Application of C6orf163 or its derivatives

Abstract

The present invention discloses the application of C6orf163 or its derivatives. The exosome-derived protein C6orf163 can improve bone aging by regulating the osteogenic-adipogenic fate differentiation of bone marrow mesenchymal stem cells (BMSCs) and inhibiting the aging process of BMSCs, thereby improving osteoporosis, providing a research basis and theoretical basis for the development of drugs for treating OP, and can be used to prepare drugs for preventing and / or treating osteoporosis. The present invention first discovered that C6orf163 can promote the transformation of BMSCs into osteoblasts, inhibit their transformation into adipocytes, and promote bone formation, providing new ideas and methods for the prevention and treatment of osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine and biochemistry, and in particular, relates to an application of an EC-Exos-derived protein C6orf163 and derivatives thereof in the preparation of drugs for treating osteoporosis and related diseases. Background Art

[0002] Osteoporosis (OP) is a metabolic bone disease characterized by decreased bone mass and destruction of bone microstructure, which leads to increased bone brittleness and increased fracture risk. Currently, about 83.9 million people in my country suffer from osteoporosis. As the aging population becomes more serious, the number of patients will continue to increase, and it is expected to increase to about 212 million by 2050, significantly increasing the risk of disability and all-cause death in the elderly, bringing heavy economic and medical burdens to society.

[0003] At present, the main drugs for the clinical treatment of osteoporosis are still bone resorption inhibitors (such as bisphosphonates). However, long-term treatment has problems such as high price, poor patient compliance, severe side effects on the gastrointestinal mucosa of oral preparations, and intravenous preparations causing mandibular osteonecrosis and atypical fractures.

[0004] EC-Exos have the effect of promoting bone formation. C6orf163 is the protein with the greatest differential expression between EC-Exos and their parent cell HMEC-1. So far, there has been no report on the use of EC-Exos-derived C6orf163 in the preparation of drugs for the treatment of osteoporosis. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the defects of the existing drugs for treating osteoporosis, such as high price, poor patient compliance, large gastrointestinal mucosal side effects of oral preparations, and osteonecrosis and atypical fractures of the mandible caused by intravenous preparations, and to provide a protein C6orf163 or its derivatives as a potential application in the preparation of new drugs for treating osteoporosis. The protein C6orf163 in the present invention can provide a research basis and theoretical basis for the development of drugs for treating osteoporosis.

[0006] The specific technical solution is as follows.

[0007] Use of C6orf163 or its derivatives in the preparation of a preparation for preventing and / or treating osteoporosis; the amino acid sequence of the protein is shown in SEQ ID No.1; the gene sequence of the protein is shown in SEQ ID No.2.

[0008] Furthermore, derivatives of C6orf163 include proteins having similar functions after any truncation or mutation of the protein.

[0009] The second object of the present invention is to provide the use of C6orf163 or its derivatives in the preparation of a preparation having an effect of promoting osteogenic differentiation.

[0010] The third object of the present invention is to provide the use of C6orf163 or its derivatives in the preparation of a preparation having an inhibitory effect on adipogenic differentiation.

[0011] The fourth object of the present invention is to provide the use of C6orf163 or its derivatives in the preparation of a preparation having an inhibitory effect on aging.

[0012] The fifth object of the present invention is to provide the use of the protein C6orf163 or its derivatives in the preparation of an expression promoter for at least one of the genes Runx2, OPN, and BMP2.

[0013] The sixth object of the present invention is to provide the use of the protein C6orf163 or its derivatives in the preparation of an expression inhibitor of at least one of the genes mPPAR-γ, mFABP4, and Perilipin.

[0014] The seventh objective of the present invention is to provide the use of the protein C6orf163 or its derivatives in the preparation of an expression inhibitor of at least one of the genes P16, I16, and I18.

[0015] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0016] The reagents and raw materials used in the present invention are commercially available.

[0017] The positive and progressive effects of the present invention are:

[0018] C6orf163 in the present invention has a promoting effect on bone metabolism, thereby significantly improving bone mass and delaying the development of osteoporosis. Specifically:

[0019] (1) C6orf163 promotes osteogenic differentiation of BMSCs. The mRNA levels of Runx2, OPN, and BMP2 in BMSCs treated with C6orf163 were significantly increased compared with those in the PBS treatment group.

[0020] (2) C6orf163 inhibits the adipogenic differentiation of BMSCs. The mRNA levels of mPPAR-γ, mFABP4, and Perilipin in BMSCs treated with C6orf163 were significantly lower than those in the PBS treatment group.

[0021] (3) C6orf163 has an inhibitory effect on the aging process of BMSCs. The mRNA levels of P16, I16, and I18 in BMSC cells treated with C6orf163 were significantly lower than those in the PBS treatment group.

[0022] (4) C6orf163 can improve bone mass, increase bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and bone formation rate, and reduce trabecular separation (Tb.Sp);

[0023] (5) C6orf163 can increase OCN + osteoblasts, reduce Trap + osteoclasts and reduce the number and area of ​​fat cells.

[0024] The present invention provides new potential drugs and treatment approaches for the prevention or treatment of osteoporosis and its related diseases, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A is the protein spectrum analysis of HMEC-1 and EC-EXOs;

[0026] Figure 1 B is a bar graph analysis of differentially expressed proteins between HMEC-1 and EC-EXOs;

[0027] Figure 1 C is the volcano plot analysis of differentially expressed proteins between HMEC-1 and EC-EXOs;

[0028] Figure 1 D is GO analysis of differentially expressed proteins between HMEC-1 and EC-EXOs;

[0029] Figure 1 E is the biological process analysis of differentially expressed proteins between HMEC-1 and EC-EXOs;

[0030] Figure 1 F is the protein with the largest differential expression between HMEC-1 and EC-EXOs. Figure 2 A is a schematic diagram of alkaline phosphatase (ALP) staining of BMSCs treated with C6orf163 and PBS respectively and induced osteogenic induction;

[0031] Figure 2 B is a schematic diagram of Alizarin red staining of BMSCs treated with C6orf163 and PBS respectively and induced osteogenic induction;

[0032] Figure 2 C is a schematic diagram of the qPCR results of C6orf163 promoting osteogenic differentiation;

[0033] Figure 2 D is a schematic diagram of the WB results of C6orf163 promoting osteogenic differentiation.

[0034] Figure 3 A is a schematic diagram of Oil Red O staining of BMSCs treated with C6orf163 and PBS respectively and induced adipogenesis;

[0035] Figure 3 B is a schematic diagram of the qPCR results of C6orf163's inhibitory effect on adipogenic differentiation.

[0036] Figure 4 A is a schematic diagram of treating aged BMSCs with C6orf163 and PBS respectively and performing β-galactosidase staining of senescent cells;

[0037] Figure 4 B is a schematic diagram of the qPCR results of C6orf163's inhibitory effect on aging.

[0038] Figure 5 A is a three-dimensional reconstruction of bone scan showing the increase in bone mass after C6orf163 was injected into the tail vein of a 15-month-old mouse.

[0039] Figure 5 B is a schematic diagram of micro-CT bone parameter analysis of bone mass increase in 15-month-old mice after tail vein injection of C6orf163.

[0040] Figure 6 A is a schematic diagram of osteocalcin (OCN) staining after C6orf163 was injected into the tail vein of 15-month-old mice. The arrow indicates OCN-positive (OCN + ) cells;

[0041] Figure 6 B is a statistical analysis of the number of OCN+ cells in 15-month-old mice after tail vein injection of C6orf163.

[0042] Figure 7 A is a schematic diagram of tartrate-resistant acid phosphatase (Trap) staining after C6orf163 was injected into the tail vein of 15-month-old mice. The arrow indicates Trap-positive (Trap + ) cells;

[0043] Figure 7 B is a statistical analysis of the number of Trap-positive (Trap+) cells after tail vein injection of C6orf163 in 15-month-old mice.

[0044] Figure 8 A is a schematic diagram of hematoxylin-eosin (HE) staining of 15-month-old mice after tail vein injection of C6orf163;

[0045] Figure 8 B is a statistical analysis of the number and area of ​​adipocytes in 15-month-old mice after tail vein injection of C6orf163 and HE staining. DETAILED DESCRIPTION

[0046] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0047] Embodiment 1:

[0048] Figure 1 In order to screen out the results of C6orf163 protein, a key factor affecting the fate differentiation of BMSCs by EC-EXOs. The proteomic spectra of endothelial cell exosomes (EC-Exos) and their parental cells HMEC-1 were compared by high-throughput sequencing. A total of 5952 proteins were identified through spectrum analysis, of which 5253 were quantifiable. The expression of 1725 proteins in EC-Exos was significantly higher than that of its parental cells HMEC-1, and the expression of 1042 proteins was significantly lower than that of its parental cells HMEC-1. (e.g. Figure 1 A~C)

[0049] GO analysis revealed that differentially expressed genes in endothelial cell exosomes were mainly involved in biological processes such as cell processes, biological regulation and metabolic processes, especially in the biological processes of the skeletal system. The expression difference of C6orf163 was the most significant, and the expression level of C6orf163 in EC-Exos was about 442 times higher than that in HMEC-1 cells. There was no report on the use of EC-Exos-derived C6orf163 in the preparation of osteoporosis drugs.

[0050] C6orf163 full gene synthesis, plasmid vector construction, protein purification:

[0051] The whole gene synthesis, plasmid construction and protein sequencing of C6orf163 were all commissioned to China Qiangyao Biotechnology Co., Ltd.

[0052] The protein purification steps are as follows:

[0053] (1) Competent transformation and positive clone screening

[0054] Take out the BL21 (DE3) competent cells from the ultra-low temperature refrigerator and place them on ice to melt; add the plasmid expressing C6orf163 constructed by pET28a, gently blow and pipette to mix thoroughly, and place on ice for 30 minutes; heat shock in a water bath at 42°C for 90 seconds, and place on ice for 10 minutes; add 800μL of non-antibiotic LB liquid culture medium, and culture at 37°C and 180rpm for 60 minutes; centrifuge at 6000rpm for 4 minutes, discard the supernatant, add 200ul of non-antibiotic LB liquid culture medium, mix well, and apply to the kanamycin resistance plate; invert the plate, and culture at 37°C for 12 to 16 hours to see the appearance of single colonies (positive clones). The recombinant protein gene can be expressed in the E. coli expression system after IPTG induction.

[0055] (2) Small-scale expression and identification of positive clones

[0056] Single colonies containing recombinant plasmids were selected and placed in 5 mL LB liquid culture medium (kanamycin resistance), and cultured at 37°C with shaking overnight before preservation (stored at -20°C); multiple colonies containing recombinant plasmids were selected and placed in 5 mL LB liquid culture medium (kanamycin resistance), and cultured at 37°C with shaking for 3 h; part of the bacterial solution was taken as the control group, and the remaining bacterial solution was added with IPTG inducer (final concentration 1 mM), and cultured at 37°C with shaking for 3 h; 0.5 mL of the bacterial solution from the two groups was taken respectively, centrifuged at 12000 rpm for 2 min, the supernatant was discarded, the bacterial pellet was resuspended and lysed with 40 μL 1× loading buffer, and detected by SDS-PAGE.

[0057] (3) Protein mass expression and bacterial lysis detection

[0058] Take the bacteria stored at -20℃ and streak them at 37℃ for overnight culture; pick 2 test tubes and culture them at 37℃ and 180rpm overnight; transfer the bacterial solution in the test tubes to 300ml LB medium, add 1mM IPTG inducer (final concentration is 1mM) after 3h, and continue to culture in a 37℃ shaker for 4-5h; collect the bacteria by centrifugation at 8000rpm for 2min, discard the supernatant, and resuspend the bacterial pellet in 50mL pre-cooled NTA-0 buffer; sonicate the bacterial resuspension (ultrasound for 30min); centrifuge at 12000rpm at 4℃ for 20min, collect the supernatant and pellet; take a small amount of supernatant and pellet for SDS-PAGE detection, and keep the remaining supernatant and pellet at 4℃ for later use.

[0059] (4) Recombinant protein purification (nickel column affinity chromatography)

[0060] The equilibration buffer is used to equilibrate the chromatography column. After the fermentation broth supernatant or cell lysis supernatant after centrifugation is added, it is washed with the equilibration buffer to remove some impurities, and then the target protein is eluted with a buffer containing different eluent (NaCl or imidazole) concentrations. After collecting the eluted fractions, the protein is desalted into the storage buffer. For insoluble proteins, denaturants such as urea may be added during the cell lysis process to dissolve the target protein and then purify it with a chromatography column. The eluted fraction containing the target protein is eluted from the chromatography column using a step-by-step dialysis method to remove the denaturant and renature the protein, and finally the renatured protein is desalted into the storage buffer.

[0061] (5) Recombinant protein purity detection and quantification

[0062] The protein samples were subjected to SDS-PAGE and UV OD 280 Purity detection and quantification are performed by methods such as the above. The desired protein C6orf163 is produced, and its protein sequence is shown in SEQ ID No.1.

[0063] Table 1 Basic physicochemical properties of C6orf163 protein

[0064] length 329 Molecular weight (KDa) 46 kDa Isoelectric point 6.1 Instability Index 41.48 Grand average of hydropathicity (GRAVY) 0.4

[0065] The following is the C6orf163 protein and gene sequence

[0066] Uncharacterized protein C6orf163 protein sequence (SEQ ID No.1)

[0067] MIRNSDYKNFVCCAVCNKIIPPAPFGKTFKRIHEYKPLKTRFYTHKDILDIG

[0068] ANILKKEEQFQEDILREHIAKAEAEVWAQANERQKQAVEKALEEANDRH

[0069] KIEIQILKEEHQKDLQEVTAKTKTEMYQNMDDEMKREHLAAEQRMVHRI

[0070] QRIMMECHREKVEAVEKARAEERHIAQEAIQAQKSKAVEEIVNTGVTVIK

[0071] DEKTSVARLMREKEHEMSILYGIAQRQRQEEVQEVLQEAEKTHQATLGN

[0072] MMDKLANTQGELLSIAKQLGIMTNWKDFLEEELQETRMAFQKYINYTFP

[0073] KLSPGHADFILPERKKTPSNLVIKENKTTLD Uncharacterized protein C6orf163 gene sequence (SEQ ID No.2)

[0074] ATGATCAGAAATTCAGATTACAAAAACTTTGTTTGCTGTGCTGTTTGTA

[0075] ATAAAATAATTCCACCAGCCCCTTTTGGAAAAACCTTCAAACGGATCCA

[0076] TGAATACAAGCCACTTAAGACACGCTTTTACACTCACAAAGACATACT

[0077] AGATATTGGGGCAAATATTCTGAAAAAAGAAGAGCAGTTTCAAGAAGA

[0078] TATACTCAGAGAACACATTGCGAAAGCAGAAGCTGAAGTATGGGCTCA

[0079] GGCTAATGAACGTCAAAAACAAGCAGTGGAGAAAGCACTTGAAGAAG

[0080] CAAATGACAGACACAAAATTGAAATTCAGATTTTGAAAGAGGAACATC

[0081] AGAAAGATTTACAGGAAGTGACAGCTAAAACTAAGACAGAGATGTATC

[0082] AAAACATGGATGACGAAATGAAGAGAGAGCACTTGGCTGCAGAACAG

[0083] CGCATGGTCCACAGAATCCAAAGGATTATGATGGAATGCCACAGAGAG

[0084] AAGGTCGAAGCTGTGGAGAAAGCCAGGGCTGAAGAAAGACACATCGC

[0085] CCAGGAAGCAATCCAGGCCCAGAAAAGCAAAGCCGTGGAGGAAATTG

[0086] TGAATACTGGTGTCACAGTTATTAAGGATGAGAAGACCAGTGTGGCCC

[0087] GACTGATGAGGGAAAAAGAACATGAAATGAGCATCCTCTATGGCATAG

[0088] CTCAGAGGCAGAGGCAAGAAGAGGTACAGGAAGTGCTTCAAGAAGC

[0089] AGAGAAAACACATCAGGCCACTCTTGGCAATATGATGGATAAACTGGC

[0090] TAACACCCAGGGGGAGCTGCTGTCTATAGCAAAACAACTGGGAATCAT

[0091] GACAAATTGGAAAGATTTCCTAGAGGAGGAATTACAGGAAACCAGGAT

[0092] GGCATTTCAAAAATACATCAATTATACCTTTCCTAAGCTTTCACCAGGA

[0093] CATGCAGATTTTATTCTGCCAGAAAGAAAGAAACACCTTCTAATCTTG

[0094] TTATTAAGGAGAACAAAACAACTCTTGATTAG

[0095] Example 2: Effect of C6orf163 on promoting osteogenic differentiation of bone marrow mesenchymal stem cells

[0096] The cell density was 5×10 3 / cm 2Bone marrow mesenchymal stem cells (BMSCs) were inoculated in α-MEM medium containing 0.1 mM dexamethasone, 10 mM β-glycerophosphate and 50 mM ascorbic acid-2-phosphate, and 10% fetal bovine serum, and divided into a control group (PBS) and a C6orf163 treatment group (C6orf163 protein prepared in Example 1, 50 ng / mL). After 6-9 days of differentiation culture, the expression levels of osteogenic-related genes were analyzed by qPCR, the expression levels of osteogenic-related proteins were analyzed by WB, and the osteogenic ability of the cells was evaluated by ALP staining; after 3 weeks of differentiation, the mineralization ability of the cells was evaluated by Alizarin red staining.

[0097] from Figure 2 A shows that compared with the PBS-treated control group, the differentiation of BMSC cells into osteoblasts was significantly increased after the addition of C6orf163.

[0098] from Figure 2 B shows that compared with the PBS-treated control group, the number of mineralized nodules increased after the addition of C6orf163, that is, the ability of BMSC cells to transform into osteoblasts was significantly increased.

[0099] from Figure 2 C It can be seen that compared with the PBS control group, the mRNA levels of osteogenic-related genes Runx2, OPN, and BMP2 in BMSC cells were significantly increased after treatment with C6orf163.

[0100] from Figure 2 D shows that compared with the PBS control group, the level of osteogenic-related protein OPN in BMSC cells was significantly increased after treatment with C6orf163.

[0101] These results suggest that C6orf163 can promote the osteogenic differentiation of bone marrow mesenchymal stem cells.

[0102] Example 3: Effect of C6orf163 on Inhibiting Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

[0103] The cell density was 5×10 3 / cm 2 Bone marrow mesenchymal stem cells (BMSCs) were inoculated in α-MEM medium containing 1 μM dexamethasone, 10 μg / ml insulin, 200 μM indomethacin, 50 mM ascorbic acid-2-phosphate, 0.5 mM IBMX and 10% fetal bovine serum, and divided into a control group (PBS) and a C6orf163 treatment group (C6orf163 protein prepared in Example 1, 50 ng / mL). After 6-9 days of differentiation culture, the expression levels of adipogenic-related genes were analyzed by qPCR; after 3 weeks of differentiation, the adipogenic ability of the cells was evaluated by Oil Red O staining.

[0104] from Figure 3 A: As can be seen, the adipocyte formation of BMSC cells was significantly reduced after the addition of C6orf163 compared with the PBS-treated control group.

[0105] from Figure 3 B shows that compared with the PBS control group, the mRNA levels of adipogenesis-related genes mPPAR-γ, mFABP4, and Perilipin in BMSC cells were significantly decreased after treatment with C6orf163.

[0106] These results suggest that C6orf163 can inhibit the adipogenic differentiation of bone marrow mesenchymal stem cells.

[0107] Example 4: Effect of C6orf163 on inhibiting the aging process of bone marrow mesenchymal stem cells

[0108] The cell density was 5×10 3 / cm 2 Aged bone marrow mesenchymal stem cells (BMSCs) were inoculated in α-MEM medium containing 10% fetal bovine serum and divided into a control group (PBS) and a C6orf163 treatment group (C6orf163 protein prepared in Example 1, 50 ng / mL). After 6-9 days of culture, the expression levels of aging-related genes were analyzed by qPCR, and the aging of BMSC cells was evaluated by β-galactosidase staining.

[0109] from Figure 4 A: As can be seen, compared with the PBS-treated control group, the formation of BMSC senescent cells was significantly reduced after the addition of C6orf163.

[0110] from Figure 4 B shows that compared with the PBS control group, the mRNA levels of senescence-related genes P16, I16, and I18 in BMSC cells were significantly decreased after treatment with C6orf163.

[0111] These results suggest that C6orf163 can inhibit the aging process of bone marrow mesenchymal stem cells.

[0112] Example 5: C6orf163 can improve bone aging by affecting bone turnover and fat accumulation

[0113] Bone aging is a complex process characterized by decreased bone formation, increased bone resorption and bone marrow fat, and a deviation in the fate of stem cells. Since C6orf163 was observed to promote osteogenic differentiation of bone marrow mesenchymal stem cells in the early stage, it was further speculated whether it had an improvement effect on bone aging. 15-month-old mice were divided into a control group and a treatment group. The control group was injected with PBS through the tail vein of the mouse, and the treatment group was injected with C6orf163 (100ug / kg) prepared in Example 1 through the tail vein of the mouse, 3 times a week, for 2 months. After 2 months, the two groups of mice were killed, and bilateral femurs were collected. One side of the femur was fixed with formaldehyde and placed in a sample tube for CT scanning, and then micro-CT analysis and three-dimensional reconstruction were performed to observe the bone mass; the other side of the femur was placed in 10% EDTA decalcification solution for decalcification for 2-3 weeks, and after paraffin embedding and sectioning, OCN, Trap and HE staining were performed respectively.

[0114] See also Figures 5 to 8 , Each value is expressed as the mean ± SD of three tests. *, P < 0.01 compared with the PBS-treated control group.

[0115] from Figure 5 The bone scan reconstruction in A shows that the bone mass in the C6orf163-treated group was significantly increased compared with that in the PBS control group. Figure 5 From the mirco-CT analysis parameters of B, it can be seen that the bone volume fraction (BV / TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) of mice in the C6orf163 group were significantly increased, while the trabecular separation (Tb.Sp) was reduced.

[0116] from Figure 6 A and Figure 6 B shows that compared with the PBS control group, the number of OCN+ osteoblasts increased significantly after C6orf163 was injected into the tail vein of 15-month-old mice.

[0117] from Figure 7 A and Figure 7 As can be seen from B, the number of Trap+ osteoclasts in the C6orf163 group was less than that in the control group, and bone resorption was weakened.

[0118] from Figure 8 The results of AB showed that compared with the PBS control group, the number and area of ​​adipocytes in 15-month-old mice were significantly reduced after tail vein injection of C6orf163.

[0119] The above results indicate that C6orf163 can improve bone aging by regulating the effects of bone turnover and fat accumulation.

[0120] The above is only a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by any technician familiar with the technical field within the technical scope disclosed in the present invention falls within the protection scope of the present invention.

Claims

1. Use of protein C6orf163 in preparing a preparation for preventing and / or treating osteoporosis; the amino acid sequence of C6orf163 is shown in SEQ ID No. 1; the gene sequence of C6orf163 is shown in SEQ ID No. 2.

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