Effect Of DNA Polymerase θ In Breast Cancer Cell Line MCF-7on The Proliferation, Apoptosis And The Radiation Sensitivity

Background:Breast cancer is one of the most common malignant tumors in our country, it takes7%-8%of the malignant tumors, be only inferior to the uterine cervical cancer, it has a predisposition more than cervical cancer in recent years, and shows a rising trend year by year.In some big citys it accounts for the first female malignant tumors. From the end of the nineteenth century on, radical mastectomy(or improved radical mastectomy) in breast cancer treatment has played an irreplaceable role in the field of controling of disease progression,and the field of improving survival rate. But its postoperative complications are problems can’t be ignored.Breast-conserving surgery carried out step by step along with the ceaseless progress of the medical device, method and technology.Breast-conserving surgery possesses a important role in the preservation of physiological function, beauty and little complications compared with the traditional radical cystectomy,which greatly improved the quality of patients’life,and make an improvement in the mental disorders significantly.But with its high local recurrence rate, radiotherapy will be applied after operation based on conditions, which can effectively kill the residual minimal disease, and reduce the recurrence.An additional tumor bed irradiation dose is the key to reduce the recurrence at the same time of improving the local control rate in the breast radiotherapy,but with a high rate of leading to complications of heart, lung injury and upper limb edema.How to improve the effect of radiotherapy while reducing the impact on normal human tissues has become one of the important subjects of breast cancer adjuvant therapy.Over the past ten years, people have made a great progress in the research of tumor molecular targeted therapy, which has provided a great help to improve the prognosis of patients with malignant tumor.Interfering some molecular targets in tumor cells can improve the local effect of radiation, while won’t cause the increase of side reaction, so as to improve the quality of life.DNA polymerase θ(POLQ) was discovered to be the eighth human DNA polymerase in1990,and it is one of the DNA polymerase A family member s.The enzyme has important characteristics of DNA double strand break (DSB) repair and DNA interstrand crosslink (ICL)repair,it participates in DNA damag e tolerance,and has a close relationship in maintaining genomic integrity.Retic ulocytes absence of this enzyme in the mouse have a increased abnormal mitot ic figures or chromosome breakage.When the normal tissue cells and tumor cells have defects in the expression of the enzyme, they would become sensit ive to external factors,such as hydrogen peroxide, gamma radiation, bleom ycin and so on.Because cells with a deletion mutant in POLQ will cause chro mosomal instability, inhibite the DNA replication and transcription, which may further lead to related diseases, such as cancer, ataxia telangiectasia and Nijme gen chromosome instability syndrome.The strong cytotoxic showed by IC L is the foundation of nitrogen mustard, mitomycin, cisplatin, psoralen and so me other chemotherapy drugs.For the feature in the enzymes of tumor cells, H iggins interfered the expression of POLQ by exogenous gene in Hela, SQ20B, PSN1cells, and confirmed that the silencing of POLQ has a direct relationsh ip with the radiosensitivity of cells.Increasing the sensitivity of tumor cells t o radiation can reduce the radiation dose with a same effect of radiotherapy, a nd reduces the injury to normal tissue,which can lead a lower cancer probabili ty by radiation.Later the reseach group revealed overexpression of POLQ happ ened in a plenty of patients with early stage breast cancer using the retrospecti ve analysis method, and confirm that the overexpression of POLQ would result in adverse clinical outcomes, and had a relationship with negative estrogen re ceptor in patients and a bad pathology grade of tumor, which would both le ad to poor prognosis of patients as independent factors by the clinical research.Interferring the expression of POLQ may inhibit the proliferation of tumo r cells, reduce the risk of relapse and metastasis, and avoid the tolerance of tu mor cells or tissues to adjuvant treatment, so as to improve the prognosis of p atients.At the same time, Lemee found that POLQ is the only gene highly expressed in breast but lowly expressed in normal breast tissue in the reseach of13nucleic acid DNA polymerase genes.It suggested that interfering the expression of POLQ in breast cancer cells or tissues can improve the radiation sensitivity of breast cancer cells,but with almost no effect on normal breast tissue. Breast cancer cells has moderate sensitivity to radiation,so improving the sensitivity of cancer cells to radiotherapy will improve the curative effect greatly.Higher doses of radiation therapy will be given after local excision,it may cause certain damage to the surrounding normal tissue when kill tumor cells.The expression of POLQ protein in human breast cancer cell line MCF-7will be confirmed by Western blot.And then POLQ-siRNA be designed and transfected into MCF-7cells with LipofectamineTM2000to inhibite of the POLQ expression effectively,Later, different doses of X rays will be used to irradiate MCF-7cells before and after transfection,and the effect of radiosensitivity of POLQ silencing on the cell lines was observed. Furthermore, the mechanism of radiosensitization will be explored.Objective:RNAi technology was used to silence the expression of POLQ in human breast cancer cell line MCF-7,and then the changes of the proliferative ability, the rate of apoptosis, and the radiation sensitivity of MCF-7cells were studied. We hope to provide a new train of thought in improving the sensitivity of radiotherapy on breast cancer cells.Methods:1. Expression of POLQ protein in human breast cancer cell line MCF-7was detected by Western blot. Small interference RNA sequence (POLQ-siRNA) was designed and synthesized according to the coding region sequence of POLQ gene, and transfected into MCF-7cells with LipofectamineTM2000.2. Transfected efficiency of siRNA on MCF-7cell line was observed by fluor escence microscopic:Adherent cells MCF-7were cultured in24-well plates, when the growth density of cells reaches60～70%, Control-siRNA-FAM (fin al concentration,5,10,20,40,80nmol/L) and LipofectamineTM2000(final concentration:1.5,2,2.5,3ml/L) make siRNA lipofectamine composite ma terials acrossly, and added into the holes20minetes later, fluorescence micro scopy was used to observe the transfected efficiency in6-12h.3. MCF-7cells were divided into three groups, real-time quantitative PCR (qRT-PCR) and Western blot techniques were used to evaluate the expression of POLQ mRNA and the corresponding protein,and to confirm the effect of gene silencing.4. Colony forming ability of cells was tested by colony-forming test after the transfection:Cells of each group were digested by trypsinase6hours after transfection, and cultured in six-well plates with100cells/hole; each group contained three replica wells. Later,cells were cultured in incubator which was maintained at37℃in water-saturated5%CO2/95%air for2weeks. When each hole appears visible clones, culture was terminated, the culture liquid was abandoned,4%parafonnaldehyde was used to fix the cells for20minetes, and then cells be stained by0.1%crystal violet for15minetes.At last,count the clones>50cells, and calculate the clone formation rate (PE).5.The proliferative ability of cells was measured by Mcthylthiazolyldiphenyl-tet razolium bromide(MTT) assay:MCF-7cells were cultured in96-well plates with4.5×103cells/hole, and be transfected after24hours’incubation.MTT (5mg/ml) was added to the holes24,48and72h after transfection with20uL/hole, the supernatant was discarded after4hours’incubation, and each h ole was added with150ul of dimethyl sulfoxide (DMSO), after10min on t he oscillatory cradle, the absorbance (A) values at490nm wavelength were read by the microplate reader.6. The colony-forming experiment was carried out to test the radiosensitivity: MCF-7cells with different concentration were cultured in six-well plates, a nd irradiated with different doses of X-irradiation after24hours’incubation. Click multiple target model was used to fit the cell survival fraction accord ing to the results of colony-forming experiment, radiosensitizing ratio (SER) be used as the quantitative indicators of radiation sensitivity to observe the radiation sensitivity of cells.7. The apoptosis and cell cycle pattern was assayed by flow cytometry after exposuring to different doses of irradiation, so as to study the mechanisms of the radiosensitization when POLQ be silenced.8. Statistical analysis:survival curve was fitted according to click multiple target model by using GraphPad Prism4Demo software. SPSS13.0software was applied to analyze, CT values are shown with x, the other data is shown with x±s, each set of experimental data accords with normal distribution, protein content [average absorbance (INT) x hybridization signal area (S)] of3groups and the comparation among clone formation rate of3groups were processed with one-way analysis. Comparations between two groups be with LSD method.The comparation among the absorbance of A values, cell cycles and apoptosis rates of cells of3groups irradiated with different doses of irradiation were processed with single factor repeated measures analysis of variance. alpha=0.05.Results:1. With cervical carcinoma cell line Hela as a positive control, POLQ protein was confirmed to be highly expressed in MCF-7,and it is easily to be degraded into～170kDa and～100kDa fragments.2. With the final concentration of40nmol/L of siRNA and2.5UL/ml of Lipof ectamineTM2000respectively, POLQ-siRNA can effectively transfected into MCF-7cells tested by thefluorescence microscopy, the transfection efficienc y is of more than90%calculated via the method of cell counting;3. qRT-PCR and Western Blot revealed that expression of POLQ mRNA and protein decreased to12.158%and (32.013±0.654)%, respectively, as compared with the Control cells.4. The change of the proliferative ability of MCF-7cells after the transfection:The colony-forming experiment show the colony forming ability of cells decreased. MTT assay showed the absorbance of the POLQ-siRNA group was significantly lower than that of the blank control group(P=0.000), and there is no significantly differences between the Control-siRNA cells and the blank control group cells(P=0.167). 5. Click multiple target model was used to fit the cell survival fraction according to the results of colony-forming experiment, the radiobiological parameters display, D0(Gy) and Dq(Gy) of POLQ-siRNA group was significantly decreased, the radiosensitization ratio (SER) is equal to1.24,demonstrating the radiation sensitivity of POLQ-siRNA cells increased obviously.6. POLQ-siRNA cells showed a lower rate of GapS than that that of the Control cells(P=0.001), but a higher GapO/Gapl arrest than that that of the Control cells after exposure to0,4or8Gy irradiation.When the dose of irradiation increased, the rate of Gap2/GapM in every groups increased obviously.The rate of apoptosis of POLQ-siRNA cells increased obviously compared with Control cells (P<0.01) exposuring to0,4or8Gy irradiation, but there were no significantly differences between the Control-siRNA cells and the Control cells(P>0.05). When the dose of irradiation increased from4Gy to8Gy, the apoptosis rate of3groups cells decreased (P<0.01).Conclusions:POLQ highly expressed in human breast cancer MCF-7cells.SiRNA can effectively silence the expression of POLQ in MCF-7. Inhibiting the expression of the gene can lead to a reduction in cell proliferation, and increase the rate of GapO/Gapl and the rate of apoptosis of POLQ-siRNA cells,and can significantly increase the radiosensitivity of MCF-7cells. When the doses of irradiation increased,the rate of Gap2/M in every groups would increase,but the rate of apoptotic won’t continue to increase.