The Functions And Mechanism Of MiR-139-5p In The Development And Progression Of Osteosarcoma

Osteosarcoma is a derived from mesenchymal tissue of the skeletal system malignant tumor, its incidence and mortality are in the lead in the primary bone tumors. Osteosarcoma happens in teenagers under the age of 15 to 25, the fast development process, transfer and the recurrence rate is high, low survival brigade, 5-year survival rate of patients underwent surgical resection of a single treatment is only 15-20%, has become a threat of adolescent health. With the rapid development of medical technology, neoadjuvant chemoradiation joint limb-salvage surgery instead of amputation became the main surgical method of treatment of osteosarcoma. Although patients prognosis level improved, radiation and chemotherapy does not have a target, don't effectively remove tumor cells, patients with postoperative recurrence rate is high, and limb-salvage surgery still need auxiliary bone revascularization, expensive, to bring certain economic pressure to patients. At present, the new treatment such as osteosarcoma immune therapy, interventional therapy, Chinese medicine therapy and gene targeting therapy become the breach of the conquering osteosarcoma. Targeted therapy can be targeted with gene plays an important function in the process of development of osteosarcoma cancer related gene, thus inhibiting tumor cell proliferation, migration and invasion of malignant biological characteristics, and become effective healing of bone sarcoma new hope. Therefore, further reveals its molecular mechanisms in the development of osteosarcoma, explore new molecular targets for treatment, to provide theoretical basis and data support for gene targeting treatment, has important clinical significance and scientific value.As the second generation of high-throughput sequencing technologies mature and widely used, Sequencing data against tumor tissues and cells, such as exon sequencing, transcriptome sequencing, miRNAs sequencing, lnc RNA sequencing and unicellular sequencing data for the research of gene targeting therapy such as provide reliable data support, oncology studies ushered in the era of "data explosion". In today's era of big data, a series of gene sequencing data integration of a large amount of tumor samples and clinical data of database such as TCGA, Oncomine also arises at the historic moment, has been widely applied to the study of oncology. TCGA database is the largest database, and its cover worldwide 29 kinds of more than 10000 cases of tumor samples of exons sequencing data, SNP data, CMV data and complete clinical data, become a sword of oncology research. Currently revolves around TCGA database related research published in "Science" and "Nature", "PNS" and a series of major scientific publications. But using the TCGA big data for the study of osteosarcoma were not reported. Therefore, in today's era of big data, how to use the tumor big data to do some in-depth study of osteosarcoma, to further improve the molecular mechanism of osteosarcoma development, become a trend in today's oncology research.Micro RNAs are a length of about 21-23 nt between species are highly conservative, non-coding RNA, Previous studies that the miRNA is a kind of don't have the function of gene transcription "spam", With in-depth research, people gradually found that the miRNA can 6-8 incomplete complementary base pairing method combined with target gene 3 'UTR region, play a role of negative regulation of gene expression. Therefore, micrornas in the regulation of various physiological functions such as cell proliferation, differentiation, apoptosis age has important function.And "Science" magazine in 2002, ten major technology breakthrough in the first place. Currently in mammals has identified as many as thousands of miRNA sequences, and continues to have a new miRNAs were found. In recent years, with the deepening of the research of micrornas in oncology, people gradually found that there are a lot of micrornas in tumor is important for cancer related gene regulation effect, in the development of tumor play an important role, is considered a potential molecular targets for the treatment of tumor. Therefore, further exploration and research in development in osteosarcoma miRNAs in one of the key control, to further clarify the molecular mechanism of occurrence and development of osteosarcoma with, and to explore new therapeutic targets has important scientific significance.Part I Based on analysis of tumor TCGA data miR-139 in osteosarcoma expression and its apparent regulation mechanism researchIn this part of the study, we used the TCGA tumor osteosarcoma of micrornas chip data in the database, filtering in osteosarcoma occurred significantly differentially expressed miRNAs, found 70 have significantly lower expression of miRNAs, markedly increase expression of miRNAs there are 115 reasons. In a significant increase of miRNAs, miR- 139-5 p is the most significant cut without reports of micrornas in osteosarcoma. We adopt q RT- PCR detection in osteosarcoma tissues and cell lines are the expression of miR- 139-5 p, the results further illustrate miR- 139-5 p cut happened in osteosarcoma expression. Explore miR- 139-5 p in the molecular mechanism of cut in osteosarcoma expression, we adopt CHIP experiments tested the miR-139 promoter region of histone methylation and acetylation modification situation, found that miR-139 in the osteosarcoma is composed of miR-139 promoter region set cut expression in protein H3K27 and H3K9 methylation level rise, H3K4 methylation level lowered and H3 acetylation levels mediated by cut. In addition, we combine TCGA database osteosarcoma sample clinical data, analyzes the miR-139-5p expression and patient surial, found that the relationship between miR-139-5p surial in patients with high expression is significantly higher than patients with lower expression.Through the above results, we preliminarily concluded that miR-139-5p play an important role in the occurrence and development of osteosarcoma.Part II miR-139-5p in osteosarcoma evil deeds biology phenotypic regulation function and its molecular mechanismIn order to study the miR-139-5p in its role in the development of osteosarcoma, we first adopt q RT PCR to detect the miR-139-5p mimics the transfection efficiency.Results show that the transfection miR-139-5p mimics can significantly improve the osteosarcoma MG63 cells and U20 S in miR-139-5p levels.Next, we adopt CCK8 experiment tested the miR-139-5p influence on the proliferation of osteosarcoma, found that miR-139-5p can significantly inhibit the proliferation of osteosarcoma.Applying the Transwell miR-139-5p on osteosarcoma cell migration and invasion, found that the influence of miR-139-5p can significantly inhibit cell migration and invasion of osteosarcoma.In addition, build the nude mice subcutaneously inhibit tumor model, after expressing miR-139-5p in the body, found that miR-139-5p can significantly inhibit the growth of tumors.Therefore, we conclude that miR-139-5p can significantly inhibit the development of osteosarcoma.To explore miR-139-5p play the molecular mechanism of tumor suppression function, we first adopt Targetscan predicted the target genes of miR-139-5p found that miR-139-5p can be targeted Cdc42 m RNA 3 'UTR sequence.In order to further explore the miR-139-5p Cdc42 m RNA targeting the function of 3 'UTR sequences, we construct the Cdc42 m RNA 3' UTR region 45 to 52 loci Luciferase double fluorescent report carrier, transfection miR-139-5 p after found that miR-139-5p can significantly inhibit fluorescence generated.The above results suggest that miR-139-5p can be targeted Cdc42 m RNA 3 'UTR 45 to 52 loci.In order to further explore the miR-139-5p Cdc42 whether there is a negative regulation role and its mechanism of action, we tested the transfection miR-139-5p, osteosarcoma cells Cdc42 m RNA and protein expression of found that miR-139-5p does not inhibit Cdc42 m RNA level, can significantly inhibit Cdc42 protein expression.Through the above results, we conclude that miR-139-5p can inhibit Cdc42 m RNA translation of Cdc42 plays a role of negative regulation of gene expression.Part III Cdc42 of osteosarcoma deeds phenotypic regulation role in biology studyIn this part, we explore the Cdc42 gene expression in osteosarcoma and its regulation function of malignant osteosarcoma biological phenotype.TCGA is firstly analyzed, found that RNA- Seq osteosarcoma data in the database Cdc42 m RNA was not happen differentially expressed in osteosarcoma, the q RT- PCR and Westernblot respectively tested the osteosarcoma tissues and cell lines of Cdc42 m RNA and protein expression, found that Cdc42 m RNA was not happen differentially expressed in osteosarcoma, but Cdc42 protein produced significant increases in osteosarcoma expression.Above results indicate that Cdc42 markedly increases in osteosarcoma expression, its higher expression mainly in the transcription regulation of miR- 139-5 p as Cdc42 the transcription regulatory factors, plays an important role in regulating the Cdc42 expression.Next, we explored the Cdc42 effect on cell proliferation and migration of osteosarcoma.CCK8, according to the results of silence Cdc42 expression can significantly inhibit bone sarcoma cell proliferation;Scratch test, according to the results of Cdc42 silence, migration of osteosarcoma cells.Through the above results, we concluded that Cdc42 can promote the proliferation and differentiation of osteosarcoma cells.To sum up, miR-139-5p in the occurrence and development plays an important role in osteosarcoma, embodied in the negative control Cdc42 gene expression, thus inhibiting the development of osteosarcoma, can be used as molecular targets for the treatment of osteosarcoma potential.