Studies On The Intervention Of Wip1 Gene In Radiosensitivity Of Cervical Cancer Line HeLa

Wild type p53-induced protein phosphatases(Wip1) is a memberof the Ser/Thr PP2 C family, which abnormally expressed in brain glioblastoma, breast cancer, membrane gland neuroendocrine tumor and other malignant tumors. Wip1 is a new discovery proto-oncogenes in cancer cells. Studies have shown that the expression of Wip1 gene depended on the activation of p53 protein, and the overexpression of Wip1 protein could activate p53 protein dephosphorylation and reduce the mutation selective of p53 gene. The abnormal expression of Wip1 could affect prognosis and increase tumor resistance to anticancer drugs in breast cancer, glioblastoma and other malignant cancer. However, the correlation between Wip1 and cervical cancer was rarely studied, especially in its radiosensitivity. Based on the above status quo, this study investiaged the role of Wip1 in the biological behavior and radiation sensitivity of He La cells, and elucidatedits molecular mechanisms.This study provided experimental basis and theoretical support for cervical cancer radiosensitization by targeted Wip1, and also provided new therapeutic targets for personalized treatment of cervical cancer patients.Objective: To explore the impact of intervention of Wip1 Gene in radiosensitivity of cervical cancer line He La and identify the related molecular mechanisms in human breast cancer cells, the effect of intervention of Wip1 Gene in breast cancer therapy will be also demonstrated.Methods:(1) the irradiation conditions used in this study: GC3000 Elan irradiator(MDS Nordion, Canada), a single γ- rays radiation dose is 2 ~ 3 Gy.(2) Western bolt was used to detect the expression level of Wip1 in cervical cancer cell lines;(2) MTT assay was used to detect the proliferation activity of different cervical cancer cell lines(3) The pc DNA3.1-Wip1 and si RNA-Wip1 recombinant plasmid were constructed and transfected into human cervical carcinoma cell line He La cells, empty vector cells,si Wip1 cells and si Wip1+overexpressing Wip1 cells was constructed;(4) real-time quantitative PCR assay was used to detect the m RNA expression of stably transfected Wip1 cells;(5) Western blot assay was used to detect the protein expression of stable transfection Wip1 cells;(6) colony formation assay was used to detect the radiosensitivity of He La cells;(7) the apoptosis level of He La cells was detected by flow cytometry;(8) comet assay was used to detect the DNA fragment distribution and observe cell DNA damage in He La cells after γ-ray irradiation;(9) Western blot assay was used to detect the protein expression of p38, p-p38, p53 and p-p53 in He La cells;(10) p38 MAPK inhibitor SB203580 was used to evaluate the relationship between p38 MAPK signal pathway and He La radiation sensitivity;(11) software Image J was used to do grayscale analysis for protein electrophoresis bands;(12) Student’s t-test was used to data analysis, p <0.05 represents the difference Statistical significance.Results: 1. The expression level of Wip1 in various cervical cancer cells with different radiosensitivity Wip1 have different expression level in different cervical cancer cell lines. The expression level Wip1 in He La cells were the highest, the expression level Wip1 in Si Ha, C-33 A, Me180 were decreased in turn; after γ-ray irradiation,the proliferative activity of He La cells were strongest, and had the strongest radiation resistent, the proliferative activity of Si Ha, C-33 A, Me180 were decreased in turn. 2. The construction of Wip1 gene silencing He La cells Wip gene and its si RNA were designed. Molecular cloning technique was used to construct pc DNA3.1-Wip1 and si RNA-Wip1 Recombinant plasmid by restriction enzyme digestion and sequencing confirmed. Empty vector cell, si Wip1 cell and si Wip1 + overexpressing Wip1 He La cells were constructed by transfected with pc DNA3.1 empty vector, si Wip1 and si Wip1+ pc DNA3.1-Wip1 plasmid. After transfection 48 h, compared to the empty vector cells and si Wip1+overexpression Wip1 cells, The Wip1 m RNA and protein expression of si Wip1 cells were significantly reduced, the gap were statistically significant(P<0.05), the difference between empty vector cells and si Wip1+overexpression Wip1 cells was no significant difference(P>0.05). 3. The gene silencing of Wip1 increase the radiosensitivity of He La The proliferation activity was significantly decreased in si Wip1 cells after transfection 48 h,and the cell number were significantly lower than empty vector cells and si Wip1+overexpression Wip1 cells at 72 h and 96 h, the difference were statistically significant(P<0.05), the difference between empty vector cells and si Wip1+overexpression Wip1 cells were no significant differenceat all times(P>0.05); the results of colony-forming number and radius were similarly with proliferation activity experiment; the apoptosis level were opposite with proliferation activity experiment. With the joint of γ- ray radiation, the colony-forming number and radius of si Wip1 cells were significantly lower than empty vector cells and si Wip1+overexpression Wip1 cells, the difference were statistically significant(P<0.05), and the difference between empty vector cells and si Wip1+overexpression Wip1 cells was no significant difference(P>0.05); the apoptosis level were opposite with colony-forming experiment. 4. The gene silencing of Wip1 reduced the He La cells DNA damage repair under γ-ray irradiation Compared with empty vector and si Wip1+overexpression Wip1 cells, the DNA damage degree of si Wip1 cells was severe, The comet head DNA percentage were significantly reduced, but the percentage of small fragments of comet tail were significantly higher, the difference were statistically significant(P<0.05), and the difference between empty vector cells and si Wip1+ overexpression Wip1 cells was no significant difference(P>0.05) 5. The relationship between Wip1 and p38 MAPK signaling pathway protein expression Compared to the empty vector cells and si Wip1+overexpression Wip1 cells, the expression level of p-p38, p53 and p-p53 in si Wip1 cells were significantly reduced(P<0.05, P<0.01, P<0.05), the difference between empty vector cells and si Wip1+overexpression Wip1 cells was no significant difference(P>0.05). And the p38 level were no significant difference among the three groups(P>0.05). When SB203580 was pretreated, compared to si Wip1 cells, the expression level of p-p38, p53 and p-p53 in si Wip1+SB20358 cells were significantly lower, the difference were statistically significant(P<0.05, P<0.01, P<0.05), and were similar with empty vector cell(P>0.05), the p38 level were no significant difference among the three groups(P>0.05). 6. SB203580 reverse the radiosensitivity benefit which is acquired by Wip1 decreased. When SB203580 was pretreated, compared to empty vector cells, the colony-forming number and radius were no difference between si Wip1+SB203580 cells and empty vector cells(P>0.05), the apoptosis level were similar with colony-forming results(P>0.05); when combined with γ-ray irradiation, the colony-forming number and radius were also no difference between si Wip1+ SB203580 cells and empty vector cells(P>0.05), the apoptosis level were similar with colony-forming results(P>0.05); showed that SB203580 reversed the radiosensitivity benefit which was acquired by Wip1 decreased.Conclusion: 1. Wip1 play an important regulatory role in the radiosensitivity of He La cells, the Wip1 of gene silencing increases the proliferation activity, anti-apoptosis and colonyforming capacity of He La cells under γ-ray irradiation; 2. Wip1 can enhance the DNA damage repair of He La cells induced by γ-ray radiation, thereby enhancing He La cells radiation tolerance; 3. The gene silencing of Wip1 in He La cells can participate in formation of radiation tolerance by activating p38 MAPK signaling pathway, the action site is the increasing expression of p53 and the increasing phosphorylation of P38; In summary, this study enriches the Wip1-tumor radiosensitivity network, found Wip1 regulation action to the radiosensitivity of He La cells and p38 MAPK signaling pathway. Wip1, as a therapeutic target, could provide new theoretical and practical perspectives for cervcial irradiation therapy, and provide theoretical support for clinical application of radiotherapy-tumorgene. But more work should be done to further explore the regulatory mechanisms of Wip1 in radiation-sensitive, especially coregulation mechanisms and animal experiment.