CN110607365B

CN110607365B - Application of nucleic acid molecule in preparation of product for diagnosing orthopedic diseases

Info

Publication number: CN110607365B
Application number: CN201911068636.9A
Authority: CN
Inventors: 田清松; 邢光杰; 吴韦强
Original assignee: Changle County Peoples Hospital
Current assignee: Changle County Peoples Hospital
Priority date: 2019-05-22
Filing date: 2019-11-05
Publication date: 2020-06-30
Anticipated expiration: 2039-11-05
Also published as: CN110129429A; CN110607365A

Abstract

The invention discloses an application of nucleic acid molecules in preparing products for diagnosing orthopedic diseases. The invention screens non-coding RNA with differential expression in male osteoporosis patients and normal people through a chip, and then proves that the content of LOC105374848 in the blood of the male osteoporosis patients is higher than that of the normal people through QPCR verification of a large sample. LOC105374848 can be used as a biomarker for diagnosing male osteoporosis. The research result of the invention provides a non-invasive method for clinically carrying out early diagnosis of the male osteoporosis, and is suitable for clinical popularization. In addition, in vitro cell experiments prove that the inhibition of the expression of LOC105374848 can promote the proliferation and differentiation of osteoblasts, so that the medicine for treating osteoporosis can be designed and synthesized according to LOC 105374848.

Description

Application of nucleic acid molecule in preparation of product for diagnosing orthopedic diseases

Technical Field

The invention belongs to the field of biomedicine, relates to application of nucleic acid molecules in preparation of products for diagnosing orthopedic diseases, and particularly relates to application of LOC105374848 in diagnosis of male osteoporosis.

Background

Less than 2% of the genes in the nucleotide sequence of human genomic DNA are used to encode proteins, and at least 75% of the genes are transcribed as non-coding RNAs (ncRNAs). According to the length of the nucleotide sequence, the nucleotide sequence can be divided into two types, wherein more than 200nt are long non-coding RNAs (1 ncRNAs), and less than 200nt are short non-coding RNAs. The 1ncRNAs are divided into five types of sense, antisense, bidirectional, intragenic and intergenic according to the position relation of the 1ncRNAs relative to the coding genes. They are abundant and active at various levels of cellular biological function, such as cell differentiation, cell proliferation, DNA damage response, dose compensation, chromosomal imprinting, and the like. Research results show that the genes play an important role in physiological processes such as gene expression regulation and the like of organisms, and the change of the lncRNA expression profile in various diseases lays a position of serving as a diagnosis and treatment target point of emerging diseases.

The incidence of osteoporosis (osteoporotis in men) in men is much lower than that in postmenopausal women, so that importance on osteoporosis in men has been far less than that in women. Recent research results show that men in the young age achieve higher peak bone mass than women, and the initial time of bone mass loss is obviously later than women, but both men and women have a bone loss process in phase with the age, namely senile osteoporosis (type II). Male osteoporosis shares many similarities with female osteoporosis, but there are still significant gender differences in etiology, pathology, and color pathology. Men over age 65 have a common occurrence of osteoporosis of varying degrees, with a severe complication of fracture.

In the United states, the incidence rate of hip fracture in men older than 65 years old is 4-5 per mill, and the incidence rate in women of corresponding ages is 8-10 per mill. Hip fractures occur at a rate of 1: 2 for men and women in northern europe, and relatively low in both sexes and other areas. Of all patients with hip fractures worldwide, about 30% are men, 51 million in 1990, and it is expected that the number of male hip fractures will increase to 116 million by 2025, and this rising trend will be more prominent in asia.

Cooper reported that men over age 50 had a lifetime risk of hip, vertebral and forearm fractures of 13.1% and women 39.7%. Neill reports 20% prevalence of vertebral fractures in 19 European countries 36 central 15570 cases of men 50-79 years old. According to the xushangzhong report about the prevalence rate of osteoporosis of male in China, the prevalence rate of male over 60 years old is 13.9%; wuqing in beijing area reported 13.4% (lumbar vertebrae); shenhuilan is reported to be 23.8% (lumbar).

Osteoporotic fractures cause loss of living ability and life risk to patients, and Poor reports that mortality and morbidity of fractures are higher in men than in women.

Diagnosis of osteoporosis at the molecular level has become a future trend, and more inventors have made patent layouts in this field, such as patent: 201510627056.4, 201510628042.4, 201711468712.6, 201711469092.8, 201510725389.0, and the like. The research direction of the above patent is the relationship between the gene and the osteoporosis diagnosis, but the relationship between the non-coding RNA and the osteoporosis diagnosis is not yet studied.

Disclosure of Invention

The invention aims to provide a molecular marker for early diagnosis of male osteoporosis.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention provides an application of a LOC105374848 detection reagent in preparation of a product for diagnosing male osteoporosis.

Further, the reagent comprises: the reagents used by LOC105374848 are detected by reverse transcription PCR, real-time quantitative PCR, in situ hybridization or microarray chip.

Further, the reagent for detecting LOC105374848 by reverse transcription PCR at least comprises a pair of primers for specifically amplifying LOC 105374848; the reagent for detecting LOC105374848 by real-time quantitative PCR at least comprises a pair of primers for specifically amplifying LOC 105374848; the reagent for detecting LOC105374848 by in situ hybridization comprises: a probe that hybridizes to a nucleic acid sequence of LOC 105374848; the reagent for detecting LOC105374848 by using the microarray chip comprises: a probe that hybridizes to a nucleic acid sequence of LOC 105374848.

In a specific embodiment of the invention, the primer sequence of the specific amplification LOC105374848 is shown as SEQ ID NO.1 and SEQ ID NO. 2.

Further, the product comprises a chip or a kit.

The invention also provides a product for diagnosing male osteoporosis, which can diagnose the male osteoporosis by detecting the level of LOC105374848 in a sample.

Further, the product comprises a chip, or a kit.

The chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes aiming at LOC 105374848; the kit comprises reagents for detecting LOC 105374848.

Further, the reagent for detecting LOC105374848 in the kit comprises a primer for specifically amplifying LOC 105374848.

The present invention provides a pharmaceutical composition for the treatment of osteoporosis comprising an agent that inhibits the expression of LOC 105374848.

Further, the agent is not limited as long as it can inhibit the expression level of LOC 105374848.

The reagent comprises siRNA or shRNA of LOC 105374848. In a specific embodiment of the invention, the siRNA sequence of LOC105374848 is shown in SEQ ID NO.5 and SEQ ID NO. 6.

The pharmaceutical composition of the present invention may be administered alone or together with other drugs as a medicine. The other drug that can be administered together with the pharmaceutical composition of the present invention is not limited as long as it does not impair the effect of the therapeutic or prophylactic pharmaceutical composition of the present invention.

The pharmaceutical composition of the invention can be prepared into various dosage forms according to requirements. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.

The route of administration of the pharmaceutical composition of the present invention is not limited as long as it can exert the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, cutaneous, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic. In some cases, the administration may be systemic. In some cases topical administration.

The dosage of the pharmaceutical composition of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained, and can be appropriately determined depending on the symptoms, sex, age, and the like. The dose of the therapeutic or prophylactic pharmaceutical composition of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.

The invention also provides the application of the long-chain non-coding RNA in preparing a medicament for treating osteoporosis.

The invention also provides the application of the reagent for inhibiting the expression of the long-chain non-coding RNA in preparing a medicament for treating osteoporosis.

In the present invention, the term "probe" refers to a molecule that is capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "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.

In the present invention, the term "microarray" is an ordered arrangement of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides), 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.

Various probe arrays have been described in the literature and can be used in the context of the present invention to detect markers that may be associated with the phenotypes described herein. For example, a DNA probe array chip or a larger DNA probe array wafer (otherwise, individual chips may be obtained by breaking the wafer) is used in one embodiment of the present invention. The DNA probe array wafer generally comprises a glass wafer on which a high-density array of DNA probes (short DNA fragments) is placed. Each of these wafers may hold, for example, about 6000 million DNA probes for identifying longer sample DNA sequences (e.g., from an individual or population, e.g., containing a marker of interest). The identification of sample DNA by the DNA probe set on the glass wafer was performed by DNA hybridization. When a DNA sample is hybridized to an array of DNA probes, the sample binds to those probes whose sample DNA sequences are complementary. By assessing that the individual sample DNA hybridizes more strongly to those probes, it is possible to determine whether a known nucleic acid sequence is present in the sample, and thus whether a marker found in the nucleic acid is present.

The probe has a base sequence complementary to a specific base sequence of the 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 may be polynucleotides in which part or all of the nucleotides are substituted with artificial nucleic acids such as PNA, LNA, ENA, GNA, TNA, etc.

In the context of the present invention, "LOC 105374848" includes the human LOC105374848 sequence and variants thereof. A "variant" of the sequence is a biologically active sequence that has a nucleotide sequence that differs from the native sequence or wild-type sequence (or its complement) due to insertion, deletion, modification and/or substitution of one or more nucleotides in the native sequence. Such variants typically have less than 100% sequence identity to the native sequence or its complement. However, a biologically active variant typically has at least about 70% sequence identity, typically at least about 75%, more typically at least about 80%, even more typically at least about 85%, even more typically at least about 90%, and even more typically at least about 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding naturally occurring sequence or complement thereof. Variant nucleotide fragments of any length retain the biological activity of the corresponding native sequence. Variants also include sequences in which one or more nucleotides are added at the 5 'or 3' end or within the native sequence or its complement. Variants also include sequences in which some nucleotides are deleted and optionally replaced by one or more different nucleotides.

LOC105374848, gene ID 105374848, and the LOC105374848 transcript in GeneBank is XR _940324.2 at present.

LOC105374848 of the present invention may be natural or synthetic, or may be obtained by transfecting cells with a vector expressing a DNA fragment of LOC 105374848. The vector comprises a viral vector and a eukaryotic expression vector.

The viral vector may be any suitable vector, including but not limited to retroviral vectors, adenoviral vectors, adeno __ virus-associated viral vectors, herpes virus (e.g., herpes simplex virus, vaccinia virus, and EB virus) vectors, alphavirus vectors. The eukaryotic expression vector may be any suitable expression vector, including but not limited to a pCMV-Myc expression vector, a pcDNA3.0 expression vector, a pcDNA3.1 expression vector, a pEGFP expression vector, a pEF Bos expression vector, a pTet expression vector, a pTRE expression vector, or a vector modified based on known expression vectors, such as pBin438, pCAMBIA1301, and the like.

In the context of the present invention, "diagnosing male osteoporosis" includes both determining whether a subject has suffered from male osteoporosis and determining whether a subject is at risk of suffering from male osteoporosis.

The term "sample" may be blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes or monocytes. The sample may be obtained from the patient or subject for direct use, or may be pretreated, e.g., by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the properties of the sample in some manner as described herein or as known in the art.

In a particular embodiment of the invention, the sample is blood.

As used herein, the terms "LncRNA", "Long non-coding RNA", "Long non-coding RNA" and "Long non-coding RNA" are synonymous and used interchangeably and refer to a fragment of RNA transcribed by RNA polymerase II that does not encode a protein and is generally greater than 200bp in length.

As used herein, "treatment" encompasses treatment-related diseases or disease states in a mammal, such as a human, having the associated disease or disorder, and includes:

(1) preventing the occurrence of a disease or condition in a mammal, particularly when the mammal is susceptible to said disease condition but has not been diagnosed as having such a disease condition;

(2) inhibiting a disease or disease state, i.e., preventing its occurrence; or

(3) Alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "treatment" generally refers to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition (including slowing the progression, stopping the progression), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".

Drawings

FIG. 1 shows a statistical chart of the detection of differential expression of LOC105374848 by QPCR;

FIG. 2 shows a statistical plot of the effect of LOC105374848 expression on cell proliferation using MTT;

FIG. 3 shows a statistical plot for detecting the effect of LOC105374848 expression on ALP activity.

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 screening for differentially expressed non-Long coding RNAs

1. Case collection

Male 6 persons over the age of 50 years of BMD were collected from the hospital physical examination team DXA and excluded patients with secondary osteoporosis and who had received anti-osteoporosis therapy. An additional 5 physical-examined healthy men were collected as controls. There was no statistical difference in age between patients with disease and normal persons.

BMD measurement the left femoral neck (left femoral neck was not measured) and lumbar L1-4BMD were measured using DXA model ASY-00409, Hologic, USA. Osteoporosis was diagnosed using the criteria "diagnosis and treatment guidelines for primary osteoporosis (2011)": the BMD value is lower than 1 standard deviation (T value is more than or equal to-1) of the peak value of the bone of a normal adult with the same sex and the same ethnic group, the BMD value is normal, the BMD value is reduced by 1-2.5 standard deviations (-2.5 < T value is less than-1) to be low in bone mass, the BMD value is reduced by 2.5 standard deviations (T value is less than or equal to-2.5) to be osteoporosis, and the lumbar vertebra is calculated by the lowest value in L1-4.

2. Blood Total RNA extraction

Total RNA was extracted using an RNA extraction kit from Promega corporation. The method comprises the following specific steps:

1) taking 1ml of whole blood collected in a test tube treated by heparin or EDTA, and putting the whole blood into a sterile centrifuge tube;

2) centrifuging at 3000rpm for 5min, and carefully sucking the supernatant from the top of the sample;

3) adding 1ml of blood cell lysate, carefully sucking and placing for 4-5 times, re-suspending the precipitate, and centrifuging at 3000rpm for 5 min;

4) repeating the step 3 twice;

5) avoid cell pellet, carefully aspirate almost all supernatant, retain only 100 μ l supernatant; confirming that BME has been added to the RNA lysate, then adding 175. mu.l of RNA lysate to the cells, pipetting, resuspending and lysing the cells;

6) adding 350 μ l of RNA diluent, mixing by inversion for 3-4 times, centrifuging at 13000g for 10min at room temperature, and transferring clear lysate to a sterile centrifuge tube;

7) adding 200 μ l 95% ethanol into the clarified lysate, and mixing by pipetting for 3-4 times; transferring the mixture into a centrifugal column assembly, and centrifuging for 1min at 13000 g;

8) taking down the centrifugal column from the centrifugal column assembly body, discarding the liquid in the collecting pipe, putting the centrifugal column back on the collecting pipe, adding 600 mu l of RNA washing liquid into the centrifugal column assembly body, and centrifuging for 1min at 13000 g;

9) discarding the liquid from the collection tube, loading the column back onto the collection tube, and adding 50 μ l of freshly prepared DNase incubation mixture directly onto the membrane inside the column;

10) incubating at room temperature for 15min, adding 200 μ l DNase termination buffer solution into the centrifugal column, and centrifuging at 13000g for 1 min;

11) adding 600 μ l RNA washing solution, 13000g, centrifuging for 1 min;

12) emptying the collection tube, adding 250 μ l of RNA washing solution (added with ethanol) into the centrifugal column, and centrifuging at high speed for 2 min;

13) transferring the centrifugal column from the collecting tube to an elution tube, adding 100. mu.l of nuclease-free water to the membrane, placing the assembly of the centrifugal column into a centrifuge with the cap of the elution tube facing outwards, centrifuging at 13000g for 1min, discarding the centrifugal column, capping the elution tube with RNA, and storing at-70 ℃.

3. Mass analysis of RNA samples

The concentration and purity of the extracted RNA were determined using Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN was determined by Agilent 2100. The concentration is more than or equal to 200 ng/mul, and the OD260/280 is between 1.8 and 2.2.

4. Double-stranded cDNA was synthesized and labeled using the QuickAmp Labeling Kit, One-Color (Agilent p/n 5190-.

5. The labeled double-stranded cDNA was purified and hybridized to a Human 8X60K LncRNAexpression array information chip from Arraystar.

6. The hybridization rinse was followed by a scanning analysis by an Agilent Microarray Scanner (Agilent p/n G2565 BA).

7. The analysis of the original data is completed by Agilent Feature Extraction Software, the screening of the differential genes uses a method of pairing random variance models, the standards of the differential genes are more than 2 times of variable expression, and the P value is less than or equal to 0.05.

8. Results

The sequencing result shows that: the differentially expressed LncRNA present in the blood of male osteoporosis patients was 256 compared to healthy males, with 175 being up-regulated and 81 being down-regulated.

Example 2 validation of Large samples the differentially expressed LncRNA selected

Based on the results of previous chip sequencing, we chose LOC105374848 for validation based on the size of P value.

1. Sample collection

Osteoporosis patients and male healthy controls were collected in 30 cases as in example 1.

2. Validation at the RNA level

Reagent: the reverse transcription kit (DDR037A) was purchased from Bao bioengineering (Dalian) Co., Ltd. SYBR Premix Ex Taq for fluorescent Real-time (Real-time) quantitative PCR (polymerase chain reaction)^TM(Tli RNaseHPlus) kit was manufactured by Takara, Japan.

2.1 extraction of blood Total RNA

The procedure is as in example 1.

2.2 reverse transcription

Using the extracted total RNA (1. mu.g) as a template, adding the following reaction system, specifically 5 ×

Buffer4μL，

RT Enzyme Mix 1. mu.L, Oligo dT Primer (50. mu.M) 1. mu.L, Random 6mers (100. mu.M) 1. mu.L, as RNase-free ddH₂O make up the reaction volume to 20. mu.L. The mixture was incubated at 37 ℃ for 15min and 85 ℃ for 5s to obtain cDNA. The cDNA can be used for IncRNA Real-time PCR detection.

2.3 QPCR

According to Takara, Japan

Premix Ex Taq^TM(Tli RNaseH Plus) kit instructions. Reaction system:SYBR Premix Ex Taq^TM(2 ×) 25. mu.L, ROX Reference Dye (50 ×) 1. mu.L, PCR forward primer (10. mu.M) 1. mu.L, PCR reverse primer (10. mu.M) 1. mu.L, cDNA 4. mu.L, sterilized ddH₂O18. mu.L. Reaction procedure: pre-denaturation at 95 ℃ for 20s, and circulation for 43 times according to denaturation at 95 ℃ for 10s, annealing at 46.5 ℃ for 20s and extension at 70 ℃ for 10s to obtain Ct value.

The result is obtained by a relative quantitative method using formula 2^-△△ctAnd (4) calculating. The experiment was repeated 3 times.

Designing a primer: primers were designed by the primer design tool of NCBI (primerbast) based on the sequence of the LOC105374848 transcript, the primer sequences are shown below: an upstream primer: 5'-CATTAGTTAAGAGATGGA-3' (SEQ ID NO. 1); a downstream primer: 5'-TTATGATGAAGACTGTAG-3' (SEQ ID NO. 2). Primers were designed based on the GAPDH (internal reference gene) sequence, the upstream primer: 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID NO. 3); 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID NO. 4).

3. Results

The results show that the LOC105374848 levels in the blood of 30 male osteoporosis patients were significantly higher than the healthy controls compared to the average levels in healthy men. Statistical results as shown in figure 1, LOC105374848 levels were significantly elevated in blood of male osteoporosis patients compared to healthy males, with statistical significance of the difference (. about.p < 0.05).

Example 3 Effect of LOC105374848 on osteoblast proliferation and differentiation

1. Cell culture

The hFOB1.19 cells were cultured in DMEM (high sugar)/F12 complete medium in 5% CO₂34 ℃ incubator. The DMEM (high sugar)/F12 complete medium formula was as follows: DMEM (high sugar)/F12 medium + fetal bovine serum at 10% final concentration + G418 at 0.3% final concentration.

2. Interfering RNA synthesis

According to the sequence of LOC105374848, small interfering RNA aiming at LOC105374848 and universal negative control RNA are designed and synthesized by Shanghai Jima pharmaceutical technology Limited.

The sequence of small interfering RNA (siR-LOC105374848) aiming at LOC105374848 is as follows:

forward sequence: 5'-ACAAUUUAGUUAUCUUUGGCUTT-3' (SEQ ID NO. 5);

reverse sequence: 5'-CCAAAGAUAACUAAAUUGUGUTT-3' (SEQ ID NO. 6).

3. Cell transfection

siRNA transfection Using Lipofectamine^TM2000, see instructions for the use of transfection reagents for specific transfection procedures.

4. Detection of the interfering Effect of SiR-LOC105374848 at the transcriptional level

4.1 cellular RNA extraction

Extracting Total RNA of human osteoblasts by using an RNAnts Total RNA Isolation System kit, dissolving the Total RNA in RNase-free water, taking out a small amount of the Total RNA for dilution, measuring the concentration and the purity of the Total RNA by using an ultraviolet spectrophotometer, and adjusting the concentration of an RNA sample to be 1g/L according to the measured value.

4.2 reverse transcription

The procedure is as in example 2.

4.3QPCR

The procedure is as in example 2.

5. Cell proliferation assay (MTT method)

The experiment was divided into 2 groups, the group of cells transfected with siR-LOC105374848, and the group of cells transfected with a negative control. The siRNA transfection was performed in a 96-well plate according to the method described above, and MTT solution (5 mg. multidot.mL) was added at 24h, 48h, 72h and 96h of transfection^-1) After 20. mu.L of the culture solution was incubated in the incubator for 4 hours, the culture solution in each well was carefully aspirated and 200. mu.L of DMSO was added to each well, and the mixture was allowed to stand at room temperature and shaken for 20min to sufficiently dissolve the crystals and mix them well. The microplate reader measures the optical density value (A value) of each well at 490nm wavelength, the A value represents the proliferation of cells, and the stronger the cell proliferation ability is inhibited, the smaller the A value.

6. ALP (alkaline phosphatase) Activity assay

ALP activity was measured by PNPP azo method.

The experiment was divided into 2 groups, the group of cells transfected with siR-LOC105374848, and the group of cells transfected with a negative control. siRNA transfection was performed in 96-well plates as described above, and 48h after transfection, the plates were washed 2 times with PBS and added with 0.2% Triton X-100 overnight at 4 ℃. And (3) incubating 40 mu L of cell lysate and 100 mu L of reaction substrate for 30min at 37 ℃ in a dark place, adding 0.4mol/L NaOH100 mu L to terminate the reaction, measuring the A value by using a microplate reader at the wavelength of 405nm, and reading the enzyme activity value on an ALP standard curve according to the A value of the sample.

7. Statistical method

Statistical analysis was performed using SPSS15.0 statistical software and the data obtained from the experiment are expressed as mean. + -. standard deviation (x. + -.s). Adopting one-way anova, carrying out statistical analysis by using an SNK method when the variance is homogeneous, and carrying out statistical analysis by using a Dunnett's T3 method when the variance is heterogeneous. Differences of P <0.05 were statistically significant.

8. Results of the experiment

8.1 interference Effect detection

The expression level of LOC105374848 in the negative control group is set to be 1, the relative expression level of LOC105374848 in the siR-LOC105374848 group is 0.357 plus or minus 0.055, P is less than 0.05, and the difference has statistical significance.

8.2 cell proliferation assay

The results are shown in figure 2, the proliferation capacity of the cells in the siR-LOC105374848 group is obviously enhanced, the P is less than 0.05, and the difference has statistical significance.

8.3ALP Activity assay

The results are shown in fig. 3, where the ALP activity of the siR-LOC105374848 group was significantly enhanced with P <0.05, and the difference was statistically significant.

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.

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Claims

1. The application of the reagent for detecting the level of nucleic acid molecules in preparing products for diagnosing orthopedic diseases, wherein the nucleic acid molecules are LOC105374848, and the orthopedic diseases are male osteoporosis.

2. The use according to claim 1, wherein the agent comprises: the reagents used by LOC105374848 are detected by reverse transcription PCR, real-time quantitative PCR, in situ hybridization or microarray chip.

3. The use according to claim 2, characterized in that said reagents for detecting LOC105374848 by real-time quantitative PCR comprise at least one pair of primers for the specific amplification of LOC 105374848.

4. The use according to claim 3, wherein the primer sequence for specific amplification of the LOC105374848 gene is shown in SEQ ID NO.1 and SEQ ID NO. 2.

5. The use of claim 1, wherein the product comprises a chip or a kit.

6. The use according to claim 1, wherein the product is diagnostic of male osteoporosis by detecting the level of LOC105374848 in a sample.

7. The use of claim 6, wherein the sample source is blood.

8. The medicine for treating the male osteoporosis is characterized by comprising a substance for inhibiting the expression of LOC105374848, wherein the substance is siRNA aiming at LOC105374848, and the sequences of the siRNA are shown as SEQ ID No.5 and SEQ ID No. 6.

9. The application of a substance inhibiting the expression of LOC105374848 in preparing a medicine for treating male osteoporosis is characterized in that the substance is siRNA aiming at LOC105374848, and the sequence of the siRNA is shown as SEQ ID No.5 and SEQ ID No. 6.