Research On Effects And Mechanism Of MiRNA-194 In Human Osteosarcoma

BackgroundOsteosarcoma is the most common malignant tumor of bone(44.6%).It is regarded as a hotspot and difficulty in osteosarcoma clinical treatment because of its high malignant degree, metastasis rate and fatality rate. With the developing in osteosarcoma therapy over the past several decades including neoadjuvant and adjuvant chemotherapy coupled with local control, patient outcomes have been obviously enhanced. However, It is still difficult to solve two major problems: the recurrence and metastasis. About 80%- 90% of the causes of death in patients with osteosarcoma is metastasis(mainly pulmonary metastasis). Therefore,it is extremely urgent to find effective early diagnostic tools and treatment in osteosarcoma.miRNAs are a class of endogenous non-coding small RNAs approximate 21 to 23 nucleotides in length. They negativly regulate their target genes expression post-transcriptionally by base-pairing with the complementary sites in the 3’-untranslated region(3’-UTR) of the m RNA of their cognate target m RNAs. Thousands of mi RNA genes have been found from they were first discovered in 1993. Studies have demonstrated that mi RNAs are associated with a series of the important process of life including development, cell proliferation, differentiation and apoptosis. mi RNAs are regarded as biomarkers and therapeutic targets for tumorand because they are associated with a variety of tumor and play a key role in controlling the metastasis of tumor in addition to the high degree of scheduling, conservative and tissue specificity.Refer to a large number of literature, we found that the mi RNA-194 is low in many human tumors, especially in the digestive tract tumors, which declare that mi RNA-194 may play the role of tumor suppressor genes in human tumors. However, the function of mi RNA-194 in human osteosarcoma has not been totally elucidated at home and abroad. In this study, we mainly research the functions of mi RNA-194 in both osteosarcoma cell lines SOSP-9607 and U2-OS, and do further research and exploration through nude mice and clinical samples. ObjectiveTo describe the effects and the mechanism of mi RNA-194 function in human osteosarcoma and establish some theoretical basis for osteosarcoma treatment and element experience for further research. Methods(1) To verify the differential expression of mi R-195 in the different metastatic potential osteosarcoma cell lines F4 and F5M2 by using real-time quantitative PCR.(2) To investigate the function of mi RNA-194, we transfected human osteosarcoma cell lines SOSP-9607 and U2-OS by using recombinant lentiviruses. After the transfection and the selection of the cells, experiments with SOSP-9607 cells and u2-os cells were divided into four groups as blank group(untransfected cells), control group(cells transfected with the control lentivirus),up-regulation group(stable cells transfected with up-regulation lentivirus) and down-regulation group(stable cells transfected with down-regulation lentivirus).(3) In both cell lines, we investigate the function of mi RNA-194 affected cell survival, apoptosis, and cell cycle by using mi RNA-194 stable cell lines.(4) We examined the effects of mi RNA-194 on the capacities of tumor formation and pulmonary metastasis of osteosarcoma in nude mice.(5) Using quantitative reverse transcription-polymerase chain reaction(q RT-PCR), we evaluated the expression of mi RNA-194 and two known mi RNA-194 target genes, CDH2 and IGF1 R in osteosarcoma samples from 107 patients and 99 formalin- or paraformalin-fixed, paraffin-embedded(FFPE) tissues.(6) Potential target genes of mi RNA-194 were predicted using bioinformatics and were verified in gene and protein levels.Moreover, luciferase reporter assay was used to confirm whether CDH2 and IGF1 R were direct targets of mi RNA-194.The expression of the target genes was also examined in osteosarcoma samples by immunohistochemistry. Results(1) The Realtime RT-PCR results showed that the expression of mi RNA-194 was lower expressed in F5M2 cells as compared with F4 cells.(p<0.01).(2) Transfection efficiency(more than 90%) of mi RNA-194 in the two cells were observed by fluorescence microscopy analysis. Real-time quantitative PCR showed that mi RNA-194(up or down) expression has the significant difference(p<0.001) compared with the blank group.(3) In vitro, Over-expression of mi RNA-194 partially inhibited proliferation, migration and invasion of osteosarcoma cells(4) In vivo, mi RNA-194 could significantly inhibit the capacities of tumor formation and pulmonary metastasis.(5) Potential target genes of mi RNA-194 were predicted by using bioinformatics. Moreover, luciferase activity of the wild-type PTEN-3’-UTR reporter was significantly suppressed in mi RNA-194 up-regulation group compared with other groups in SOSP-9607 cells(p<0.01) while down-regulation group could significantly increase it(p<0.01). At the same time, western blot analysis revealed that mi RNA-194 expression could affect the expression of CDH2 and IGF1 R protein while there was no significant difference on m RNA level.(6) Real-time quantitative PCR analysis were used in clinical osteosarcoma samples which were followed up. We found that there were statistically significant relationships between mi RNA-194 expression and some clinicopathological features of osteosarcoma, including age(P = 0.0015), clinical stage(P = 0.019), distant metastasis(P = 0.0251) and patient mortality(P=0.0065). The immunohistochemistry analysis showed that the expression of the IGF1 R and N-cadherin proteins were both inversely correlated with mi RNA-194 expression in osteosarcoma. Conclusion(1) The expression of mi RNA-194 in osteosarcoma cells was closely correlated with the capacity of metastasis.(2) The four transfected and selected groups of mi RNA-194 provided the experimental basis for further study of mi RNA-194 function and mechanism in osteosarcoma.(3) mi RNA-194 could significantly inhibit the capacity of cell survival and metastasis and induce cell apoptosis.mi RNA-194 works as an anti-oncogene in osteosarcoma cells.(4) In vivo, mi RNA-194 could significantly inhibit the capacities of tumor formation and pulmonary metastasis.(5) CDH2 and IGF1 R, two targets gene of mi RNA-194, were predicted and verified, which provided the theoretical basis for the next study of mi RNA-194 in the osteosarcoma.(6) In clinical specimens, mi RNA-194 expression levels were correlated with some clinicopathological features of osteosarcoma, including age, clinical stage, distant metastasis and patient mortality and there was an inverse correlation of expression of mi RNA-194 and CDH2 and IGF1 R in human osteosarcoma samples.