The Mechanisms Of MiR-125b-STAT3Feedback Regulation Loop In The Genesis And Development Of Osteosarcoma And Serum MicroRNAs As Potential Biomarkers For Osteosarcoma

Osteosarcoma is the most common primary malignant bone tumor in children and adolescent. It is also one of the most difficult diseases treated in orthopaedics. According to statistic data in our country, the incidence rate of osteosarcoma is the top one of primary malignant bone tumors. Osteosarcoma is a poor prognosis malignancy.Lung metastasis can occur within a few months, the3to5-year survival rate after the amputation is only60%.Although there is some improvement in pathogenesis, diagnosis and treatment of osteosarcoma in recent years, the mechanisms underlying the development of the osteosarcoma remain obscure. Furthermore, there is currently no an effective target for diagnosis and treatment of osteosarcoma available. There is an urgent need for identification of biological targets.Meantime the discovery of the serum markers of osteosarcoma will give far-reaching impact on the diagnosis and treatment of osteosarcoma.MicroRNA is non-protein coding RNA, which is about21-25nucleotides in length. It regulates the expression of target genes by specific binding. From1993,the first microRNA:lin-4was discovered, in the past few decades, numerous microRNAs were discovered, and played as a critical role in cells differentiation, proliferation and apoptosis. Recent studies have shown that, the changes of microRNA expression are associated with tumor occurrence, development, diagnosis and prognosis. In different tumor types, microRNAs played acting either as Tumor suppressor gene or oncogene.The mechanisms included deletions, amplifications,or mutations in miRNA loci,epigenetic change, the dysregulation of transcription factors that target specific miRNAs, or the inhibition of processing. MicroRNA has an important property, that is exceptional stable in serum and plasma, and is unlikely to be degraded by RNA enzyme.So the actual value is consistent with what is obtained from patient. The discovery of microRNA in blood samples with specific expression patterns has significant meaning for the early diagnosis and treatment of cancer.STAT3protein is an important transcription factor. In the physiological state, STAT3can be activated quickly and last for a short time,which plays a key role in physiological function in normal cells. STAT3is the point of convergence for numerous oncogenic tyrosine kinase pathway such as EGFR, IL26/JAK、Src.STAT3could be found with excessive activation in a variety of tumor cells.Excessive activation of STAT3will induce abnormally overexpression of key genes,which are closely related with proliferation, differentiation and apoptosis.These processes will promote cell proliferation or malignant transformation and inhibit apoptosis.Activation of STAT3can induce the expression of anti-apoptotic genes (c-myc), cell cycle regulatory genes (cyclin D1/D2), etc. Furthermore, it can promote tumor metastasis and angiogenesis by inducing the expression of MMP and VEGF.Additionally, STAT3is related with clinical biological behavior of tumor, such as tumor invasion, lymph node metastasis, and drug resistance of tumor cells, etc.This project was conducted to compare six osteosarcoma tissue samples with adjacent tissue area with high differentiation and maturation by using of microRNA expression microarray, and found that the expression of miR-125b significantly decreased in six osteosarcoma patients’tumor tissue samples. We speculated that miR-125b plays an important role in the occurrence and development of osteosarcoma, which has the potential function of tumor suppressor genes. Our previous data demonstrated that miR-125b can inhibit osteosarcoma cell proliferation and migration, but the mechanisms are still unknown. Through bioinformatics analysis, we found that miR-125b binding sites located on the3’UTR of STAT3.It was supposed that STAT3may be candidate downstream target gene of miR-125b.Meantime, We also found there were CAAT elements, two STAT3binding sites and STAT6binding site in core area of the promoter, suggesting that STAT3can specifically bind to core promoter region of miR-125b. In summary, miR-125b might be actived by STAT3at the transcription level and in turn regulates the STAT3expression. We postulate that there maybe a feedback regulation loop between STAT3-miR-125b.Under physiological conditions, cells do not need much STAT3,STAT3is inhibited by miR-125b at post-transcription level; excessive activation of STAT3can produce more miR-125b at transcription level, which can inhibit STAT3to maintain the balance of miR-125b-STAT3.In osteosarcoma cells, STAT3expression is high while miR-125b expression is low, why sustained activation of STAT3cannot activate miR-125b at transcriptional level? Further analysis showed that miR-125b promoter has a CpG island, this feedback regulation loop may be interrupted by tumor cell epigenetic regulation. This project consisted of two parts,which were the investigation of STAT3-miR-125b regulation loop feedback in the occurrence and Identification of serum microRNA as biomarkers in osteosarcoma patients. Part I:The mechanisms of miR-125b-STAT3feedback regulation loop in the genesis and development of osteosarcoma:(1)the expression of miR-125b-STAT3loop in osteosarcoma tissues and cells;(2) miR-125b targets downstream gene:STAT3;(3)STAT3function in activating miR-125b at transcription level;(4) DNA methylation status in the miR-125b promoter region in osteosarcoma. Part II:Identification of serum microRNAs as osteosarcoma biomarkers.These studies revealed the mechanisms of miR-125b-STAT3feedback regulation loop involved in osteosarcoma’s genesis and development, aberrant serum miRNAs expression are expected to provide new strategies and methods to the clinical diagnosis and treatment of osteosarcoma.Part I:The mechanisms of miR-125b-STAT3feedback regulation loop in the genesis and development of osteosarcomaSection I The expression of miR-125b-STAT3loop in osteosarcoma tissues and cellsObjective:To investigate the expression levels of miR-125b and STAT3in osteosarcoma tissues and cells.Methods:Levels of miR-125b expression level was detected by real-time quantitative PCR method in two teams:osteosarcoma and adjacent normal tissues. Furthermore, levels of STAT3protein in osteosarcoma tissue and cell lines were detected by immunoblotting (Western blot).Results:Compared with the adjacent tissues,miR-125b expression levels in osteosarcoma tissues were significantly decreased. Compared with HEK-293cell lines,miR-125b expression levels in the three osteosarcoma cell lines (MG-63,Saos-2,U2-OS)were significantly decreased. Compared with the adjacent normal tissues, STAT3protein expression levels in osteosarcoma were significantly increased; compared with HEK-293cell lines, STAT3protein expression levels in the three osteosarcoma cell lines (MG-63,Saos-2, U2-OS) were significantly increased.Conclusion:miR-125b’s expression level was significantly decreased in osteosarcoma cells,while the expression of STAT3was significantly increased. This results indicated that these two factors may be involved in mechanisms of the genesis and development of osteosarcoma。 Section II miR-125b targets downstream gene STAT3Objective:To validate STAT3as a downstream target gene of miR-125b.Methods:1.We used target gene prediction software Targetscans/PICTAR etc to predict downstream target genes of miR-125b, screening candidate target genes that may be associated with the occurrence and development of tumor, we selected STAT3as downstream target gene of miR-125b.2.A fragment sequence from3’UTR of STAT3containing putative miR-125b binding sites was amplified by PCR from the blood lymphocyte genomic DNA, then was cloned into the vector pMIR-REPORTER Luciferase vector to construct STAT3wild-type (STAT3WT-luc) luciferase reporter gene plasmids. Then use a point mutation method to make mutation in the3’UTR region of STAT3in the (STAT3WT-luc) luciferase reporter gene plasmid to construct mutant STAT3(STAT3MUT-luc) luciferase reporter gene plasmid. STAT3WT-luc, STAT3MUT-luc, miR-125b mimics and negative control were simultaneously transfected into MG-63cells by lipofectamine method. After48hours’transfection, firefly luciferase activity was measured by using Dual luciferase reporter system. Renilla luciferase was used as an internal control, the ratio of the two relative expression values (Firefly/Renilla) is the final value.3.Different concentrations of miR-125b mimics were transfected into Saos-2cell, Western blot was used to detect the expression of STAT3protein levels. CKK8assay was used to measure cell proliferative activity.4.miR-125b mimics, anti-miR-125b and negative control were transfected into the MG-63and Saos-2cells.Total protein was isolated with protein isolation kits, then STAT3protein expression levels were detected by Western blot.Results: 1.STAT3was the target gene of miR-125b, which inhibited the expression of target genes by binding to STAT33’UTR region.2.miR-125b attenuated the growth of Saos-2cell’s proliferation and inhibited STAT3protein expression in a dose-dependent manner.3The STAT3protein expression was decreased in MG-63and Saos-2cells transfected with miR-125b mimics.4The STAT3protein expression was increased in MG-63and Saos-2cells transfected with anti-miR-125b.Conclusion:STAT3is miR-125b’s target gene, miR-125b inhibited the expression of STAT3by binding to3’UTR region. Section III:STAT3function in activating miR-125b at transcription levelObjective:To investigate whether the transcription factor STAT3can activate miR-125b or not.Methods:1.Mature miR-125b has two gene loci in the human genome located on Chromosome11(miR-125b-l) and Chromosome21(miR-125b-2). We first predicted the promoter region using a bioinformatics analysis. Then,10kilobases (KB)upstream of both pre-miR-125b were searched for predicted promoter regions using CONSITE and PromoterInspector software.2The fragments with different length of5’end of miR-125b-1(-1600to-900bp,-1400to-900bp,-1175to-900bp) were cloned into the PGL3vector. Then, the construsted plasimd were simultaneously transfected into MG-63cells and Saos-2by lipofectamine method. After48hours’transfection, firefly luciferase activity was measured by using Dual luciferase reporter system. Renilla luciferase was used as an internal control, the ratio of the two relative expression values (Firefly/Renilla) is the final value.3.STAT3,STAT6and CCAAT elements in the core promoter region were identified by using MatInspector software (Genomatrix).4.Mutations were made at STAT3,STAT6and CAAT sites in the region from-1175to-900bp to construct PGL3-mSTAT3, PGL3-mSTAT6PGL3-mCAAT mutational luciferase reporter gene plasmids.Then, the construsted plasimd were simultaneously transfected into MG-63cells and Saos-2by lipofectamine method. After48hours’ transfection, firefly luciferase activity was measured by using Dual luciferase reporter gene system. Renilla luciferase was used as an internal control, the ratio of the two relative expression values (Firefly/Renilla) is the final value.5.Electrophoresis mobility shift assay(EMSA) was performed to identify the interaction between STAT3protein and premiR-125b promoter region.The oligonucleotides containing wild-type or mutated putative transcription factor binding sites like STAT3-wt, STAT3-consensus, STAT3-mut, STAT6-wt were synthesized and end-labeled with [c-32P]ATP using T4polynucleotide kinase and purified over a G-25column. The specific binding was determined according to the location, characteristics and intensity of the DNA-protein complexes.Results:1.Through CONSITE and PromoterInspector software, we found miR-125b-1had an RNA polymerase II core promoter, which was located at the-1600to-900bp upstream of the MiR-125b precursor sequence.2.Analyzing luciferase activities of different fragments, the core promoter sequence was identified locating at-1175bp to-900bp region.3.Based on MatInspector software analysis,we forecasted that there were CAAT element, STAT3and STAT6at the core promoter region.4.The luciferase activity significantly decreased after mutation at both the STAT3and CCAAT sites, whereas there was no obvious change after the mutation at the STAT6site.5.EMS A showed that DNA-protein complexes can be detected after incubating [32p]-STAT3wt probe with MG-63’s nuclear extraction.As a positive control, a classic STAT3binding oligonucleotide probe (consensus sequence) was also shown to form a protein-DNA complex that migrated to the same position. The complex formation was dramatically inhibited by competitive binding of the STAT3consensus, unlabeled probe and the addition of STAT3antibody. This binding was not affected by competition from a mutant probe.Conclusion:STAT3protein can activate miR-125b at transcription level by binding to the core promoter region. Section IV:DNA methylation status in the miR-125b promoter region in osteosarcomaObjective:To investigate DNA methylation status in miR-125b promoter region of osteosarcoma cellsMethods:Demethylating agent5-Aza-2’-deoxycytidine was added into osteosarcoma cell line MG-63and Saos-2,after that cell proliferation and the expression levels of miR-125b were detected. DNA was isolated from MG-63and Saos-2by kit, and then under bisulfite treatment, DNA methylation status of miR-125b promoter region was confirmed by methylation specific PCR.Results:1.With increasing concentration of5-Aza-2’-deoxycytidine, cell proliferation was significantly inhibited.2.With increasing concentration of5-Aza-2’-deoxycytidine, miR-125b expression level was significantly increased.3.DNA hypermethylation of miR-125b promoter region was detected by methylation-specific PCR.Conclusion:Down-regulation of miR-125b might be caused by hypermethylation in miR-125b promoter region. Part II Identification of serum microRNAs as osteosarcoma biomarkersObjective:Serum samples of patients with osteosarcoma were screening to find differentially expressed microRNA.Methods:Osteosarcoma patients and normal control group were screening by microRNA PCR microarray. qRT-PCR was applied to evaluate the expression of seleted microRNA in20cases of tumor samples and20normal controls, ROC analysis was used to evaluate the diagnostic accuracy.Results:The initial selection of fourteen miRNAs was further validated in the20patients cohort and20controls cohort.7miRNAs were selected in the OS pre-therapeutic group when compared to the control group.The AUC of the expression of7miRNAs in serum for OS diagnosis were0.7255(95%CI0.5435-0.9075),0.7686(95%CI0.6067-0.9306),0.8471(95%CI0.7155-0.9786)and0.7961(95%CI0.6438-0.9484) for miR-106a-5p, miR-16-5p, miR-20a-5p and miR-25-3p respectively, while the AUC for miR-425-5p, miR-451a and miR-139-5p were0.7765(95%CI0.6163-0.9366),0.7961(95%0.6420-0.9501) and0.7098(95%0.5308-0.8888)respectively.Conclusion:Altered serum miRNAs profile was detected and those miRNAs have potential to be molecular markers in diagnosis and prediction of osteosarcoma.