Silencing Of XRCC4 Expressionwith ShRNA Increased Radiosensitivity Of Triple-negative Breast Cancer Cells

Background:Triple Negative Breast Cancer(TNBC)is a type of aggressive breast cancer that does not express estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(Her-2).Although TNBC is more sensitive to radiotherapy and chemotherapy,it is not sensitive to traditional endocrine therapy and targeted therapy against Her2 antigen.Therefore,the clinical prognosis of patient with TNBC is poor.As an important part of TNBC therapy,radiation therapy is one of the important local treatments,but with its own strong side effects.Radiation therapy used low doses of radiation can not achieve a good effect of killing tumor cells,but high doses of radiation may cause serious complications such as skin damage,dysfunction or even loss of sensitive tissue(such as bone marrow,testis,ovary,etc.),even the serious radiation injury(such as radioactive paraplegia,brain necrosis and other extremely serious complications).Therefore,it is necessary and urgent to explore how to improve the sensitivity of TNBC to radiation,so as to achieve a smaller radiation dose to achieve the purpose of treatment of TNBC and to reduce the complications of treatment.X-ray repair cross complementing gene 4(XRCC4)is a very important member of Non-homologous end joining(NHEJ),a DNA double-strand break repair pathway.The gene expression of the protein can interact with ligase IV to form a complex,to repair the DNA break,so as to maintain the stability and integrity of the genome.The malfunction of XRCC4 can lead to mistakes of DNA repair pathways so that it causes tumor cell with DNA damage to death.Studies have shown that patients with low expression of XRCC4 in patients with esophageal cancer who receive radiation therapy have a longer survival time than patients with high expression of XRCC4,suggesting that the low expression of XRCC4 may have a certain relationship with the increase in radiotherapy sensitivity.In TNBC patients,XRCC4 expression was significantly lower in cancer tissues than that in adjacent normal tissues.There was no correlation between XRCC4 expression and survival of TNBC patients.To our knowledge,the relationship between expression status of XRCC4 and the radiotherapy sensitivity of TNBC has not been investigated.Objective:The aim of the present study is to investigate whether silencing of XRCC4 expression gene can increase the sensitivity of triple negative breast cancer to radiotherapy.Methods:The RNAi technique was used to establish the TNBC stable cell line with XRCC4 gene knockdown.The effect of XRCC4 knockdown on the proliferation of MDA-MB-231 and MDA-MB-468cells was detected by MTT colorimetric assay.Colony formation assay was performed to detect the effect of XRCC4 knockdown on the colony formation ability of MDA-MB-231 cells.Post treated with different doses(2Gy,4Gy,6Gy)of radiation,colony formation assay was used to detect the effect of XRCC4knockdown on the radiotherapy sensitivity of MDA-MB-231cells.The colony formation ability among the nottransduced(NT),emptyvector-transduced(Vector)andXRCC4-shRNA-transduced MDA-MB-231cellswas compared.Results:1.The positive rate of XRCC4 expression in the three-negative breast cancer tissues was34.38%,and the positive rate of XRCC4 expression in the adjacent tissues was 62.50%,the difference was statisticallysignificant(P<0.05).2.TheshRNAexpressionlentiviralvector pLenti-U6-EF1a-cop GFP-P2A-Puro,which was carrying both green fluorescent protein and puromycin gene,was successfully constructed.3.The lentiviral vector pLenti-U6-XRCC4-EF1a-copGFP-P2A-Puro expressingXRCC4-sh RNAwassuccessfullyconstructedbyusingtheconstructed pLenti-U6-EF1a-copGFP-P2A-Puro vector.After the lentivirus was packaged,the triple-negative breast cancer cell lines MDA-MB-231-XRCC4~-and MDA-MB-468-XRCC4~-with stable knockdown of XRCC4were successfully established.4.MTT assay showed that there was no significant difference in proliferation among the not transduced(NT),the empty vector-transduced(Vector)and the XRCC4-shRNA-transduced MDA-MB-231cells(P>0.05);There was no significant difference in proliferation rate among the three MDA-MB-231 cells at anytime points(24h,48h,72h;all P>0.05).The proliferation rate among the three MDA-MB-468 cells was also not statistically significant(P>0.05).At each time point(24h,48h,72h),the proliferation of the three MDA-MB-468 cells was not statistically significant(P>0.05).5.With no radiation,there was no significant difference of colony formation numbers among the XRCC4-shRNA-transduced,the non-transduced and Vector-transduced MDA-MB-231 cells(P>0.05).Neither did the survival rate of colony formation.However,clone's diameter of XRCC4-shRNA transduced MDA-MB-231cells was larger than that of control cells,the difference was statistically significant(all P<0.01).The survival rate and the numbers of all three MDA-MB-231cells decreased with the increasing of radiation dose.The survival rate of XRCC4-shRNA-transduced MDA-MB-231cells decreased more gradually than the other two cells,and the difference among the three cells was statistically significant(P<0.01).So did the number of colonies.There was no significant difference between the non-transduced and Vector-transduced cells(P>0.05).The colony numbers of the XRCC4-shRNA-transduced MDA-MB-231 cells was significantly lower than that of the non-transduced and Vector-transduced cells at the corresponding dose of 4Gy and 6Gy(P<0.01 and P<0.05 respectively).The survival rate of cloned colonies of XRCC4-shRNA-transduced MDA-MB-231cells was lower than that of non-transduced cells at each irradiation doses(2Gy,4Gy,6Gy,all P<0.01).Compared with vector-transduced cells,the survival rate of cloned colonies of XRCC4-shRNA-transduced MDA-MB-231 cells was lower,there was significant difference at the irradiation dose of 2Gy,4Gy(all P<0.01)and 6Gy(P<0.05).There were no significant differences in the survival rate of clonal colonies between the the non-transduced cells and the vector-transduced cells at various irradiation doses(2Gy,4Gy,6Gy,all P>0.05).Conclusions:1.The positive expression rate of XRCC4 in cancer tissues of TNBC patients was lower than that in the adjacent tissues.2.XRCC4 gene knockdown has no effect on the proliferation of TNBC cells.3.XRCC4 gene knockdown has no effect on the number of clone formation of MDA-MB-231,but it can increase the size of clones.4.XRCC4 gene knockdown can increase the radiation sensitivity of the MDA-MB-231TNBC cells.