CN110923313B

CN110923313B - Detection of LncRNA and application thereof in osteoporosis diagnosis and treatment

Info

Publication number: CN110923313B
Application number: CN201911369287.4A
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: 2019-12-26
Filing date: 2019-12-26
Publication date: 2021-06-25
Anticipated expiration: 2039-12-26
Also published as: CN110923313A

Abstract

The invention discloses detection of LncRNA and application of the LncRNA in osteoporosis diagnosis and treatment, wherein the LncRNA is LOC 643072. The invention discovers that the expression of LOC643072 is obviously up-regulated in osteoporosis for the first time, and the expression level of LOC643072 can be detected to effectively distinguish osteoporosis patients from normal controls. And simultaneously, LOC643072 can be used as a potential molecular target to be applied to the treatment of osteoporosis.

Description

Detection of LncRNA and application thereof in osteoporosis diagnosis and treatment

Technical Field

The invention belongs to the field of biological medicine, and relates to detection of lncRNA and application of lncRNA in osteoporosis diagnosis and treatment, wherein the lncRNA is LOC 643072.

Background

Osteoporosis is one of the most common age-related diseases and also one of the leading causes of fractures. There are currently many international standards for the assessment and diagnosis of this disease, but there are several details that require further refinement and improvement. Research on methods for preventing and treating osteoporosis has also progressed. However, since the pathogenesis of osteoporosis still needs to be further studied, and the current mechanistic exploration remains in the basic research level, the development of new therapeutic approaches is limited. At present, the pathogenesis of osteoporosis is deeply elucidated, the key target point of osteoporosis is discovered, a reliable theoretical basis is provided for the application of osteoporosis in clinical treatment, and a foundation is provided for the conversion of osteoporosis into clinical application.

Long non-coding RNAs (1ncRNAs) are RNA molecules with transcript lengths greater than 200bp that do not encode proteins. As research progresses, 1ncRNA plays an important role in body development and disease development, is involved in the regulation and control of various biological processes at epigenetic, transcriptional and post-transcriptional levels, and has close connection with disease occurrence, development and prevention (Fatica A, Bozzoni I.Long non-coding RNAs: new planes in cell differentiation and development, 2014nat Rev. Gene.15 (1): 7-21.). The 1ncRNA is similar to mRNA in structure, and different from the coding gene, the expression of the 1ncRNA has tissue specificity and space-time property, the expression quantity of the same 1ncRNA in different tissues is different, and the expression quantity of the same 1ncRNA in different biological processes is changed even in the same tissue. Related studies have in recent years reported that 1ncRNA has an important role in orthopedic diseases, including knee osteoarthritis, rheumatoid arthritis, osteoporosis, bone tumors, and the like (Xie Z, Li J, Wang P, et al. differential Expression Profiles of Long non-coding RNA and mRNA of osteoprogenic v Differentiated mental Stem Cells in orthopedic stress. J Rheumatous. 2016.43(8): 1523-31.).

Long non-coding RNA is used as the main transcript of the gene, and the direction is guided for the deep research of disease mechanisms. The invention deeply discusses the expression condition of lncRNA in osteoporosis for the first time and the influence of lncRNA on osteoblasts, provides a solid foundation and a brand-new insight for the deep research of osteoporosis diseases, and provides a new molecular target and an important theoretical basis for clinically preventing and treating osteoporosis diseases.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide a biomarker for osteoporosis in order to achieve a specific diagnosis, prevention and/or treatment of osteoporosis.

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

the invention provides an application of a reagent for detecting LOC643072 in preparing a product for diagnosing osteoporosis.

Further, the product comprises a reagent for detecting the expression level of LOC643072 gene in a sample by a sequencing technology, a nucleic acid hybridization technology and a nucleic acid amplification technology.

The invention provides a product for diagnosing osteoporosis, which comprises a chip, a preparation, a kit or a nucleic acid membrane strip, wherein the product comprises a reagent for detecting the expression level of LOC643072 gene in a sample.

Further, the reagent comprises:

a probe that specifically recognizes LOC 643072; or

Primer for specific amplification of LOC 643072.

Further, the primer sequence of the specific amplification LOC643072 is shown in SEQ ID NO. 1-2.

The invention provides application of LOC643072 in construction of a calculation model for predicting osteoporosis.

The invention provides application of LOC643072 in preparation of a medicine for treating osteoporosis.

Further, the medicament comprises an inhibitor of LOC 643072.

Further, the inhibitor is siRNA.

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

The invention provides a medicine for treating osteoporosis, which comprises an inhibitor of LOC 643072.

Further, the inhibitor is siRNA.

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

Detailed Description

LOC643072 in the invention is located on the zone 4 of the No. 2 staining long arm 2 of human, and LOC643072 in the invention comprises a wild type, a mutant type or a fragment thereof. In the examples of the present invention, the nucleotide sequence of a representative human LOC643072 gene is shown in the current International public nucleic acid database GeneBank LOC643072 gene (NR _ 110587.1). The full-length sequence of LOC643072 nucleotide or its fragment can be obtained by PCR amplification, recombination or artificial synthesis.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level.

Detection techniques

The lncrnas of the invention are detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

Nucleic acid hybridization techniques of the invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

The nucleic acid amplification technique of the invention is selected from the group consisting of Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Chip, preparation, nucleic acid membrane strip and kit

The invention provides products for detecting the expression level of LOC643072 gene in the test, and the products comprise (but are not limited to) chips, preparations, nucleic acid membrane strips or kits. Wherein the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown by LOC 643072.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

The term "probe" refers to a molecule that binds 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.

Exemplary probes in the present invention include PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.

In the present invention, a nucleic acid membrane strip comprises a substrate and oligonucleotide probes immobilized on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

The invention provides a kit which can be used for detecting the expression of LOC 643072. The kit comprises a specific primer pair for amplifying LOC 643072; a standard DNA template; and (3) PCR reaction liquid. In a preferred embodiment, the specific primer pair comprises an upstream primer and a downstream primer, and the sequences are shown as SEQ ID NO. 1-2.

In a more preferred embodiment, the PCR reaction solution in the kit is a fluorescent quantitative PCR reaction solution, and further comprises a fluorescent dye.

In a more preferred embodiment, the fluorescent quantitative PCR reaction solution comprises dNTP and Mg²⁺The fluorescent dye is SYBR Green II, and the Taq enzyme is hot start enzyme.

Inhibitors and pharmaceutical compositions

Based on the findings of the present invention, the present invention provides a pharmaceutical composition comprising an inhibitor of LOC 643072.

Said inhibitor of LOC643072 refers to any substance that can lower the level of LOC643072 gene, and these substances can be used in the present invention as a substance useful for down-regulating the expression of LOC643072 gene, thereby being useful for treating osteoporosis. For example, the inhibitor of the present invention may be an interfering molecule that uses LOC643072 as a target sequence and is capable of inhibiting LOC643072 gene, including: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

As a preferred mode of the invention, the inhibitor of LOC643072 is a LOC643072 specific small interfering RNA molecule. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, which is the RNA interference (RNA interference) process. Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

As an alternative of the present invention, the inhibitor of LOC643072 may be a "Small hairpin RNA (shRNA)" which is a non-coding Small RNA molecule capable of forming a hairpin structure, and the Small hairpin RNA can inhibit gene expression through an RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.

Statistical analysis

In the specific embodiment of the present invention, the experiments were performed by repeating at least 3 times, the data of the results are expressed as mean ± standard deviation, and the statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is considered to have statistical significance by using t test when P is less than 0.05.

The invention has the advantages and beneficial effects that:

the invention firstly discovers that a molecular marker LOC643072 is related to the occurrence and development of osteoporosis, diagnoses whether a patient has the osteoporosis and risks of the osteoporosis by detecting the change of LOC643072 of a subject, and simultaneously verifies that the targeted LOC643072 can change the cell proliferation activity, thereby providing a molecular means for the diagnosis and treatment of the osteoporosis.

Drawings

FIG. 1 is a graph showing the expression of LOC643072 in osteoblasts;

fig. 2 is a diagram of the interference situation of LOC 643072.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 QPCR detection of LOC643072 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 LOC643072 gene and GAPDH gene in Genebank, and specific primer sequences are shown in Table 1.

TABLE 1 specific primer sequences

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

TABLE 2 PCR reaction System

3) And (3) PCR reaction conditions: : 95 ℃ for 15min, (95 ℃ for 10s, 55 ℃ for 30s, 72 ℃ for 30s) x 42 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

LOC643072 gene was significantly up-regulated in osteoporosis patients with statistical significance (P <0.05) compared to the control group, where LOC643072 was up-regulated about 2.67-fold and up-regulated about 3.57-fold in the tissues in the blood samples.

The expression of LOC643072 in the blood samples was up-regulated in 44 samples, and was not significantly different in 36 samples, and the expression of LOC643072 in the tissue samples was up-regulated in 47 samples, and was not significantly different in 33 samples, as shown in table 3.

TABLE 3 Positive in disease

Example 2 expression of LOC643072 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 bone with physiological saline, after washing liquid is clarified, washing and shaking with PBS liquid for 3 times, and shearing femoral neck into 1mm with sterilized rongeur and scissors³The 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. 1, the expression of LOC643072 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 LOC643072 on osteoblasts

1. Design and synthesis of siRNA directed against LOC643072, siRNA-LOC643072 and general negative control siRNA-NC were purchased from Shanghai Jima Genenco chemical technology, Inc., and the sequences of interfering RNAs directed against LOC643072 are shown in Table 4.

TABLE 4 sequences of siRNA against LOC643072

2. Cell culture

Con group and OP group bone cells were cultured to logarithmic growth phase, and concentration measurement and dilution were performed, and 24h before transfection, logarithmic phase cells were cultured at 2X 10⁵The 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. 2, compared to the control group (OP) not transfected, the expression level of LOC643072 was not significantly changed in siRNA-NC-transfected osteoporotic osteoblasts, whereas the expression level of LOC643072 was significantly decreased in siRNA-1-transfected and siRNA 2-transfected osteoporotic osteoblasts, the difference was statistically significant (P <0.05), wherein siRNA1 was able to more effectively inhibit the expression of LOC643072, 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 mu l of MTT (5mg/m1) into each well, placing the well in an incubator for further culturing for 4h, carefully discarding the supernatant, adding 150 mu l of dimethyl sulfoxide, placing the well on a horizontal oscillator for oscillation for 10min to fully dissolve crystals, placing a 96-well plate in an enzyme-linked immunosorbent assay (ELISA) detector, selecting a 490nm wavelength, adjusting a blank well to be zero, detecting the absorbance value of each well and recording the result.

As shown in table 5, the cell proliferation was more significant in the experimental group of osteoporotic osteoblasts transfected with siRNA1 (group 3) compared to the control group of osteoporotic osteoblasts (group 2), and there was no significant difference in the experimental group of osteoporotic osteoblasts transfected with siRNA1 compared to the normal control group (group 1).

TABLE 5 determination of OD values in osteoblast MTT assay

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

1. Application of a reagent for detecting LOC643072 in preparing a product for diagnosing osteoporosis.

2. The use according to claim 1, wherein said product comprises reagents for detecting the expression level of LOC643072 gene in a sample by sequencing, nucleic acid hybridization or nucleic acid amplification techniques.

3. Use according to claim 1 or 2, wherein the product comprises a chip, a preparation, a kit or a nucleic acid membrane strip.

4. The use according to claim 2, wherein the agent comprises:

a probe that specifically recognizes LOC 643072; or

Primer for specific amplification of LOC 643072.

5. The use according to claim 4, wherein the primer sequence for specific amplification of LOC643072 is shown in SEQ ID No. 1-2.

Application of LOC643072 in building a calculation model for predicting osteoporosis.

7. Use of an inhibitor of LOC643072 in the manufacture of a medicament for the treatment of osteoporosis, wherein said inhibitor is siRNA.

8. The use of claim 7, wherein the siRNA has a sequence as shown in SEQ ID No. 5-8.

9. A medicament for treating osteoporosis, comprising an inhibitor of LOC643072, wherein the inhibitor is siRNA.

10. The drug of claim 9, wherein the siRNA has a sequence as shown in SEQ ID No. 5-8.