| Objective:To investigate the expression of miR-503 in humanesophageal squamous carcinoma tissue samples and in the serum,and to analyze the influence of miR-503 expression on clinicopathologic correlation;To research the biological function and mechanism of miR-503 on the esophageal squamous cancer cells Eca109 and Eca9706.Methods:1.Collection from March 2014 to September 2014 in SiChuan medical university affiliated hospital the cardiac surgery resection of thoracic esophageal squamous carcinoma and adjacent tissues of 40 cases of fresh.2.To analyze the expression of miR-503 in patients with ESCC tissue,the tissue adjacent to carcinoma,expression of serum and normal serum with Taqman probe method.3.Basis On the expression of miR-503,using t test analysis of the patients with esophageal squamous cell carcinomas of gender,age,degree of tumor differentiation,T staging,and presence of the correlation between lymph node metastasis.4.Synthesis of miR-503 in vitro mimics and miR-503 inhibitor,by using combined training method transfection the esophageal squamous cancer cells Eca109,Eca9706.5.To detect level of miR-503 expression in esophageal squamous cancer cells with Taqman probe method.6.To detect esophageal squamous cancer cells Eca109,Eca9706 proliferation ability of change with CCK 8 kits.7.To detect Vimentin,E-cadherin,CyclinD1 and CyclinE1 protein in transfection of miR-503 mimics Eca109 expression level using Western Blot method.9.To detect Eca109,Eca9706 cell invasion and metastasis ability with Transwell experimentation.8.Using flow cytometry analysis the influence of miR-503 in the Eca109?Eca9706 cell apoptosis,cell cycle on stress condition.9.Using Western Blot method to detect Vimentin,E-cadherin,CyclinD1 and CyclinE1 protein in transfection miR-503 expression level of Eca109 mimics and NC group.10.Statistical analysis:all experimental data uses SPSS 17.0 statistical software package,using single factor analysis of variance between a number of indicators,with a further comparison between the two indicators using LSD-t test;Measurement data using t test to On the basis of a=0.05.Results:1.The results showed Taqman probe method,according to the results of 40 cases of esophageal squamous carcinoma tissues of miR-503 expression level was significantly higher than that of tissue adjacent to carcinoma;2.The result of t test analysis:miR-503 expression in esophageal squamous carcinoma tissue level and presence of lymph node metastasis,esophageal squamous carcinoma differentiation and TNM staging(P<0.05),and has no impact on the patient's age,sex and tumor location(P>0.05).3.The Taqman probe method results show that:the relative expression of miR-503 in MiR-503 mimics group is 180.84±3.07,compared with blank control group,expression level rise significantly(P<0.05);the relative expression of miR-503 in MiR-503 inhibitor groups is 0.14±0.02,compared with blank control group,expression level significantly lowered(P<0.01),indicating that liposome transfection success,lay a foundation for the back of the cell experiment;4.The CCK 8 experimental results:Compared with NC group,miR-503 mimics group Eca 109,Eca9706 cell proliferation and slowed(P<0.05),miR-503 cell proliferation inhibitor group speed(P<0.05).Show that miR-503 can inhibit the proliferation of esophageal squamous cancer cells.5.The result of Transwell experiment shows:miR-503 in Eca109 cells migrating mimics group average cell number was 41.3±41.3,compared with the control group decreased significantly(P<0.05).MiR-503 the average migration inhibitor group cell number was 147.2±4.8,compared with the control group significantly increased(P<0.05).MiR-503 in Eca9706 cells migrating mimics group average cell number was 37.2±2.6,compared with the control group decreased significantly(P<0.05).MiR-503 the average migration inhibitor group cell number was 93.4±3.6,compared with the control group significantly increased(P<0.05).Show that miR-503 can inhibit Eca 109,Eca9706 cell migration and invasion.6.The result of Flow cytometry test shows:Eca109 miR-503 in G0/G1 cell mimics group increased to 68.41±1.04%,S cells,reduce to 23.59±1.58%have significant difference compared with the control group(P<0.05),while proportion of G2/M phase cells have no obvious change;MiR-503 inhibitor group of G0/G1 cell proportion to reduce 44.61 ± 1.15%,S cells increased to 47.39± 1.32%,with significant difference compared with the control group(P<0.05),whereas did not change significantly G2/M phase cells proportion;In Eca9706 cells also showed a trend of the same results,show that miR-503 has blocked the esophageal squamous cancer cells Eca109,Eca9706 in G1 phase function.7.The result of Western blot shows:the Eca109 transfection miR-503 mimics cell groups compared with NC group reduced expression of Vimentin,E-cadherin higher expression,CyclinDl and CyclinEl protein expression levels.This showed that miR-503 can be suppressed by promoting the expression of E-cadherin and Vimentin expression to regulate Eca109 invasion and metastasis of esophageal squamous cancer cells,can inhibit the expression of CyclinD1 and CyclinEl adjust the cell proliferation of Eca109 esophageal squamous cancer cells.Conclusion:1.MiR-503 expressed in esophageal squamous carcinoma tissue,the tissue adjacent to carcinoma low expression;MiR-503 expression in esophageal squamous carcinoma tissue level and presence of lymph node metastasis,esophageal squamous carcinoma differentiation and T staging,and has no impact on the patient's age,sex and tumor size;2.MiR-503 expressed in esophageal squamous carcinoma tissue,the tissue adjacent to carcinoma low expression;MiR-503 expression in esophageal squamous carcinoma tissue level and presence of lymph node metastasis,esophageal squamous carcinoma differentiation and TNM staging,and has no impact on the patient's age,sex and tumor size. |
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