| Background:Osteosarcoma is the most common primary malignant bone tumors, is derived from embryo mesenchymal tissues malignant degree high ontic tumor.Most of osteosarcoma for primary tumor, only a few are secondary, common location is located in the rich blood supply of four limbs which stem epiphyseal end, with lower limbs distal femoral and proximal tibia, the most common followed by proximal humerus and fibula.Osteosarcoma is dissolved osseous changes more, a few of osseous changes, osteosarcoma tissues contain different composition of bone, cartilage, fibrous connective tissue and bone tissue.Osteosarcoma pathology characteristic is tumor tissues can produce highly malignant spindle stromal cells, and the formation of bone tissue, tumor cells can also directly osteogenesis and bone matrix, can be spread to the bone cortex and medullary cavity.Now also have a new study shows that the malignant cells of osteosarcoma are not fully from bone stromal cells, bone mesenchymal pluripotent stem cells malignant value-added can also lead to the occurrence of osteosarcoma.Incidence of osteosarcoma in primary malignant bone tumor occupies the first place, osteosarcoma onset ages 15 to 25 years old, in more incidence of 4.5/100, new each year in the United States is about 900 patients with osteosarcoma. Osteosarcoma much early distant metastasis, and the rapid development, the transfer rate and case fatality rate is very high. Pure line treatment or amputation limb-salvage surgery and tumor prosthesis replacement surgery can not effectively control the transfer of tumor, patients often died of tumor metastasis in the short term, how to treat pulmonary metastasis osteosarcoma become one of the most important problems faced in the treatment of osteosarcoma. With the constant improvement of the treatment, at present USES the comprehensive treatment such as neoadjuvant chemotherapy plus limb-salvage surgery has become a major way for the treatment of osteosarcoma, can effectively improve the short-term survival, for the treatment of osteosarcoma has made obvious progress. According to the centre of the American cancer patients with osteosarcoma of the 5-year survival rate of about 53.9% on average, as the surgery and the rapid development of technology, such as the new adjuvant chemotherapy is not metastatic osteosarcoma patients 5-year survival rate was increased to 70%, but for osteosarcoma metastasis and recurrence effectively solve the problems such as there is still no, osteosarcoma patients with lung metastasis short-term survival rate did not significantly improve. About 80% of the patients with osteosarcoma, at the time of diagnosis of osteosarcoma has small asymptomatic lung metastases, other parts of the transfer such as liver, kidney and other parts of the transfer of common memory. Lung metastases in patients with osteosarcoma, indicating poor prognosis, even with a variety of treatments, the 5-year survival rate is around 25%-30%.Patients with osteosarcoma of himself, family and social cause great pain and economic pressure.Along with the advance of molecular genetics and molecular biology, studies of oncogene and tumor stem cell development gradually, interventional therapy, immune therapy, gene therapy, targeted treatment, the deepening of the stem cell treatment, provided a new possibility for the treatment of malignant tumor.Although great progress has been made in the treatment of osteosarcoma, but there is still no ideal breakthrough progress.There are still many key issues in its treatment, such as middle-late, recurrent and metastatic bone tumors lack of effective targeted therapy drugs.How to realize the early discovery, early diagnosis of osteosarcoma of the early treatment and prevention of osteosarcoma metastasis has crucial significance.So as soon as possible and prevent the malignant osteosarcoma proliferation become it is need urgently to solve the problem.Tumor cell proliferation is a malignant tumor occurred one of the most important and most basic biology. Despite the means to cover in the clinical treatment of tumor resection, radiotherapy and chemical medication, and tumor remains easy to relapse, however, serious problems such as easy to transfer. Studies have shown that the generation of tumor is a complex, multi stage process. Cancer monoclonal theory points out that when the cancer cells, cells gain the ability to divide and split speed accelerated at the same time, in the organizational environment and growth advantage, eventually damage the normal function of the organization. Many studies have shown that the tumor is actually in the early stage-the evidence in clinical imaging findings before-have uncontrolled rapidly dividing. Therefore, to clarify the mechanism of tumor proliferation, find tumor proliferation of molecular markers and therapeutic targets, so as to find an effective way to prevention and treatment of cancer research is currently one of the biggest problem. Key events cause rapid tumor proliferation, the cell division cycle conversion regulation, mainly including the cell cycle checkpoint (checkpoint) and the decreasing of the cycle dysfunction [6]. Cell cycle regulation is the main way of G1/S phase transformation. Proliferation, in the Gl phase of the cell division cycle decreased significantly, while the percentage of S phase cells in DNA synthesis. Main molecular mechanism characterized by cycle factor CyclinD1, CyclinA higher expression, such as protein and cycle dependent kinase inhibitor p21 and p27. Event study, therefore, regulating cell proliferation and its molecular mechanism, is one of the hotspots in the study of tumor development. In addition, due to the urgent demand for tumor growth inhibition of drug clinical, therefore to clarify the molecular mechanism of tumor cell proliferation events, explores the causes of the cell cycle transformation, even looking for the targeted of the tumor proliferation, will be for a variety of malignant tumors has important guiding significance to the prevention and drug development.micrornas (miRNA) is a newly discovered in recent years, a class of about 20 to 25 nt of non-coding small RNA molecules. In the present study has proved the miRNA participate in a variety of biological processes, and exert important function, including cell proliferation, apoptosis, invasion and metastasis, differentiation and stem cells, etc. MiRNA can by combining the target gene mRNA, regulate the translation of target genes. But the latest report points out that miRNA can also regulate gene expression in transcription level. Human heritage related miRNA gene pool has found that more than 1000 kinds of the human body. miRNA in evolution quite conservative, found in plants, animals and fungi miRNA only expressed in specific organizations and developmental stage, the miRNA tissue specificity and scheduling, decide the function of the tissue and cell specificity, suggests that micrornas in cell growth and development process a variety of adjustment. Most of micrornas genes located in non-coding regions, only a few in the coding regions, generally exist independently, a small number of clusters pattern, clusters of arrangement has the characteristics of collaborative expression of multiple genes. Micrornas closely related to the occurrence of a wide variety of tumor development. The miRNA can be used as oncogenes, cut the activity of tumor suppressor genes, Also can be used as a tumor suppressor gene, cut the activity of proto-oncogene. Esquela-Kerscher existing form and for the study of tumor associated microRNAs, think small RNA is closely related to the occurrence of a variety of malignant tumor development, and put forward the "oncology microRNAs" new concept of small RNA (tumor), opened up the applications of miRNA in oncology research of a new chapter. A growing number of studies have found that small RNA biological several important life activities:the regulation of cell growth, differentiation, proliferation, adhesion, monitoring, and apoptosis; Adjust the organization structure, balance, identification. Small RNA is known as "biology of dark matter, in a barely escaped all around us", the central dogma of RNA is not secondary intermediary role. A variety of micrornas mutation or content change and the deterioration of malignant bone tumors of osteosarcoma, and migration are closely related.Fork head transcription factor (FOXO) can inhibit tumor growth, restrictions on cell proliferation and induce cell apoptosis, expression of FOXO correlation human cancers including rhabdomyosarcoma, osteosarcoma, leukemia and lymphoma. Phosphatidylinositol 3 kinase pathway of FOXO related inactivated proteins are the main signals of cancer, a variety of small RNA by inhibiting FOXO genes contributing to cell carcinoma. FOXO as a tumor suppressor factor can bring new treatments for cancer, its downstream target genes is a serine/threonine protein kinase B (PKB/Akt), a kind of protein kinases is able to adjust the process of cell proliferation and survival. New evidence suggests that FOXO involved in different intracellular signaling pathways and may be many physiological and pathological states, including the key role of cancer FOXO interact with these signaling pathways, and participate in other intracellular protein phosphorylation and post-translational modifications. FOXO multiple promote cancer by inducing expression Bcl2 family members to actively participate in mitochondrial targeting proteins stimulate death receptor ligands such as Fas ligand and tumor necrosis factor related apoptosis inducing ligand (TRAIL), or improve the level of all kinds of cell cycle protein dependent kinase inhibitors (CDKIs), promote cell growth inhibition and/or apoptosis. FOXO expression spectrum has been identified in several human cancers, and that has the potential molecular therapy.This study will be looking for a high correlation between osteosarcoma development and small RNA as the research object, explore its on the formation of the cancer to promote cancer or tumor suppressor role, and tries to analyze the target genes and the mechanism of action. Now an article about miR-664 high expression in liver cancer tissue and associated with the expression of MATA in liver cancer. MiR-664 lymph nodes in papillary thyroid carcinoma samples low expression, miR-664 high expression of myocardial injury patients. And the research situation, is just a test sample is not the function and specific mechanisms to make a detailed discussion. We found that miR-664 in chip platform GSE39040 chip high expression in osteosarcoma, and through the Targetscan databases to forecast the differential expression of target genes, and the prediction results input software to analyze biological pathways, found that there is strong correlation between miR-664 and FOXO4 gene. MiR-664 in osteosarcoma biological mechanism there is no article related article reports.MiRNA expression at the early stage of the project by bioinformatics method, determine the miR-664 in osteosarcoma tissues expression, on the basis of mimic transfection miR-664, miR-664 inhibitor osteosarcoma cells as the model, by studying the miR-664 induced the molecular mechanism of cell proliferation, miR-664 cut FOXO4 activated PI3K/AKT/FOXO signaling pathways that regulate p21, CyclinDl etc. The change of the signal molecules. Verified through miR-664 through targeted combination of FOXO4 3’UTR FOXO4 translation inhibition, which induced rapid cell proliferation assumptions provide direct evidence and new molecular targets for treatment of osteosarcoma. This study will be divided into three parts cut FOXO4 miR-664 genes promote proliferation of malignant osteosarcomaPart one The abnormal expression of MiR-664 in osteosarcoma(1) miR-664 expression in human osteosarcoma fresh tissueObjective:to verify the miR-664 in the amount of expression in human osteosarcoma fresh tissueMethods:by using the method of real-time fluorescent quantitative PCR, Sybrgreen dye method, internal genes for U6, the miR-664 genes in human osteosarcoma fresh pathological tissue and osteosarcoma cell lines express the amount of testing. (P<0.05)Results:the expression of miR-664 osteosarcoma was 47.55 times more than the tissue adjacent to carcinoma (P<0.05)Conclusion:miR-664 in osteosarcoma tissue increased.(2) miR-664 in osteosarcoma cell line expressingobjective:Validation of miR-664 in the amount of expression in human osteosarcoma cellsmethod:to observe the expression of human osteosarcoma cells by the method of real-time fluorescent quantitative PCR Sybrgreen dye method, internal genes for U6, the miR-664 genes in SOSP-9607, U2-OS, SAOS-2, HOS, MG-63 cell lines and normal development hFOB1.19 comparing the expression of quantity detection.Results:the five groups of osteosarcoma cell lines in miR-664 in a Normal bone cells cell lines significantly increase 9-26 times (P<0.05)Conclusion:miR-664 in osteosarcoma cells increased, select an object to U2-OS osteosarcoma strains as transfection and platforms.Part two miR-664 expression disorders for US-OS osteosarcoma cells malignant proliferation effect(1) Overexpression of miR-664 promoting rolepurpose:malignant osteosarcoma cells proliferation studied expression effect on the osteosarcoma cell proliferation.Methods:By liposome transfection miR-664 mimic and NC control U2-OS osteosarcoma cells as the model, through 1, determined by MTT colorimetric method detecting osteosarcoma cell proliferation rate; 2, cell cloning experiments to detect osteosarcoma cell proliferation ability; 3, Soft AGAR cloning experiments (Soft AGAR assay) to detect the cancer cells than the growth of the anchor malignization ability; 4, BrdU infiltration method (5-bromine deoxidization uracil nucleoside method) technology to detect miR-664 for regulating cell division cycle.results:1, determined by MTT colorimetry experiment:miR-664 osteosarcoma cell proliferation rate mimic group was obviously higher than that of control group (P< 0.05); 2, cell cloning experiments:miR-664 osteosarcoma cell proliferation ability to mimic group was obviously higher than that of control group (P< 0.05); 3, soft AGAR cloning experiments:miR-664 osteosarcoma cells mimic group of anchor malignization ability of growth was obviously higher than that of control group (P< 0.05); 4, BrdU infiltration experiment:miR-664 S period osteosarcoma cells mimic group was obviously higher than that of control group (P< 0.05).Conclusion:overexpression of miR-664 significantly promote U2-OS osteosarcoma cell proliferation rate, proliferation and malignant division.ant change, cell division cycle number is significantly higher than the control group.(2)By miR-664 malignant proliferation inhibition of osteosarcoma cellsobjective:Inhibition of miR-664 inhibitory effect of U2-OS malignant osteosarcoma cells proliferationmethod:1, determined by MTT colorimetric method detecting osteosarcoma cell proliferation rate; 2, cell cloning experiments to detect osteosarcoma cell proliferation ability; 3, Soft AGAR cloning experiments (Soft AGAR assay) to detect the cancer cells than the growth of the anchor malignization ability; 4, BrdU infiltration method (5-bromine deoxidization uracil nucleoside method) technology to detect miR-664 for regulating cell division cycle.Results:1, determined by MTT colorimetry experiment:miR-664 osteosarcoma cell proliferation rate inhibitor group was obviously lower than the control group (P< 0.05); 2, cell cloning experiments:miR-664 osteosarcoma cells proliferation inhibitor group was obviously lower than the control group (P<0.05); 3, soft AGAR cloning experiments:miR-664 inhibitor group of anchor the growth of malignant osteosarcoma cells the ability significantly lower than the control group (P<0.05); 4, BrdU infiltration experiment:miR-664 S period osteosarcoma cells inhibitor group was obviously lower than that of control group (P<0.05).Conclusion:down regulate miR-664 expression significantly inhibited U2-OS osteosarcoma cell proliferation rate, proliferation and malignant division.Part three Down regulation of FOXO4 promote U2-OS osteosarcoma cells malignant proliferation(1)To study the effect of miR-664 to FOXO4 signal wayobjective:research on osteosarcoma cells malignant proliferation whether internal molecular mechanism by FOXO4 signaling pathways regulating.Methods:mimic transfection miR-664 and miR-664 inhibitor U2-OS cells as the model, by western blot method to detect FOXO4 gene protein expression levels, and USES the method of real-time quantitative PCR and western blot detection FOXO4 target gene P21, Rb, p-Rb, CyclinDl mRNA and protein expression.Results:1, the detection of the western blot method, mimic U2 FOXO4 in miR-664-OS expression in cells is reduced, while in miR-664 inhibitor U2-OS increased expression in cells(P<0.05).2, in the same way, the target gene p21 FOXO4, p-Rb mimic U2 in miR-664-OS expression in cells is reduced(P<0.05), while in miR-664 inhibitor U2-OS increased expression in cells(P<0.05).3, instead of CyclinDl gene expression and FOXO4 expression instead(P<0.05).Conclusion:miR-664 can inhibit FOXO4 and the expression of target genes.(2) Discuss miR-664 for target genes FOXO4 targeted regulating the relationship betweenobjective:determine the miR-664 for target genes FOXO4 targeted adjustmentmethod:using Targetscan software tools analysis showed that FOXO4 is a potential target genes of miR-664, using western blot analysis showed that the U2-OS osteosarcoma cells in control group, miR-664 mimic group and miR-664 inhibitor FOXO4 exist significant differences in gene expression, mimic group protein expression is lower than the control group, while the inhibitor protein expression is higher than the control group. In order to further verify the miR-664 and FOXO4 targeted adjustment relations, building contains FOXO4-3’UTR luciferase report plasmid, will be in the direct synthesis of miR-664 mutation transfection into cells, the liposome transfection method is adopted, in the control group, miR-664 mimic, miR-664 inhibitor, miR-664 mutation U2-OS cell of the communist party of China transfection FOXO4-3’UTR luciferase report plasmid,48 hours after cracking cells, detect luciferase activity, determine to FOX04 miR-664-3’UTR regulating relations.Results:luciferase detection showed that FOXO4 gene in the control group, miR-664 mimic, miR-664 inhibitor U2-OS cells, the expression differences(P<0.05); And in miR-664 mutation U2-OS cells but had no difference. Conclusion:FOXO4 genetic gene regulation is the goal of miR-664.(3) FOXO4 gene is miR-664 target genes that promote the proliferation of malignant osteosarcomaobjective:to study FOXO4 in value-added role of osteosarcoma.Methods:in order to further analyze FOXO4 in value-added role of osteosarcoma, through specific siRNA blocking FOXO4 two targets for inhibition of the expression of endogenous genes, in miR-664 inhibitor U2-OS cells, by determined by MTT, soft AGAR cloning experiments, BrdU technology, observe the cell proliferation.Results:in the miR-664 inhibitor, U2-OS cells by endogenous FOXO4 gene expression, western blot prompt downgrade miR-664, and through specific siRNA blocking FOXO4 after two target genes, endogenous FOXO4 expression is decreased obviously(P<0.05). Cell proliferation ability and speed, the cancer cells of anchor growth ability of malignant change, cell division cycle number is significantly higher than the control group(P<0.05).Conclusion:FOXO4 genes in miR-664 promoting malignant osteosarcoma proliferation plays a key role.(4) Osteosarcoma miR-664 and FOXO4 expression correlation analysisobjective:To analysis the clinical osteosarcoma tissues FOXO4 expression associated with miR-664.methods:immune real-time fluorescence PCR detection of 8 cases of osteosarcoma tissue and tissue adjacent to carcinoma in miR-664 expression level; Western Blot-detection of 8 cases of osteosarcoma tissue FOXO4 expression level. Next do the correlation analysis.Results:statistical result showed that the expression of miR-664 levels and negatively correlated with FOX04 linear relationship (r2= 0.951, p< 0.05).Conclusion:to further verify the expression levels of miR-664 and the proliferation of osteosarcoma were positively correlated, clinical and FOXO4 expression levels and its negative correlation.Through the above three parts experiments, we can draw the conclusion of this experiment:miR-664 promotes U2-OS osteosarcoma cells proliferation via down regulating of FOXO4. |
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