NKD1参与介导miR-195对骨肉瘤转移的抑制作用
摘要
骨肉瘤（Osteosarcoma，OS）好发于儿童和青少年，是临床上最常见的原发性恶性骨肿瘤之一。其恶性程度高、血行转移发生早、肺转移发生率高、患者病情发展快且死亡率十分高。准确地对骨肉瘤做出早期诊断和有效地抑制或阻断骨肉瘤转移，已经成为骨肉瘤治疗领域的研究重点。深入认识骨肉瘤转移的分子机制对其基因靶向治疗具有十分重要的指导作用。因此，科研工作者致力于寻找更为重要的骨肉瘤转移的相关分子并试图阐明其分子机制。
miRNA (microRNA)是一类进化上高度保守的、非编码蛋白质的小分子RNA。它在多种生物体中广泛表达。通过对靶基因的转录后调控，miRNA参与多种肿瘤转移的调控。前期研究表明，miR-195能够显著抑制骨肉瘤细胞的转移能力，但是其具体的作用机制仍不十分清楚。
Wnt信号通路是目前肿瘤研究领域的热点之一。Wnt信号通路参与调控包括骨肉瘤在内的多种肿瘤的发生、发展、转移等病理过程。课题组前期基因芯片检测结果显示，Wnt信号通路分子成员NKD1在高转移能力骨肉瘤细胞系F5M2中高表达，提示NKD1可能促进骨肉瘤的转移。同时，生物信息学分析结果显示NKD1是miR-195的潜在靶基因。这为进一步研究miR-195的作用机制奠定了良好的实验基础。结合上述―miR-195能够显著抑制骨肉瘤转移‖的研究基础，课题组提出了NKD1调控骨肉瘤转移机制的推论，即：NKD1参与介导miR-195对骨肉瘤转移的抑制作用。基于以上推论，研究NKD1在骨肉瘤转移中的作用和阐明其调控骨肉瘤转移的分子机制具有十分重要的临床指导意义。
研究目的：
本实验拟通过研究：阐明NKD1在骨肉瘤组织中的表达及其与骨肉瘤分级分期、病理类型、预后、患者生存率等临床表型之间的相关性；阐明NKD1在骨肉瘤转移中的调控作用及其分子机制；证明NKD1是miR-195的直接靶基因，并参与介导miR-195对骨肉瘤转移的调控。以期最终阐明NKD1在miR-195调控骨肉瘤转移中的作用及机制，为其在骨肉瘤转移的基因靶向治疗、早期诊断以及预后判断等方面的临床应用提供新的研究思路和切入点。
研究方法：
1.分别采用Realtime RT-PCR分析及Western Blot分析的方法，检测NKD1在不同转移能力骨肉瘤细胞系（F4和F5M2）中的表达情况，以验证NKD1在以上细胞中的差异表达。
2.构建NKD1的干涉质粒pSil-NKD1-sh，将其转染入骨肉瘤细胞F5M2，并用G418筛选出稳定下调NKD1表达水平的骨肉瘤细胞系F5M2+NKD1-sh，为进一步研究NKD1在骨肉瘤转移中的作用及机制奠定实验基础。
3.利用稳定下调NKD1表达水平的骨肉瘤细胞系F5M2+NKD1-sh，进行NKD1的功能缺失实验。在细胞水平，检测骨肉瘤细胞F5M2+NKD1-sh增殖、迁移及侵袭能力的变化情况；在裸鼠模型中，检测骨肉瘤细胞F5M2+NKD1-sh原位成瘤能力和肺转移能力的变化情况。
4.首先，对NKD1和miR-195之间的调控关系进行生物信息学分析。然后，分别采用Realtime RT-PCR及Western Blot技术检测在骨肉瘤细胞中改变miR-195水平后NKD1的表达情况。最后，采用双荧光素酶报告基因系统证明NKD1mRNA3’UTR和miR-195之间直接结合，NKD1是miR-195的直接靶基因。
5.收集福尔马林固定石蜡包埋（Formalin-fixed, paraffin-embedded, FFPE）的骨肉瘤组织标本，用免疫组织化学（immunohistochemistry, IHC）的方法检测NKD1蛋白的表达水平，并统计分析其与骨肉瘤转移、分级分期、病理类型、患者预后及生存率等临床表型之间的相关性。
研究结果：
1.分别采用Western Blot分析和Realtime RT-PCR分析的方法，检测骨肉瘤细胞系F4和F5M2中NKD1的表达情况，结果显示：NKD1在F5M2中的表达较F4中明显降低，与基因芯片检测结果一致。
2.成功构建了NKD1的干涉质粒pSil-NKD1-sh，并筛选出稳定下调NKD1表达水平的骨肉瘤细胞系F5M2+NKD1-sh及其对照骨肉瘤细胞系F5M2+NC。Real-timeRT-PCR及Western Blot检测显示：NKD1在F5M2+NKD1-sh细胞中的表达较对照组明显降低。
3.在体外细胞水平，质粒pSil-NKD1-sh对骨肉瘤细胞F5M2的侵袭和迁移能力有明显的抑制作用，而对增殖能力无显著影响。在裸鼠体内，质粒PSil-NKD1-sh对骨肉瘤细胞的肺转移能力和原位成瘤能力均有显著的抑制作用。
4.生物信息学分析结果显示：NKD1是miR-195的潜在直接靶基因。在骨肉瘤细胞中，上调（下调）miR-195水平可以抑制（促进）NKD1的表达。双荧光素酶报告基因系统证明NKD1mRNA3’UTR和miR-195之间直接结合。
5.骨肉瘤组织中NKD1蛋白表达阳性率明显高于骨样骨瘤组织和骨软骨瘤组织；而骨样骨瘤组织NKD1蛋白表达阳性率与骨软骨瘤NKD1蛋白表达阳性率相比无显著差异。同时，NKD1的表达与骨肉瘤是否转移及患者3年生存率显著相关，而与性别、年龄、肿瘤大小、组织学分级、组织学分型及肿瘤发生部位等临床表型无明确相关性。
研究结论：
1. NKD1的表达水平与骨肉瘤细胞的转移能力呈正相关性，这提示NKD1可能促进骨肉瘤的转移。因此，有必要进一步深入研究NKD1在骨肉瘤细胞转移中的功能及其分子机制。
2.稳定下调NKD1表达水平的骨肉瘤细胞系F5M2+NKD1-sh及其对照骨肉瘤细胞系F5M2+NC构建成功。这为进一步研究NKD1在骨肉瘤细胞中的功能及其分子机制奠定了实验基础。
3.在体外细胞水平及裸鼠体内，pSil-NKD1-sh均可以显著抑制骨肉瘤细胞的转移能力。提示，NKD1有可能成为骨肉瘤转移基因靶向治疗的新靶点。
4.证明了NKD1是miR-195的直接靶基因。结合NKD1促进骨肉瘤转移以及miR-195抑制骨肉瘤转移的前期研究结果，NKD1可能介导miR-195对骨肉瘤转移的抑制。这为NKD1在骨肉瘤基因靶向治疗中的临床应用提供了一定的理论依据和实验基础。
5. NKD1在骨肉瘤组织中的表达与是否转移及患者3年生存率显著相关。这提示，NKD1在骨肉瘤的转移过程中发挥着重要作用，有可能作为新的反映骨肉瘤转移及预后的重要分子标志物。
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.
miRNA (microRNA)是一类进化上高度保守的、非编码蛋白质的小分子RNA。它在多种生物体中广泛表达。通过对靶基因的转录后调控，miRNA参与多种肿瘤转移的调控。前期研究表明，miR-195能够显著抑制骨肉瘤细胞的转移能力，但是其具体的作用机制仍不十分清楚。
Wnt信号通路是目前肿瘤研究领域的热点之一。Wnt信号通路参与调控包括骨肉瘤在内的多种肿瘤的发生、发展、转移等病理过程。课题组前期基因芯片检测结果显示，Wnt信号通路分子成员NKD1在高转移能力骨肉瘤细胞系F5M2中高表达，提示NKD1可能促进骨肉瘤的转移。同时，生物信息学分析结果显示NKD1是miR-195的潜在靶基因。这为进一步研究miR-195的作用机制奠定了良好的实验基础。结合上述―miR-195能够显著抑制骨肉瘤转移‖的研究基础，课题组提出了NKD1调控骨肉瘤转移机制的推论，即：NKD1参与介导miR-195对骨肉瘤转移的抑制作用。基于以上推论，研究NKD1在骨肉瘤转移中的作用和阐明其调控骨肉瘤转移的分子机制具有十分重要的临床指导意义。
研究目的：
本实验拟通过研究：阐明NKD1在骨肉瘤组织中的表达及其与骨肉瘤分级分期、病理类型、预后、患者生存率等临床表型之间的相关性；阐明NKD1在骨肉瘤转移中的调控作用及其分子机制；证明NKD1是miR-195的直接靶基因，并参与介导miR-195对骨肉瘤转移的调控。以期最终阐明NKD1在miR-195调控骨肉瘤转移中的作用及机制，为其在骨肉瘤转移的基因靶向治疗、早期诊断以及预后判断等方面的临床应用提供新的研究思路和切入点。
研究方法：
1.分别采用Realtime RT-PCR分析及Western Blot分析的方法，检测NKD1在不同转移能力骨肉瘤细胞系（F4和F5M2）中的表达情况，以验证NKD1在以上细胞中的差异表达。
2.构建NKD1的干涉质粒pSil-NKD1-sh，将其转染入骨肉瘤细胞F5M2，并用G418筛选出稳定下调NKD1表达水平的骨肉瘤细胞系F5M2+NKD1-sh，为进一步研究NKD1在骨肉瘤转移中的作用及机制奠定实验基础。
3.利用稳定下调NKD1表达水平的骨肉瘤细胞系F5M2+NKD1-sh，进行NKD1的功能缺失实验。在细胞水平，检测骨肉瘤细胞F5M2+NKD1-sh增殖、迁移及侵袭能力的变化情况；在裸鼠模型中，检测骨肉瘤细胞F5M2+NKD1-sh原位成瘤能力和肺转移能力的变化情况。
4.首先，对NKD1和miR-195之间的调控关系进行生物信息学分析。然后，分别采用Realtime RT-PCR及Western Blot技术检测在骨肉瘤细胞中改变miR-195水平后NKD1的表达情况。最后，采用双荧光素酶报告基因系统证明NKD1mRNA3’UTR和miR-195之间直接结合，NKD1是miR-195的直接靶基因。
5.收集福尔马林固定石蜡包埋（Formalin-fixed, paraffin-embedded, FFPE）的骨肉瘤组织标本，用免疫组织化学（immunohistochemistry, IHC）的方法检测NKD1蛋白的表达水平，并统计分析其与骨肉瘤转移、分级分期、病理类型、患者预后及生存率等临床表型之间的相关性。
研究结果：
1.分别采用Western Blot分析和Realtime RT-PCR分析的方法，检测骨肉瘤细胞系F4和F5M2中NKD1的表达情况，结果显示：NKD1在F5M2中的表达较F4中明显降低，与基因芯片检测结果一致。
2.成功构建了NKD1的干涉质粒pSil-NKD1-sh，并筛选出稳定下调NKD1表达水平的骨肉瘤细胞系F5M2+NKD1-sh及其对照骨肉瘤细胞系F5M2+NC。Real-timeRT-PCR及Western Blot检测显示：NKD1在F5M2+NKD1-sh细胞中的表达较对照组明显降低。
3.在体外细胞水平，质粒pSil-NKD1-sh对骨肉瘤细胞F5M2的侵袭和迁移能力有明显的抑制作用，而对增殖能力无显著影响。在裸鼠体内，质粒PSil-NKD1-sh对骨肉瘤细胞的肺转移能力和原位成瘤能力均有显著的抑制作用。
4.生物信息学分析结果显示：NKD1是miR-195的潜在直接靶基因。在骨肉瘤细胞中，上调（下调）miR-195水平可以抑制（促进）NKD1的表达。双荧光素酶报告基因系统证明NKD1mRNA3’UTR和miR-195之间直接结合。
5.骨肉瘤组织中NKD1蛋白表达阳性率明显高于骨样骨瘤组织和骨软骨瘤组织；而骨样骨瘤组织NKD1蛋白表达阳性率与骨软骨瘤NKD1蛋白表达阳性率相比无显著差异。同时，NKD1的表达与骨肉瘤是否转移及患者3年生存率显著相关，而与性别、年龄、肿瘤大小、组织学分级、组织学分型及肿瘤发生部位等临床表型无明确相关性。
研究结论：
1. NKD1的表达水平与骨肉瘤细胞的转移能力呈正相关性，这提示NKD1可能促进骨肉瘤的转移。因此，有必要进一步深入研究NKD1在骨肉瘤细胞转移中的功能及其分子机制。
2.稳定下调NKD1表达水平的骨肉瘤细胞系F5M2+NKD1-sh及其对照骨肉瘤细胞系F5M2+NC构建成功。这为进一步研究NKD1在骨肉瘤细胞中的功能及其分子机制奠定了实验基础。
3.在体外细胞水平及裸鼠体内，pSil-NKD1-sh均可以显著抑制骨肉瘤细胞的转移能力。提示，NKD1有可能成为骨肉瘤转移基因靶向治疗的新靶点。
4.证明了NKD1是miR-195的直接靶基因。结合NKD1促进骨肉瘤转移以及miR-195抑制骨肉瘤转移的前期研究结果，NKD1可能介导miR-195对骨肉瘤转移的抑制。这为NKD1在骨肉瘤基因靶向治疗中的临床应用提供了一定的理论依据和实验基础。
5. NKD1在骨肉瘤组织中的表达与是否转移及患者3年生存率显著相关。这提示，NKD1在骨肉瘤的转移过程中发挥着重要作用，有可能作为新的反映骨肉瘤转移及预后的重要分子标志物。
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.
引文
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