WO2019033468A1 - 与绝经后骨质疏松相关的miRNA标志物和试剂盒 - Google Patents

与绝经后骨质疏松相关的miRNA标志物和试剂盒 Download PDF

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WO2019033468A1
WO2019033468A1 PCT/CN2017/100135 CN2017100135W WO2019033468A1 WO 2019033468 A1 WO2019033468 A1 WO 2019033468A1 CN 2017100135 W CN2017100135 W CN 2017100135W WO 2019033468 A1 WO2019033468 A1 WO 2019033468A1
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postmenopausal osteoporosis
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osteoporosis
cluster
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刘欢
林楚娇
陈智
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武汉大学
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to the field of genetic engineering and, more particularly, to miRNA markers and kits associated with postmenopausal osteoporosis.
  • MicroRNAs represent a specific small molecule of non-coding RNA that regulates gene expression through transcription.
  • a single miRNA can interact with transcription factors by inhibiting the expression of a series of related functional genes.
  • blocking miRNA can block the progression of osteoporosis.
  • Multiple independent animal model experiments have also confirmed that many miRNAs have altered expression in osteoporosis and are involved in the pathological process of osteoporosis. Since the first discovery of miRNA in 1993, miRNA has received more and more attention from researchers. The role of miRNAs in the development and progression of various diseases has also been extensively and deeply explored in recent years. More than 2000 miRNAs in humans have been reported to be tissue or cell specific.
  • miRNAs can be stable, replicable, and persist in serum or plasma, making miRNAs possible for the diagnosis or treatment of a variety of diseases, including osteoporosis. Diagnosing osteoporosis by analyzing the expression of miRNA in peripheral blood is non-invasive, stable, and real-time.
  • Osteoporosis is a metabolic bone lesion with reduced bone mass, destruction of bone tissue microstructure, increased bone fragility, and increased incidence of fractures.
  • Postmenopausal osteoporosis is a high-risk disease in postmenopausal women. It is a multifactorial disease: estrogen deficiency, genetic factors, nutritional status, lifestyle habits, physical exercise, menstrual cycle disorders, menopause as early as 40 years old, etc. Related, but postmenopausal estrogen deficiency is an important cause of postmenopausal osteoporosis. In postmenopausal women, bone loss in the first 5 to 7 years increases at a rate of 1% to 5% per year.
  • the determination of bone mineral density by dual-energy X-ray absorptive technique is the gold standard for early detection and diagnosis of osteoporosis, and can also be carried out by biochemical indicators of bone metabolism such as BAP, OC, Tracp5b, PINP, NTX, CTX, etc. Determination to reflect bone metabolism. Bone density testing can predict bone density in normal, low, and osteoporotic conditions and predict the risk of certain fractures. However, the process of osteoporosis is very slow, the change is not obvious in a short time, it takes a long time to observe and continuous detection, and once bone loss is found, the bone density is reduced, it is difficult to reverse, giving early diagnosis of clinical patients. It is a great difficulty.
  • a miRNA cluster refers to two or more clustered miRNAs that are similar in distance on the genome (usually less than 2 kb).
  • the miR-338 cluster is a miRNA that is similar in genome to the genome encoding miR-338 (less than 2 kb), and miR-338-3p and miR-3065-5p belong to the miR-338 cluster, encoding both The positional relationship of genes is shown in Figure 1.
  • the miR-338 cluster is preferably miR-338-3p and/or miR-3065-5p.
  • Postmenopausal osteoporosis is early postmenopausal osteoporosis, which can detect osteoporosis when the T value is between -2.5 and -1.
  • the existing osteoporosis imaging test can be detected, and the miR-338 cluster of the present invention can be used to detect early postmenopausal osteoporosis, for example, when osteoporosis occurs.
  • the miRNA cluster of the present invention can be clearly detected within 8 weeks, preferably within 1 week.
  • the present invention detects the expression levels of miR-338 clusters such as miR-338-3p and miR-3065-5p in the serum of patients with early postmenopausal osteoporosis, and the results show that the expression level of miR-338 cluster is significantly higher than that of the control healthy group. At the same time, the results of ROC analysis showed that the miR-338 cluster has a high diagnostic value.
  • the miR-338 cluster is miR-338-3p and miR-3065-5p, it is more effective as a marker of postmenopausal osteoporosis.
  • the invention also provides the use of a biomaterial comprising a miR-338 cluster as a marker for postmenopausal osteoporosis.
  • the miR-338 cluster is preferably miR-338-3p and/or miR-3065-5p.
  • the biological material may be one or more of a vector, a host cell, a transgenic cell line, and an engineered bacteria.
  • the invention also provides the use of the miR-338 cluster in the preparation of a postmenopausal osteoporosis auxiliary diagnostic kit.
  • the miR-338 cluster is highly expressed in the serum of postmenopausal osteoporosis patients.
  • “High expression” and “significant” as used in the present invention mean that, in a statistical sense, the p value is usually less than 0.05.
  • the miR-338 cluster is preferably miR-338-3p and/or miR-3065-5p.
  • the diagnostic kit of the present invention it is only necessary to determine the content of the miR-338 group in the serum of the patient, and the advantage of the previous diagnostic method is that it is non-invasive and sensitive, and can be diagnosed if the patient suffers from postmenopausal osteoporosis in the early stage.
  • the patient has early postmenopausal osteoporosis, ie osteoporosis when the T value is between -2.5 and -1, it can be diagnosed.
  • the invention also provides the use of an inhibitor of miR-338, such as miR-338-3p and/or miR-3065-5p, in the manufacture of a medicament for the treatment or prevention of postmenopausal osteoporosis.
  • an inhibitor of miR-338 such as miR-338-3p and/or miR-3065-5p
  • a drug for treating or preventing postmenopausal osteoporosis is a drug for promoting osteogenic differentiation.
  • the present invention also provides a medicament for treating or preventing postmenopausal osteoporosis, which comprises an inhibitor of miR-338 cluster, preferably an inhibitor of miR-338-3p and/or miR-3065-5p.
  • the present invention surprisingly finds that the miR-338 cluster, preferably miR-338-3p and/or miR-3065-5p, is highly expressed in postmenopausal osteoporosis patients and can be used as a marker for postmenopausal osteoporosis for preparation.
  • the expression level of miR-338 cluster, preferably miR-338-3p and/or miR-3065-5p, in the serum of the tissue can be specifically detected, and used early and effectively for clinical menopause. Early diagnosis of post-osteoporosis, and the above diagnosis can be achieved in serum without the need for additional wounds.
  • the present invention also finds that the inhibitor of miR-338 cluster can effectively block the progression of osteoporosis and promote osteogenesis, thereby effectively treating or preventing postmenopausal osteoporosis, which can be used as a treatment or prevention of postmenopausal osteoporosis.
  • the drug has good clinical application value and broad application prospects.
  • Figure 1 is a diagram showing the positional relationship of genes encoding miR-338-3p and miR-3065-5p in accordance with a preferred embodiment of the present invention
  • 2A is a graph showing the content of miR-338-3p in a postmenopausal osteoporosis patient group and a healthy group according to Example 1 of the present invention
  • 2B is a graph showing the content of miR-3065-5p in a postmenopausal osteoporosis patient group and a healthy group according to Example 1 of the present invention
  • 2C is a ROC graph of miR-338-3p and miR-3065-5p in Example 1 according to the present invention
  • 3A is a mouse, and a control thereof at 0 weeks, 1 week, 4 weeks, 8 weeks, 12 weeks, 16 weeks, and 18 weeks after bilateral ovarian ablation according to Example 1 of the present invention (Sham group: untreated) Group) micro-CT images of the femur;
  • 3B is a mouse and a control group at 0, 1, 4, 8 weeks, 12 weeks, 16 weeks, and 18 weeks after bilateral ovarian ablation according to Example 1 of the present invention (Sham group: untreated group) a femoral section and stained with HE staining;
  • 3C is a femur bone marrow mesenchymal stem cell of mice and their control groups at 0, 1, 4, 8 weeks, 12 weeks, 16 weeks, and 18 weeks after bilateral ovarian ablation according to Example 1 of the present invention.
  • BMSCs staining diagram of alizarin red staining after 14 days of mineralization induction;
  • 4A is a graph showing the expression content of miR-338-3p and the castration time according to Example 1 of the present invention.
  • 4B is a graph showing the expression content of miR-3065-5p and the time of castration according to Example 1 of the present invention
  • 5A is a flow chart of an experimental scheme for injecting an anti-formulation according to Embodiment 1 of the present invention.
  • Figure 5B is a graph showing the expression levels of miR-338-3p or miR-3065-5p in mouse serum after injection of miR-338-3p or miR-3065-5p inhibitor according to Example 1 of the present invention;
  • the white column was the castration group 3 months after ovarian ablation, and the gray column was the inhibitor group after castration;
  • 5C is a micro-CT analysis of a femur of a mouse, a three-dimensional reconstruction map and a HE staining map of a trabecular bone after injection of a miR-338-3p inhibitor according to Example 1 of the present invention
  • Figure 5D is the trabecular volume ratio, trabecular thickness, trabecular bone volume per unit volume, and trabecular bone spacing in the ovx, con, and ovx+anti-miR-338-3p groups of the trabecular bone reconstruction in Figure 5C.
  • 5E is a micro-CT analysis, a trabecular reconstruction and a HE staining of a mouse femur after injection of a miR-3065-5p inhibitor according to Example 1 of the present invention
  • Figure 5F is the trabecular volume ratio, trabecular thickness, trabecular bone volume per unit volume, and trabecular bone spacing in the ovx, con, and ovx+anti-miR-338-3p groups of the trabecular bone reconstruction in Figure 5E.
  • Figure 6A is a diagram showing the establishment of miR-338-3p and miR-3065-5p from healthy cells in healthy cells using the gene editing method of crispr-cas9 in Example 1 according to the present invention.
  • Figure 6B is a flow chart showing the culture of a cell line that induces miR-338-3p/miR-3065-5p knockdown and a wild-type osteoblast cell line according to Example 1 of the present invention
  • 6C is a miR-338- in the supernatant of the WT group (wild type osteoblast cell line group) and the 3065ko group (miR-338-3p/miR-3065-5p knockout cell line) according to Example 1 of the present invention. Comparison of the expression levels of 3p and miR-3065; wherein, the black column indicates the WT group and the white column indicates the 3065ko group;
  • Figure 6D shows Alp, Ocn, Opn, Alp in the WT group (wild type osteoblast cell line group) and the 3065ko group (miR-338-3p/miR-3065-5p knockout cell line) according to Example 1 of the present invention.
  • WT group wild type osteoblast cell line group
  • 3065ko group miR-338-3p/miR-3065-5p knockout cell line
  • Figure 7 is a representation of miR-338-3p, miR-3065-5p, and overexpression of miR-338 and miR-3065, and downstream genes Col1a, Wnt5a, usp20, Igf1, respectively, in mc3T3, according to Example 1 of the present invention. Comparison of expression levels of Msx1.
  • mice were fixed in the prone position, and the left side of the spine was cut with an ophthalmology to cut an outwardly inclined length of about 1 cm.
  • the skin and muscle layers were peeled off, the ovarian tissue was found, and the ovarian mesangial vessels were ligated with a 4th wire to remove the ovaries. Stitch the skin.
  • the same method was used to remove the other ovary; the uterus was resected and sutured.
  • Micro-CT and HE staining of the mouse femur showed that, as shown in Figures 3A and 3B, a significant reduction in bone mass was observed 3 months after castration, while bone mass in the OVX group was lower than that in the CON group.
  • the trabecular bone density was higher from 0 weeks to 8 weeks, and there was no difference between the Sham group and the ovx group.
  • the number of trabecular bone in the ovx group was significantly lower than that in the sham group.
  • the trabecular bone density gradually decreased, and the ovx group was lower than the sham group.
  • FIG 3C shows that the mineralization capacity of BMSCs (bone marrow mesenchymal stem cells) in mouse femurs was significantly reduced from three months after castration, and the OVX mineralization ability was lower than that of the CON group.
  • BMSCs bone marrow mesenchymal stem cells
  • the expression levels of miR-338-3p and miR-3065-5p in the serum of mice increased with the extension of castration time. It is worth noting that the expression level of miR-338-3p was one week after castration. Significantly higher than the control group, the expression level of miR-3065-5p was significantly higher than that of the control group eight weeks after castration, significantly earlier than the imaging test (micro-CT), as shown in Figures 4A and 4B. This indicates that miR-338-3p and miR-3065-5p can be used as early diagnostic markers for postmenopausal osteoporosis.
  • mice after three months of castration were injected with inhibitors of miR-338-3p and miR-3065-5p by injection into the tail vein, once a week, for a total of 4 injections. 5A is shown.
  • bilateral ovarian ablation was performed on 8-week-old Balb/c mice, and miR-338-3p or miR-3065-5p inhibitor was injected through the tail vein three weeks after surgery, once a week. Inject four times, fight for 1 month, and then collect the specimen. The contents of miR-338-3p and miR-3065-5p in blood and femur bone marrow mesenchymal stem cells were detected.
  • BV/TV (%), Tb.Th (mm), and Tb.N (1/mm) are both ovx group lower than con group, ovx+anti-miR-338-3p group and The ovx+anti-miR-3065-3p group was higher than the ovx group.
  • Tb.Sp(mm) was higher in the ovx group than in the con group, and the ovx+anti-miR-338-3p group and the ovx+anti-miR-3065-3p group were lower than the ovx group.
  • the gene editing method of cristr-cas9 was used to establish miR-338-3p and miR-3065-5p in healthy cells.
  • Schematic map: miR-338-3p and miR-3065-5p in healthy cells are derived from miR-338-3p and miR-3065-5p, which are highly expressed in unhealthy cells.
  • the mR-338-3p and miR-3065-5p knockout osteoblasts were established using the gene editing method of cristr-cas9, which was used to represent healthy osteoblasts, and the wild type mc3T3-E1 represented unhealthy osteoblast.
  • the supernatant was assayed for the content of miRNA, as shown in Figures 6C and 6D, wherein, in Figure 6C, D0 represents mineralization for 0 days, d7 represents culture with mineralization inducing solution for 7 days, and d7co is represented in Figure 6B.
  • the culture method was cultured for 7 days. The results showed that the mineralization of D0 days, the content of miR-338-3p in the WT group was significantly higher than that in the 3065ko group, and the difference was increased in mineralization for 7 days. After 7 days of culture in the exchange medium, this difference was reduced.
  • the osteogenic ability of the WT group (wild type osteoblast cell line group) and the 3065ko group (miR-338-3p/miR-3065-5p knockout cell line) was examined in Fig. 6D.
  • the effects of miR-338-3p/miR-3065-5p content on osteogenic capacity were examined by detecting the expression levels of different groups of Alp, Ocn, Opn, Alp and Runx2. Consistent with the 6C results, mineralization d0 days, WT group Due to the high expression of miR-338-3p/miR-3065-5p, the osteogenic gene expression level was lower than that of the 3065 ko group.
  • miR-338-3p, miR-3065-5p, and miR-338 and miR-3065 were overexpressed in mc3T3, and the downstream genes Col1a, Wnt5a, usp20, Igf1, Msx1 were detected.
  • the expression level of the gene encoding the positively related gene showed that, as shown in Figure 7, overexpression of miR-338-3p, miR-3065-5p, and simultaneous overexpression of miR-338 and miR-3065 significantly down-regulated downstream Gene expression.
  • overexpression of miR-338-3p and miR-3065-5p down-regulated the downstream gene significantly more than the over-expression of miR-338-3p or miR-3065-5p, indicating that miR-338-3p was used simultaneously.
  • Inhibitors of miR-3065-5p which act to promote osteogenic differentiation, to better treat or prevent postmenopausal osteoporosis.
  • Runx2 acts as an upstream gene for the miR-338-3p and miR-3065-5p genes, making miR-338-3p and miR-3065-
  • the expression level of 5p decreased, while Runx2 acts as a downstream target gene for miR-338-3p and miR-3065-5p, with fewer miR-338-3p and miR-3065-5p binding downstream of their Runx2 3
  • the 'UTR region which allows Runx2 to have a higher level of expression, then more Runx2 binds to the promoter regions of miR-338-3p and miR-3065-5p to inhibit miR-338-3p and miR-3065-
  • the present invention provides a miRNA marker and kit associated with postmenopausal osteoporosis, and in particular to the use of the miR-338 cluster as a marker for postmenopausal osteoporosis.
  • the present invention finds that miR-338 clusters such as miR-338-3p and/or miR-3065-5p are highly expressed in postmenopausal osteoporosis patients and can be used as markers for postmenopausal osteoporosis for preparation of menopause.
  • the post-osteoporosis auxiliary diagnostic kit can specifically detect the expression level of miR-338 cluster in tissues, and can be used early and effectively for early diagnosis of postmenopausal osteoporosis, and is non-invasive.
  • inhibitors of the miR-338 cluster have an effect of promoting osteogenesis, and effectively treating or preventing postmenopausal osteoporosis.
  • the marker provided by the invention can be applied to the field of postmenopausal osteoporosis detection on a large scale, and is suitable for industrial application, and has broad market prospects.

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Abstract

本发明提供了miR-338簇在作为绝经后骨质疏松标志物中的应用。本发明发现miR-338簇如miR-338-3p和/或miR-3065-5p在绝经后骨质疏松病人中的高表达，可作为绝经后骨质疏松病的标志物，用于制备针对绝经后骨质疏松病辅助诊断试剂盒，可以特异地检测组织中miR-338簇的表达量，用于临床绝经后骨质疏松病的早期辅助诊断，且无创。另外，miR-338簇的抑制剂有促进成骨类的功效，可用于治疗或预防绝经后骨质疏松。

Description

与绝经后骨质疏松相关的miRNA标志物和试剂盒
相关申请的交叉引用
本申请要求2017年8月17日提交、申请号为201710708188.9的中国专利申请的优先权，其所公开的内容作为参考全文并入本申请。
技术领域
本发明涉及基因工程领域，更具体地，涉及与绝经后骨质疏松相关的miRNA标志物和试剂盒。
背景技术
microRNA(miRNAs)代表了特定的一种小分子的非编码RNA，它通过转录后调控基因的表达。单独的一个miRNA可以通过抑制一系列相关功能基因的表达，因此可以与转录因子相互作用。至今已有多个研究证明，阻断miRNA可以阻滞骨质疏松的进程。多个独立的动物模型实验也证实，有许多miRNA在骨质疏松中表达出现改变，也参与了骨质疏松的病理过程。自1993年miRNA被首次发现以后，miRNA受到越来越多科研工作者的重视。miRNA在多种疾病的发生发展过程中起到的作用也在近年来进行了广泛而深入的探索。人体内有超过2000个miRNA被报道具有组织或细胞特异性。越来越多的研究证明人的体液如尿液、汗液、唾液等中存在稳定表达的miRNA，尤其是血清或血浆中的miRNA。miRNA可以稳定，可复制，持续地存在于血清或者血浆中，从而使得miRNA作为诊断或者治疗多种疾病成为可能，包括骨质疏松。通过分析外周循环血中miRNA的表达情况诊断骨质疏松是无创，稳定，实时的。
通过生物信息学的技术和实验手段可以寻找任意一个miRNA的多个与疾病相关的靶点基因，而寡聚核苷酸化学技术的合成的发展促使我们可以直接的抑制某一特定miRNAs的表达，自由的穿梭于细胞之 间，通过碱基互补配对的方式特异地结合于特定的miRNAs,从而阻断该miRNAs的功能。因此与转录因子不同的是，在人体和动物内，特定的miRNAs更易于通过寡聚核苷酸的方式被特异性的靶定。
骨质疏松是一种以骨量减少，骨组织微结构发生破坏，骨脆性增加，骨折发生发生率增加的代谢性骨病变。绝经后骨质疏松症是绝经后妇女的高发病症，为多因素性疾病：雌激素的缺乏、遗传因素、营养状况、生活习惯、体格锻炼、月经周期紊乱、绝经早于40岁等均与发病有关，但绝经后雌激素缺乏则是绝经后骨质疏松症发生的重要原因。在绝经后的妇女，第一个5～7年中骨的丢失以每年1％～5％的速度递增。
目前，通过双能X线吸收技术对骨密度进行测定是骨质疏松早期发现和诊断的黄金标准，此外还可通过对骨代谢生化指标，如BAP，OC，Tracp5b，PINP，NTX，CTX等进行测定来反映骨代谢情况。骨密度检测可以在正常、较低以及骨质疏松情况下推测骨密度，并预测某些骨折发生的风险。然而骨质疏松发生过程中是非常缓慢的，短时间内变化不明显，需要长时间的观察和连续的检测，并且一旦发现骨质流失，骨密度降低则难以逆转，给临床病人的早期诊断带来极大的困难。
发明内容
本发明的目的在于提供miR-338簇在作为绝经后骨质疏松标志物中的应用。
其中，miRNA簇是指在基因组上距离相近(通常为小于2kb)的两个或多个成簇存在的miRNA。
本发明中miR-338簇为在基因组上与编码miR-338的基因组上距离相近(小于2kb)的miRNA，miR-338-3p和miR-3065-5p均属于miR-338簇，编码两者的基因的位置关系如图1所示。
其中，miR-338簇优选为miR-338-3p和/或miR-3065-5p。
绝经后骨质疏松为早期绝经后骨质疏松，即可以检测当T值在-2.5和-1之间时的骨质疏松。
例如当小鼠的骨质疏松发生12周后，现有的骨质疏松影像检测才能检测出来，而使用本发明的miR-338簇可以检测小鼠早期绝后骨质疏松，例如当骨质疏松发生8周以后，优选当骨质疏松发生1周以后，即使用本发明的miRNA簇可以在8周内可明显检测出来，优选1周内即可明显检测出来。
本发明检测了早期绝经后骨质疏松病人血清中miR-338簇如miR-338-3p和miR-3065-5p的表达含量，结果显示miR-338簇的表达水平显著高于对照健康组样本。同时ROC分析的结果显示miR-338簇有较高的诊断价值。
当miR-338簇为miR-338-3p和miR-3065-5p时，在作为绝经后骨质疏松标志物中效果更好。
本发明还提供了一种含有miR-338簇的生物材料在作为绝经后骨质疏松标志物中的应用。
其中，miR-338簇优选为miR-338-3p和/或miR-3065-5p。
优选地，生物材料可以为载体、宿主细胞、转基因细胞系、工程菌中的一种或多种。
本发明还提供了miR-338簇在制备绝经后骨质疏松辅助诊断试剂盒中的应用。
其中，所述miR-338簇在绝经后骨质疏松患者的血清中高表达。本发明中所指的“高表达”以及“显著”均是指从统计学意义上讲，p值通常小于0.05。
其中，miR-338簇优选为miR-338-3p和/或miR-3065-5p。
使用本发明的诊断试剂盒，只需要测定患者的血清中miR-338族的含量，与以往的诊断方法好处在于，无创且灵敏，若患者在早期患有绝经后骨质疏松，即可被诊断出，优选地是，若患者患有早期绝经后骨质疏松，即当T值在-2.5和-1之间时的骨质疏松，即可被诊断出。
本发明还提供了一种miR-338簇如miR-338-3p和/或miR-3065-5p的抑制剂在制备治疗或预防绝经后骨质疏松药物中的应用。
其中，治疗或预防绝经后骨质疏松的药物为促进成骨分化类药物。
本发明还提供了一种用于治疗或预防绝经后骨质疏松药物，该药物中含有miR-338簇的抑制剂，优选为miR-338-3p和/或miR-3065-5p的抑制剂。
本发明首次意外发现miR-338簇优选miR-338-3p和/或miR-3065-5p在绝经后骨质疏松病人中的高表达，可作为绝经后骨质疏松病的标志物，用于制备针对绝经后骨质疏松病辅助诊断试剂盒，可以特异地检测组织的血清中miR-338簇优选miR-338-3p和/或miR-3065-5p的表达量，及早且有效地用于临床绝经后骨质疏松病的早期辅助诊断，且在血清中即可实现上述诊断，不需要额外的创伤口。本发明还发现miR-338簇的抑制剂可以有效地阻滞骨质疏松的进程，促进成骨，从而有效地治疗或预防绝经后骨质疏松，可以将其作为治疗或预防绝经后骨质疏松药物，具有较好的临床应用价值和广阔的应用前景。
附图说明
图1为根据本发明一个优选实施方式中编码miR-338-3p和miR-3065-5p的基因的位置关系图；
图2A为根据本发明实施例1中绝经后骨质疏松病人组和健康组中miR-338-3p的含量曲线图；
图2B为根据本发明实施例1中绝经后骨质疏松病人组和健康组中miR-3065-5p的含量曲线图；
图2C为根据本发明实施例1中miR-338-3p和miR-3065-5p的ROC曲线图；
图3A为根据本发明实施例1中双侧卵巢摘除术后0周，1周，4周，8周，12周，16周和18周的小鼠及其对照(Sham组：未做处理 组)股骨的micro-CT图；
图3B为根据本发明实施例1中双侧卵巢摘除术后0周，1周，4周，8周，12周，16周和18周的小鼠及其对照(Sham组：未做处理组)股骨切片并进行HE染色的染色图；
图3C为根据本发明实施例1中双侧卵巢摘除术后0周，1周，4周，8周，12周，16周和18周的小鼠及其对照组的股骨骨髓间充质干细胞(BMSCs)，矿化诱导14天后进行茜素红染色的染色图；
图4A为根据本发明实施例1中miR-338-3p的表达含量与去势时间的曲线图；
图4B为根据本发明实施例1中miR-3065-5p的表达含量与去势时间的曲线图；
图5A为根据本发明实施例1中注射抵制剂的实验方案流程图；
图5B为根据本发明实施例1中注射miR-338-3p或miR-3065-5p抑制剂后小鼠血清中miR-338-3p或miR-3065-5p的表达含量曲线图；黑色柱子为未做处理的con组，白色柱子为卵巢摘除术后3个月的去势组，灰色柱子为去势后打抑制剂组；
图5C为根据本发明实施例1中对注射miR-338-3p抑制剂后小鼠股骨micro-CT分析图、骨小梁的三围重建图和HE染色图；
图5D为图5C中骨小梁重建的ovx组、con组、ovx+anti-miR-338-3p组中骨小梁体积比、骨小梁厚度、单位体积骨小梁数目以及骨小梁间距的曲线图；
图5E为根据本发明实施例1中对注射miR-3065-5p抑制剂后小鼠股骨进行micro-CT分析、骨小梁的三围重建和HE染色图；
图5F为图5E中骨小梁重建的ovx组、con组、ovx+anti-miR-338-3p组中骨小梁体积比、骨小梁厚度、单位体积骨小梁数目以及骨小梁间距的曲线图；
图6A为根据本发明实施例1中采用crispr-cas9的基因编辑手段建立健康细胞中的miR-338-3p和miR-3065-5p来自于不健康细胞中高表 达的miR-338-3p和miR-3065-5p实验假设模式图，其中，星行细胞代表健康的成骨细胞，方形细胞代表不健康的成骨细胞；
图6B为根据本发明实施例1中培养诱导miR-338-3p/miR-3065-5p敲除的细胞系和野生型成骨细胞系的流程图；
图6C为根据本发明实施例1中WT组(野生型成骨细胞系组)和3065ko组(miR-338-3p/miR-3065-5p敲除的细胞系)上清液中miR-338-3p和miR-3065的表达含量对比图；其中，黑色柱子表示WT组，白色柱子表示3065ko组；
图6D为根据本发明实施例1中WT组(野生型成骨细胞系组)和3065ko组(miR-338-3p/miR-3065-5p敲除的细胞系)中Alp、Ocn、Opn、Alp以及Runx2表达含量对比图；其中，黑色柱子表示WT组，白色柱子表示3065ko组；
图7为根据本发明实施例1中在mc3T3中分别过表达miR-338-3p，miR-3065-5p，以及同时过表达miR-338和miR-3065，下游基因Col1a、Wnt5a、usp20、Igf1以及Msx1的表达含量对比图。
具体实施方式
下面结合具体实施例，对本发明作进一步的详细说明。以下实施例用于说明本发明，但不用来限制本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规技术手段。若未特别指明，实施例中所用的试剂为市售。
实施例1
实验过程：收集15例绝经后骨质疏松病人和15例健康样本的血样，离心后收集血清，并使用试剂盒提取血清中的总RNA(包括miRNA)，逆转后进行RT-PCR，根据CT值进行统计学分析。
检测了绝经后骨质疏松病人血中miR-338-3p和miR-3065-5p的表达含量，如图2A所示和2B所示，结果显示miR-338-3p和miR-3065-5p的表达水平显著高于对照健康组样本，其中，每一个黑点表示每一个 样本，点的位置越高表示含量越多。同时如图2C所示，ROC分析的结果显示miR-338-3p和miR-3065-5p有较高的诊断价值。
建立双侧卵巢摘除术的小鼠骨质疏松模型：
小鼠取俯卧位固定，在脊柱两侧背部用眼科剪剪一向外倾斜的长约1cm切口，剥开皮肤和肌层，找到卵巢组织，用4号丝线结扎卵巢系膜血管后摘除卵巢。缝合皮肤。同法摘除另侧卵巢；将子宫复位后缝合。
小鼠股骨的micro-CT和HE染色结果显示，如图3A和3B所示，去势后3个月呈现明显的骨量减少，同时OVX组骨量低于CON组。如图3A所示，0周到8周骨小梁密度较高，且Sham组和ovx组没有差异。至术后12周，ovx组骨小梁数量显著低于sham组，之后随着术后时间的增加，骨小梁密度逐渐降低，且ovx组低于sham组。图3C表明去势后三个月起小鼠股骨的BMSCs(骨髓间充质干细胞)矿化能力呈现显著降低，且OVX矿化能力低于CON组。同时，发现随着去势时间的延长，小鼠血清中miR-338-3p和miR-3065-5p的表达水平升高，值得注意的是，去势后一周，miR-338-3p的表达含量显著高于对照组，去势后八周，miR-3065-5p的表达含量显著高于对照组，明显早于影像学检测(micro-CT)，如图4A和4B所示。这表示miR-338-3p和miR-3065-5p可以作为绝经后骨质疏松的早期诊断标记物。
通过尾静脉注射的方式，给去势后三个月的小鼠分别注射了miR-338-3p和miR-3065-5p的抑制剂，每周注射一次，共注射4次，其流程图如图5A所示。具体为：对8周大的Balb/c小鼠进行双侧卵巢摘除术，术后三个月通过尾静脉注射miR-338-3p或miR-3065-5p的抑制剂，每周注射一次，共注射四次，打药共1个月，然后收集标本。检测小鼠血中和股骨骨髓间充质干细胞中的miR-338-3p和miR-3065-5p的含量，结果显示去势组中miR-338-3p和miR-3065-5p的含量显著高于对照组，注射抑制剂后，miRNA的含量明显下降，如图5B所示。如图5C和5E所示，microCT和染色结果显示，抑制剂组 骨小梁和骨密度显著高于去势组，这表示抑制miR-338-3p和miR-3065-5p可以阻滞骨质疏松的进程，对绝经后骨质疏松有明显的治疗效果。如图5D和5F所示，BV/TV(％)，Tb.Th(mm)，Tb.N(1/mm)均是ovx组低于con组，ovx+anti-miR-338-3p组和ovx+anti-miR-3065-3p组均高于ovx组。Tb.Sp(mm)则为ovx组高于con组，ovx+anti-miR-338-3p组和ovx+anti-miR-3065-3p组均低于ovx组。这些结果进一步支持了micro-CT和HE染色的结果。
如图6A所示，采用crispr-cas9的基因编辑手段建立健康细胞中的miR-338-3p和miR-3065-5p来自于不健康细胞中高表达的miR-338-3p和miR-3065-5p实验假设模式图：健康细胞中的miR-338-3p和miR-3065-5p来自于不健康细胞中高表达的miR-338-3p和miR-3065-5p。采用crispr-cas9的基因编辑手段建立了miR-338-3p和miR-3065-5p敲除的成骨细胞系，将其用来代表健康的成骨细胞，把野生型的mc3T3-E1代表不健康的成骨细胞。
提取野生型成骨细胞系分化诱导后0天，3天和7天的细胞上清液，上清液中miR-338-3p和miR-3065-5p表达呈下降趋势。与其在成骨细胞分化过程中的表达趋势一致。随后我们分别诱导了miR-338-3p/miR-3065-5p敲除的细胞系和野生型成骨细胞系，并每两天交换一次培养基上清液，7天后，收集了两种细胞系的上清液并检测了其中miRNA的含量，如图6C和6D所示，其中，图6C中，D0表示矿化0天，d7表示用矿化诱导液培养7天，d7co表示照图6B中的培养方式培养7天。结果显示矿化D0天，WT组中miR-338-3p的含量显著高于3065ko组，矿化7天则这种差异增加。而交换培养基培养7天后，这种差异则降低。
图6D中检测WT组(野生型成骨细胞系组)和3065ko组(miR-338-3p/miR-3065-5p敲除的细胞系)成骨能力。通过检测不同组Alp,Ocn,Opn,Alp,Runx2表达含量来检测miR-338-3p/miR-3065-5p含量的改变对成骨能力的影响。与6C结果一致的是，矿化d0天，WT组 中由于高表达miR-338-3p/miR-3065-5p，成骨基因表达含量低于3065ko组，d7组中，这种差异增加，而交换培养基矿化诱导7天后，3065ko组中由于受到3wt组中培养基高表达的miR-338-3p/miR-3065-5p影响，成骨基因表达水平下调，与wt组的差异变小。上述结果显示，野生型中高表达的miR-338-3p和miR-3065-5p在用miR-338-3p/miR-3065-5p敲除的细胞系上清液共培养之后，表达水平下降，而miR-338-3p/miR-3065-5p敲除的细胞系中低表达的miR-338-3p和miR-3065-5p在与野生型成骨细胞系上清液共培养之后，表达水平上升。该结论也验证了图6B的实验假设。
同时，在mc3T3中分别过表达miR-338-3p，miR-3065-5p，以及同时过表达miR-338和miR-3065,检测下游基因Col1a,Wnt5a,usp20,Igf1,Msx1(均为与成骨分化正向相关的基因)的表达含量，结果显示，如图7所示：过表达miR-338-3p，miR-3065-5p，以及同时过表达miR-338和miR-3065均可显著下调下游基因的表达。同时，过表达miR-338-3p和miR-3065-5p对下游基因的下调幅度显著大于只过表达miR-338-3p或miR-3065-5p的下调幅度，即说明同时使用miR-338-3p和miR-3065-5p的抑制剂，两者协调作用，促进成骨分化，从而更好地治疗或是预防绝经后骨质疏松。
在正常妇女的体内，雌激素水平处于比较高的正常水平，使得Runx2水平上升，Runx2作为miR-338-3p和miR-3065-5p基因的上游基因，使得miR-338-3p和miR-3065-5p的表达水平下降，同时Runx2又作为miR-338-3p和miR-3065-5p的下游靶点基因，有较少的miR-338-3p和miR-3065-5p结合在它们的下游Runx2的3’UTR区，使得Runx2有更高水平的表达，那么就有更多的Runx2结合在miR-338-3p和miR-3065-5p的启动子区域，去抑制miR-338-3p和miR-3065-5p的表达，使得miR-338-3p和miR-3065-5p在外周循环血中保持较低水平的表达。而在绝经后骨质疏松病人体内，雌激素水平急剧下降，使得Runx2水平降低，较少的Runx2结合在miR-338-3p和miR-3065-5p的 启动子区域，促进miR-338-3p和miR-3065-5p的表达，较多的miR-338-3p和miR-3065-5p会结合在它们的下游基因Runx2上，使得Runx2水平进一步下降，导致更少的Runx2结合在miR-338-3p和miR-3065-5p的启动子区域，使得miR-338-3p和miR-3065-5p在外周循环血中维持一个较高水平的表达。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
工业实用性
本发明提供了一种与绝经后骨质疏松相关的miRNA标志物和试剂盒，具体涉及miR-338簇在作为绝经后骨质疏松标志物中的应用。本发明发现miR-338簇如miR-338-3p和/或miR-3065-5p在绝经后骨质疏松病人中的高表达，可作为绝经后骨质疏松病的标志物，用于制备针对绝经后骨质疏松病辅助诊断试剂盒，可以特异地检测组织中miR-338簇的表达量，及早且有效地用于临床绝经后骨质疏松病的早期辅助诊断，且无创。另外，miR-338簇的抑制剂有促进成骨类的功效，有效地治疗或预防绝经后骨质疏松。本发明提供的标志物能被大规模地应用于绝经后骨质疏松检测领域，适用于工业化应用，具有广阔的市场前景。

Claims (10)

  1. miR-338簇在作为绝经后骨质疏松标志物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述绝经后骨质疏松为早期绝经后骨质疏松。
  3. 含有miR-338簇的生物材料在作为绝经后骨质疏松标志物中的应用。
  4. 根据权利要求3所述的应用，所述生物材料为载体、宿主细胞、转基因细胞系、工程菌中的一种或多种。
  5. miR-338簇在制备绝经后骨质疏松辅助诊断试剂盒中的应用。
  6. 根据权利要求5所述的应用，其特征在于，所述miR-338簇在血清中高表达。
  7. miR-338簇的抑制剂在制备治疗或预防绝经后骨质疏松药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述治疗或预防绝经后骨质疏松的药物为促进成骨分化类药物。
  9. 一种用于治疗或预防绝经后骨质疏松药物，其特征在于，所述药物中含有miR-338簇的抑制剂。
  10. 根据权利要求1-8中任一项所述的应用或权利要求9中所述的药物，所述miR-338簇为miR-338-3p和/或miR-3065-5p。
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