Research On Effects And Mechanism Of MiR-221in Human Osteosarcoma

Background miRNAs are a class of endogenous non-coding small RNAsapprexumate18to25nucleotides in length that negative regulate their target genesexpression post-transcriptionally by base-pairing with the complementary sites in the3’-untranslated region (3’-UTR) of the mRNA of their cognate target mRNAs. They areimportant regulatory molecules and play an important role in vivo. Thousands of miRNAgenes have been found in diverse animals and plants. More and more studies have shownthat miRNAs play a key role in diverse biological processes, including development, cellproliferation, differentiation, and apoptosis. Cell differentiation and abnormal proliferationare the most important link in the process, so the studies about the efforts of miRNAs instem cell and cancer become the hot spot of resent research.Osteosarcoma is the most common primary malignant bone tumor that is usuallycontracted by children and young adult with a poor prognosis. Advances in osteosarcomatherapy over the past several decades have enhanced patient outcomes, with most effectiveregimens currently including neoadjuvant and adjuvant chemotherapy coupled with localcontrol that usually consists of limb-sparing surger. However,outcome remains poor formost patients with metastatic or recurrent osteosarcoma. The frequent acquisition ofdrug-resistant phenotypes and the occurrence of second malignancies often associatedwith chemotherapy remain serious problems. Therefore, the identification of the effectormolecules and/or signal pathways responsible for regulating chemotherapy resistant and malignant development is crucial for improving the osteosarcoma treatment level. In ourstudy, we combine the miRNAs which are the star molecules in resent years andosteosarcoma which is our laboratory features and especially we focus on the effects ofmiR-221in osteosarcoma and the possible mechanism by which miR-221affected thesurvival, apoptosis, and cisplatin resistance of osteosarcoma. Accumulating evidenceindicates that miR-221is overexpressed in many human cancers and it functions as anoncogene. However, the function of miR-221in human osteosarcoma has not been totallyelucidated. In this study, we mainly research the functions of miR-221in bothosteosarcoma cell lines SOSP-9607and MG63.Objective To describe the effects and the mechanism of miR-221function in humanosteosarcoma and establish some theoretical basis for osteosarcoma treatment and elementexperience for further research.Methods (1) To elucidate the role of miR-221in human osteosarcoma, we evaluatedthe expression levels of miR-221in five human osteosarcoma cell lines, SOSP-9607,U2OS, Saos-2, MG63and SOSP-9901, as well as hFOB1.19osteoblasts by usingreal-time quantitative PCR. The hFOB1.19osteoblasts was used as control.(2) Toinvestigate the function of miR-221, we transfected human osteosarcoma cell linesSOSP-9607and MG63with chemically synthesized double-stranded oligonucleotides(miR-221mimic) that mimic the endogenous mature miR-221function and modifiedantisense oligonucleotides that inhibit miR-221function (miR-221inhibitor) or scrambleoligonucleotides. The cells transfected with nothing (blank) were used as control.(3) Inboth cell lines, we investigate the function of miR-221affected cell survival, apoptosis,and cell cycle by using miR-221mimics or inhibitor.(4) We examined whether miR-221was capable of inhibiting cisplatin-induced apoptosis. Cisplatin-induced cell cytotoxicityin both cells is determined via MTT assay.(5) Potential target genes of miR-221werepredicted using bioinformatics. Moreover, luciferase reporter assay and western blot wereused to confirm whether PTEN was a direct target of miR-221. Furthermore, introductionof PTEN cDNA lacking3′-UTR or PI3K inhibitor LY294002were used to verify thecorrelation between miR-221and its target gene.(6) Both miR-221and PTEN expression levels in osteosarcoma samples were examined by using real-time quantitative PCR andimmunohistochemistry.Results (1) Although MG63had the highest one and SOSP-9607had the lowest one,the expression levels of miR-221in investigated osteosarcoma cell lines were allsignificantly higher compared with that of hFOB1.19osteoblasts cells.(each foldchange>2.5, p<0.001).(2) Transfection efficiency (more than80%) of miR-221mimic inthese two cells were observed by fluorescence microscopy analysis. Real-time quantitativePCR showed that miR-221mimic increased the expression of miR-221by about360-fold(p<0.001) while miR-221inhibitor decreased the expression by about65-fold inSOSP-9607cells (p<0.001) compared with the blank group. The expression of miR-221was increased by about290-fold (p<0.001) in miR-221mimic group while the expressionof miR-221was decreased by about45-fold (p<0.001) in miR-221inhibitor groupcompared with the blank group.(3) In both SOSP-9607and MG63cells, MTT assaydemonstrated that both miR-221inhibitor transfected cells proliferate at a significantlylower rate as compared with blank cells, respectively. In contrast, both cells withoverexpression of miR-221by transfected with miR-221mimic significantly enhancedproliferation as compared with blank cells, respectively. Both in SOSP-9607and MG63cells, transfection with miR-221mimic resulted in the lowest percentage of cells in G0/G1phase (p=0.001for SOSP-9607cells and p=0.017for MG63) but highest fraction in Sphase (p=0.001for SOSP-9607cells and p=0.005for MG63cells). In contrast, bothSOSP-9607and MG63cells treated with miR-221inhibitor had the higher percentage ofcells in G0/G1phase (p=0.009for SOSP-9607and p=0.032for MG63) but lower fractionin S phase (p=0.005for SOSP-9607and p=0.008for MG63) as compared with the blankgroup, respectively.(4) MTT assay showed that overexpression of miR-221markedlyinhibited cisplatin-induced cell cytotoxicity in both SOSP-9607and MG63cells.Moreover, overexpression of miR-221decreased cisplatin-induced cytotoxicity andincreased cisplatin resistance under various concentrations of cisplatin treatment in bothSOSP-9607and MG63cells. In contrast, downexpression of miR-221increasedcisplatin-induced cytotoxicity and decreased cisplatin resistance under various concentrations of cisplatin treatment in these two cells. And more, lower caspase3activitywas found in miR-221mimic transfected cells compared with blank control groups whilehigher caspase3activity was found in miR-221inhibitor transfected cells compared withthe blank control groups.(5) Potential target genes of miR-221were predicted by usingbioinformatics. Moreover, luciferase activity of the wild-type PTEN-3’-UTR reporter wassignificantly suppressed in miR-221mimic group compared with scramble group inSOSP-9607cells (p=0.002). And more, luciferase activity of the wild-type, but not mutant,PTEN-3’-UTR reporter was significantly increased in miR-221inhibitor transfected MG63cells (p=0.009). At the same time, western blot analysis revealed that miR-221mimics andinhibitor could affect the expression of PTEN protein and PI3K/AKT pathway. MTT assayshowed restoring expression of PTEN and decreasing expression of phospho-Akt byco-transfecting pcDNA3.1-PTEN recovered the cisplatin sensitivity in both miR-221mimic transfected SOSP-9607and MG63cells. And more, PI3K/Akt inhibitor LY294002significantly abrogated miR-221-activated Akt and significantly inhibitedmiR-221-induced cisplatin resistance in both SOSP-9607and MG63cells.(6) Real-timequantitative PCR analysis showed miR-221expression levels in recurrent and primaryosteosarcoma tissues were significantly higher than that in normal adjacent tissues (eachp<0.001). In addition, we also observed that miR-221is significantly increased in theserecurrent tissues when compared with primary tissues (p<0.05). Immunohistochemicalstaining showed that there was an inverse correlation of expression of miR-221and PTENin human osteosarcoma samples.Conclusion (1) miR-221was significantly upregulated in osteosarcoma cell lines.(2)miR-221mimic and miR-221inhibitor could be transfected into cells and regulatemiR-221expression effectively in both SOSP-9607and MG63cells.(3) miR-221mimicsinduced cell survival and reduced cell apoptosis while miR-221inhibitor inhibited cellgrowth and induced cell apoptosis. miR-221works as an oncogene in osteosarcoma cells.(4) Overexpression of miR-221markedly inhibited cisplatin-induced cell cytotoxicity andincreased cisplatin resistance in both SOSP-9607and MG63cells.(5) PTEN is a directtarget of miR-221and miR-221reduced PTEN expression leading to activation of PI3K/Akt pathway. Introduction of PTEN cDNA lacking3’-UTR or PI3K/Akt inhibitorLY294002abrogated miR-221-induced cisplatin resistance.(6) In clinical specimens,miR-221expression levels in recurrent and primary osteosarcoma tissues weresignificantly higher than that in normal adjacent tissues and there was an inversecorrelation of expression of miR-221and PTEN in human osteosarcoma samples.