| ObjectiveThis study was to detect the expression of RUNX3 in tumor tissues and the corresponding esophageal cancer cell lines, and to explore its correlation with clinical cases of parameters, and analyzes changes in the expression of RUNX3 and Aktl esophageal cancer cell biology after cisplatin influence behavior, to explore the reversal of RUNX3 esophageal carcinoma cells to cisplatin resistance mechanisms aimed at clinical diagnosis of esophageal cancer targeted therapy and provide a reliable basis.Methods(1) 103 cases of esophageal squamous cell carciuoma(stage Ⅱa-Ⅲb) resection tissue and corresponding adjacent normal tissues chant acquisition in January 2009-January 2012 period in Thoracic Surgery, Affiliated Hospital of Shandong University. RUNX3 mRNA and protein expression were detected by fluorescence quantitative PCR, and the expression of RUNX3 protein Aktl was detected by immunohistochemistry method, analyze the correlation between RUNX3 and resistance to chemotherapy in patients with clinical data, analyzed the correlation between RUNX3 and Akt1.(2) We lentiviral vector containing the RUNX3 gene or whole siAktl, to be packed a viral infection ESCC cell lines. PCR and western blot to detect the expression of RUNX3; Apoptosis was detected by flow cytometry; CCK-8 cell proliferation assay and cell cycle; Hoechst staining assay apoptosis rate; western blot detection of RUNX3, Aktl p-Akt1 Bcl-2 Bcl-xl P21 expression,,,, CyclinD1,, Bax protein.(3) Nude mice were injected using cell RUNX3 and the corresponding control group ESCC cell expression, followed by coating the inner injection of cisplatin. tumor size in each group of mice inhibits tumor inhibition rate.Results(1) General information analysis showed that male patients compared with female patients, low expression of RUNX3 difference was not statistically significant (P> 0.05); in esophageal squamous cell carcinoma in patients less than 56 years of age, with a low expression of RUNX3 is greater than 56-year-old patients compared to no significant difference (P> 0.05); the degree of tumor infiltration of T1-T2 among patients, RUNX3 was the proportion of patients with low expression was significantly lower than the T3-T4 patients (P<0.05):the proportion of patients in clinical stage I～Ⅱ patients were stage. RUNX3 expression was low significantly lower than the III～IV patients (P<0.05):in patients with lymph node metastasis were, RUNX3 expression was significantly lower proportion of patients with high in patients without lymph node metastasis (P<0.05). Chemotherapy sensitive esophageal squamous cell carcinoma patients in the expression level of RUNX3 mRNA and the corresponding protein was significantly higher than those who are not sensitive to chemotherapy (P<0.05), in patients not receiving adjuvant chemotherapy for their cancer tissue RUNX3 mRNA and the corresponding protein the expression level was significantly higher than before surgery received adjuvant chemotherapy (P<0.05). In RUNX3 expression-positive tissues, the expression of Aktl significantly decreased (P <0.05); in RUNX3 expression was detected in tissues, the expression of Aktl is significantly higher (P<0.05); RUNX3 and Akt1 expression was negatively correlated (r=-0.296, p=0.0042<0.01).(2) Cisplatin resistance Eca109 and TE-1 cells, the expression of RUNX3 mRNA and protein was significantly lower than non-resistant Eca109 and TE-1 cells (P <0.01). Eca-109 PR R3 and Eca-109 PR R3 cell expression of RUNX3 mRNA and protein were significantly higher than Eca-109 PR vector and TE-1 PR vector cells (P<0.01). With increasing concentration of cisplatin, Eca-109 PR vector group and Eca-109 PR R3 group. TE-1 PR vector group and TE-1 cell activity PR R3 groups were decreased in a dose-dependent manner, wherein when cisplatin at a concentration of 100μg mL. the lowest cell activity; the activity of Eca-109 PR R3 group and TE-1 PR R3 group of cells in each concentration was significantly lower than TE-1 PR vector group and TE-1 PR vector group (P<0.05). With the extension of cisplatin time. Eca-109 PR vector group and Eca-109 PR R3 group, TE-1 PR vector group and TE-1 PR R3 group activity of cells were gradually increased in a time-dependent manner. wherein when when cisplatin highest cell viability after 96 h:Eca-109 PR R3 group cell activity cisplatin each time point was significantly lower than Eca-109 PR vector group (P<0.05). With the extension of cisplatin time. Eca-109 PR R3 group and TE-1 PR R3 group group activity of cells were gradually increased in a time-dependent manner, with the highest cellular activity when cisplatin after 96 h; Eca-109 PR R3 group and TE-1 PR R3 group cell activity each time point cisplatin significantly lower than the TE-1 PR vector group and TE-1 PR vector (P<0.05). Eca-109 PR R3 group and IC50 concentrations of TE-1 PR R3 group were significantly lower than Eca-109 PR vector group and TE-i PR vector group (P<0.05). Eca-109 PR R3 group and the number of TE-1 PR apoptosis was significantly higher than R3 Eca-109 PR vector group and TE-1 PR vector group (P<0.01). Eca-109 PR R3 group and TE-1 PR R3 group were significantly higher than the number of cells in G1 phase Eca-109 PR vector group and TE-1 PR vector group (P<0.01), G2/M phase cells was significantly less than the number of Eca-109 PR vector group and TE-1 PR vector group (P<0.05). With increasing concentration of cisplatin, Eca-109 PR vector group and Eca-109 PR siAktl group, TE-1 PR vector group cell activity and TE-1 PR siAktl groups were decreased in a dose-dependent manner, wherein when cisplatin at a concentration of 100μg/mL, the lowest cell activity; Eca-109 PR siAktl group and TE-1 PR siAktl activity of each cell group was significantly lower than the concentration of Eca-109 PR vector and TE-1 PR vector (P<0.05). Eca-109 PR siAktl group and IC50 concentrations of TE-1 PR siAktl group were significantly lower than Eca-109 PR vector group and TE-1 PR vector group (P<0.05) Eca-109 PR siAktl group and TE-1 PR siAktl the number of apoptotic cells was significantly higher than Eca-109 PR vector group and TE-1 PR vector group (P<0.01). Eca-109 PR siAktl group and TE-1 PR siAktl set the number of cells in G1 phase cells was significantly higher than Eca-109 PR vector group and TE-1 PR vector group (P<0.01). G2/M phase cells was significantly less than the number of Eca-109 PR vector group and TE-1 PR vector group (P<0.05). Eca-109 PR R3 and Eca-109 PR siAktl cells in Aktl, Bcl-2, Bcl-xl, CyclinDl protein expression level was significantly lower than Eca-109 PR vector and TE-1 PR vector group (P<0.05); the Eca-109 PR R3 and Eca-109 PR siAktl cells in RUNX3, P21, Bax protein expression level was significantly higher than Eca-109 PR vector and TE-1 PR vector group (P<0.05).(3) Eca-109 PR vector group and Eca-109 PR R3 groups of nude mice were tumor, tumor formation rate was 100%. During the treatment of the animals were tumor volume over time is increasing, but Eca-109 PR R3 groups of nude mice in the first 18 days of each measurement time point of tumor volume was significantly smaller than Eca-109 PR vector group (P< 0.05). Over time, each group of mice tumor volume inhibition rate increased gradually, but Eca-109 PR R3 groups of nude mice at each measurement time point of tumor volume inhibition was significantly greater than the Eca-109 PR vector group (P<0.05).ConclusionsRUNX3 reduced expression in esophageal squamous cell carcinoma may lead to cisplatin resistance, RUNX3 could reverse esophageal squamous cell carcinoma resistant to cisplatin by inhibiting Akt signaling pathway. |
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