Effects Of Knockdown Of STAT3 On Autophagy And Radiosensitivity Of Esophageal Cancer Cells

Objectives:1.To explore the expression and clinical significance of sequestosome-1(P62),Microtubule-associated protein light chain 3(LC3)and Signal transduction and activators of transcription factor(STAT)3 in esophageal squamous cell carcinoma.2.The effect on proliferation,migration,invasion and the expression of P62 and LC3 in esophageal cancer EC 109,TE1,KYSE150 cells by knocking-down STAT3.3.To study whether knocking down STAT3 can increase the sensitivity of ionizing radiation in esophageal cancer EC109,TE1 and KYSE150 cells.Methods:1.Collected 48 cases of esophageal squamous cell carcinoma tissues and adjacent tissues after radical resection in the Affiliated Hospital of North Sichuan Medical College from July 2018 to April 2019.The real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)and Western blot were used to detect the mRNA and protein expression of P62,LC3 and STAT3 in esophageal squamous cell carcinoma tissues and adjacent tissues.2.Analyzed the relationship between the expression of STAT3,P62,LC3 and clinicopathological characteristics and the correlation among of the three.Through telephone follow-up to obtain the patient’s survival time.3.Western blot was used to detect the expression of Janus kinase 2(JAK2)/STAT3 pathway protein,P62 and LC3 in esophageal cancer EC 109,TE1,KYSE150 cells and normal esophageal epithelial Het-1A cells.4.Transfected esophageal cancer EC 109,TE1 and KYSE150 cells with knockdown plasmids(ShSTAT3-1,ShSTAT3-2)and empty plasmids(ShNC),knocking down STAT3 in those cells,RT-qPCR was used to detect knockdown efficiency,Western blot was used to test the expression of LC3,P62,STAT3,JAK2 and phosphorylation signal transducer and activator of transcription 3(p-STAT3)in the ShSTAT3-group and the ShNC-group.5.The Cell Counting Kit-8(CCK-8)Assay and the Colony Formation Assay were used to detect the proliferation of EC 109,TE1 and KYSE150 cells after knocking down STAT3.Wound-Healing Assay and Transwell Assay were used to detect the effect of knocking down STAT3 on the migration and invasion of EC 109,TE1 and KYSE150 cells.6.The three transfected esophageal cancer cell lines were divided into ShSTAT3-group,radiotherapy+ShNC group and radiotherapy+ShSTAT3group.The radiotherapy dose was 6GY.Western blot was used to detect the expression of LC3,P62,STAT3,JAK2 and p-STAT3 in the three groups.7.Cell Counting Kit-8(CCK-8)Assay and Colony Formation Assay were used to test the effect of knocking-down STAT3 on the proliferation of EC 109,TE1 and KYSE150 cells after radiotherapy.Wound-Healing Assay and Transwell Assay were used to measure the influence of knocking-down STAT3 on the migration and invasion of EC109,KYSE150 and TE1 cells after radiotherapy.The data were analyzed by χ2 test,t test and Spearman correlation analysis method.Results:1.The positive expression rate of P62 and STAT3 in esophageal squamous cell carcinoma tissue were higher than that in adjacent tissues(62.5%to 37.5%,66.7%to 33.3%,P<0.05).The positive expression rate of LC3 in esophageal squamous cell carcinoma tissue was lower than that of adjacent tissues((35.4%to 64.6%,P<0.05);the expression levels of P62 and STAT3 in esophageal squamous cell carcinoma tissues were higher than those of adjacent tissues(P<0.05),the expression level of LC3 was lower than that of adjacent tissues(P<0.05).2.The expression of STAT3 was positively correlated with theespression of P62(rs=0.438,P<0.05)and negatively correlated with the expression of LC3(rs=-0.400,P<0.05),The expression of P62 was negatively correlated with the expression of LC3(rs=-0.585,P<0.01).The expression of P62,LC3 and STAT3 were closely related to tumor T staging,clinical staging and lymph node metastasis(P<0.05).3.Survival analysis shows that high expression of STAT3 and low expression of LC3 are closely related to the poor prognosis of patients(P<0.05).4.The expression of JAK2/STAT3 pathway protein and P62 in esophageal cancer EC109,TE1 and KYSE150 cells was significantly higher than those in normal esophageal epithelial HET1A cells(P<0.05),the protein expression of LC3 was significantly lower than that in HET-1A cells(P<0.05).5.Knocking down STAT3 in EC109,TE1 and KYSE150 cells,the expression level of JAK2/STAT3 pathway protein and P62 in the ShSTAT3 group were lower than those in the ShNC group,and the protein expression level of LC3 was higher than that in the ShNC group(P<0.05).6.The abilities of proliferation,migration and invasion in the ShSTAT3 group of the three cell lines were significantly lower than those in the ShNC group(P<0.05).7.Among the three cell lines,the expression level of JAK2/STAT3 pathway protein in the radiotherapy+ShSTAT3 group were higher than those in the ShSTAT3 group,and lower than those in the radiotherapy+ShNC group;the protein expression of P62 was gradually decreased in the ShSTAT3 group,the radiotherapy+ShNC group and the radiotherapy+ShSTAT3 group,while the protein expression of LC3 gradually increased in the ShSTAT3 group,radiotherapy+ShNC group and radiotherapy+ShSTAT3 group(P<0.05).8.In the three cell lines,the cell proliferation,migration and invasion ability of the ShSTAT3 group,radiotherapy+ShNC group and radiotherapy+ShSTAT3 group were gradually decreased(P<0.05).Conclusions:1.STAT3 may participate in the progression of esophageal squamous cell carcinoma by inhibiting autophagy.2.The expression of STAT3 and LC3 is closely related to the overall survival of esophageal squamous cell carcinoma patients.3.Knockdown of STAT3 can inhibit the activity of JAK2/STAT3 pathway,activate autophagy,and hinder the growth of esophageal cancer cells.4.Knockdown of STAT3 can increase the sensitivity of esophageal cancer cells to radiotherapy.