CN110951869A

CN110951869A - Biomarker for osteoporosis diagnosis and treatment

Info

Publication number: CN110951869A
Application number: CN202010003381.4A
Authority: CN
Inventors: 吴成爱; 田伟; 王超; 王莹; 王倩倩; 张砚卓; 陶剑锋; 袁越
Original assignee: BEIJING RESEARCH INSTITUTE OF TRAUMATOLOGY AND ORTHOPAEDICS
Current assignee: BEIJING RESEARCH INSTITUTE OF TRAUMATOLOGY AND ORTHOPAEDICS
Priority date: 2020-01-02
Filing date: 2020-01-02
Publication date: 2020-04-03
Anticipated expiration: 2040-01-02
Also published as: CN110951869B

Abstract

The invention discloses a biomarker for diagnosing and treating osteoporosis, which is LOC 101927627. The invention discloses an application of LOC101927627 in preparation of osteoporosis diagnosis products, and simultaneously discloses an application of LOC101927627 as a molecular target in preparation of a medicine for treating osteoporosis.

Description

Biomarker for osteoporosis diagnosis and treatment

Technical Field

The invention belongs to the field of biological medicines, and relates to a biomarker for osteoporosis diagnosis and treatment, wherein the biomarker is LOC 101927627.

Background

Osteoporosis is a systemic skeletal disease characterized by low bone mass and deterioration of the microstructure in bone tissue, resulting in increased bone fragility and increased risk of fracture. Osteoporotic fractures result in a significant decrease in quality of life, increased morbidity, mortality, and disability.

Osteoporosis is classified into primary osteoporosis and secondary osteoporosis according to its cause. Primary osteoporosis is associated with the aging process, with a decrease in sex hormones. Bone has deteriorated in microstructure leading to loss of bone mineral density and increased risk of fracture. Secondary osteoporosis can be a consequence of endocrine and metabolic disorders (e.g., hypogonadism, hypercortisolism, hyperparathyroidism, hyperthyroidism, anorexia), lymphoproliferative disorders, intestinal malabsorption, rheumatoid arthritis, renal failure, and the like. In addition, the use of some drugs may also cause Osteoporosis, such as glucocorticoids, selective 5-hydroxytryptamine reuptake inhibitors, anticoagulants and antidiabetic drugs (Society Italiana delfOstoporsi dMMedMdS. [ Guidelidinines for the diagnosis, prevention and treatment of Osteoporosis. Italian osteoporotosis, Mineral Metabolism and Mineral Diseases Society ]. Minervera Endocrinol.2013; 38(1supp 1):1-30.), other drugs such as chemotherapeutic drugs, proton pump inhibitors and thiazolidine drugs have less research on the effect of Osteoporosis, so the use of these drugs has not been completely identified as a risk factor for secondary Osteoporosis. Osteoporosis can occur due to a variety of etiologies, however, regardless of the etiology, all patients with osteoporosis share a common feature of imbalance between bone formation and resorption, the rate of bone formation by osteoblasts being often normal, and the rate of bone resorption by osteoclasts being increased (Akesson K. New aproacids to pharmacological characterization of osteoporosis, Bull World Health Organ. 2003; 81(9): 657-64.).

Although the current research considers that the onset of osteoporosis is related to age and sex, and is closely related to genetic factors, environmental factors and the like, the pathogenesis of osteoporosis is still unclear, and the early diagnosis, the monitoring of the treatment effect and the prognosis evaluation of osteoporosis play an important role in preventing and treating osteoporosis. In recent years, with the development of biotechnology, the research of genetic factors becomes a hot topic in the field of osteoporosis diagnosis and treatment, and the search for osteoporosis-related genes is of great significance in disclosing osteoporosis pathogenesis and realizing early diagnosis and treatment of osteoporosis.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a molecular marker for early diagnosis of osteoporosis, so as to realize specific diagnosis and prevention of osteoporosis.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an application of LOC101927627 gene, which is used for preparing products for diagnosing osteoporosis.

Further, the product comprises a reagent for detecting the expression level of LOC101927627 in the sample.

Further, LOC101927627 is up-regulated in osteoporosis.

Further, the reagent is used for detecting the LOC101927627 expression level by RT-PCR, real-time quantitative PCR, in-situ hybridization or chip.

In the present invention, a "sample" is a sample containing cells or cellular material from which nucleic acids, polypeptides, or other analytes can be obtained. Examples of biological samples include, but are not limited to: urine, blood, serum, plasma, cerebrospinal fluid, pleural fluid, bronchial lavage, sputum, peritoneal fluid, bladder wash, secretions (e.g., breast secretions), oral wash, swabs (e.g., oral swabs), isolated cells, tissue samples, touch preps, and fine needle punctures.

The invention provides a product for diagnosing osteoporosis, which comprises a preparation, a chip or a kit, wherein the preparation, the chip or the kit comprises a reagent for detecting the expression level of LOC 101927627.

Further, the reagent comprises a primer for specifically amplifying LOC 101927627.

Further, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.

The invention provides an application of LOC101927627 gene in preparing a medicine for treating osteoporosis.

Further, the medicament comprises an inhibitor of LOC 101927627.

Further, the inhibitor down-regulates the expression level of LOC 101927627.

Further, the inhibitor is siRNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 5-8.

The invention provides a medicament for treating osteoporosis, which comprises an inhibitor of LOC 101927627.

Further, the inhibitor is siRNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 5-8.

Term(s) for

The term "biomarker", also referred to as "molecular marker", is any gene whose expression level in a tissue or cell is altered compared to the expression level of a normal or healthy cell or tissue.

Any method available in the art for detecting expression of a molecular marker is encompassed herein. Expression of the molecular markers of the invention can be detected at the nucleic acid level (e.g., RNA transcript) expression level. By "detecting expression" is intended the determination of the amount or presence of an expression product of an RNA transcript or its molecular marker gene. Thus, "detecting expression" includes instances where a molecular marker is determined to be not expressed, not to be detected, expressed at a low level, expressed at a normal level, or overexpressed. To determine overexpression, the body sample being tested can be compared with a corresponding body sample from a healthy person. That is, the "normal" level of expression is the level of expression of the molecular marker. This sample may be presented in a normalized form. In some embodiments, the determination of the overexpression of the molecular marker is performed by comparing a body sample with a corresponding body sample from a healthy person.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. LOC101927627 is located on chromosome 13 with gene ID 101927627, and includes the LOC101927627 gene and homologs, mutations, and isoforms thereof. The term encompasses full length, unprocessed LOC101927627, as well as any form of LOC101927627 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of LOC 101927627. A representative human LOC101927627 gene sequence is shown in XR-001750012.1.

The term "chip", also referred to as an "array", refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

A "microarray" is an ordered array of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

As the probe, a labeled probe in which a disease-detecting polynucleotide is labeled, such as a fluorescent label, a radioactive label, or a biotin label, can be used. Methods for labeling polynucleotides are known per se. The presence or absence of the test nucleic acid in the sample can be checked by: immobilizing the test nucleic acid or an amplification product thereof, hybridizing with the labeled probe, washing, and then measuring the label bound to the solid phase. Alternatively, the polynucleotide for disease detection may be immobilized, a nucleic acid to be tested may be hybridized therewith, and the nucleic acid to be tested bound to the solid phase may be detected using a labeled probe or the like. In this case, the polynucleotide for detecting a disease bound to the solid phase is also referred to as a probe. Methods for assaying test nucleic acids using polynucleotide probes are also well known in the art. The process can be carried out as follows: the polynucleotide probe is contacted with the test nucleic acid at or near Tm (preferably within ± 4 ℃) in a buffer for hybridization, washed, and the hybridized labeled probe or template nucleic acid bound to the solid phase probe is then measured.

The size of the polynucleotide used as a probe is preferably 18 or more nucleotides, more preferably 20 or more nucleotides, and the entire length of the coding region or less. When used as a primer, the polynucleotide is preferably 18 or more nucleotides in size, and 50 or less nucleotides in size. These probes have a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, or they may be polynucleotides in which part or all of the nucleotides are substituted with artificial nucleic acids such as PN, LNA, ENA, GNA, TNA, etc.

The term "kit" includes one or more sterile containers, which may be in the form of a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments.

In the present invention, the term "including" is used to mean, and is used interchangeably with, the phrase "including but not limited to".

Statistical method

In the present invention, the experiment is repeated at least 3 times, the result data are expressed in the form of mean value ± standard deviation, statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is determined by t test, and the statistical significance is considered when P is less than 0.05.

The invention has the advantages and beneficial effects that:

the invention discovers that the molecular marker LOC101927627 is related to the occurrence and development of osteoporosis for the first time, and diagnoses whether a patient has osteoporosis and the risk of having the osteoporosis by detecting the change of the LOC101927627 of a subject, thereby realizing the early discovery and early treatment of the osteoporosis patient and improving the life quality of the patient.

The invention discovers the relevance of the molecular marker LOC101927627 and the proliferation of osteoblasts for the first time, and the molecular marker LOC101927627 can be used as a molecular target for intervention in the treatment of osteoporosis.

Drawings

FIG. 1 is a graph showing the expression of the LOC101927627 gene in different samples; wherein panel a is a blood sample and panel B is a tissue sample;

FIG. 2 is a graph showing the expression of LOC101927627 in osteoblasts;

fig. 3 is a diagram of the interference situation of LOC 101927627.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 QPCR detection of LOC101927627 expression in osteoporosis

1. Sample collection

80 patients who underwent hip replacement surgery, aged 50-75 years, 47 patients with osteoporosis and 33 normal controls were selected, and blood and tissue samples were collected from the patients.

Inclusion and exclusion criteria for osteoporotic patients:

inclusion criteria were: the patient is diagnosed to determine that hip replacement surgery is needed; the patients are 50-75 years old and determined to be patients with osteoporosis through double-energy X-ray bone density examination.

Exclusion criteria: the patient takes the medicine which affects the bone metabolism within about 6 months, and the patient also suffers from other diseases.

Inclusion and exclusion criteria for control group:

inclusion criteria were: the patient is diagnosed to determine that hip replacement surgery is needed; the patients are 50-75 years old and determined to be non-osteoporosis patients by double-energy X-ray bone density examination.

2. Preparation and Mass analysis of RNA samples

2.1 extraction of tissue sample RNA

Tissue RNA was extracted using Trizol method as follows:

1) cutting tissue with scissors, adding 1ml Trizol, and shaking on oscillator for 1 min; standing at room temperature for 10min to completely decompose nucleoprotein;

2) adding 200 μ l chloroform (chloroform), covering the tube cover, shaking vigorously for 15s, standing at room temperature for 10min, centrifuging at 4 deg.C and 11000rpm for 15 min;

3) transferring the water sample layer into a new centrifuge tube, adding 500 μ l isopropanol, reversing, mixing, standing at normal temperature for 10min, centrifuging at 4 deg.C and 11000rpm for 15 min;

4) carefully sucking away the liquid with a gun, leaving the precipitate at the bottom of the tube, adding 1ml of 75% ethanol, shaking on an oscillator for 5s, washing the precipitate once, and centrifuging at 4 ℃ and 8000rpm for 5 min;

5) carefully removing the supernatant, drying the precipitate for 10min, and adding a proper amount of water to dissolve the precipitate for 10 min;

6) and detecting the concentration of the RNA, and identifying the yield and purity of the RNA.

2.2 extraction of blood sample RNA

1) Adding 3 times volume of erythrocyte lysate into blood, mixing uniformly, and standing at room temperature for 10 min;

2) centrifuging at 10000rpm for 1min, sucking and removing supernatant, and collecting leukocyte precipitate;

3) 1ml of TRIZOL was added to every 100-;

4) centrifugation was carried out at 12,000rpm at 4 ℃ for 10 minutes, and the supernatant was collected, and the other steps were the same as 2) to 6) in 2.1.

3. Reverse transcription

Reverse transcription of mRNA was performed using FastQuant cDNA first Strand Synthesis kit (cat # KR106) from Tiangen

Add 5 XgDNA B. mu.ffer 2.0. mu.l, total RNA 1. mu.g, RNase Free ddH to the tube₂O to make the total volume to 10 μ l, heating in water bath at 42 deg.C for 3min, and placing on ice.

10 XFast RT B. mu.ffer 2.0. mu.l, RT Enzyme Mix 1.0. mu.l, FQ-RT Primer Mix 2.0. mu.l, RNase Free ddH₂O5.0 μ l, mixing, adding into the above test tube, mixing to give 20 μ l, heating in water bath at 42 deg.C for 15min, heating at 95 deg.C for 3min, and placing on ice.

4、QPCR

Amplification was carried out using SuperReal PreMix Plus (SYBR Green) (cat # FP205) and the experimental procedures were performed according to the product instructions.

1) Primer design

QPCR amplification primers are designed according to the coding sequences of LOC101927627 gene and GAPDH gene in Genebank, and the specific primer sequences are as follows:

LOC101927627 gene:

the forward primer is 5'-ATGTTGTTGCTATTCTCT-3' (SEQ ID NO. 1);

the reverse primer was 5'-AATGTGGTTATTATTGTTGT-3' (SEQ ID NO. 2).

The primer sequence of housekeeping gene GAPDH is as follows:

a forward primer: 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID NO.3)

Reverse primer: 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID NO.4)

2) Prepare 20 μ l PCR reaction system according to Table 1:

TABLE 1 PCR reaction System

3) And (3) PCR reaction conditions: : 95 ℃ for 15min, (95 ℃ for 10s, 55 ℃ for 30s, 72 ℃ for 30s) x 40 cycles, 95 ℃ for 15s, 60 ℃ for 60s, 95 ℃ for 15 s. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, relative quantification is carried out by a delta-CT method, and each sample is subjected to 3 times of repeated experiments.

5. Results

As shown in fig. 1, LOC101927627 gene is significantly up-regulated in osteoporosis patients compared to the control group, and the difference is statistically significant (P <0.05), wherein 48 samples in blood samples are up-regulated, 32 samples are not significantly different, 44 samples in the up-regulated samples are osteoporosis patients, 4 samples in normal controls are normal controls, 3 samples in the samples without significant difference are osteoporosis patients, and 29 samples in the normal controls are normal controls; in the tissue samples, 49 samples were up-regulated, 31 samples were not significantly different, 46 osteoporosis patients were present in the up-regulated samples, 3 normal controls were present in the up-regulated samples, 1 osteoporosis patient was present in the samples without significant differences, and 27 normal controls were present in the samples without significant differences.

Example 2 expression of LOC101927627 in cells

1. Isolation and culture of osteoblasts

Taking cancellous bone from femoral head of human being taken down from hip joint replacement, removing redundant periosteum and soft tissue, repeatedly washing sclerotin with physiological saline, and washing liquidAfter clarification, the femoral neck was cut to 1mm using sterilized rongeur and scissors with 3 washes of PBS solution³The osteoblasts are isolated and purified from the fragments by enzymatic digestion.

The cell suspension obtained by the enzyme digestion was counted on a hemocytometer and diluted to a concentration of 5X 10⁷mL-1, inoculating it in sterile petri dish, placing CO₂Incubator (5% CO)₂Humidity 95%, temperature 37 ℃) for 48h, changing the liquid every two days, and leading the cells to approach the fusion passage (trypsin: 0.2% EDTA ═ 1: 2).

2. Extraction of Total RNA from cells

The total RNA of the cells was extracted from osteoblasts that grew well in the logarithmic phase, and the cellular RNA was extracted by the Trizol method.

3. Reverse transcription was performed as in example 1

4. QPCR same as example 1

5. Results

As shown in FIG. 2, the expression of LOC101927627 was up-regulated in Osteoblasts (OP) of the osteoporosis group compared to osteoblasts (Con) of the normal group, and the difference was statistically significant (P < 0.05).

Example 3 Effect of LOC101927627 on osteoblasts

1. Design synthesis of siRNA against LOC101927627, siRNA-LOC101927627 and general negative control siRNA-NC purchased from Shanghai Ji code Gene chemistry, Inc., the sequence of interfering RNA against LOC101927627 is shown below.

sequence of siRNA 1:

the sense strand is 5'-UUUGAUCUUUAUCUAGAAGGC-3' (SEQ ID NO.5)

The antisense strand is 5'-CUUCUAGAUAAAGAUCAAACC-3' (SEQ ID NO.6)

sequence of siRNA 2:

the sense strand is 5'-ACAACAUCAUUAUAACCAGAU-3' (SEQ ID NO.7)

The antisense strand is 5'-CUGGUUAUAAUGAUGUUGUGA-3' (SEQ ID NO.8)

2. Cell culture

Culturing Con group and OP group bone cells to logarithmic growth phase, and performingConcentration determination and dilution, cells in log phase were cultured at 2X 10 for 24h before transfection⁵The cells were inoculated in 6-well plates and the corresponding medium was added to a final volume of 2m1, placed at 37 ℃ in 5% CO₂Culturing in an incubator.

3. Transfection

Lipofectamin from Invitrogen was used^TM2000, the transfection was carried out with the kit, the transfection method being as described in the specification, and the blank control group being serum-free medium only. After 6h of transfection, the liquid in the 6-well plate was replaced with freshly prepared medium and incubation was continued for 48 h.

4. QPCR (quantitative polymerase chain reaction) experiment for detecting interference effect of siRNA (small interfering ribonucleic acid)

Extracting total RNA of cells in the same manner as in example 2; reverse transcription and QPCR were then performed as in example 1.

As a result, as shown in fig. 3, the expression level of LOC101927627 was not significantly changed in the osteoporotic osteoblasts transfected with siRNA-NC compared to the control group (OP) without transfection, while the expression level of LOC101927627 was significantly decreased in the osteoporotic osteoblasts transfected with siRNA1 and siRNA2, the difference was statistically significant (P <0.05), wherein siRNA1 was able to more effectively inhibit the expression of LOC101927627, and thus siRNA1 was selected for the subsequent experiments.

5. MTT assay for cell proliferation

The transfected cells were cultured for 24 hours, 48 hours, and 72 hours, and the proliferation of the transfected cells was detected by MTT colorimetry.

The experiment was divided into 3 groups, group 1: normal human osteoblasts are transfected with siRNA-NC; group 2: transfecting siRNA-NC to osteoblasts of a patient with osteoporosis; group 3: osteoblasts of patients with osteoporosis were transfected with siRNA 1.

Trypsinizing the transfected osteoblasts to form a cell suspension at 2X 10⁴The cells were plated at a concentration of 200. mu.l/ml in 96-well plates, each containing 6 wells. Transfecting for 24h, 48h and 72h, adding 20 μ l MTT (5mg/m1) into each well, culturing for 4h in an incubator, carefully discarding the supernatant, adding 150 μ l dimethyl sulfoxide, placing on a horizontal oscillator, oscillating for 10min to dissolve the crystal, placing a 96-well plate in an enzyme linked immunosorbent assay detector, selecting 490nm wavelength,blank wells were zeroed, absorbance values were measured for each well and the results recorded.

The results are shown in table 2, compared with group 2, the cell proliferation of group 3 is obvious, and the cell of group 3 has no obvious change compared with group 1, which shows that the over-expression of LOC101927627 inhibits the proliferation of osteoblasts of osteoporosis patients, and the proliferation of osteoporosis cells is obvious after the interference of the expression of LOC101927627, and suggests that LOC101927627 can be used as a molecular target for the treatment of osteoporosis.

TABLE 2 osteoblast MTT assay determination of OD values

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

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Claims

Use of the LOC101927627 gene for the preparation of a product for the diagnosis of osteoporosis.
2. The use according to claim 1, wherein the product comprises a reagent for detecting the expression level of LOC101927627 in a sample.
3. Use according to claim 2, characterized in that LOC101927627 is up-regulated in osteoporosis.
4. The use according to claim 3, wherein the reagent is a reagent for detecting the expression level of LOC101927627 by RT-PCR, real-time quantitative PCR, in situ hybridization or chip.
5. A product for diagnosing osteoporosis, which comprises a preparation, a chip or a kit, wherein the preparation, the chip or the kit comprises a reagent for detecting the expression level of LOC 101927627.
6. The product according to claim 5, characterized in that said reagents comprise primers for the specific amplification of LOC101927627, preferably the primer sequences are shown in SEQ ID No.1 and SEQ ID No. 2.
The application of LOC101927627 gene is characterized in that the LOC101927627 gene is used for preparing medicine for treating osteoporosis.
8. Use according to claim 7, characterized in that the medicament comprises an inhibitor of LOC101927627, preferably said inhibitor down-regulates the expression level of LOC 101927627.
9. The use according to claim 8, wherein the inhibitor is siRNA, preferably the siRNA has a sequence as shown in SEQ ID No. 5-8.
10. The medicine for treating osteoporosis is characterized by comprising an inhibitor of LOC101927627, preferably, the inhibitor is siRNA, and preferably, the sequence of the siRNA is shown in SEQ ID No. 5-8.