NKD1Mediates The Anti-metastatic Effects Of MiR-195in Osteosarcoma Cells

Osteosarcoma (OS) is one of the most common human primary malignant bone tumors inchildren and young adults. Osteosarcoma has a high metastatic potential. The clinicaltreatment for osteosarcoma is very difficult, and patients treated with amputation aloneoften died of pulmonary metastasis within one year. Therefore, it is of great importance tomake accurate early diagnosis of osteosarcoma and to block or inhibit osteosarcomametastasis effectively. Understanding of the molecular mechanism of osteosarcomametastasis plays an important guiding role for the gene therapy of osteosarcoma.Therefore, many studies have been carried out to investigate the genes that are involved inthe metastasis of osteosarcoma.MiRNAs (microRNAs) is a class of endogenous, noncoding, single stranded small regulatory RNA molecules, which is highly conserved in evolution. Studies have shownthat miRNAs participate in the regulation of tumor metastasis through regulating theirtarget genes at the post-transcriptional level. It has been reported that miR-195significantly inhibits the pulmonary metastasis of osteosarcoma. However, the molecularmechanism by which miR-195regulates osteosarcoma metastasis has not been totallyelucidated.Wnt signaling pathway is one of the hotspots in cancer research. Wnt signaling pathwayinvolves in the occurrence, development and metastasis of various tumors, includingosteosarcoma. Previously, we have detected the expression of NKD1, which is one of themembers in the Wnt signaling pathway, in osteosarcoma cells with different metastaticcapacities by using gene microarray. The results demonstrated that the expression ofNKD1was up-regulated in osteosarcoma cell with high metastatic potential. Meanwhile,the bioinformatics analysis showed that NKD1is a potential target gene of miR-195.Combined with the previous report that miR-195significantly inhibits the pulmonarymetastasis of osteosarcoma. We put forward the hypothesis that NKD1mediates theanti-metastatic effects of miR-195in osteosarcoma cells.Objective:On the basis of this hypothesis, studies were carry out①to demonstrate the relationshipbetween NKD1expression levels in osteosarcoma tissues and the clinical phenotypes,such as classifications, stages, pathologic types, prognosis and the survival rates;②todemonstrate the role of NKD1in regulating osteosarcoma metastasis and to investigate itsmolecular mechanism;③to prove NKD1is a direct target of miR-195, and NKD1mediates the anti-metastatic effects of miR-195in osteosarcoma cells. We hope that thesework will facilitates the clinical application of NKD1in the early diagnosis, prognosis andgene targeted therapy of osteosarcoma.Methods:1. To validate the differential expression of NKD1in osteosarcoma cells with differentmetastatic capacities (F4and F5M2) by using Realtime RT-PCR and Western Blotanalysis. 2. To construct the NKD1RNA interfere plasmid pSil-NKD-sh. To transfectpSil-NKD-sh into osteosarcoma cells with high metastatic potentials F5M2, and togenerate G418-selected stable cell lines.3. To investigate the effects of pSil-NKD1-sh on proliferation, migration and invasion ofosteosarcoma cells in vitro, and on the capacities of pulmonary metastasis and tumorformation of osteosarcoma cells in vivo.4. To analysis the relationship between NKD1and miR-195by using bioinformaticssoftwares. To detect the NKD1expression levels after changing the miR-195levels inosteosarcoma cells. And finally, to validate NKD1is a direct target of miR-195byusing the dual luciferase reporter gene system.5. To investigate the expression of NKD1in osteosarcoma tissues by using IHC staining.And then, to analyze the associativity between NKD1expression levels inosteosarcoma tissues and the clinical phenotypes, such as Classifications, stages,pathologic types, prognosis and survival rates.Results:1. By using Realtime RT-PCR and Western Blot analysis, the NKD1expression levels inosteosarcoma cells (F4and F5M2) were investigated. The results demonstrated thatNKD1was higher expressed in osteosarcoma cells F5M2as compared withosteosarcoma cells F4, which were consistent with the previous results from genemicroarray.2. The NKD1RNA interfere plasmid pSil-NKD-sh was successfully constructed.PSil-NKD-sh can inhibit the expression of NKD1in osteosarcoma cells with highmetastatic potentials F5M2. Finally, we obtained stable osteosarcoma cells in whichNKD1was down-regulated.3. NKD1significantly inhibited the capacities of migration and invasion of osteosarcomacells F5M2in vitro. However, NKD1had no effect on its proliferation. Meanwhile,NKD1significantly inhibited the capacities of tumor formation and pulmonarymetastasis of osteosarcoma cells in vivo.4. By using bioinformatics softwares, NKD1was predicted to be a potential target gene of miR-195. In osteosarcoma cells, the NKD1expression was inhibited (promoted)after up-regulating (down-regulating) the miR-195levels. Finally, the results from dualluciferase reporter gene system demonstrated that NKD1is a direct target of miR-195,and miR-195could bind directly to the3’UTR of NKD1.5. The Positive rate of NKD1expression in osteosarcoma tissues was higher than that inosteochondroma tissues. Meanwhile, the NKD1expression level in osteosarcomatissue was closely correlated to the clinical phenotypes, such as pulmonary metastasisand the3years of survival rate.Conclusion:1. The expression levels of NKD1in osteosarcoma cells were positively associated totheir metastatic potential. This indicates that NKD1may promote osteosarcomametastasis. Therefore, it is of great importance to further investigate the function andmolecular mechanism of NKD1in osteosarcoma metastasis.2. The stable osteosarcoma cell, in which NKD1was down-regulated, was successfullyconstructed. This facilitated the further investigations of function and molecularmechanism of NKD1in osteosarcoma metastasis.3. PSil-NKD1-sh significantly inhibits the metastatic potential of osteosarcoma cellsF5M2both in vitro and in vivo. This indicated that NKD1may be used as a potentialtarget for the gene therapy of osteosarcoma.4. NKD1is a direct target gene of miR-195and NKD1may mediate the anti-metastaticeffects of miR-195in osteosarcoma cells. This facilitates the clinical application ofNKD1in the targeted therapy of osteosarcoma metastasis.5. The NKD1expression levels in osteosarcoma tissues were closely correlated to theclinical phenotypes, such as pulmonary metastasis and the3years of survival rates.These indicate that NKD1play an important role in the process of osteosarcomametastasis. Therefore, NKD1may be regarded as new biomarkers for the earlydiagnosis and prognosis of osteosarcoma.