| Objective: To investigate the influence of Huntington protein 1(HAP1)gene on radiation biological characteristics of breast cancer(BC)MCF-7 cell line.Methods: Constructed HAP1-expressing stabled MCF-7/HAP1 and MCF-7/Pb cell lines.then the Breast cancer cell line MCF-7 was infected to construct HAP1-expressing stable cell line and vector control group.All cells were screened by puromycin.HAP1 gene was confirmed by quantitative reverse transcription-polymerase chain reaction analysis(qRT-PCR)and Western blot in vitro.Colony formation assay was applied to assess the changes of cell radiosensitivity in MCF-7,MCF-7/Pb and MCF-7HAP1 cells.Apoptosis were examined by flow cytometry.Western blot was used to dectect apoptosis related protein levels of Bcl-2and Bax.Results: Our data showed that HAP1 gene expression was significantly increased in HAP1-transfected MCF-7 cells in comparison with the parental cells or negative control cells.Colony formation assay showed that the survival rate in MCF-7/HAP1 cells was significantly decreased after irradiation(0,2,4,6,8 Gy),compared to cells in MCF-7 and MCF-7/Pb groups in vitro.The flow cytometer anslysis result indicated apoptosis rates incresed in alone irradiation group and individually transfection with HAP1 gene group,but the apoptosis rate in HAP1 gene combined with irradiationgroup was significantly higher than that in other groups,there are significant differences between the groups(P<0.05).Therefore,we found that the expression of Bcl-2 protein were reduced in alone irradiation group,individually transfection with HAP1 gene group and combination group,but the expression of Bax protein were incresed.And Bcl-2 in combination group was significantly lower than those in other groups,Bax was significantly higher than those in other groups.Conclusion: The present study indicated that HAP1 gene expression was related to the radiosensitivity of breast cancer cells and may play an important role in the regulation of cellular radiosensitivityObjective: To study the effect and mechanism of HAP1 gene on radiosensitivity of human breast Carcinoma cell MCF-7 xenograft in nude mice.Methods: Constructed MCF-7/HAP1 and MCF-7/Pb stably transfected cells(5×106)were subcutaneously injected into nude mice to establish implanted tumor model of human breast cancer in nude mice.The nude mice were divided into 4 groups(20days,5 mice per group)for MCF-7/Pb group,MCF-7/HAP1 group,MCF-7/Pb +radiation group,MCF-7/HAP1 + radiation group.The dose of IR was 5-Gy X-ray(once)and followed for about 3 weeks.The long diameter and short diameter of the tumor were measured every 4 days.The tumor was weighted after 6 weeks.The Bcl-2and Bax expression were analyzed by Immunohistochemical method.And apoptosis was analyzed by TUNEL assay.Results: The tumor volume of MCF-7/HAP1 + radiation group was smallest than that in other three groups(all P<0.01).The weight of tumor in MCF-7/HAP1 + radiation group was lowest than that in other three groups(all P<0.01).TUNEL showed that apoptosis rate of MCF-7/Pb group,MCF-7/HAP1 group,MCF-7/Pb + radiation group and MCF-7/HAP1+ radiation group was 8.90±1.37%,34.43±1.67%,55.03±0.49%and 81.73±1.87% respectively,the apoptosis rate in MCF-7/HAP1 + radiation group was significantly higher than that in other three groups(P<0.05).Immunohistochemistry showed that the expressions of Bcl-2 in MCF-7/Pb group,MCF-7/HAP1 group,MCF-7/Pb+ irradiation group and MCF-7/HAP1+ radiation group was 13.33±1.89,7.67±1.25,8.67±0.47 and 4.67±1.89,respectively;and the expression of Bax was 1.67±0.47,4.00±1.63,7.00±1.41 and 14.67±1.89,respectively.The expressions of Bcl-2 in MCF-7/HAP1 + radiation group was significantly lower than those in other three groups(P<0.05),but the expression of Bax was significantly higher than those in other three groups(P<0.05).Conclusion: HAP1 gene might enhance the sensitivity of human breast cancer cells to radiotherapy by regulating apoptosis in vivo. |
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