Application of m6A methyltransferase METTL16 in disuse osteoporosis

Abstract

The invention discloses an application of m6A methyltransferase METTL16 in disuse osteoporosis, and the m6A methyltransferase METTL16 is used as a potential biomarker of the disuse osteoporosis, can be used as a new target spot to resist the disuse osteoporosis, and has a good transformation medical prospect.

Description

Technical Field

[0001] The present invention relates to the field of molecular biomedicine technology, in particular to m 6 Application of A methyltransferase METTL16 in the diagnosis and treatment of disuse osteoporosis. Background Art

[0002] Disuse osteoporosis is an abnormal bone metabolism disease caused by a continuous reduction or absence of mechanical load on the bones. It is commonly seen clinically in pathological conditions where the mechanical stimulation of the bones is significantly reduced, such as space flight, long-term bed rest, limb immobilization, and spinal cord injury. Its core pathological manifestations are progressive loss of bone mass, destruction of trabecular microstructure, and decline in bone biomechanical properties. Studies have found that patients can lose 1% to 2% of bone mass per month under complete immobilization, significantly increasing the risk of fractures. Clinicians face challenges such as delayed early intervention, difficulties in multi-system coordinated intervention, and the lack of an accurate efficacy evaluation system. Therefore, exploring the nature of disuse osteoporosis at the molecular level is of key significance for advancing the research of prevention and treatment technologies.

[0003] Skeleton is a homeostatic organ that achieves self-renewal through cyclical bone remodeling. This process is precisely coupled by bone resorption by osteoclasts and bone formation by osteoblasts, thereby maintaining the functional homeostasis of bone tissue. Normally, bones have a strong self-repair ability. However, with aging and changes in the pathological microenvironment, the body gradually develops an imbalance in the osteoblast-osteoclast dynamic balance. This bone metabolism disorder eventually leads to osteoporotic pathological changes. As the main functional cell for bone formation, abnormal osteoblast function is closely related to the occurrence and development of disuse bone loss.

[0004] Currently, the treatment of disuse osteoporosis primarily relies on a combination of physical interventions (whole-body vibration therapy, electrical stimulation) and medications (bisphosphonates, parathyroid hormone). However, controversy remains regarding the effectiveness of these treatments in preventing and treating bone loss, the drugs used, and their targets. There are concerns about insufficient efficiency of deep bone stimulation, drugs that only delay bone loss, and side effects such as hypercalcemia and heterotopic ossification. Therefore, it is necessary to focus on the molecular pathway regulatory networks underlying bone metabolic imbalance, systematically elucidate their pathophysiological mechanisms, and, based on this, develop new intervention strategies that adapt to changes in the mechanical environment to provide a theoretical basis for the prevention and treatment of disuse osteoporosis.

[0005] Studies have shown that a probiotic consisting of Bacillus coagulans BC99 and Pediococcus acidilactici PA53 can improve osteoporosis symptoms, with the two strains potentially interacting and synergizing in their osteoporosis-improving effects (Authorization Announcement No. CN117887644B). Furthermore, other studies have developed an exercise device suitable for osteoporosis patients, making exercise more convenient for them (Authorization Announcement No. CN116392773B). However, neither of these two probiotics can mechanistically improve osteoblast differentiation disorders and bone loss. Summary of the Invention

[0006] In order to overcome the above technical problems, the object of the present invention is to provide m 6 Application of A methyltransferase METTL16 in the diagnosis and treatment of disuse osteoporosis, 6 A methyltransferase METTL16, as a potential biomarker of disuse osteoporosis and a new target for combating disuse osteoporosis, has good prospects in translational medicine.

[0007] The technical solution adopted in the present invention is:

[0008] m 6 Application of A methyltransferase METTL16 in the diagnosis and treatment of disuse osteoporosis.

[0009] m 6 A methyltransferase METTL16 is a potential biomarker for disuse osteoporosis and a new target for combating disuse osteoporosis.

[0010] METTL16 can significantly promote the differentiation and mineralization ability of MC3T3-E1 cells.

[0011] Overexpression of Mettl16 can partially alleviate the weakening of osteogenic differentiation ability caused by rotational weightlessness.

[0012] Beneficial effects of the present invention:

[0013] The expression of METTL16 of the present invention is significantly reduced in both the cell unloading model (2D rotation MC3T3-E1 cells) and the animal unloading model (mouse tail suspension), suggesting that it may serve as a potential biomarker for disuse osteoporosis.

[0014] The METTL16 of the present invention can significantly promote the differentiation and mineralization ability of MC3T3-E1 cells, and overexpression of Mettl16 can partially alleviate the weakening of osteogenic differentiation ability caused by rotational weightlessness, indicating its importance to osteogenic differentiation and providing a reference for the treatment of disuse osteoporosis.

[0015] The present invention delivers pEX-Mettl16 to the bone formation area of mice through the bone-targeting material (DSS) 6-liposome, and finds that it can alleviate the new bone formation disorder, bone microstructure destruction and impaired biomechanical properties of mice caused by unloading, further establishing the diagnostic role of METTL16 in disuse osteoporosis.

[0016] The present invention conducts an in-depth and systematic study on the relationship between METTL16 and disuse osteoporosis; based on the above findings, the expression level of METTL16 can be used as a new biomarker to assist in the diagnosis of disuse osteoporosis, and can be used as a new target to combat disuse osteoporosis, with good prospects for translational medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the expression of METTL16 in osteoblasts and mouse bone tissue after unloading.

[0018] Figure 2 Schematic diagram of the function of METTL16 in promoting osteogenic differentiation and mineralization.

[0019] Figure 3 Schematic diagram of how METTL16 can promote bone formation in mice.

[0020] Figure 4 Schematic diagram of the effect of overexpression of Mettl16 on the differentiation function of unloaded MC3T3-E1 cells.

[0021] Figure 5 Schematic diagram of how bone-targeted Mettl16 overexpression plasmid can promote bone formation in HLU mice. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] (I) Application of METTL16 in the diagnosis of disuse osteoporosis.

[0024] The present invention simulated weightlessness treatment of mouse preosteoblasts MC3T3-E1 cells using a 2D rotator, and established a hind limb tail-hanging weightlessness bone loss model using adult male C57BL / 6J mice. The mRNA and protein of MC3T3-E1 cells and mouse hind tibia were detected by qRT-PCR and western blotting. It was found that compared with the control group, Mettl16 mRNA and protein were significantly downregulated in the recombinant cells and the hind tibia bone tissue of tail-hanging mice (P<0.001) ( Figure 1 AD). In addition, immunohistochemical staining revealed that the number of METTL16-positive cells in the hind limb bone tissue of tail-suspended mice was significantly reduced ( Figure 1 E) The above results suggest that the expression of METTL16 in osteoblasts and mouse bone tissue was significantly reduced after unloading treatment.

[0025] Figure 1 Schematic diagram of the expression of METTL16 in osteoblasts and mouse bone tissue after unloading.

[0026] (2) Effect of METTL16 on osteogenic differentiation and mineralization function.

[0027] To investigate the regulatory effect of METTL16 on osteoblasts, MC3T3-E1 cells were transfected with si-Mettl16 or pEX-Mettl16. The osteoblast differentiation ability was assessed by detecting osteogenic differentiation-related indicators such as RUNX2, OSX, and OCN, as well as ALP activity and staining; the osteoblast mineralization ability was assessed by Alizarin red staining. qRT-PCR results showed that the mRNA expression levels of Runx2, Osx, Ocn, and Alp were decreased after knockdown of Mettl16 compared with the negative control group; western blotting results showed a similar trend, with the protein levels of Runx2, OSX, and OCN in the si-Mettl16 group significantly decreased compared with the si-NC group (P<0.05, P<0.01, or P<0.001) ( Figure 2 A, B). In addition, ALP staining and ALP activity of osteoblasts were weakened after knockdown of Mettl16 (P<0.01 or P<0.001) ( Figure 2 C, D). Alizarin red staining was used to detect the mineralization ability of osteoblasts. It was found that the mineralized nodules decreased after knocking down Mettl16, indicating that si-Mettl16 can inhibit osteoblast mineralization. In contrast, the differentiation and mineralization ability of MC3T3-E1 cells were significantly improved after overexpression of Mettl16 ( Figure 2E). These results suggest that METTL16 can promote osteoblast differentiation and mineralization. The siRNA-Mettl16-1 sequence is: sense: 5'-GGAAGGCAUAGUGGUUGUATT-3'; antisense: 5'-UACAACCACUAUGCCUUCCTT-3'. The siRNA-Mettl16-2 sequence is: sense: 5'-CCACCUAGUUCUGUAAACATT-3'; antisense: 5'-UG UUUACAGAACUAGGUGGTT'. Figure 2 METTL16 can promote osteogenic differentiation and mineralization function.

[0028] (III) Effect of METTL16 on bone formation in mice.

[0029] Mettl16 conditional knockout mice were constructed using the Cre-Loxp system. To study the effect of Mettl16 knockout on bone formation in mice, mice were intraperitoneally injected with tamoxifen for 7 consecutive days at 8 weeks of age to induce Mettl16 knockout. Then, empty plasmids or pEX-Mettl16 were delivered to Mettl16 cells using the (DSS)6-liposome system. f / f The osteogenic area of Osx-CreERT2 mice was harvested 30 days later and bone-related indicators were detected. Micro-CT was used to observe the microstructure and bone mass of the mice, the calcein double-labeling test was used to evaluate the new bone formation, and the three-point bending test was used to evaluate the biomechanical properties of the bones. The results showed that Mettl16-specific deletion inhibited bone formation in mice. f / f Osx-CreERT2 mice are more sensitive to Mettl16 f / f The bone mass and trabecular microstructure of mice were damaged, the new bone formation ability was weakened, and the bone biomechanical properties were reduced. After the Mettl16 overexpression plasmid was delivered to the bone formation area of mice, Mettl16 f / f The bone loss in Osx-CreERT2 mice was significantly alleviated (P<0.05, P<0.01 or P<0.001) ( Figure 3 ). Figure 3 METTL16 can promote bone formation in mice.

[0030] (IV) Effect of overexpression of Mettl16 on the differentiation function of unloaded MC3T3-E1 cells.

[0031] MC3T3-E1 cells were transfected with an empty vector or a Mettl16 overexpression plasmid, and then subjected to 2D rotation. RNA and protein were extracted for detection after 48 hours of simulated weightlessness. The results showed that compared with the CON group, the mRNA and protein expressions of osteogenic differentiation indicators Runx2, Osx, and Ocn in MC3T3-E1 cells were reduced after simulated weightlessness, and the mRNA and activity of Alp were also significantly reduced (P<0.01 or P<0.001) ( Figure 4 ); overexpression of Mettl16 could partially alleviate the weakening of osteogenic differentiation ability caused by unloading (P<0.05, P<0.01 or P<0.001) ( Figure 4 ). Figure 4 Overexpression of Mettl16 can alleviate the unloading-induced osteogenic differentiation disorder of MC3T3-E1 cells.

[0032] (V) Effect of bone-targeted Mettl16 overexpression plasmid on bone formation in HLU mice.

[0033] The experiment was divided into four groups and the Mettl16 overexpression plasmid was delivered to the bone formation area of HLU mice: CON group, HLU group, HLU+(DSS)6-liposome group and HLU+(DSS)6-liposome-Mettl16 group. After 21 days of tail suspension, the hind limb bones of each group of mice were taken for examination. Micro-CT was used to examine the microstructure and bone mass of the mouse femur; the calcein double-labeling experiment was used to examine the growth rate of cortical bone; and the three-point bending test was used to examine the biomechanical properties of the femur. Micro-CT results showed that bone-targeted supplementation of Mettl16 can alleviate the bone loss and bone microstructure damage caused by HLU ( Figure 5 A, B); Calcein double labeling experiments revealed that supplementation with Mettl16 could alleviate the inhibition of new bone formation induced by tail suspension ( Figure 5 C). In addition, a three-point bending test revealed that bone-targeted supplementation of Mettl16 could improve the bone biomechanical properties of HLU mice ( Figure 5 D, E). The above results indicate that METTL16 can promote osteogenesis under unloading conditions. Figure 5 Bone-targeted Mettl16 overexpression plasmid can promote bone formation in HLU mice.

[0034] In the application of the drugs in (2), (3), (4) and (5) above, the drugs include one or more pharmaceutically acceptable carriers, and the carriers include diluents, binders, adsorption carriers, fillers and disintegrants; the drugs also include additives, and the additives include stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents and surfactants.

[0035] This invention discovered for the first time that m6 The changes of A methyltransferase METTL16 in disuse osteoporosis were investigated, and its effects on osteoblast differentiation, mineralization function and bone formation were explored. 6 A methyltransferase METTL16 is a potential biomarker for disuse osteoporosis and can be used as a new target to combat disuse osteoporosis, with good prospects in translational medicine.

[0036] The present invention METTL16 is independent of the METTL3 / 14 complex 6 A methyltransferase, whose methyltransferase domain contains the Rossmann-like coil of class I methyltransferases and uses S-adenosylmethionine as a methyl donor. METTL16 can bind to and methylate MAT2A mRNA, U6 snRNA, MALAT1 lncRNA, and multiple other RNAs. Unlike METTL3 / 14, METTL16 can play both methyltransferase activity-dependent and -independent roles in gene regulation. In the nucleus, METTL16 plays a methyltransferase activity-dependent role, catalyzing the mRNA of downstream target genes. 6 A modification, which is bound by reader proteins and read to regulate downstream pathways; in the cytoplasm, METTL16 recruits eIF3a / b and rRNAs in a methyltransferase activity-independent manner to promote the formation of the 43S transcription preinitiation complex and the 80S translation initiation complex, thereby promoting the translation of RNA in the cytoplasm.

[0037] The present invention discovered that m 6 A methyltransferase METTL16 can promote osteoblast differentiation and mineralization and bone formation in mice, and can effectively alleviate the weakening of osteoblast differentiation ability and bone loss in tail-suspended mice caused by simulated weightlessness under unloading conditions. It has potential clinical application value in the diagnosis and treatment of disuse osteoporosis.

Claims

1. m 6 The application of A methyltransferase METTL16 in the diagnosis and treatment of disuse osteoporosis is characterized in that m 6 Application of A methyltransferase METTL16 in the diagnosis and treatment of disuse osteoporosis.

2. The m according to claim 1 6 The application of A methyltransferase METTL16 in the diagnosis and treatment of disuse osteoporosis is characterized in that m 6 A methyltransferase METTL16 is a potential biomarker for disuse osteoporosis and a new target for combating disuse osteoporosis.

3. The m according to claim 1 6 The application of A methyltransferase METTL16 in the diagnosis and treatment of disuse osteoporosis is characterized in that METTL16 can significantly promote the differentiation and mineralization ability of MC3T3-E1 cells.

4. The m according to claim 1 6 The application of A methyltransferase METTL16 in the diagnosis and treatment of disuse osteoporosis is characterized in that Overexpression of Mettl16 can partially alleviate the weakening of osteogenic differentiation ability caused by rotational weightlessness.

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