| ackground:Osteosarcoma (OS) is the most common primary malignant bone tumors in orthopaedics, children from 15 to 25 years are easy to occur at good location for the stem epiphyseal end of the long tubular bones of limbs, which accounts for about 50% of the total number of cases in the distal femoral and proximal tibia. Due to the local destructive of osteosarcoma, morbidity and mortality is high, distant metastasis is easily happened, almost involving the lung, therefore which attracts extensive attention by researchers. With the rapid development of new technology, combined with the operation of osteosarcoma, and use of osteosarcoma drug resistance, making the 5-year survival rate for patients with primary site of the OS has reached about 70%. Although through a variety of active treatment, however, the long-term survival of patients with metastasis or recurrence is still less than 20%. Such situation has never changed for the past 30 years. Thus, through studying the heterogeneity, drug resistance and metastasis of osteosarcoma of the basic molecular mechanisms, it’s necessary to look for new markers for patients with osteosarcoma accurately diagnosis, treatment and prognosis evaluation.Micro RNA(miRNA) is a kind of small molecular endogenous single noncoding RNA molecules, which combine the 3’end of the messenger RNA(mRNA) district (3’ UTR) with translation, regulate the expression of target genes in the transcription level, mainly inhibit the target genes of the degradation of mRNA translation. Currently, more and more evidence indicates that microRNAs play an important role in processes of physiological and pathological of many diseases, especially in tumor growth, proliferation, differentiation, apoptosis, metastasis and invasion. Due to the diversity of microRNAs, it can be either as a tumor suppressor or as a tumor promoter. The present study found that abnormal expression of specific microRNAs associated with the growth of many kinds of cancer and clinical prognosis, but the miRNA disorders and its role in cancer is still unknown. On osteosarcoma, although some studies have shown that microRNAs associated with osteosarcoma, such as miR-34a, miR-183 and miR-132, but we still lack of understanding about the role of these microRNAs in osteosarcoma.According to the latest research we found that the expression of miR-135b may be a tumor promoting factor among many human tumors, such as:expression level in colon, breast and lung cancer and. Wu et al. found in colorectal cancer,lymph node and distant metastasis group, miR-135b expression level, and miR-135b inhibitors can effectively reduce metastasis and invasion of this kind of tumor cells. In addition, miR-135b promote tumor progression among head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma and lymphoma. The recent research also suggests that miR-135b is activated in the developmental process of the neural ectoderm, abnormal expression of miR-135 in human embryonic stem cells can inhibit TGF-beta and BMP signaling pathway to accelerate the transformation of neural ectoderm hESC. miR-135b is also involved in the osteogenic differentiation, negative regulation of osteogenesis effect appears through effecting human bone marrow mesenchymal stem cells.Above all of these studies suggest miR-135b played an important role. in the process of human physiological and pathological physiology But it is still unclear that role and mechanism of miR-135b in osteosarcoma. The FOXO1 is a member of the family of FOXO transcription factors, its characteristic is to have a conservative fork structure containing DNA binding domain. Existing research shows that FOXO1 plays a key role in a variety of biological processes including cell proliferation, apoptosis, differentiation and DNA damage repair and stress reaction. For example FOXO1 can lower the cell cycle regulatory proteins and increase the cell cycle D1 protein dependent kinase inhibitor p21 and p27 protein which can lead to the stagnation of the cell cycle. A growing number of studies found FOXO1 is related to many kinds of cancer. Regulate the expression of FOXO1 miR-107 is associated with the proliferation of human gastric cancer cells. Have been confirmed in nuclei and transcription of FOXO1 both decrease in prostate cancer, so that FOXO1 expression can significantly induce apoptosis. In lung cancer cell model and in vivo, FOXO1 inhibits the activation of EGFR signal. Curcumin, through induced the expression of FOXO1,can inhibit the progression and metastasis of lung cancer. In addition, the FOXO1 inhibits the proliferation of osteosarcoma cells.This study regard miR-135b as the main research goal, research the mechanism in osteosarcoma and confirmed that FOXO1 for their targets, and by influencing the FOXO1 to achieve regulation of the development of osteosarcoma.Objective:to study the human osteosarcoma tissue of miR-135b and the expression of FOXO1 situation and whether there is a correlation between them, discuss the role of miR-135b in progress; In U2OS and Saos-2 osteosarcoma cell line, whether validation of miR-135b directly regulate the expression of FOXO1, the expression of miR-135b and FOXOl in osteosarcoma cells and the role relationship between each other. For the clinical treatment of osteosarcoma, diagnosis and prognosis judgement, providing treatment targets, and some experimental basis for future use.Methods:The first part:to collect a total of 7 patients with osteosarcoma (wuhan university people’s hospital)in bone tumor tissue and normal tissue around the tumor, organize collection will be organized immediately after putting into liquid nitrogen tank, by Rt-pcr and Western-blot experimental detection of osteosarcoma tissue and normal tissue around the miR-135b protein expression level. Through a public database to find the target of miR-135b, and select the FOXO1 as possible targets, by Rt-pcr and Western-blot experimental detection of osteosarcoma tissue and normal tissue around the FOXO1 protein expression level, and study if there is a correlation in next experiments.The second part:to transfect miR-135b inhibitors in osteosarcoma cell U2OS and Saos-2 cells line, protein content of miR-135b and FOXO1 were measured by experiment of Rt-pcr and Western blot, evaluate the impact of miR-135b inhibitors to expression of miR-135b and FOXO1. Saos-2 in osteosarcoma cell line and U2OS cells respectively transfect wild type gene carrier FOXO1 and mutant gene carrier FOXO1, after transfection with luciferase reporter gene carrier pMir, luciferase carrier Prl-TK and Renilla, inhibitors miR-135b, together with luciferase reporter gene test miR-135b inhibitors for transfection respectively wild type gene carrier FOXO1 and protein expression level of FOXO1 in mutant gene carrier FOXO1, at the same time, compared with protein expression level of FOXO1 in transfection osteosarcoma cells of miR-135b inhibitorsThe third part:the mainly study whether miR-135b influnce on the biological characteristics of osteosarcoma cells or not, and whether through affecting the expression of target of FOXO1 to achieve its impact on osteosarcoma cells proliferation and invasive ability.1) after transfection of the miR-135b inhibitor and contrast to osteosarcoma cells, making use of MTS to inspect effects of miR-135b on osteosarcoma cell proliferation, making use of Trans well experimental to detect miR-135b on osteosarcoma cell invasion.2) whether miR-135b has impact on osteosarcoma cells proliferation and invasion by FOXO1, firstly, we can enhance the expression of FOXO1 by transfection of pLEX-FOXO1, and verify the effect of FOXO1 on the proliferation and invasion of osteosarcoma cells. and then through transfection specific regulation FOXO1 plasmid siRNA and miR-135b inhibitor or parallels in osteosarcoma cell line U20S and Saos-2, and to examine the influence of osteosarcoma cells in appreciation and the attack by MTS and Transwell experiment tests.Results:The first part:expression of miR-135b raised obviously in bone sarcoma tissues, and negative correlation with the expression of FOXO1:we tested the 7 cases of bone sarcoma tissues and adjacent non tumor tissue in the expression of miR-135b.We found the osteosarcoma tissue of miR-135b expression is significantly higher than normal tissue near the tumor. Through a public database (http://www.targetscan.org) to find the target of miR-135b, and ultimately chose a highly conservative binding sites of FOXO1 to detect the expression and correlation with miR-135b. by detecting FOXO1 expression in these organizations, We found FOXO1 mRNA and protein expression in the tissue of osteosarcoma quantity are lower than those of the corresponding non-cancerous normal bone tissue. And the expression of miR-135b level and protein expression level of FOXO1 mRNA negatively correlated. As a result there is statistical significance.The second part:miR-135b directly regulated FOXO1:we will transfect miR-135b inhibitors and proven miR-135b expression product to OS cell line Saos-2, and U20S. Results show that the join miR-135b inhibitors transfection of osteosarcoma cell line Saos-2 and protein expression level of FOXO1 mRNA in U20S cells increased significantly. Respectively constructed area contain FOXO13’end of translation accepted downstream of miR-135b binding sites of luciferase reporter gene vector (pMir-FOXO1-Wt, setting wild type) and in addition to the seed zone 8 mutant carrier of base pairs (pMir-FOXO1-Mut, setting mutant type), in osteosarcoma cell line Saos-2 and of U20S of transfection miR-135b inhibitors we found that wild type carrier luciferase activity was significantly enhanced, and the mutant carrier luciferase activity compared with not transfection miR-135b inhibitors of osteosarcoma cell line U20SandSaos-2, luciferase activity, there is no significant changes, but the results is in statistical differences The experimental results proved that miR-135b, with the translation section recognized FOXO13’end of binding site,directly combine to achieve negative regulatory role of expression of FOXO1.The third part:miR-135b, through regulating the expression of FOXO1, has impact on osteosarcoma cells proliferation and invasive ability:Firstly,we can enhance the expression of FOXO1 specificity by transfection pLEX-FOXO1, and verify whether FOXO1 has the impact on osteosarcoma cell proliferation and invasive ability, found FOXO1 can significantly inhibit bone sarcoma cells proliferation and invasive ability; Secondly, we choose the most specific lower siRNA plasmid of FOXOl expression, through transfection siRNA plasmid and miR-135b inhibitor or contrast to osteosarcoma cells, testing its impact on osteosarcoma cells proliferation and invasive ability, and we found that after the lower expression of FOXOl, the impact of miR-135b inhibitor on osteosarcoma cell proliferation and invasive ability of inhibitory effect was offset.Conclusion:Our experiments proved that the expression of miR-135b exist in normal tissue adjacent to carcinoma in patients with osteosarcoma and with osteosarcoma tissue, and compared with the normal tissue around the tumor, expression level of concurrent osteosarcoma tissues miR-135b has increased significantly. Through bioinformatics analysis and the experiment, We proves FOXO1 is direct targets of miR-135b in osteosarcoma cells, at the same time, there is a negative correlation between osteosarcoma cells of miR-135b expression level and the expression of FOXO1 level. So we found that miR-135b can promote and enhance the bone sarcoma cells on the proliferation and invasion of local normal tissue by the direct effect of FOXO1 targets, the experiment showed that miR-135b of FOXOl involved in the regulation of the development process of osteosarcoma, which played a role in occurrence and developmen of osteosarcoma... |
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