| Objective: To explore the expression of micro RNA(Micro RNA) 26a(miR-26a) and MTDH of esophageal squamous cell carcinoma(ESCC). To verify the regulatory relationships between miR-26 a and MTDH, and to explore whether the miR-26a/MTDH pathway could become one of the molecular targets for diagnosis and treatment of esophageal cancer.Methods:(1) In situ Hybridization(ISH) analysis was performed to detected the expression of miR-26 a in ESCC using tissue array contained 86 cases of ESCC tissues [including 78 paired normal adjacent tissues(NAT) ] in tissue levels.Analyzed the relationship of the expression of miR-26 a with clinicopathological parameters(age, sex, pathological grade, differentiation, lymph node metastasis).The relationship between miR-26 a expression and prognosis of patients was analyzed.Real-time quantitative PCR(qRT-PCR) was performed to detect the basic miR-26 a expression in six kinds of esophageal cancer cell lines and human esophageal epithelial cells(HEEC). Cells were transfected with miR-26 a, mi R-26a-inhibitor and miR-Con lentiviral. qRT-PCR was used to detect the expression levels of miR-26 a after lentivirus transfection.MTT assay, cell migration assay, cell cycle and apoptosis assay were used to detect cell proliferation, migration ability. We also measured the apoptosis cell death assay using flow cytometic analysis.(2) Immunohistochemistry(IHC) was performed to examine MTDH expression using an ESCC tissue array consisting of 86 ESCC and 78 paired normal adjacent tissues(NAT). The clinicopathological parameters(age, sex, pathological grade, differentiation, lymph node metastasis) were analyzed. The relationship between the expression of MTDH and prognosis were analyzed.Meta-analysis was used to analyze the MTDH expression in squamous cell carcinoma(SCC) and its clinical pathological significance.(3) The expression of MTDHin squamous cell carcinoma(SCC) and clinical pathological significance was analized by Meta analysis.(4) Potential binding sites miR-26 a and people MTDH gene mRNA3’untranslated region was analyzed by bioinformatics software Target Scan.qRT-PCR and Westem blot were used to detect the transfection effect.The wild-type and the mutant-type of the miR-26 a binding site in MTDH mRNA 3’untranslated region were cloned into reporter gene vectors, respectively. Then the luciferase assay was used to detect the luciferase activity of the wild-type MTDH 3’UTR.(5) 5 to 8-week-old BALB/c female nude mice were randomized into six groups(n=5). miR-26 a, miR-Con,and miR-26 a inhibitor lentivirus were transfected to ESCC cell lines.2×107cells/0.2mL/mouse was injected to nude mice.At day 28, all mice were sacrificed and tumors were dissected. Tumor volumes and body weights were measured every week. Tumor volumes were calculated with the using the equation: volume=(longest diameter×shortest diameter2)/2. We used situ hybridization and immunohistochemistry assay to detect the express miR-26 a and MTDH in mice tumor mass organization.Results:(1) mi R-26 a was located in cytoplasm and nucleus of cancer cells, there was low expression of miR-26 a in ESCC tissues, the expression of miR-26 a in ESCC tissues was significantly lower than the NAT, the difference was statistically significant(2? =4.572, P=0.033). miR-26 a expression was inversely correlated to pathological grade, N classification, tumor volume(P< 0.003).Spearman correlation analysis proved that miR-26 a expression level was correlated with lymph node metastasis(r=0.609, P=0.001) and pathological grade(r=0.262, P=0.018).Kaplan-Meier survival analysis indicated the low expression of miR-26 a was significantly associated with poorer overall survival(log-rank test, P=0.019).In univariate analysis showed miR-26 a expression, N classification, gross classification were significantly associated with the overall survival time. The final multivariate model revealed that overall survival time significantly depended on N classification, gross classification. miR-26 a expression levels in six kinds of esophageal cancer cell lines were significantly lower than human esophageal epithelial cells(HEEC). Eca109 cells, KYSE450 cells, with the lowest and highest miR-26 a expression level were assigned for the following function study.In order to verify the implications for the ESCC cells after expression of miR-26 a, cells transfected with lentivirus were divided into three groups:scramble Eca109 group(Eca109 normal cultured cells), LV-con group(transfected random sequence lentivirus) and LV-miR-26 a group(transfected overexpressed miR-26 a lentivirus). In order to verify the impact of miR-26 a interference effect, the cellstransfected according to the type of the lentivirus were divided into three groups:scramble KYSE450 group(normal KYSE450 cultured cells), LV-con group(ransfected random sequence lentivirus) and LV-miR-26a-inhibitor group(transfection interference lentivirus miR-26a). After transfection of miR-26 a overexpression lentivirus, mRNA levels of LV-mi R-26 a group was lower than scramble Eca109 group and LV-con group(P< 0.05); after miR-26 a interfered ovexpression lentivirus, mRNA levels of LV-miR-26a-inhibitor group was significantly increased than scramble KYSE450 group(P < 0.05). The proliferation ability in cells transfected with miR-26 a was significant lower than that cells transfected with miR-Con. Meanwhile, miR-26 a inhibitor induced growth in KYSE450 cells, which indicated miR-26 a induced growth inhibition in ESCC cells. The cells transfected with miR-26 a showed a significant accumulation of cells in the G1 phase, whereas the S phase population decreased compared cells transfected with the miR-Con and scramble cells.The results suggested that miR-26 a could inhibit ESCC cell growth partly due to G1-phase arrest.We found miR-26 a and miR-26 a inhibitor had no significant different changes on apoptosis.(2) MTDH was expressed in esophageal cancer cells and normal esophageal epithelium cells, the positive cells were mainly located in the cytoplasm, a few localized in the nucleus, pale yellow, brown or brown granules.MTDH expression was positively detected in 54.65%(47/86) in ESCC samples and detected in 14.10%(11/78) in ESCC samples. MTDH was significantly up-regulated in ESCC compared with the NAT. Differentiation(P=0.005) and lymph node metastasis(P=0.044) were found to be significantly correlated to MTDH expression. Spearman correlation analysis proved that high MTDH expression level was strongly correlated with lymph node metastasis(r=0.609, P=0.044) and differentiation(r=0.296, P=0.009).Univariate Cox regression analyses showed that MTDH expression(P=0.017), lymph node metastasis(P=0.004), gross classification(P=0.008) were prognostic factors for ESCC. By multivariate analysis, we further examined prognostic parameters of ESCC that were significant in univariate analysis. MTDH expression(P=0.045) and gross classification(P=0.013) were independent prognostic factors influencing 5-year overall survival.Overexpression of MTDH was significantly associated with the lymph node metastasis, advanced clinical stage and T classification in tissues of SCC. MTDH mainly localized in the cytoplasm.(3) miR-26 a can negatively regulated the expression of MTDH by binding to the binding sites of 3’-UTR of miR-26 a. qRT-PCR and Western blot showed that after interference with miR-26 a, the expression of MTDH significantly increased after overexpression of mi R-26 a, and significantly reduced the expression ofMTDH. The tumor volume of Eca109 cells stably expression miR-26 a was significantly lower than that of the stably expression miR-Con. The tumor volume of KYSE 450 cells stably expression miR-26 a inhibitor was significantly higher than that of the stably expression miR-Con.miR-26 a suppressed the tumorigenesis in vivo.miR-26 a was low expresion in nude mice tumor tissues.Overexpresion of miR-26 a showed low expression of MTDH which proved negative regulation of the relationship between MTDH and miR-26 a. Spearman correlation analysis proved there was negtive correlation between MTDH and miR-26a(r=-0.249, P < 0.05).Conclutions: miR-26 a expression is low expression in esophageal cancer, miR-26 a inhibits the cell proliferation, migration and cell cycle distribution of esophageal squamous cell carcinoma, but has no effect on apoptosis. MTDH mainly localized in the cytoplasm. miR-26 a can act as a tumor suppressor in culture and in nude mice and has a key role in regulating physiological and physiological angiogenesis by targeting MTDH in ESCC. These results suggest miR-26a/MTDH pathway may be one of the molecular targets for diagnosis and treatment of esophageal cancer. |
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