The Research Of The Expression Of MiR-148a In Patients With Osteosarcoma And Its Function Mechanism

Background:Osteosarcoma is a tumor which grows rapidly with invading the adjacent tissues. This high malignant tumor develops rapidly and is prone to metastasize to lung in the early stage with high recurrence rate and poor prognosis. As osteosarcoma could not be cured by effective treatment, it is now a worldwide heath problem. With the development of medicine, researchers have found some osteosarcoma-related genes including miRNAs. The miRNA is a kind of non-coding RNAs with 19-25 nucleotides, and they could regulate target genes by binding to the 3’-UTR, inhibiting the translation of the target genes. Hundreds of miRNAs have been founded in human cells and they could be involved in cells’growth, apoptosis and differentiation with close relationships with diseases. Some miRNAs were found to be abnormally expressed in osteosarcoma, and miR-34 was donw-regulated and it could regulate target genes with p53depended manner. miR-34c could inhibit Notch pathway and regulate the differentiation of osteoblasts, promoting the development of osteosarcoma. miR-24 was up-regulated in osteosarcoma and involved in osteosarcoma progression by regulating LPAATβ.Down-regulated miR-16 could inhibit miR-148a cells’ growth by repressing MAPK/ ERK pathway and IGF1R. miR-148a is one member of the miR-148/152 family and was correlated with tumor growth and metastasis with tissue-specific expression. Tens of abnormal miRNAs have be identified in osteosarcoma by microarray including miR-148a with up-regulated expression level. Our investigation also confirmed that miR-148a was up-regulated osteosarcoma tissues compared to non-tumor tissues and was up-regulated in metastatic osteosarcoma tissues by comparison with no- metastatic osteosarcoma tissues. The results suggested that miR-148a might be involved in osteosarcoma progression, but the function and mechanism were unclear. miRNAs not only play an important role in tumor progression but also could be a bio-marker in peripheral blood. The studies had demonstrated that the expression of miR-184 in blood plasma was down-regulated after the tumor was excised in gastric cancer patients. miR-141 was up-regulated in the blood plasma of prostate cancer patients compared to the expression in the blood plasma of heath people. However, the expression of miR-148a in the peripheral blood of the osteosarcoma patients is unknown.Purpose:To explore the relation between the expression miR-148a and clinicopathological factors for deeply understanding the mechanism of osteosarcoma progression. To study whether the expression of miR-148a in peripheral blood could be an effective bio-marker in osteosarcoma patients. To clarify the function of miR-148a in osteosarcoma cells and identify the target gene to provide proof for therapy.Methods1. The expression of miR-148a in osteosarcoma tissue was investigated the by RT-qPCR and the relationship with clinicopathological factors was analyzed. The formalin-fixed paraffin-embedded osteosarcoma tissues were collected from the second hospital of Shandong university from July 2004 to December 2007. miRNAs were extracted according to the instructions of the miRNAs extraction kit and were evaluated by their absorbance ratio at 260/280 nm. The miRNAs were firstly reverse-transcripted and then RT-qPCR was performed to detect the expression of miR-148a. The relationship between miR-148a expression and clinicopathological factors such as gender, tumor size, metastasis was analyzed.2. The expression of miR-148a in osteosarcoma tissue was investigate the by RT-qPCR and the relationship with clinicopathological factors was analyzed. The peripheral blood of the osteosarcoma patients was drawn, and the blood plasma was collected after the blood being centrifuged at 12000rpm/min for 5min. miRNAs were extracted according to the instructions of the miRNAs extraction kit. RT-qPCR was performed and the relationship between miR-148a expression and clinicopathological factors was analyzed as previously described.3.The correlation of expression of miR-148a in osteosarcoma tissues with the expression in peripheral blood was analyzed.4. The function of miR-148a in osteosarcoma cells was examined in vitro. The osteosarcoma cells, and also negative control cells, were seeded in 12 well-plate, and then were transfected with miR-148a. The expression of miR-148a was detected after 24,48 and 72h to comfirm that miR-148a was successfully be overexpressed. In vitro migration and invasion was performed after miR-148a being transfected. MTS assay was also be performed to investigate the proliferation of osteosarcoma cells.5. Dual-luciferase assay was performed to identify the direct target genes of miR-148a. The potential target genes were predicted by some softwares, and then the 3’-UTR of the predicted genes were constructed in to dual-luciferase plasmids. The plasmids were co-transfected with miR-148a into osteosarcoma cells and then dual-luciferase assay was performed to identify the direct target genes.Results1. The relation between miR-148a expression in osteosarcoma tissues and clinicopathological factors. The miR-148a was up-regulated in 106 osteosarcoma tissues compared with 89 non-tumor tissues and was up-regulated in 40 metastatic osteosarcoma tissues. miR-148a expression was also up-regulated in the osteosarcoma tissues of patients with age≥20 years old compared to the steosarcoma tissues of patenits with age <20 years old. No significant difference was found in different groups classfied by tumor size, gender, response to chemotherapy, histological subtype or tumor site. The patients with higher miR-148a expression had poorer overall survival and disease-specific survival. Tumor size, metastasis and miR-148a expression were independent prognostic factors of osteosarcoma patients.2.The relation between miR-148a expression in peripheral blood and clinicopathological factors. miR-148a expression in the serum of the patients was up-regulated and higher expression was detected in metastatic samples. The miR-148a expression in the serum of the patients who response to chemotherapy good was also higher than those response to chemotherapy poor. The patients with higher miR-148a expression also had poorer overall survival and disease-specific survival. Tumor size, metastasis and miR-148a expression were also independent prognostic factors of osteosarcoma patients.3. The correlation analysis demonstrated that the expression of miR-148a in osteosarcoma tissues had a positive correlation with its expression in blood plasma.4. miR-148a promoted osteosarcoma cells migration and invasion in vitro. Compared to controls, the ability of migration and invasion of the cells trasfected with miR-148a were significantly promoted in transwell assay. However, miR-148a had no effect on cells’ proliferation ability.5. SMAD2 was the direct target of miR-148a. Four potential target genes of miR-148a were predicted by softwares. miR-148a significantly affected the luciferase expression of SMAD2, but not the other predicted genes. SMAD2 was so be identified as the direct target gene of miR-148a.Conclusion:Our results demonstrated that miR-148a was up-regulated in osteosarcoma tissues and blood plasma and the expression was higher in metastatic samples. Patients with higher miR-148a expression had poorer prognosis. miR-148a expression metastasis were independent prognostic factors of osteosarcoma patients. It is suggested that miR-148a could be a biomarker of osteosarcoma patients. miR-148a could promote osteosarcoma cells’ migration and invasion ability and could directly regulate target gene SMAD2, so we suppose that it could be a new therapy target of osteosarcoma in future.