The Study On The Function And Mechanism Of EGFL6 In Esophageal Cancer

Objective: 1.To study the expression level of EGFL6 in Esophageal Cancer(EC)patients and its relationship with clinicopathological data.2.To study the effect of EGFL6 silencing on proliferation,invasion,migration,and apoptosis of EC cells.3.To study the effect of EGFL6 overexpression on proliferation,invasion,migration,and apoptosis of EC cells.4.To explore the mechanism of inducing and promoting proliferation,invasion and migration of EC cells.Methods: 1.Collect the First Hospital of Shanxi Medical University,in 2017-2019 in patients with EC tissues and adjacent non-tumor tissues(ANT)specimens,Comparison of EGFL6 expression in different tissues by immunohistochemical staining,and the correlation between the expression level of EGFL6 and clinicopathological data and survival was analyzed in combination with the clinicopathological data of EC patients.The TCGA(The Cancer Genome Atlas)database was used to detect the expression level of EGFL6 gene in human EC tissues.2.The expression of EGFL6 in normal esophageal epithelial cell line(Het-1A)and different EC cell lines(EC9706,KYSE150,KYSE450)was detected by Real-time PCR and Westernblot.3.The KYSE450 cell line with high EGFL6 gene expression and the KYSE150 cell line with low expression were taken as research objects,and EGFL6 gene silencing and overexpression EC cell lines were constructed by transfecting EGFL6 small interfering RNA and overexpression plasmids.KYSE450 cells were divided into 3 groups: blank group(blank),control group(si NC)and small interference group(si RNA);KYSE150 cells were divided into 2 groups: empty plasmid group(Pc DNA3.1)and EGFL6 overexpression group(Pc DNA3.1 + EGFL6),detect transfection efficiency by Real-time PCR and Western-blot method.4.Detect the effect of EGFL6 silencing and overexpression on the proliferation of EC cells through CCK8 assay and Colony formation assay.5.Through Wound healing assays and transwell assays to detect the effect of EGFL6 silencing and overexpression on the invasion and migration of EC cells.6.Detect the effect of EGFL6 on apoptosis of EC cells by flow cytometry,and use real-time PCR and Western-blot to detect the changes of apoptosis-related markers(Bcl-2,Bax,and Casepase3).7.Detect the effect of EGFL6 silencing and overexpression on EMT related markers(Ecadherin,Vimentin,Snail,Fibronectin,Twist,and N-cadherin)by real-time PCR and Western-blot method.8.Detect the effects of EGFL6 silencing and overexpression on esophageal cancer related stem cell markers(Bmi1,OCT,SOX2,NANOG,and CD44)by real-time PCR.9.Western-blot method was used to detect the effect of EGFL6 on the expression of key proteins(p-?-catenin,?-catenin,GSK3?,and c-myc)in the Wnt/?-catenin signaling pathway.10.Animal experiment: Nude mice were injected subcutaneously with sh EGFL6-KYSE450 group and sh NC-KYSE450 group cells to establish a nude mouse EC transplantation tumor model,and the volume and quality of the transplantation tumor were measured and statistically analyzed.Results: 1.The expression level of EGFL6 protein in EC tissues is significantly higher than that in adjacent non-tumor tissues(ANT);The expression level of EGFL6 protein is related to the size of EC tumor(P=0.042),T classification(P=0.014),lymph node metastasis(P=0.002),distant metastasis(P < 0.001)and cancer differentiation degree(P=0.01),but not to the patient's age(P=0.541)and gender(P=0.496).TCGA database shows that the expression of EGFL6 gene in human EC tissue is obviously high.The overall survival rate of patients with high expression of EGFL6 protein in EC tissue is lower than that of patients with low expression of EGFL6 protein(P <0.05).2.The expression of EGFL6 in esophageal epithelial cells(Het-1A)is significantly lower than that in EC cells(EC9706,KYSE150,and KYSE450),and EGFL6 is expressed highest in KYSE450 cell line and relatively lower in KYSE150 cell line.3.CCK-8 assay showed that when EGFL6 gene was silenced,the activity of EC cells was significantly reduced compared with the control group.When EGFL6 gene is overexpressed,the activity of EC cells is significantly enhanced compared with the control group.4.The colony formation assay showed that when EGFL6 gene was silenced,compared with the control group,EC cells formed significantly fewer clone masses.When EGFL6 gene is overexpressed,compared with the control group,the number of clonal masses formed by EC cells is significantly increased.5.Wound healing assays and transwell assays showed that when EGFL6 gene was silenced,the invasion and migration ability of EC cells was significantly reduced compared with the control group.When EGFL6 gene was overexpressed,the invasion and migration ability of EC cells is significantly enhanced compared with the control group.6.Flow cytometry showed that the apoptosis of EC cells was significantly increased when EGFL6 was silent,and significantly decreased when EGFL6 was overexpressed.Real-time PCR and Western-blot results were consistent with flow cytometry.7.Real-time PCR and Western-blot detection of EMT-related markers show that EGFL6 silencing inhibited the conversion of EC cells to EMT and increased the expression of EMTrelated marker E-cadherin.Vimentin,Snail,Fibronectin,Twist,and N-cadherin protein expression decreased;Overexpression of EGFL6 promotes the transition of EC cells to EMT,and the expression of EMT-related marker E-cadherin decreases.Vimentin,Snail,Fibronectin,Twist,and N-cadherin protein expressed higher.8.Real-time PCR was used to detect the expression of esophageal cancer stem cell related genes when EGFL6 gene was silenced and overexpressed.The results showed that when EGFL6 gene was silenced,the expression of esophageal cancer stem cell gene is lower,and when EGFL6 gene was overexpressed,the expression of esophageal cancer stem cell gene is increased,indicating that EGFL6 may maintain the expression of esophageal cancer stem cell-like cell population.9.Western-blot assays the expression of the marker genes P-?-catenin,?-catenin,GSK3?,and c-myc of WNT /?-catenin signal.The results showed that in KYSE450 cells,compared with si NC group,the expression of p-?-catenin,total ?-catenin,and c-myc protein in si RNA group is down-regulated,and the expression of GSK3? protein is up-regulated.In KYSE150 cells,compared with Pc DNA3.1 group,In Pc DNA3.1+EGFL6 group,the expression of p-?-catenin,total ?-catenin and c-myc protein is up-regulated while the expression of GSK3? protein is down-regulated.10.The subcutaneous tumor model of nude mice is successfully established.Compared with sh NC-KYSE450 group,the subcutaneous tumor of sh EGFL6-KYSE450 group is significantly smaller in volume and lower in quality,which is consistent with our results in cell experiments.Conclusion: 1.The expression of EGFL6 in EC tissue is significantly higher than that in adjacent nontumor tissues,and it is related to poor prognosis of patients.2.EGFL6 can promote the proliferation,invasion and migration of EC cells and inhibit the apoptosis of EC cells.3.The effect of EGFL6 on proliferation,invasion and migration of EC cells may be related to EGFL6 inducing EMT of tumor cells and maintaining the characteristics of cancer stem cells,and Wnt/?-catenin signaling pathway may participate in the whole process.4.Knocking EGFL6 out of EC cells can inhibit the growth of subcutaneous transplanted tumor in nude mice.