| ObjectiveRadiation therapy is one of the common treatments for cervical cancer,so improving the radiosensitivity of tumor cells is essential to enhance the efficacy of radiotherapy.IL-11 is a member of the IL-6 cytokine family.Previous studies have shown that IL-11 is involved in the occurrence and development of a variety of tumors.However,the role of IL-11 in tumor radiosensitivity remains unclear.The purpose of this study was to explore the effect of IL-11 expression on the radiosensitivity of cervical cancer and the related molecular mechanism,in order to provide a new idea for improving the efficacy of radiotherapy in patients with cervical cancer.Methods1.TCGA public database was used to analyze the expression of IL-11 in pan-cancer tissues and para-cancerous tissues2.TCGA public database was used to analyze the expression of IL-11 in normal cervical tissue and cervical cancer tissue,and analyzed the relationship between the expression level of IL-11 and the clinical stage of cervical cancer patients.The receiver operating characteristic curve(ROC curve)was conducted to the diagnostic value of IL-11 for patients with cervical cancer.Kaplan-Meier(K-M)was used to evaluate its effect on the prognosis of cervical cancer patients.3.The expression of IL-11 in the supernatant of cervical cancer cells(HeLa,Siha,Caski and C33A)and normal cervical epithelial cells(Ect1/E6E7)was detected by ELISA.4.The expression level of IL-11 mRNA in cervical cancer cell lines(HeLa,Siha,Caski and C33A)was detected by real-time quantitative PCR(qPCR).The radiosensitivity of four cervical cancer cell lines was detected by colony formation and apoptosis assay.And the relationship between the expression level of IL-11 and radiosensitivity of cervical cancer cells was analyzed.5.IL-11 shRNA was used to silence the expression of IL-11 in HeLa cells,and human recombinant IL-11(rhIL-11)was used to stimulate C33 A cells.The protein and mRNA expression of IL-11 in HeLa cells were transfected with IL-11 shRNA.The effects of silencing IL-11 or rhIL-11 treatment on the proliferation and colony formation ability of cervical cancer cells irradiated by 0,2,4,6,8Gy X-ray were detected by CCK8 and colony formation assay.The effects of silencing IL-11 or rhIL-11 treatment on the cell cycle and apoptosis of cervical cancer cells irradiated by 6Gy X-ray were detected by flow cytometry6.Western Blot experiment was used to detect the effects of silencing IL-11 or rhIL-11 treatment on the phosphorylation levels of STAT3,ERK,and AKT in cervical cancer cells and the expression levels of downstream target genes(Bcl-2,Bcl-xl,Bax)in the PI3K/Akt pathway.7.Cervical cancer cell were treated with AKT inhibitor LY294002,after determining the inhibitory efficiency by western Blot.Cervical cancer cells were treated with 40 μM LY294002 and 25,50,100ng/ m L rhIL-11.The apoptosis level and colony formation ability of cervical cancer cells were detected by colony formation assay and flow cytometry after 6Gy X-ray irradiationResults1.TCGA database analysis showed that the expression level of IL-11 in tumor tissues such as bladder urothelial carcinoma(BLCA),breast cancer(BRCA),cervical cancer(CESC),Cholangiocarcinoma(CHOL),Stomach adenocarcinoma(STAD)was higher than that in para-cancerous tissue.In contrast,the expression level of IL-11 in skin cutaneous melanoma(SKCM)and kidney renal clear cell carcinoma(KIRC)was lower compared to para-cancerous tissue.However,IL-11 expression showed no significant difference in brain lower grade glioma(LGG)and prostate adenocarcinoma(PRAD)compared to paracancerous tissue.2.TCGA database analysis showed that the expression level of IL-11 in cervical cancer tissue was significantly higher than that in normal cervical epithelial tissue,and was correlated with the clinical stage of cervical cancer patients.The ROC Curve showed that the Area Under the Curve(AUC)was0.899,and the 95% confidence interval(95%CI)was 0.821-0.977,suggesting that IL-11 is a valuable marker in the clinical diagnosis of cervical cancer patients.K-M survival curve analysis showed that the overall survival time and diseasefree survival time of IL-11 high expression cervical cancer patients significantly shorter than that of the lower expression patients.3.ELISA results showed that the expression level of IL-11 in four cervical cancer cells(HeLa,Siha,Caski and C33A)was significantly higher than that in normal cervical epithelial cells(Ect1/E6E7).4.The expression level of IL-11 mRNA in cervical cancer cells from high to low was HeLa,Siha,Caski and C33 A cells.After radiation treatment,the clone formation ability of four cervical cancer cells was HeLa,Siha,Caski and C33 A cells from high to low,and the apoptosis rate was C33 A,Caski,Siha and HeLa cells from high to low,suggesting that the expression level of IL-11 was positively correlated with the radioresistance of cervical cancer cells.5.The knockdown effect of IL-11 shRNA-3 was the most effective among the designed three shRNAs.Interfered IL-11 expression reduced the proliferative ability and colony formation ability of HeLa cells when exposed to various doses of X-ray irradiation(0,2,4,6 and 8Gy),rhIL-11 upregulated cell proliferative ability of C33 A cells after exposure to X-ray irradiation at doses ranging from 0to 8Gy.Silencing IL-11 significantly promoted HeLa cell apoptosis and G2/M phase arrest after 6Gy X-ray irradiation,and rhIL-11(25,50,100ng/m L)significantly inhibited C33 A cell apoptosis and G2/M phase arrest.6.Silencing IL-11 significantly reduced AKT phosphorylation expression,but STAT3 and ERK phosphorylation were not affected in HeLa cells.Rh IL-11 treatment significantly increased AKT phosphorylation expression,but STAT3 and ERK phosphorylation were not affected in C33 A cells.Silencing IL-11 can reduce the expression levels of Akt downstream target genes Bcl-2 and Bcl-xl,and upregulate the expression level of Bax.Rh IL-11 treatment can increase the expression levels of Akt downstream target genes Bcl-2 and Bcl-xl,and reduce the expression level of Bax.7.Akt phosphorylation was effectively inhibited by 40μΜ LY294002 in C33 A cells.40μΜLY294002 significantly decreased the survival fraction and promoted apoptosis in C33 A treated by rhIL-11 after exposure to 6 Gy X-ray irradiationConclusionIL-11 is highly expressed in cervical cancer tissues and is correlated with a poor prognosis in cervical cancer.The expression level of IL-11 is positively correlated with the radioresistance of cervical cancer cells.IL-11 may promote the radioresistance of cervical cancer cells through the activation of PI3K/Akt pathway.This study is expected to provide a potential therapeutic target to solve the radiation resistance problem of cervical cancer. |
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