The Mechanism Of WNT4 Inhibiting The Progression Of Esophageal Cancer

BackgroundEsophageal cancer(ESC)is a common malignancy in the digestive system.There were about 604,000 new cases and 544,000 deaths worldwide in 2020,and the 5-year overall survival rate was only 15%-25%.The number of new cases of ESC in China in 2020 was 324,000,and there were obvious regional differences,among which the high incidence areas were concentrated in the Taihang Mountains and northern Jiangsu regions.Esophageal squamous cell carcinoma(ESCC)and adenocarcinoma(AC)are the major histopathological types of ESC.A recent study found that Abnormal activation of NF-κB signaling pathway participates in the pathogenesis of malignant tumors by promoting the cellular proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT).The WNT4 protein belongs to one of the 19 members of the human WNT protein family and has been demonstrated to participate in the development of several malignancies by activating or inhibiting the canonical and non-canonical WNT signaling pathways.It has been reported that the WNT4 gene suppresses the development of osteoporosis by inhibiting the NF-κB signaling pathway,but it is unclear whether there is a correlation between them during the development of ESC.Therefore,this study will reveal the role of WNT4 in ESC lesions and whether it acts through regulating NF-κB signaling pathway,which help to enrich the theoretical basis of ESC pathogenesis and may provide new biomarkers and drug targets for the early diagnosis and treatment of ESC.ObjectiveTo explore the expression of WNT4 gene in ESC tissues and the effect of WNT4 gene expression changes on ESC progression;to explore whether WNT4 gene regulates NF-κB signaling during the development ESC.Method1.To detect the expression levels of WNT4 protein in ESC tissues(1)Western Blot was used to detect the difference in WNT4 protein expression level between ESC tissues and paired adjacent tissues.(2)ESC related data in TCGA database were mined,and the differences in the transcription levels of WNT4 gene between ESC tissues and normal esophageal tissues,in ESC tissues with different clinical grading and TNM stages and in tissues from survival and death cases were compared.The relationship between WNT4 gene transcription level and the overall survival of ESC patients was analyzed,and the survival curve was drawn.2.ESC cell lines with stable knockdown of WNT4 gene were constructed,and the changes in their proliferation and migration abilities were detected(1)ESC cell lines EC9706 and Eca-109 were infected with LV3 lentivirus targeting WNT4 gene(WNT4-1057-LV3)and control virus(LV3-NC),respectively,and then screened by puromycin for 10 days.The clones with the reduction of WNT4 gene in m RNA and protein levels were considered as successful ones.(2)The effects of WNT4 knockdown on the proliferation,colony formation,migration and tumorigenesis of EC9706 and Eca-109 cells in vitro and in vivo were detected.3.ESC cell lines stably overexpressing WNT4 gene were constructed,and the changes in their proliferation and migration abilities were detected.(1)ESC cell lines EC9706 and Eca-109 were infected with LV5 lentivirus that can overexpress human WNT4 gene(HOMO-WNT4-LV5)and control virus(LV5-NC)respectively,and then screened by puromycin for 10 days.The clones with the overexpression of WNT4 gene in m RNA and protein levels were considered as successful ones.(2)The effects of WNT4 overexpression on the proliferation,colony formation,migration and tumorigenesis of EC9706 and Eca-109 cells in vitro and in vivo were detected.4.The effect of stable knockdown or overexpression of WNT4 gene on the signal transduction ability of canonical WNT pathway in ESC was detected.(1)q RT-PCR and Western Blot were used to detect the effect of knockdown or overexpression of WNT4 gene in ESC on the m RNA and protein levels of key genes in canonical WNT pathway in ESC,including CTNNB1(encoding β-catenin protein)gene and some downstream target genes such as MYC,CCND1 and CD44.(2)Since Li Cl could induce an increase in the transcriptional activity of TCF/LEF,the effect of WNT4 overexpression on the transcriptional activity of TCF/LEF was further examined using the TCF/LEF luciferase reporter assay.5.The correlation between WNT4 gene and NF-κB signaling pathway-related genes expression levels in ESC tissues was analyzed.The data of ESC in TCGA database were mined to analyze the correlation between WNT4 gene and some key target genes of NF-κB signaling pathway,such as TNFA,CCL2,IL6,CXCL10 and ICAM1,at m RNA level.6.The effect of WNT4 gene knockdown or overexpression on TNF-α induced NF-κB signaling pathway was detected.(1)EC9706 and Eca-109 cells with WNT4 gene knockdown were treated with TNF-α to activate the NF-κB signaling pathway.The phosphorylation of IκBα protein(p-IκBα)was detected by Western Blot.The m RNA levels of the downstream target genes of NF-κB signaling pathway including TNFA,MCP1,IL6,CXCL13 and LIF were detected by q RT-PCR.(2)The above experiments were repeated in EC9706 and Eca-109 cells with the overexpression of WNT4 gene.7.The effect of WNT4 gene overexpression on the transcriptional activity of NF-κB induced by TNF-αwas detected by luciferase reporter assay.Result1.The WNT4 gene was downregulated in ESC tissuesThe expression level of WNT4 protein in ESC tissues and paired adjacent normal tissues was tested by Western Blot.The results showed that WNT4 protein level was significantly reduced in ESC tissues compared with adjacent normal ones.In addition,the m RNA level of WNT4 gene was significantly decreased in ESC tissues compared with normal tissues after analyzing ESC-related dataset in TCGA.2.Correlation between the m RNA levels of WNT4 gene and clinicopathological parameters of ESCThe analysis of ESC-related dataset in TCGA showed that there was no significant difference in the m RNA level of WNT4 gene in ESC tissues with different clinical grading.WNT4 m RNA levels were decreased in T3 and T4 ESC tissues compared with T1(tumor ≤ 3cm without peripheral metastasis)and T2ones(tumor ≥ 3cm with hilar spread with atelectasis).In addition,the WNT4 m RNA levels were relatively low in ESC tissues with lymph node involvement(N1/N2/N3)compared with those without lymph node involvement.There was no significant difference in WNT4 m RNA levels between ESC tissues without distant metastasis(M0)and those with distant metastasis(M1).In addition,the m RNA level of WNT4 gene was relatively low in ESC tissues from patients aged ≥ 65 years.3.WNT4 suppressed the proliferation and migration abilities of ESC cells in vitro and the tumorigenic ability in vivoEC9706 and Eca-109 cells were infected with WNT4-LV5 or its negative control(NC-LV5)to establish stable WNT4 overexpression cell lines.q RT-PCR and Western Blot results revealed that WNT4 gene was obviously increased at both m RNA and protein levels.The CCK8 cell proliferation assay,colony forming assay and scratch assay were shown that overexpression of WNT4 inhibited the proliferation,colony formation and migration of EC9706 and Eca-109 cells in vitro.The m RNA levels of genes related to cellular proliferation and migration,such as CCNE1,N-cadherin,ROCK1,CDK2,CDK4 and CDK6,were decreased in different degrees in WNT4 overexpressing ESC cells.In addition,to explore the effect of WNT4 overexpression on the tumorigenic ability of ESC cells in vivo,WNT4-LV5 or control EC9706 cells were injected into the axillary and inguinal subcutaneous tissues of nude mice,respectively.The results showed that WNT4 overexpressing EC9706 cells grew slower and formed smaller tumors,indicating that overexpression of WNT4 gene could inhibit the growth rate of ESC cells in vivo.In contrast,EC9706 and Eca-109 cells were infected with WNT4-LV3 or its negative control(NC-LV3)to establish stable WNT4 knockdown cell lines.The results showed that knockdown of WNT4 gene promoted the proliferation,colony formation and migration of EC9706 and Eca-109 cells in vitro.The m RNA levels of CCNE1,N-cadherin,ROCK1,CDK2,CDK4 and CDK6 genes in EC9706 and Eca-109 cells were increased,and the growth rate of ESC cells was faster in vivo.4.WNT4 inhibited signal transduction of the canonical WNT pathway in ESC cellsTo elucidate the underlying mechanism of WNT4 overexpression in inhibiting ESC progression,we examined the canonical WNT signaling in EC9706 and Eca-109 cells.The results from q RT-PCR and Western Blot showed that overexpression of WNT4 gene inhibited the m RNA and protein levels of the CTNNB1(encoding β-catenin protein)gene and some main downstream target genes,such as MYC,CCND1 and CD44.In contrast,inhibiting WNT4 gene had the opposite effect.This indicates that WNT4 is able to interfere with the normal signal transduction of the canonical WNT pathway.Subsequently,TCF/LEF luciferase reporter assay was used to evaluate the effect of WNT4 overexpression on the transcriptional activity of TCF/LEF.Li Cl treatment increased the TCF/LEF transcriptional activity,whereas overexpression of WNT4 gene could antagonize Li Cl-induced upregulation of TCF/LEF transcriptional activity,indicating that WNT4 could inhibit the signal transduction of the canonical WNT pathway by interfering with the transcriptional activity of TCF/LEF.5.The m RNA levels of WNT4 gene was negatively correlated with the m RNA levels of target genes in NF-κB signaling pathwayWe analyzed the m RNA levels of WNT4 gene and NF-κB signaling pathway target genes including TNFA,CCL2,IL6,CXCL10,ICAM1 in 251 ESC tissues after mining TCGA data.The results showed that the m RNA levels of WNT4 gene were significantly negatively correlated with these target genes,suggesting that WNT4 also inhibits the NF-κB signaling pathway.6.WNT4 inhibited TNF-α-induced signal transduction of the NF-κB signaling pathwayTo clarify the regulatory effect of WNT4 on NF-κB signaling pathway,we activated the NF-κB signaling pathway in EC9706 and Eca-109 cells by TNF-α treatment.Western Blot results showed that the expression of p-IκBα protein was significantly increased after TNF-α treatment,indicating that the NF-κB signaling pathway was activated,and simultaneous inhibition of WNT4 gene expression further up-regulated the expression of p-IκBα protein.Moreover,knockdown of WNT4 gene could further upregulate the m RNA levels of TNFA,MCP1,IL6,CXCL13 and LIF induced by TNF-α.In contrast,overexpression of WNT4 not only inhibited TNF-α induced p-IκBα protein expression,but also reduced TNF-α induced increase in the m RNA levels of TNFA,MCP1,IL6,CXCL13 and LIF genes.The result of NF-κB luciferase reporter assay also revealed that overexpression of WNT4 could significantly inhibit the NF-κB transcriptional activity induced by TNF-α,which indicates that WNT4 decreased the expression of its downstream target genes by interfering with the transcriptional activity of NF-κB.7.WNT4 enhanced the sensitivity of ESC cells to cisplatinTo test whether changes in WNT4 gene expression could affect the sensitivity of ESC cells to chemotherapeutic drugs,WNT4 overexpressing EC9706 and Eca-109 and corresponding controls were treated with different concentrations of cisplatin,and the cell viability was measured by CCK-8 assay.The result revealed that the WNT4 overexpressing ESC cell lines were more sensitive to cisplatin treatment.On the contrary,both WNT4 knockdown ESC cell lines were more resistant to cisplatin than control ones.These results indicated that WNT4 could enhance the sensitivity of ESC cells to cisplatin treatment.In addition,q RT-PCR and Western Blot results showed that WNT4 overexpressing ESC cell lines had higher m RNA and protein levels of apoptosis-related genes when treated with the same concentration of cisplatin,and knockdown of WNT4 gene showed the exactly opposite results.These results suggest that knockdown of WNT4 may induce chemoresistance in ESC cells.Conclusion1.The expression of WNT4 gene was lower in ESC tissues and negatively correlated with clinical T stage.2.Overexpression of WNT4 gene could reduce the malignant phenotype of ESC in vitro and in vivo,while knockdown WNT4 gene had the opposite effect.3.WNT4 could inhibit the canonical WNT signaling pathway in ESC cells.4.WNT4 can inhibit TNF-α induced NF-κB signaling pathway in ESC cells.5.WNT4 can enhance the sensitivity of ESC cells to cisplatin.