| Background and ObjectiveCervical cancer is the most common malignancy in the female reproductive system,which seriously endangers the health and life of women.Radiotherapy is one of the most common therapeutic intervention for cervical cancer.How to improve the effectiveness of radiation therapy has always been a difficult problem in the treatment of cervical cancer.So far the cellular resistance to radiotherapy is the main cause of the treatment failure in cervical cancer patients.Therefore,it is urgent to find new targets to improve the cellular radio sensitivity in cervical cancer.Researches demonstrated that long non-coding RNAs(lncRNAs)are implicated in radiation sensitivity of various cancers.lncRNA growth arrest special 5(GAS5),a tumor suppressor,was down-regulated in many kinds of cancers,including cervical cancer.There is study showed that GAS5 expression was an independent prognostic factor for patients with cervical cancer,and overexpression of GAS5 increased cisplatin resistance in cervical cancer cells by regulating Akt phosphorylation.Another study showed accumulation of GAS5 contributes to human prostate cancer cell radio sensitivity by inhibiting cell survival and facilitating apoptosis and DNA damage.However,whether GAS5 can regulate the radio sensitivity of cervical cancer cells has not been reported.MicroRNA(miRNA)is involved in many pathological processes such as tumor occurrence and development.MiR-106 b plays a carcinogenic role in a variety of malignancies,regulating apoptosis,proliferation,differentiation and cell cycle progression.Several studies confirmed that miR-106 b was highly expressed in cervical cancer tissues compared with normal cervical tissues.The researchers found that miR-106 b is expressed in cervical cancer tissue and that miR-106 b regulates the radio sensitivity of various cancers.However,whether miR-106 b is involved in the regulation of radiotherapy sensitivity of cervical cancer has not been reported.In the current study,we found that GAS5 was decreased and miR-106 b was increased in tumor tissues from cervical cancer patients who were insensitive to radiation therapy.By bioinformatics-based target prediction analysis(DIANA,LncBase Predicted v.2),GAS5 was found to be a molecular sponge of miR-106 b.Therefore,we speculated that GAS5 might participate in the radio sensitivity of cervical cancer cells by targeting miR-106 b.The purpose of our study is as follows :1.To investigate the biological role of GAS5/miR-106 b in radio sensitivity of cervical cancer in vitro and in vivo.2.To explore the relationship between GAS5 / mir-106 b and the regulatory mechanism of radiotherapy sensitivity of cervical cancer.3.These findings might help establish new strategies for the sensitivity of cervical cancer cells to radiotherapy.Materials and Methods1.Twenty cervical cancer patients(FIGO stage IIB-IV)included in this study.According to the results of the evaluation,samples were divided into radio-sensitive group(11 cases)and radio-resistant group(9 cases).Human cervical cancer cell lines SiHa and ME180 cells were purchased.2.The relative expression of GAS5 and miR-106 b between the two groups of tumor tissues were detected by qRT-PCR.The survival fractions of SiHa cells and ME180 cells were detected after the radiation treatment,and then the relative expression of GAS5 and miR-106 b were detected.To explore the relationship between the GAS5 expression and radiosensitivity of cervical cancer.3.The overexpression and interference of GAS5 in cervical cancer cell lines were constructed by transfecting cells.After transfection,the expression of GAS5 was detected by qRT-PCR,and the cell survival fraction was detected after further radiation treatment.To investigate the effect of GAS5 expression on the radiosensitivity of cervical cancer cells.4.LncBase prediction Predicted v.2 was used to analyze its possible target genes.Bioinformatics software analysis revealed a potential combination of GAS5 and miR-106 b,and then using RIP assay and RNA pull down assay to verify the interaction between GAS5 and miR-106 b.5.The change in survival fraction after irradiation was examined by overexpressing/inhibiting the overexpression/inhibition of GAS5 of SiHa cells and ME180 cells by ±miR-106 b.To explore whether GAS5 regulates the radiosensitivity of cervical cancer cells through miR-106 b.6.The GAS5 interference mouse model and the GAS5 overexpressing mouse model were constructed.The tumor volume was measured after radiation treatment,and the expression levels of GAS5 and miR-106 b were detected by qRT-PCR.To verify the effect of GAS5 on the radiosensitivity of cervical cancer in vivo.Results1.GAS5 was decreased and miR-106 b was increased in radio-resistant human cervical cancer tissues vs radio-sensitive(P<0.01).After irradiation treatment,the survival fraction of SiHa cells was significantly higher than that of ME180 cells(P<0.05),and the expression of GAS5 in SiHa cells was lower than that in ME180 cells(P<0.01),and the expression of miR-106 b was higher than that in ME180 cells(P<0.05).It indicated that GAS5 was decreased and miR-106 b was increased in cervical cancer tissues and cell lines of the radio-resistant group.2.The expression of GAS5 in pcDNA-GAS5-SiHa cells increased(P<0.01),and the survival fraction decreased after radiation treatment(P<0.05).The expression of GAS5 in siRNA-GAS5-ME180 cells decreased(P<0.01),radiation treatment.The post-survival score increased(P<0.05).This indicates that overexpression of GAS5 enhances the sensitivity of cervical cancer cells to radiotherapy.3.Bioinformatics software analysis showed potential combination of GAS5 and miR-106 b,RIP assay results showed that GAS5 and miR-106 b significantly increased(P<0.05)in the production precipitated by anti-AGO2 which revealed that both of them could simultaneously exist.In accordance with this,RNA pull-down assay showed that miR-106 b expression was significantly higher than loc285194(P<0.05)which revealed that miR-106 b interacted with GAS5 in SiHa cells possibly through a sequence-specific manner.4.After radiation treatment,the survival fraction of SiHa cells transfected with pcDNA-GAS5 was decreased(P<0.05),and the results were reversed after transfection of miR-106 b mimics(P<0.05).After radiation treatment,the survival fraction of ME180 cells transfected with siRNA-GAS5 was increased(P<0.01),and the result was reversed after transfection with miR-106 b inhibitor(P<0.01).This indicates that GAS5 enhances the radiosensitivity of cervical cancer cells by inhibiting miR-106 b.5.In vivo,the tumor volume increased in the lenti-siRNA-GAS5 group after radiation treatment(P<0.01);qRT-PCR results showed that the GAS5 expression was down-regulated(P<0.01)and miR-106 b was up-regulated(P<0.05).The tumor volume of the mice in the lenti-GAS5 group decreased after radiation treatment(P<0.01),and the expression of GAS5 in the tissues increased(P<0.01)and the miR-106 b was down-regulated(P<0.01).This indicates that overexpression of GAS5 in vivo can enhance the radiosensitivity of cervical cancer.ConclusionOverexpression of GAS5 enhancing the sensitivity of cervical cancer cells to radiotherapy by down regulating miR-106 b. |
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