CN107028971A - Applications of the miR 141 in preparing anti-osteoporosis, suppressing bone information preparation - Google Patents

Abstract

The invention belongs to the field of biomedical materials, and discloses the application of miR-141 in preparing anti-osteoporosis and inhibiting bone resorption preparations. Experiments have confirmed that miR-141 inhibits the differentiation process of osteoclasts by regulating the expression of genes related to osteoclast differentiation. miR-141 can inhibit the loss of bone mass and the occurrence of osteoporosis, so miR-141 can be used to prepare various biomedical preparations for anti-osteoporosis and inhibition of bone loss.

Description

Application of miR-141 in the preparation of anti-osteoporosis and anti-bone resorption preparations

technical field

The invention belongs to the technical field of biomedical materials, and more specifically relates to an application of miR-141 in preparing anti-osteoporosis and inhibiting bone resorption preparations.

Background technique

Osteoporosis is a systemic bone disease characterized by decreased bone mass, destruction of bone tissue microstructure, increased bone fragility, and susceptibility to fracture. With the increasing aging trend of the world population, the incidence of osteoporosis is increasing. According to the sixth census, the elderly over 65 years old account for 7.5% of the total population in China. It is estimated that by 2020, there will be more than 250 million osteoporosis patients in my country, accounting for more than half of the osteoporosis patients in the world. The World Health Organization has listed osteoporosis, diabetes, and cardiovascular disease as the three major killers that endanger the health of the elderly.

Osteoclasts are a myeloid-derived cell type that play an important role in bone development and bone resorption. A deep understanding of the regulatory mechanisms of osteoclast function is a prerequisite for combating osteoporosis. Osteoclasts are important targets in the prevention and treatment of osteoporosis and fractures, and bisphosphonates are the most widely used drugs in this regard. However, studies have shown that long-term use of the drug is closely related to atypical femur fractures, and patients who receive clinical intravenous bisphosphonates are prone to osteonecrosis of the jaw. Therefore, the development of anti-resorptive drugs with new targets has urgent practical significance.

microRNA (abbreviated as miRNA) is a type of endogenous non-coding small RNA with a length of 18 to 22 nucleotides. miRNA negatively regulates gene expression at the post-transcriptional level through complementary pairing with target mRNA, resulting in mRNA degradation or translation inhibition. So far, thousands of miRNAs have been reported to exist in various organisms such as animals, plants, and fungi. Each miRNA can have multiple target genes, and several miRNAs can also regulate the same gene. This complex regulatory network can not only regulate the expression of multiple genes through one miRNA, but also finely regulate the expression of a gene through the combination of several miRNAs. With the gradual deepening of research on miRNA regulation of gene expression, it will help us understand the complexity of the genome of higher eukaryotes and the complex gene expression regulatory network.

As a member of the miR-200 family, miR-141 has few reports on its function, and only abnormal expression levels in some tumors have been found. For example, abnormally expressed miR-141 is involved in the proliferation of cholangiocarcinoma cells and renal cell carcinoma cells. invasion activities. miR-141 can combine with the 3'UTR of ZEB2 to reduce the level of ZEB2 in cells, thereby inhibiting the development of liver cancer. However, there is no report about miR-141 in the process of anti-osteoporosis and bone resorption.

Contents of the invention

The purpose of the present invention is to provide a new application of miR-141, that is, the application of miR-141 in preparing anti-osteoporosis and inhibiting bone resorption preparations.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

Application of miR-141 in the preparation of anti-osteoporosis and anti-bone resorption preparations

In order to better understand the essence of the present invention, its new use in the field of pharmacy will be described below in conjunction with pharmacological experiments and results.

The study found that the overexpression of miR-141 (5'-aacacugucugguaaagaugg-3') (SEQ ID NO:I) in macaque cells can significantly reduce the synthesis of tartrate-resistant acid phosphatase (TRAP) in bone marrow mononuclear cells (BMM), and at the same time The results of real-time PCR showed that the level of TRAPmRNA in BMM cells was significantly reduced. The level of miR-141 was significantly decreased during the osteoclast differentiation of RANKL-induced BMM cells. After BMM cells were transfected with miR-141, the ability of RANKL-induced BMM cells to differentiate into osteoclasts was significantly reduced, and compared with the control group, the ability of cells to express TRAP was significantly reduced. It shows that miR-141 plays an important regulatory role in the process of osteoclast induction and differentiation.

The expression level of miR-141 was decreased in the bone tissue of female aged macaques and osteoporotic patients. After miR-141 was re-delivered into aged female Yokohama monkeys using a nucleic acid delivery system, miR-141 was able to inhibit osteoclast differentiation by detecting osteoclast differentiation indicators. And through Micro CT detection, it was found that the bone mineral density, the number and thickness of bone trabeculae in the experimental group of rhesus monkeys delivered with miR-141 were higher than those in the control group of rhesus monkeys. It shows that miR-141 has a certain regulatory effect on the osteoporosis model of female aged rhesus monkeys.

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention discovers a new medical application for the known miR-141 and opens up a new application field.

2. The miR-141 inhibitor of the present invention can down-regulate the expression abundance of miR-141, promote the differentiation of osteoclasts, enhance bone resorption, and thus cause osteoporosis. miR-141 can inhibit osteoclast differentiation and further inhibit bone resorption, and miR-141 can be used to prepare various pharmaceutical preparations for inhibiting bone loss and anti-osteoporosis.

Description of drawings

Figure 1 shows the expression levels of marker genes in the process of macaque osteoclast differentiation detected by real-time fluorescent quantitative PCR.

Figure 2 shows the expression abundance of miR-141 detected by real-time fluorescent quantitative PCR during the differentiation of macaque osteoclasts.

Figure 3 shows the indicators related to the inhibition of osteoclast differentiation by transient transfection of miR-141.

Figure 4 is a graph of TRAP staining results of miR-141 inhibiting osteoclast differentiation.

Fig. 5 is a CT scan of the rhesus monkey femur in the control group and the experimental group.

Figure 6 shows the body weight changes of macaques in the control group and the experimental group.

Fig. 7 is the histological section of the main organs of the control group and the experimental group.

Fig. 8 shows the changes of serum physiological and biochemical indexes of rhesus monkeys in the control group and the experimental group.

detailed description

The content of the present invention will be further described below in conjunction with specific examples, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

miR-141 was purchased from Shanghai Gemma Pharmaceutical Technology Co., Ltd.

In the examples, healthy aged female rhesus monkeys were used, which were purchased from Nanning Wincon Thera Cells Biotechnologies Co Ltd (Wincon). The macaques participating in the experiment were between 19 and 23 years old, and their body weight was between 5.8 and 6.5 kg. Each experimental monkey was reared in a single cage, fed with feed twice a day, supplemented with an appropriate amount of fruit, and drank water freely. The ambient temperature of the feeding room was controlled at 24-30°C, and the light was 12 hours, and the animals were divided automatically throughout the day. The use and care of experimental animals were applied to and approved by the Wincon Animal Welfare Committee in advance. Before the experiment, they were anesthetized for bone density testing (lumbar spine and hip bone), and finally 3 mL of venous blood was drawn. And the serum NTx, CTx and TRAP were detected.

For macaques participating in ovariectomy, anesthesia was first performed, 3 mL of blood was drawn, and serum was obtained by centrifugation. A bone sample is then collected and CT is used to measure bone density. Serum, bone samples, and CT were collected every one and a half months after ovariectomy.

Example 1 Induction of macaque BMM cells

The bone marrow of 2-month-old rhesus monkeys was flushed out, and cultured in α-MEM medium containing 10% FBS in a constant temperature incubator at 37°C with 5% CO ₂ for 24 hours. During induction, cells were inoculated into 24-well plates at 1×10 ⁴ /well, and 50ng/ml M-CSF was added for induction for 3 days. After 3 days, 50ng/ml M-CSF and 100ng/ml RANKL were added to induce cells, and the cells were replaced every day. After 5 days of cell culture, subsequent molecular detection and TRAP staining were performed. Osteoclast differentiation-specific gene primer sequences are shown in Table 1: (F, upstream primer; R, downstream primer)

Table 1 Osteoclast differentiation-specific gene primer sequences

Example 2 Extraction of Total Cell RNA

After the cells cover 80%-90% of each well of the 24-well cell culture plate, add 1ml Trizol to lyse the cells and repeatedly pipette with a pipette tip for 5 minutes at room temperature. Transfer the lysate into a 1.5ml centrifuge tube, add 200ul chloroform, close the cap tightly, shake the centrifuge tube vigorously up and down, let it stand at room temperature for 5 minutes, and centrifuge at 12000g/min for 15 minutes at 4°C. Transfer the colorless upper aqueous phase liquid to a new 1.5ml centrifuge tube, add 500 μL of isopropanol, mix up and down, let stand at room temperature for 10 minutes, and centrifuge at 12,000 g/min for 10 minutes at 4°C. Aspirate the supernatant with a pipette and discard, keeping the white precipitate at the bottom. Add 1ml of 75% ethanol (prepared with DEPC water), gently pop the white precipitate by hand, and then centrifuge at 7500g/min for 5 minutes at 4°C. Discard the supernatant, dry at room temperature for 10-15 minutes, add 30-40 μl LDEPC water to redissolve, store in -80°C refrigerator or use directly.

Example 3 Detection of expression levels of marker genes for osteoclast differentiation in rhesus monkey BMM cells

The extracted total RNA was reverse-transcribed into cDNA using the reverse transcription kit (PrimeScript ^TM RT reagent Kit with gDNAEraser, Code No.: RR047A) of TaKaRa Company according to the operation procedure of the kit. The expression levels of marker genes in the process of macaque BMM differentiation were detected by real-time fluorescent quantitative PCR technology. Figure 1 shows the expression levels of marker genes in the process of macaque osteoclast differentiation detected by real-time fluorescent quantitative PCR. It can be seen from Figure 1 that the relative expression levels of TRAP and CTSK both increased with the increase of differentiation days. The molecular indicators of osteoclast differentiation increased with the differentiation content of cells, and the results indicated that osteoclast differentiation was successful.

Example 4 Detection of miR-141 expression abundance during the differentiation of macaque bone marrow mononuclear cells into osteoclasts

Total RNA was extracted from rhesus monkey bone marrow mononuclear cells at different differentiation stages, cDNA was synthesized using TaKaRa's Mir-X ^TM miRNAFirst-Strand Synthesis Kit, using PCR primers for miR-141 and TaKaRa's Mir-X ^TM miRNAqRT-PCR Kit for fluorescent quantitative PCR, as shown in Table 2. The U6 gene was used as an internal reference to standardize the miR-141 detection results. Figure 2 shows the expression abundance of miR-141 detected by real-time fluorescent quantitative PCR during the differentiation of macaque BMM cells. The results showed that with the differentiation of osteoclasts, the content of miR-141 gradually decreased.

Table 2 Primer sequences of miR-141 used for real-time fluorescent quantitative PCR analysis

gene name Primer sequence Mml-miR-141 5'-AACACTGTCTGGTAAAGATGG-3' U6 5'-CGACTGCATAATTTGTGGTAGTGG-3

Example 5 Cell Transfection Experiment

Cells were seeded in 24-well plates at 1×10 ⁴ cells/well, transfected once after 2 days of cell induction, and retransfected once after 4 days of cell induction. During transfection, an appropriate amount of miR-141 was dissolved in serum-free optimized medium, and P3000 in Lip3000 was mixed with it at the same time, and finally Lip3000 was added to the mixture and allowed to stand at room temperature for 5 minutes. Aspirate the original cell culture medium, replace with fresh culture medium, add the transfection solution into the fresh culture medium, and incubate at 37°C for 24 hours. Finally, the medium containing the transfection solution was discarded, and the complete medium was added to continue the culture. Figure 3 shows the indicators related to the inhibition of osteoclast differentiation by transient transfection of miR-141. The results showed that the further differentiation of osteoclasts was inhibited after transfection with miR-141.

Example 6 Osteoclast trap staining

After 6 days of osteoclast differentiation, discard the supernatant, wash the cells with PBS, and then add fixative. Cells were fixed for 30 seconds at room temperature and then rinsed 3 times with deionized water. Mix 0.5mL Fast Garnet GBC Base solution and 0.5mL SodiumNitrite solution, invert and mix well, and let stand at room temperature for 2 minutes. Mix the above mixed solution with 45mL deionized water preheated to 37°C, 0.5mL Naphthol AS-BI phosphate solution, 2mL Acetate solution and 1mL Tartrate solution, and bathe in a 37°C water bath. Add the above solution to the cell culture dish and incubate at 37°C for 1 hour. Figure 4 is a graph of TRAP staining results of miR-141 inhibiting osteoclast differentiation. Among them, (a) is before miR-141 transfection, (b) is after miR-141 transfection, as can be seen from Figure 4, the osteoclasts in the experimental group decreased significantly, and the results showed that after miR-141 transfection, osteoclasts The number of decreased, indicating that miR-141 has an inhibitory effect on osteoclast differentiation.

Example 7 CT detection of rhesus monkey femur

At the 12th week, the macaques in the experimental group and the control group were euthanized, and the rhesus macaques were fixed with 4% paraformaldehyde cardiac perfusion. Remove the lower limb bones, lumbar spine and hip bones of the rhesus monkey. 4% paraformaldehyde fixed for 1 week, and then washed with 10% sucrose solution. Bone samples from rhesus monkeys were scanned using micro computed tomography (Micro-CT). After the scan is complete, the metaphyseal trabecular bone is analyzed. Including trabecular volume ratio (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N). Figure 5 is the CT images of the rhesus monkey femurs in the control group and the experimental group. Among them, (a) is the control group, and (b) is the experimental group. It can be seen from FIG. 5 that the bone density of the experimental group increases significantly. The results showed that the bone mass of macaques in the experimental group was higher than that of the control group, indicating that the administration of miR-141 alleviated the bone mass loss of aged macaques.

Example 8 miR-141 safety assessment

The body weight of the macaques in the experimental group and the control group was recorded and blood was drawn every week. The macaques in the experimental group and the control group were euthanized at the 12th week, and the main organs (heart, liver, spleen, kidney) were collected for histological staining. And the physiological and biochemical indicators of its serum were detected. Figure 5 shows the body weight changes of macaques in the control group and the experimental group. The experimental results showed that there was no significant difference in body weight between the experimental group and the control group. Figure 6 is the histological sections of the main organs of the control group and the experimental group. The results showed that there was no significant difference in major organs between the experimental group and the control group. Fig. 7 shows the changes of serum physiological and biochemical indexes of rhesus monkeys in the experimental group and the control group. It can be seen from Fig. 7 that miR-141 did not cause obvious tissue damage to the heart, liver, spleen, and lung tissues. The results showed that there was no significant difference between the experimental group and the control group. Summary The administration of miR-141 to rhesus monkeys did not produce significant side effects.

Fig. 8 shows the changes of serum physiological and biochemical indexes of rhesus monkeys in the control group and the experimental group. It can be seen from Figure 8 that after administration of miR-141 in rhesus monkeys, compared with the control group, the levels of alanine aminotransferase and glucose in the body, as well as the degree of amyotrophic lateral sclerosis, did not change significantly, indicating that administration of miR-141 did not give Rhesus monkeys produced significant side effects.

The above studies show that miR-141 can inhibit osteoclast differentiation and further inhibit bone resorption, so miR-141 can be used to prepare various pharmaceutical preparations for inhibiting bone loss and anti-osteoporosis.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations and modifications made without departing from the spirit and principles of the present invention Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (1)

1. Applications of the 1.miR-141 in preparing anti-osteoporosis, suppressing bone information preparation.