| Objectives: To study the role of hTERT and β-catenin in gallbladder carcinoma. Methods : The expression of hTERT and β-catenin examined by immunohistochemistry in 20 cases including normal and inflammatory gallbladders, 19 cases of non-gallbladder-carcinoma-related polyps. 16 cases of gallbladder-carcinoma-related polyps, 82 cases with gallbladder carcinoma. Results: (1) The positive rate of hTERT in normal and inflammatory gallbladders, non- gallbladder-carcinoma-related polyps, gallbladder-carcinoma-related polyps and gallbladder carcinoma were 0%, 5.3%, 37.5% and 84.1%, respectively. The prevalence and intensity of hTERT signal were higher in gallbladder-carcinoma-related polyps and gallbladder carcinoma than those in normal and inflammatory gallbladders and non-gallbladder-carcinoma-related polyps (P<0.05). The abnormal expressions of β -catenin were similar to that of hTERT. (2) The positive rate of hTERT of gallbladder cancer in T1+ T2 and T3+ T4, in group with lymph node metastasis and without lymph node metastasis were 72.7% and 92.8% (P<0.05), 93.6% and 71.4% (P<0.05) respectively. The positive rate of abnormal β -catenin were 33.3% and 63.3% (P<0.01) in T1+ T2 and T3+ T4, 57.4% and 42.9% (P<0.05) in group with lymph node metastasis and without lymph node metastasis respectively. The correlation coefficient between the expression of hTERT and differentiation degree or invasive depth or lymph node metastasis or not were 0.5, 0.4 and -0.5 (P<0.01) respectively. (3) In 82 cases of gallbladder carcinoma, the cumulative survival rate of hTERT negative and positive expression were 22.8% and 5.9% (P<0.05), that of β -catenin abnormal negative and positive expression were 17.1% and 5.7% (P<0.05), respectively. (4) The coincident expression rate of hTERT and abnormal p-catenin was 48.8 % and significant correlation existed between hTERT and abnormal P-catenin (r = 0.311,P<0.05).Conclusions: The expression of hTERT protein and abnormal P-catenin protein is strongly correlated with malignant gallbladder lesions. The expressions of hTERT protein may be involved in the progression from the gallbladder-carcinoma-related polyp to carcinoma. They may also be associated with the prognosis of gallbladder carcinoma. The correlation between the abnormal expression of P-catenin protein and hTERT protein in gallbladder carcinoma is found.Part II The Expression of hTERT and p-catenin and Cell Growthand Invasion Assay in Gallbladder Cell LinesObjectives: To detect the expression of human telomerase reverse transcriptase (hTERT) and P-catenin protein and mRNA and cell growth and invasion assay in gallbladder carcinoma cell lines and to elucidate their roles in gallbladder carcinogenesis.Methods: (1) Telomerase activity, mRNA and protein of hTERT and P-catenin in 3 human gallbladder carcinoma cell lines using telomerase TRAP , reverse transcriptase polymerase chain reaction (RT-PCR) and Western Blot techniques, respectively. (2) SDH activity by MTT, cell growth by counting, invasion assay by Transwell chamber and migration assay by lineation in culture utensil. Results: (1) The absorbency of the cell (OD value) was used to represent the telomerase activity. In TGBC1TKB, TGBC2TKB and GBC-SD, the OD value was 0.183 + 0.003, 0.257±0.002 and 0.260±0.003. p-catenin mRNA value was 51287±1818, 62346±733 and63345±1154. hTERT mRNA value was 41051 ±1073, 50812±1439 and 51302±1176. p-catenin protein value was 51956±5882 , 95843±11856 and 95960±11845. hTERT protein value was 41182±6200 , 68749±5934 and 69229±5208, respectively. The OD value of telomerase activity, mRNA and protein value of hTERT and p-catenin were greater in TGBC2TKB and GBC-SD than those in TGBC1TKB (P<0.01). (2) In TGBC1TKB, TGBC2TKB and GBC-SD, the OD value of SDH at 5th day was 1.324+0.792, 1.573±0.043 and 1.647 ±0.033. After 24 hours, the counts of cell penetrating through membrane of Transwell chamber were 60.667±3.512, 113.333 + 5.508 and 124:667±6.506. After 24 hours, the counts of cell getting over lineation in culture utensil were 23.667+1.155, 40.000+1.000 and 42.667±2.082, respectively. The OD value of SDH, the counts of cell penetrating through membrane of Transwell chamber andgetting over lineation in culture utensil were higher in TGBC2TKB and GBC-SD than those in TGBC1TKB (PO.01).Conclusions: The positive expression of telomerase activity, mRNA and protein of hTERT and p-catenin appeared in human gallbladder carcinoma cell lines that may indicate biological characteristics and play a role in cell growth, migration and invasion efficiency of gallbladder carcinoma cells.Part III Inhibition of hTERT Activity for Suppression of GBC-SD Cell Proliferation and MigrationUsing hTERT-siRNA TechniqueObjectives: To evaluate the inhibitory effect on telomerase activity, SDH activity, cell growth and invasion efficiency and to investigate the effect on hTERT mRNA, hTERT protein , P-catenin mRNA , P-catenin protein by transfecting pGCsi-Hl/GFP-hTERT vector into gallbladder carcinoma GBC-SD cells and realize partly the molecular mechanism about the gallbladder carcinoma effect of hTERT-siRNA.Methods: Telomerase activity, mRNA and protein of hTERT and P-catenin in GBC-SD using telomerase TRAP, RT-PCR and Western Blot techniques, respectively. SDH activity by MTT and cell growth by counting, invasion assay by Transwell chamber and migration assay by lineation in culture utensil. Results: pGCsi-H 1 /GFP-hTERT could inhibit telomerase activity, SDH activity, cell growth, migration and invasion efficiency, expression of hTERT mRNA and protein. The inhibition became more marked with the increase of pGCsi-Hl/GFP-hTERT vector concentrations. Concentrations of 0.25~2|ag/ml pGCsi-Hl/GFP-hTERT vector specifically reduced the telomerase activity by 5.51—79.41%, SDH activity by 6.10—78.54%, cell growth by 4.64~75.66%, migration efficiency 10.94—78.91%, invasion efficiency 14.71—81.82%, hTERT mRNA by 1.91—78.53%, hTERT protein by 2.21—76.60%, respectively. The results indicate that pGCsi-Hl/NEGative vector failed to inhibit telomerase activity, SDH activity, cell growth, migration and invasion efficiency, expression of mRNA and protein of hTERT. A statistically significant difference exists between pGCsi-Hl/GFP-hTERT vector and pGCsi-Hl/NEGative vector (P<0.01). The dose of 0.25~2^g/ml pGCsi-Hl/NEGative vector hadn't the ability to inhibit expression of mRNA and protein of P-catenin.
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