Objective: To investigate the effect of antisense integrin beta6 gene on the growth of colon cancer cells.
Methods: Expressing vector of antisense alphavbeta6 was constructed. Human colon cancer cells of the line HT29 were cultured and divided into 3 groups: Group A, remaining wild type; Group B, transfected with antisense integrin beta6 gene; and Group C, transfected with blank vector. RT-PCR was used to detect the integrin beta6 mRNA expression of in the HT29 cells. The integrinbeta6 protein expression on the surface of the cells was detected by immunohistochemistry and flow cytometry. The binding between the cells and fibronectin was examined. (3)H-labeled thymidine (T) was added into the culture fluid of the cells, and then the radiation amount was detected every 6 days so as to determine the capacity to proliferation of the cells in vitro. Thirty female nude mice were divided into 3 groups to be injected subcutaneously with suspension of HT29 cells of Groups A, B, and C as mentioned above. Six weeks later the size of tumors was measured and part of the tumor nodules were resected 5 weeks after the inoculation to undergo pathological examination.
Results: Compared with Groups A and C, no corresponding band at 141 bp was found in Group B by RT-PCR. Flow cytometry showed that the expression level of beta6 protein had was (0.30 +/- 0.051, 30%), significantly lower than those of Groups A and C [(0.80 +/- 0.038, 80%) and (0.85 +/- 0.045, 85%), both P < 0.01]. The binding between the HT29 cells and fibronectin of Group B was significantly degraded after the further addition of anti-beta1 and anti-alphav in comparison of Groups A and C (both P < 0.01). The accumulation values of (3)H-labeled T of Group B 2, 4, and 6 days after addition were all significantly lower than those of Groups A and C (all P < 0.01). The tumors in 9 of the 10 mice injected with the HT29 cells of Group B disappeared and the tumor in the only one mice in Group B was only less than 1 mm(3), significantly smaller then those in Groups A and C (15 mm(3) on average, all P < 0.01).
Conclusion: Antisense beta6 gene significantly inhibits the mRNA and protein expression of the beta6 gene, and then inhibits the growth and proliferation of colon cancer cells, thus proving that integrin beta6 plays an important role in the regulation of colon cancer cells.