MiR-421 Promotes The Development Of Osteosarcoma By Regulating MCPIP1 Expression

Introduction:Despite improvements in surgical resection and adjuvant chemotherapy,the prognosis and outcomes of patients with osteosarcoma remains poor due to the occurrence of metastasis or relapse.Monocyte chemoattractant protein-1-induced protein-1(MCPIP1),a zinc-finger RNA-binding protein,is known to regulate inflammatory responses and repress breast cancer growth.However,the regulation of MCPIP1 by microRNAs has not been clearly elucidated in osteosarcoma.In this study,we found that miR-421 expression was upregulated and MCPIP1expression was downregulated in the osteosarcoma specimens from patients.Moreover,MCPIP1 expression was inversely correlated with miR-421 expression in the clinical samples.Furthermore,the upregulation of miR-421 and downregulation of MCPIP1resulted in poor overall survival and severe disease progression,respectively,in the patients with osteosarcoma.Bioinformatics analysis and luciferase reporter gene assays confirmed that miR-421 specifically targets and binds to the 3’-UTR of MCPIP1.The overexpression of miR-421 induced cell proliferation,invasion,and migration,and the release of pro-inflammatory IL-6 in cultured human osteosarcoma cells.Additionally,the administration of miR-421 to tumor-bearing mice facilitated osteosarcoma growth by downregulating MCPIP1 expression.Taken together,these findings indicate that miR-421 is able to promote the development of osteosarcoma by regulating MCPIP1 expression,and can be a potential therapeutic target for osteosarcoma.Materials and methods:Materials1.Osteosarcoma tissue samplesTumor tissues and adjacent non-tumor bone tissues were obtained between July2011 and July 2013 from 30 patients with osteosarcoma treated at China Medical university Affiliated Hospital and Cancer Hospital.Patient consent was obtained and the experimental protocols were approved by the Ethics Committee of China Medical University.2.Cell cultureMG63 and U2OS cells were purchased from ATCC(American Type Culture Collection).The MG63 and U2OS cells were thawed,subcultured,cultured and transfected according to the instructions of ATCC cell culture3.Osteosarcoma xenografts in nude miceSix-week-old male Balb/c nude mice were purchased from the Laboratory Animal Center of Shenyang,China.MG63 cells(1×10~7)were suspended in 100μl PBS and subcutaneously injected into mice.The mice were were injected intramuscularly close to the tibia with MG63 cells(1×10~7).All experimental procedures were approved by the Institutional Animal Research Ethics Committee with reference to the Chinese Community guidelines for the use of experimental animals and the Ethics Committee of China Medical University.All experimental procedures involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.Methods1.We first evaluated the mRNA expressions of miR-15b,miR-133b,miR-140-5p,miR-25-3p,mi R-129,miR-300,miR-421,miR-422,miR-421,miR-449,miR-542in 30 samples of osteosarcoma tissues and adjacent tissues were detected by real-time PCR.2.We used Kaplan-Meier survival analysis to evaluate the relationship between the expression of miR-421 and the survival time of osteosarcoma patients,and we used Chi-square test to evaluate the correlations between the expression of miR-421 and clinic pathologic features in osteosarcoma.3.We first evaluated the mRNA expressions of MCPIP1 in 30 samples of osteosarcoma tissues and adjacent tissues were detected by real-time PCR.4.We used Kaplan-Meier survival analysis to evaluate the relationship between the expression of MCPIP1 and the survival time of osteosarcoma patients,and we used Chi-square test to evaluate the correlations between the expression of MCPIP1 and clinic pathologic features in osteosarcoma.5.After transfecting with miR-421 mimic/inhibitor,the proliferations of MG63 and U2OS cells were detected by MTT assay.We evaluated the effect of miR-421 on the proliferation of osteosarcoma cells6.After transfecting with miR-421 mimic/inhibitor,the protein and mRNA levels of cyclin D1 and bcl-2 in MG63 and cells were detected by western blotting and real-time PCR assay.We evaluated the effect of mi R-421 on the the levels of cyclin D1,bcl-2,and MMP2 of osteosarcoma cells7.After transfecting miR-421 mimic or inhibitor in MG63 cells and U2OS cells,transwell assays were performed.Cells were counted and results represent the mean±SD for three experiments.We evaluated the effect of miR-421 on the migration of osteosarcoma cells8.After transfecting miR-421 mimic/inhibitor in MG63 and U2OS cells,the expression of IL-6 was detected by ELISA.The levels of IL-6 were detected by western blot and real-time PCR.We evaluated the effect of miR-421 on the release of IL-6 of osteosarcoma cells.9.We used bioinformatics tools and luciferase reporter assay to predict a target sequence in the 3’-UTR of MCPIP1 for miR-421.We used miRDB to evaluate whether miR-421 could target MCPIP1.The interaction between miR-421 and the MCPIP1 was tested by luciferase reporter assays in MG63 and U2OS cells10.After transfecting with miR-421 mimic/inhibitor,the protein and mRNA levels of MCPIP1 in MG63 and cells were detected by western blotting and real-time PCR assay.We evaluated the effect of miR-421 on the levels of MCPIP1 in osteosarcoma cells11.MG63 Cells were divided into three groups and transfected with control,miR-421 and miR-421 with MCPIP1 respectively.The proliferation of MG63 cells was detected by MTT assay;The migration of MG63 cells was detected by transwell assay.Cells were counted and results represent the mean±SD for three experiments.The levels of MCPIP1,Cyclin D1,bcl-2,MMP2 and IL-6 in MG63 cells were detected by western blot and real-time PCR.We evaluated the effect of miR-421 on the proliferation,migration,and pro-inflammation in MCPIP1 in osteosarcoma cells,after intervention of MCPIP1 supplement.12.The tumorigenic effect of miR-421 in vivo:The tumor volumes and the levels of MCPIP1,Cyclin D1,bcl-2,MMP2 and IL-6 in tumors from controls and miR-421 agomir-treated nude mice were detected by western blot and real-time PCR.The tumor volumes and the levels of MCPIP1,Cyclin D1,bcl-2,MMP2 and IL-6 in tumors from miR-421 controls and miR-421 antagomir-treated nude mice were detected by western blot and real-time PCR.Results:1.Real-time PCR analysis showed that miR-421 expression in the tumor tissues was significantly higher than that in the control bone tissues.Compared to low expression of miR-421,Kaplan-Meier analysis revealed that patients with high expression of miR-421had a shorter overall survival(χ~2=3.842;P=0.0299).High expression of miR-421significantly correlated with the clinical TNM stage of osteosarcoma.2.Real-time PCR analysis showed that MCPIP1 expression in the tumor tissues was significantly lower than that in the control bone tissues.Compared to high expression of MCPIP1,Kaplan-Meier analysis revealed that patients with low expression of MCPIP1had a shorter overall survival(χ~2=5.840;P=0.0210).Low expression of MCPIP1significantly correlated with the clinical TNM stage of osteosarcoma.3.After MG63 cells and U2OS cells were transfected with miR-421 mimic and miR-421 inhibitor,we found that the overexpression of miR-421 promoted the proliferation of in MG63 and U2OS cells,whereas inhibition of mi R-421 attenuated proliferation.Moreover,the protein and mRNA levels of cyclin D1 and Bcl-2 were increased upon the overexpression of miR-421 in MG63 and U2OS cells.4.After MG63 cells and U2OS cells were transfected with miR-421 mimic and miR-421 inhibitor,we found that the overexpression of miR-421 increased the migration and invasion of in MG63 and U2OS cells,whereas inhibition of miR-421 attenuated the migration and invasion.Moreover,the protein and mRNA levels of MMP2 were increased upon the overexpression of miR-421 in MG63 and U2OS cells.5.After MG63 cells and U2OS cells were transfected with miR-421 mimic and miR-421 inhibitor,we found that the overexpression of miR-421 promoted the release of IL-6 in MG63 and U2OS cells,whereas inhibition of miR-421 attenuated the release of IL-6.6.We further explored that an inverse correlation between miR-421 expression and MCPIP1 expression by Spearman’s correlation analysis.Bioinformatics tools predicted a target sequence in the 3’-UTR of MCPIP1 for miR-421.The luciferase reporter gene assay demonstrated that miR-421 could directly bind to the 3’-UTR of MCPIP1.Western blotting and real-time PCR analysis showed that miR-421 could significantly decreased MCPIP1 expression of MG63 and U2OS cells.7.MTT and transwell assays demonstrated that MCPIP1 could promote cell proliferation and cell migration upon the overexpression of mi R-421.Additionally,MCPIP1 decreased the elevated mRNA and protein levels of cyclin D1,Bcl-2,MMP2,and IL-6 induced by the overexpression of miR-421.8.Nude mice treated with the miR-421 agomir exhibited increased tumor volume compared to the control nude-mice.In addition,treatment with the miR-421 agomir elevated the protein and mRNA levels of MCPIP1,and increased the levels of cyclin D1,Bcl-2,MMP2,and IL-6.However,mice treated with the miR-421 antagomir exhibited decreased tumor volume compared to the mi R-421 control nude-mice.Compared to the miR-421 control nude-mice,treatment with the mi R-421 antagomir increased the levels of MCPIP1,and decreased the levels of cyclin D1,Bcl-2,MMP2,and IL-6.Conclusion:1.The expression of miR-421 is inversely correlated with The expression of MCPIP1 in human osteosarcoma tissues2.MiR-421 promotes the proliferation in osteosarcoma MG63 and U2OS cell3.MiR-421 enhances the migration in osteosarcoma MG63 and U2OS cell4.MiR-421 stimulates IL-6 secretion from osteosarcoma MG63 and U2OS cells5.MiR-421 could target MCPIP1 to promote growth and migration in osteosarcoma in vivo and in vitro...