The Influence And Mechanism Of HER-3 Knockdown On Radiosensitivity Of HER-2 Positive Breast Cancer Cells

Background:Breast cancer（BC）is a well-known malignant tumor with a higher incidence and mortality rate in women.BC has become the world’s worst cancer.Although the clinical treatment of BC has arrived remarkable achievement over the past decade,about 20%of patients are still suffering from events such as local recurrence,distant metastasis,drug resistance and other treatment failures.Since the 20th century,radiotherapy has become an emerging cornerstone of breast cancer treatment deserving research space and value.Human epidermal growth factor receptor 3（HER-3）is an old receptor which is considered worthless in the history of tumor research.Nowadays,researchers believe that HER-3 plays an irreplaceable role in the progress of occurrence,growth,metastasis,and treatment resistance in breast cancer by regulating PI3K/AKT,MEK/MAPK and other signaling pathways.It is expected that HER-3 shall be an emerging target in radiotherapy sensitization.Objective:To investigate the effect and mechanism of HER-3 gene in regulating radiation sensitivity of HER-2 breast cancer cells.Methods:Lentivirus was used to construct the HER-3 knockdown cell model by infecting HER-2 breast cancer cells.Western Blot was used to detect the efficacy of lentivirus infection.A clinical linear accelerator was used to provide 6MV X-rays（source skin distance 100 cm）to simulate the radiotherapy environment.The clony formation experiment was implemented to detect the effect of HER-3 knockdown on the growth ability of breast cancer cells.A multitarget-single-hitting model was constructed to modify the survival curve and calculate the radiosensitivity in radiation environment.The wound healing assay was used to detect the influence of HER-3knockdown in the migration ability of HER-2 breast cancer cells.Transwell model was constructed to detect the influence of HER-3 knockdown in the invasion ability.Flow cytometry was used to determine the effect of HER-3 knockdown on the apoptosis rate of breast cancer cells under radiation environment as well as the regulation of cell cycle distribution.Immunofluorescence experiment was performed to evaluate the DNA damage of breast cancer cells at different time by countingγ-H₂AX foci after radiation.Western blot was used to detect the expression of key proteins related to the mechanism.Results:The HER-3 knockdown model in HER-2 breast cancer cells was successfully constructed by lentivirus infection.The clony formation experiment demonstrated that HER-3 knockdown inhibited the growth ability of breast cancer cells,at the same time enhanced the radiosensitivity.The results of wound healing experiment illustrated that HER-3 knockdown reduced the migration ability.Transwell experiments demonstrated that HER-3 knockdown reduced the number of cells crossing the matrigel membrane that represents down-regulation of the invasion ability.The flow cytometry assay showed that HER-3 knockdown had a synergistic effect on the induction of apoptosis with ionizing radiation,and it was concluded that HER-3 knockdown up-regulated the G2 arrest of the cell cycle.The result ofγ-H₂AX foci under immunofluorescence showed HER-3 knockdown up-regulated the DNA damage of HER-2 breast cancer cells under the radiation environment,meantime reduced DNA repair.The result of Western Blot showed that HER-3 knockdown down-regulated the expression of signaling pathway proteins related to apoptosis and cell cycle regulation.Conclusion:HER-3 knockdown is able to inhibit the growth,invasion and migration of HER-2 breast cancer cells,and enhance radiosensitivity.On the one hand,HER-3knockdown increases the DNA damage of HER-2 breast cancer cells after radiation and reduces DNA repair ability,as the result,promotes cell apoptosis.On the other hand,HER-3 knockdown increases the G2 phase distribution by down-regulating the expression of cell cycle-related protein cyclin-B1.