-regulated,and its expression level was positively correlated with tumor grade and survival status of clinical case data.2.Overexpression of LRP5 gene can reduce its sensitivity to chemotherapy drugs by activating the classical Wnt signaling pathway;Macrophages were induced to become M2-type polarized and EC was promoted.3.Knockout of LRP5 gene can increase its sensitivity to chemotherapy drugs by inhibiting classical Wnt signaling pathway;Inhibition of macrophages towards M2-type polarization and inhibition of EC progression.4.Inhibition of LRP5 gene expression is expected to be a new idea for clinical treatment of EC.