| Background and Objective:MicroRNA(mi R)-590 was found to play potential roles in cancer development,however,there is no report about the expression and function of miR-590 in human gliomas until now.The present study aimed to investigate the expression of miR-590 in human glioma tissues and radioresistant human glioblastoma cells(U251R),and to examine the effect and related molecular mechanism of miR-590-3p on the radiosensitivity of U251 R cells in vitro.The results from reverse transcription-quantitative polymerase chain reaction(RTqPCR)showed that miR-590-3p was upregulated in the human glioma tissues and the radioresistant human glioblastoma cells,and miR-590-3p expression level was higher in the high grade gliomas than that in the low grade gliomas.The in vitro experiments revealed that the miR-590-3p inhibitor enhanced the radiosensitivity of U251 R cells by suppressing cell proliferation,decreasing colony formation capacity,and increasing cell apoptosis rate,as demonstrated by MTT,colony formation and flow cytometry analyses.The luciferase reporter assay demonstrated that leucine-rich repeats and immunoglobulin-like domains protein 1(LRIG1)was a direct target of mi R-590-3p.Furthermore,we found that the effect of miR-590-3p suppression on cell viability,colony formation capacity and cell apoptosis rate could attenuated by the knockdown of LRIG1 in the U251 R cells.In conclusion,this study firstly revealed that mi R-590-3p was upregulated in the human glioma tissues and the radioresistant human glioblastoma cells,and miR-590-3p contributes to the radioresistance of human glioblastoma cells by directly targeting LRIG1.This study may provide potential therapeutic strategies to prevent radioresistance in human gliomas.Gliomas account for approximately 30% of all brain and central nervous system tumors.Glioblastoma is the most common and malignant gliomas.The median survival time of glioblastoma patients was only 4.9 months after diagnosis.Resistance ofglioblastoma cells to irradiation therapy is a major obstacle in the treatment of human glioblastoma.Therefore,it is important to explore the mechanisms underlying this resistance to irradiation therapy and find out efficient radiosensitizers.MicroRNAs(miRNAs or miRs)are a class of small,functional and non-coding RNAs which regulate the expression of target mRNAs at the post-transcriptional level.The altered expression of specific miRNAs has been implicated to play important roles in the initiation and progression of tumors,including tumor radiation resistance.The human mi R-590 family has two mature members,namely miR-590-3p and miR-590-5p.miR-590 was found to play potential roles in cancer development,including breast cancer,cervical cancer,clear cell renal carcinoma,and hepatocellular carcinoma.However,there is no report about the expression and function of miR-590 in human gliomas until now.In this study,we for the first time investigated the expression of miR-590-3p and miR-590-5p in human glioma tissues and radioresistant human glioblastoma cells(U251R),and we examined the effect of miR-590-3p on the radiosensitivity of U251 R cells in vitro.Furthermore,a novel direct target by which miR-590-3p exerts its effect on radiosensitivity was elucidated.Materials and Methods1.Through the microRNA.org database and the Database on Predicted and Published MicroRNAs(miRWalk),we previously found that the expression of MicroRNA-590-3p(miR-590-3p)is most strongly correlated with human tumors.2.Establishment of the radioresistant human glioblastoma U251 cells(U251R).3.To determine the expression of miR-590-3p in normal brain tissue and Human glioma tissue samples respectively,we used the methods of Real-time quantitative RT–PCR(qRT–PCR).4.We examined the expression of miR-590-3p,miR-590-5p and LRIG1 in the radioresistant cells U251 R using RT-qPCR and western blot analysis..5.Effect of miR-590-3p on the radiosensitivity of U251 R cells was demonstrated by MTT,colony formation and flow cytometry analyses.6.The expression of LRIG1 proteins were detected by western blot.7.Luciferase reporter assay demonstrate tha LRIG1 is a direct target of miR-590-3p.8.The data were expressed as the mean+SD.Comparison of data between 2 groupswas made using the Student's t-test.A P value less than 0.05 was considered that the difference was considered statistically significant.Results1.The expression of miR-590 in human glioma tissues.We examined the expression of miR-590-3p and miR-590-5p in human glioma tissues using RT-qPCR.The mRNA level of miR-590-3p was significantly upregulated in the glioma tissues compared with the normal brain tissues.Furthermore,we found that miR-590-3p expression level was higher in the high grade gliomas(grade ?-?)than that in the low grade gliomas(grade?-?).However,the m RNA level of miR-590-5p did not show any difference between the normal and the glioma tissues.2.Establishment of the radioresistant human glioblastoma U251 cells(U251R).As shown in Fig.2A,the U251 R cells exhibited higher cell viability compared with theU251 cells.The results from colony formation assay demonstrated that U251 R cells displayed higher colony formation capacity than U251 cells.In addition,U251 R cells had lower cell apoptosis rate than U251 cells.3.Expression of miR-590 and LRIG1 in U251 R cells.We examined the expression of miR-590-3p,miR-590-5p and LRIG1 in the radioresistant cells U251 R using RT-qPCR and western blot analysis.The results from RT-qPCR analysis showed that compared with the U251 cells,the expression level of miR-590-3p was significantly increased in the U251 R cells;however,the expression level of mi R-590-5p was not significantly different between U251 and U251 R cells.The results from western blot analysis demonstrated that the expression of LRIG1 protein was downregulated in the U251 R cells compared with the U251 cells.4.Effect of miR-590-3p on the radiosensitivity of U251 R cells.To investigate the effect of miR-590-3p on the radiosensitivity of U251 R cells,the miR-590-3p inhibitor was transfected into the U251 R cells,and the cells were exposed to 6Gy IR for 24 h.The mRNA level of miR-590-3p in the cells transfected with the miR-590-3p inhibitor decreased to 15% of the control.The results from MTT assay revealed that the cells transfected with the miR-590-3p inhibitor exhibited significantly decreased cell viability compared with the control.Colony formation capacity was also decreased in the cells transfected with the mi R-590-3p inhibitor.Moreover,FCM assay demonstrated that transfection with the miR-590-3p inhibitor markedly increased the cellapoptosis rate.5.LRIG1 was a direct target of miR-590-3p.To reveal the relationship between miR-590-3p and LRIG1,the LRIG1 3 ? UTR containing the wild type or mutant potential target site of miR-590-3p was constructed,and co-transfected with the miR-590-3p mimic into the HEK293 cells.The results from luciferase reporter assay showed that the miR-590-3p mimic significantly decreased the luciferase activity of wild type LRIG1 3?UTR,whereas the luciferase activity of mutant LRIG1 3?UTR was not affected by the miR-590-3p mimic.6.LRIG1 mediated the effect of miR-590-3p on the radiosensitivity of U251 R cells.Toinvestigate whether LRIG1 mediates the effect of miR-590-3p on the radiosensitivity of U251 R cells,LRIG1 siRNA was transfected into the U251 R cells to knock down LRIG1,and the cells were exposed to 6 Gy IR for 24 h.mi R-590-3p inhibitor significantly increased the expression of LRIG1 protein in U251 R cells,but the increased LRIG1 expression was abolished by the transfection of LRIG1 siRNA.Meanwhile,the effect of miR-590-3p on the radiosensitivity of U251 R cells was attenuated by LRIG1.As demonstrated in the study,suppression of miR-590-3p by treatment with miR-590-3p inhibitor resulted in decreased cell viability and colony formation capacity,and increased cell apoptosis rate;however,these effects were reversed by the transfection of LRIG1 si RNA.The cell viability,colony formation capacity and cell apoptosis rate were significantly different between the miR-590-3p inhibitor + scramble siRNA group and the miR-590-3p inhibitor +LRIG1 siRNA group.Conclusions:In the present study,we firstly demonstrated that miR-590-3p was upregulated in the human glioma tissues,and its expression level was higher in the high grade gliomas than that in the low grade gliomas.These data suggested that miR-590-3p may contribute to the initiation and development of human gliomas.Radiotherapy is essential for the treatment of more than half of the newly diagnosed cancer patients.Until now,some microRNAs have been demonstrated to be involved in the radiosensitivity of human glioma cells,such as miR-181 a,miR-124,miR-26 a,et al(13-17).In the present study,we firstly found that the expression level of miR-590-3p was higher in the radioresistant human glioblastoma cells than that in the parental glioblastoma cells.Then,we performed the in vitro experiments to examine the role of miR-590-3p in the radiosensitivity of glioblastoma cells.We found that downregulation of mi R-590-3p enhanced the radiosensitivity of the radioresistant human glioblastoma cells U251 R,as demonstrated by suppressed cell proliferation,decreased colony formation capacity,and increased cell apoptosis rate.These results indicated that miR-590-3p decreases radiation sensitivity of glioblastoma cells.Leucine-rich repeats and immunoglobulin-like domains protein 1(LRIG1)is atransmembrane protein which acts as a tumor suppressor in various human cancer cells.LRIG1 downregulation has been implicated to be associated with the poor prognosis of patients(18,19).Previous studies indicated that LRIG1 was frequently decreased in gliomas(20),and the expression level of LRIG1 is significantly correlated with the malignancy of glioma(20,21).It is reported that upregulation of LRIG1 expression suppresses malignant glioma cell growth and induces cell apoptosis(20,22-24),whereas downregulation of LRIG1 expression promotes the proliferation and aggressive properties of glioma cells(25,26).In addition,some studies suggested that LRIG1 was involved in the regulation of chemosensitivity of human cancer cells(27,28).It has been elucidated that LRIG1 sensitizes glioma cells to cisplatin and temozolomide(TMZ)(29-32).Recently,Yang et al reported that LRIG1 expression was related to the radiosensitivity of human glioblastoma cells,and LRIG1 overexpression enhances the radiosensitivity of radioresistant human glioblastoma U251 cells(32).Consistent with the previous study(32),we found that LRIG1 was downregulated in the radioresistant human glioblastoma cells.Furthermore,the luciferase reporter assay demonstrated that LRIG1 was a direct target of miR-590-3p.The effect of miR-590-3p suppression on cell viability,colony formation capacity and cell apoptosis rate could be attenuated by the knockdown of LRIG1 in the U251 R cells.These results elucidated that LRIG1 mediates the effect of miR-590-3p on the radiosensitivity of human glioblastoma cells.In conclusion,this study firstly identified miR-590-3p as a potential target of radioresistance in human gliomas,and LRIG1 was involved in mediating the effect of miR-590-3p on the radiosensitivity of human glioblastoma cells.Our findings improved the understanding of the relationship between microRNAs and radiosensitivity in gliomas,and may provide potential therapeutic strategies to prevent radioresistance. |
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