Expression And Significance Of Genes Related To Sternness In Cervical Cancer Preradiotherapy And Postradiotherapy

Radiotherapy is one of the most important treatment for tumor therapy. As reports by WHO in 1992, about 60-70% patients with malignant tumor need to undergo radiotherapy.About 45% tumors could be cured with different treatments, among them, 22% could be cured by surgery, 18% by radiotherapy and 5% by chemotherapy. Thus, radiation therapy played an important role in tumor treatment. With the widespread use of the precise radiotherapy facilities such as conformal raidotherapy, intensity modulated radiotherapy and Image guarded radiation therapy(IGRT) and so on, and the application of proton and heavy ion in clinic, the role of radiation therapy become more and more important in tumor therapy. However, in clinical practice, most radiation practitioners found that not every patient with tumor respond to the treatment.Moreover, some patients showed good responds at the beginning of the treatment. Radiation could well control the tumor and achieve good imaging and clinical efficacy. But local recurrence or distant metastasis occurred in a short time after treatment. Second cource irradiation usually could not possess good results. Therefore, tumor recurrence and metastasis become the difficult point in clinical work and the mechanism of which become the clinical and basic research hotspot. At present, most tumor biology researches showed that cancer stem cells might be an important reason for tumor recurrence and metastasis.Radio- and chemo-resistance are one of the important characteristics of cancer stem cells. The special biological behaviors of these cells attribute to their special gene expression profiles named stemness-related genes and molecular biological functions. The irradiation resistant mechanism of these stemness related genes is an important part of the tumor radiation biology. Until now, it is unclear that which genes are involed in redioresistance and what mechanism of these genes to play role.In this study, we screened the radioresistance related stemness genes with the whole human genome oligomicroarray in patients with cervical cancer who had undergone radical radiotherapy. Subsequently, we investigate the radioresistance mechanism of the interested stemness related genes. The relationship between these genes and prognosis is also verified.The results would provide reliable reference of prognosis and also provide a foundation for the development of individualized clinical radiotherapy. Meanwhile, we hope theinvestigation of these gene targets would be used for development of more specific radiotherapy method against cervical cancer.Research methods1、Residual tumor tissues of 3 patients undergoing a total radiotherapy dose of 50 Gy were obtained by punch biopsy. The tumor tissues before radiotherapy were also obtained.The differentially expressed genes in tissues before and after radiotherapy were screened by the whole human genome oligomicroarray.2、Quantitative real-time PCR validated the microarray results. The interested genes included CXCL12 and CD44 in this study were screened.3、CXCL12 and CD44 were validated at the protein level by immunohistochemistry in paraffin embedded cervical cancer tissues of 130 patients underwent radical radiotherapy.The relationship of CXCL12 and CD44 expression and prognosis of cervical cancer was also investigated.4、Three si RNA sequences of CXCL12 were transfected into Hela cervical cancer cells to inhibit the expression of CXCL12. We detected the expression of CXCL12 by ELISA and real-time quantitative RT-PCR. The influence of CXCL12 on the expression of CD44 and CXCR4 was investigated by flow cytometry.5、Hela cells transfected with si RNA of CXCL12 were treated with irradiation of different dose. Cell proliferation was detected by CCK8, cell apoptosis by PI double staining flow cytometry, the expression of CXCL12 by ELISA and real-time quantitative RT-PCR, the influence of on the expression of CD44 and CXCR4 by flow cytometry.Results1、There were 111 up-regulated and 127 down-regulated genes among a total of 238 differentiated genes that exhibited ≥ 3.0 –fold change and p < 0.05 were identified.The expression of CXCL12 in residual tumor tissues after 50 Gy dose of irradiation was34.37 times of that in tumor tissues before radiotherapy. The expression of CD44 in residual tumor tissues after 50 Gy dose of irradiation was 34.37 times of that in tumor tissues before radiotherapy. The real-time PCR results were in high correlation to the microarray data.2、Immunolocalization with anti-CXCL12 antibody largely showed positive staining in the cell membrane and cytoplasm of cancer cells, the positive staining of CD44 mainly in the cell membrane. In the patients with cervical cancer underwent radical radiotherapy,we did not found correlation between the expression of CXCL12 and severalclinicopathological factors including age, sex, FIGO stage, lymphatic metastasis, tumor size and treatment program. No correlation was also found between the expression of CD44 and the same clinicopathological factors. A multivariate analysis, including the prognostic factors determined by the univariate analysis to have statistical significance,revealed a positive finding for FIGO stage(p=0.017), lymph node metastasis(p=0.013),treatment program(p=0.014), CXCL12 expression(p=0.038), CD44v5 expression(p=0.039), CXCL12 and CD44v5 coexpression(p=0.000) to be independent significant prognostic factors. In the 130 patients, the significant correlation was found between the expression of CXCL12 and CD44(P = 0.028). The 5-year survival of patients with negative expression of CXCL12 was 50.6%, while only 30.0% in patients with positive expression. The 5-year survival of patients with negative expression of CD44 was 42.3%,while only 33.4% in patients with positive expression. Coexpression of CXCL12 and CD44 was found in 34 patients. The 5-year survival rate of these 34 patients was significantly lower of only 22.9%. The 5-year survival rate was 44.5% in the remaining patients(P = 0.005).3 、 In real-time quantitative PCR, the relative expression of CXCL12 m RNA was0.69±0.16 in the group of Si-RNA1, 0.27±0.05 in the group of Si-RNA2, 0.50±0.12 in the group of Si-RNA3. In the ELISA of CXCL12, the relative expression of CXCL12 protein was 0.62±0.04 in the group of Si-RNA1, 0.33±0.04 in the group of Si-RNA2, 0.52±0.05 in the group of Si-RNA3. The inhibitory effect of CXCL12 si RNA on the expression of CXCL12 was most significant in the group of Si-RNA2 no whether in the protein level or the RNA level(P<0.05).4、 The change of CD44 and CXCR4 protein in Hela cells were detected by flow cytometry 48 h after transfected in the group of Si-RNA2. The results show that the expression content of CD44 protein is 0.69±0.01, which is significant lower than that in the blank control group(1.00±0.02) and that in the negative group(1.02±0.03). The expression content of CXCR4 protein is 1.41±0.08, which is significant lower than that in the control group(1.00±0.01) and that in the negative control group(0.96±0.03).5、CXCL12 silence combined irradiation could inhibit the Hela cells proliferation and promote cell apoptosis. The cell viability rate decreased from 79% to 62% after 4 Gy dose of irradiation, from 59% to 44% after 8 Gy dose of irradiation. The results of flow cytometry with PI staining showed that the apoptosis rate increased from 21% in cells irradiated with 4 Gy dose to 27.96% in cells transfected with si RNA2 combined 4 Gy doseof irradiation. The apoptosis rate increased from 39% in cells irradiated with 8 Gy dose to51% in cells transfected with si RNA2 combined 8 Gy dose of irradiation.6、CXCL12 silence combined irradiation could inhibit the expression of CD44 protein and promote the expression of CXCR4 protein in cells transfected with si RNA2 contrast to those in cells treated with irradiation simply. The results of flow cytometry showed that the expressing quantity of CD44 protein decreased from 1.55±0.02 in cells irradiated with 4Gy dose to 1.33±0.02 in cells transfected with si RNA2, while the expressing quantity of CXCR4 protein increased from 1.13±0.02 to 1.40±0.01. The expressing quantity of CD44 protein decreased from 1.85±0.02 in cells irradiated with 8 Gy dose to 1.40±0.01 in cells transfected with si RNA2, while the expressing quantity of CXCR4 increased from1.27±0.02 to 1.48±0.02.Conclusions1、There were 111 up-regulated and 127 down-regulated genes among a total of 238 differentiated genes that exhibited ≥ 3.0-fold change and p < 0.05 were identified. The increasing expression of CXCL12 and CD44 occurred in residual tissues, with the radio of34.37 and 3.40.2、The expression of CXCL12 had correlation with the expression of CD44. CXCL12 and CD44 were the important prognosis factors for patients with cervical cancer underwent radical radiotherapy.3、Silence of CXCL12 could increase radiosensitivity of cervical cancer by preventing cell proliferation and apoptosis.4、CXCL12 might promote radiation resistance by mediating the microenvironment of CD44-positive cancer stem cell.