| BackgroundOsteosarcoma is one of the most common primary malignant tumors in youth and children,the onset of the main parts involved long bone femoral and tibial epiphyseal side.It is a serious threat to the survival of patients with osteosarcoma,due to its high malignant degree and the height tendency of the lung metastasis.Recently,the survival rate of patients with osteosarcoma is gradually improving,because of the the combination therapy of surgery and postoperative chemotherapy in clinical.However,unfortunately,the lack of early sensitive and specific tumor markers,so the diagnosis and treatment of patients with osteosarcoma often belongs to the late stage;In addition,osteosarcoma recurrence and distal metastasis after the operation still emerge in endlessly,therefore,it is of great significance to explore the early effective and high specificity and sensitivity tumor biomarkers to diagnose osteosarcoma,which is also important for post-operative disease monitoring and improvement of the survival rate and survival quality in patients with osteosarcoma.ObjectivesThe objective of this study is to explore the diagnostic value of miRNA-223 in the osteosarcoma and its impact on the biological functions of osteosarcoma cells.From the clinical research and cell level,this paper discussed the following questions: first of all,the diffirent expression level of miRNA-223 in osteosarcoma tissue and normal tissue adjacent to carcinoma,in patients with osteosarcoma and physical examination,osteosarcoma cell lines and normal osteoblast.Secondly,serum miRNA-223 was evaluated to distinguish the osteosarcoma patients and healthy people as an early diagnosis of tumor markers.Thirdly,the role of miRNA-223 in osteosarcoma cell proliferation,migration and invasion were observed,and explore its downstream target protein expression.Which might provide certain theoretical basis for the use of serum miRNA-223 in early diagnosis of osteosarcoma as a tumor biomarker,and the applied of targeted gene therapy in osteosarcoma.Materials and MethodsFirstly,osteosarcoma tissue samples collected: 100 cases of patients with osteosarcoma resection were selected from Department of orthopaedic in the first affiliated hospital of Zhengzhou university from July 2009 to July 2012,which was permissed of all the surgical patients.The primary cancer tissues and the surrounding normal tissues with no cancerous tissue invasion were resected in all the patients with osteosarcoma.The surrounding normal tissue was as a negative control,which passed the pathology confirmed.qRT-PCR was used to compare the expression level of miRNA-223 in tumor tissue and the adjacent to carcinoma tissues in patients with osteosarcoma.In addition,the clinical pathologic information of osteosarcoma patients were collected.The expression level of miRNA-223 in patients with osteosarcoma tissue was compared with tumor size,lymph node metastasis,tumor infiltration.Secondly,serum specimens from patients with osteosarcoma collected: the peripheral blood of 112 cases patients with osteosarcoma were collected from Department of orthopaedic in the first affiliated hospital of zhengzhou university since July 2008 to July 2011,which was obtain the consent of the patients,5 ml peripheral blood were collected from 112 patients prior to surgery,in addition,5 ml peripheral blood extracted from 50 cases of healthy check-up as a negative control.qRT-PCR was used to compare the expression of miRNA-223 in peripheral blood in patients with osteosarcoma and health check-up,ROC curve and multivariate survival analysis were applied to evaluate of serum miRNA-223 as tumor biomarkers in early diagnosis of patients with osteosarcoma,and monitoring the value of the survival of patients with osteosarcoma after surgery.Lastly,qRT-PCR was applied to compare the expression level of miRNA-223 in osteogenesis cell and osteosarcoma cell lines.After miRNA-223 transfection in osteosarcoma cell lines,MTT,scratches and Transwell experiments were uesd to observe cell proliferation,migration and invasion of osteosarcoma cells,in addition,Western blot was applied to detecte the protien expression level of miRNA-223 target molecules Caprin-1.Results1,the expression level of miRNA-223 in osteosarcoma patient tissueqRT-PCR was used to observate the expression level of miRNA-223 in osteosarcoma tissues and normal tissue adjacent to carcinoma,the results showed that the expression level of miRNA-223 in osteosarcoma tissue was significantly lower than that of tissue adjacent to carcinoma(P = 0.0021),suggesting miRNA-223 might be involved in tumor occurrence and development of osteosarcoma.To explor the relationship of miRNA-223 in patients with osteosarcoma tussie presented low expression patterns and clinical pathological parameters,osteosarcoma patients tissues miRNA-223 expression and different gender,age,tumor size,tumor location,different type of tumor tissue,patients with and without transfer case,different expression differences in survival condition,such as category were compared,the results showed that osteosarcoma patients tissues miRNA-223 expression in the death group,transfer osteosarcoma tissue were significantly lower than the survival group,not transfer group,respectively(P < 0.05),suggesting that miRNA-223 might play an important role in osteosarcoma patients metastasis and survival.2,the expression level of miRNA-223 in the peripheral blood of osteosarcoma patientsqRT-PCR experiment was applied to compare the serum miR-223 expression of 112 patients with osteosarcoma and 50 cases of normal healthy check-up.The results showed that compared with the normal healthy check-up,the expression of serum miRNA-223 in patients with osteosarcoma is significantly lower(P < 0.01).And further confirmed that miRNA-223 might be involved in the progress of osteosarcoma malignant tumor.To investigate whether serum miRNA-223 could be used as an early diagnostic biomarker in patients with osteosarcoma,ROC curve was draw,according to the results of serum miRNA-223 in patients with osteosarcoma and normal healthy check-up,the reslut showed that the area under the ROC curve is 0.926,while according to some numerical setting cut-off value was 1.76,miRNA-223 in the diagnosis of patients with osteosarcoma of the specific value of 89.5%,the sensitivity value of 97.2%,prompting serum miRNA-223 could be used as a potential biomarker in distinguish osteosarcoma patients and physical examination with the high specificity and sensitivity.Multivariate Cox regression analysis showed that the expression of serum miRNA-223(RR = 4.59,95% CI 1.84 11.45,P = 0.001)could be used as an overall survival of patients with osteosarcoma an independent prognostic factor.3,the preliminary discussion of miRNA-223 in osteosarcoma cell invasionOverexpressed of osteosarcoma cell line with miRNA-223,MTT method,experiment scratches and Transwell experimental were determined to observate osteosarcoma cell proliferation,migration and invasion.Compared to the HOS,MG-63 cell without transfection,and cells with the negative control transfection,MTT experiment result showed that the overexpression of miRNA-223 could significantly inhibit HOS,MG-63 cell proliferation;Scratch experimental results showed that overexpression of miRNA-223 could significantly inhibit HOS,MG-63 cell migration;In addition,Transwell experiments showed overexpression of miRNA-223 could significantly inhibit HOS,MG-63 cell invasion.Cytoplasmic the activation/proliferation associated protein 1(Cytoplasmic activation/effort-associated protein 1,Caprin-1)is the downstream directly targeting molecules of miRNA-223,presious study found that inhitbed the expression of Caprin-1 could inhibit the proliferation of tumor cells,so as to prompt the Caprin-1 might be an important molecules of regulating tumor metastasis.Western Blot experiments results showed that compared with the blank cells and negative transfection cells,overexpression of miRNA-233 significantly suppressed the protein expression level of Caprin-1 in MG-63.Conclusions1,miRNA-223 was down-regulation in the osteosarcoma patients tissue,osteosarcoma patients peripheral blood and osteosarcoma cell line.2,serum miRNA-223 could be used as an effective early diagnostic marker to distingush osteosarcoma patients with high specificity and sensitivity,in addition,the serum miRNA-223 also could serve as an indicator of disease detection after surgery in osteosarcoma patients.3,miRNA-223 inhibited osteosarcoma cell proliferation,migration and invasion,preliminary discussed the molecular mechanisms of miRNA-223 inhibition of osteosarcoma invasion and metastasis. |
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