CN107043811B - Application of the CFAP20 genes in diagnosis of osteoporosis - Google Patents

Abstract

The invention discloses that the CFAP20 gene can be used as a molecular marker for early diagnosis of osteoporosis. The present invention uses high-throughput sequencing and QPCR experiments to study differentially expressed genes in patients with osteoporosis. The research results of the present invention show that whether a subject suffers from osteoporosis can be judged by detecting the CFAP20 gene expression in blood, and the detection method can be used for early diagnosis of osteoporosis, and has the characteristics of timeliness and accuracy.

Description

The application of CFAP20 gene in the diagnosis of osteoporosis

technical field

The invention belongs to the field of molecular diagnosis, and relates to a molecular marker for diagnosing osteoporosis, in particular to the application of the molecular marker-CFAP20 gene in blood in the preparation of products for diagnosing osteoporosis.

Background technique

Osteoporosis is a common metabolic bone disease characterized by decreased bone mass, destruction of bone microarchitecture, increased bone fragility, and susceptibility to fracture. It is caused by excessive bone resorption due to increased/over-activated osteoclasts, or decreased bone formation caused by osteoblast reduction/functional defects, resulting in excessive bone loss, and is one of the most important diseases that harm middle-aged and elderly people .

The detection of osteoporosis includes the detection of laboratory examination indicators and auxiliary detection.

Laboratory inspection indicators:

Some serum biochemical indicators in patients with osteoporosis can reflect the state of bone turnover (including bone formation and bone resorption). In the state of high bone turnover (such as type I osteoporosis), these indicators can be increased, and can also be used for Monitor for early response to treatment. However, its clinical significance in osteoporosis remains to be further studied. These biochemical measurements include: bone-specific alkaline phosphatase (response to bone formation), tartrated resistant acid phosphatase (response to bone resorption), osteocalcin (response to bone formation) ), type I procollagenpeptidase (response to bone formation), urinary pyridinoline (Urinary pyridinoline) and deoxypyridinoline (Urinary deoxypyridinoline, response to bone resorption), type I collagen N-C-terminal cross-linked peptide (cross- linked N-and C-telopeptide of type I collagen, in response to bone resorption). As mentioned earlier, detection of osteoporosis using biochemical markers is not precise enough.

Auxiliary examination: including bone imaging examination and bone density detection. The objects of auxiliary examination are generally patients with advanced osteoporosis, and cannot be used for the screening of patients with early osteoporosis.

Based on the limitations of the methods for detecting osteoporosis in the prior art, finding specific molecular markers related to the early diagnosis and prognosis of osteoporosis has far-reaching significance for the early diagnosis and individualized treatment of osteoporosis.

Contents of the invention

In order to make up for the deficiencies in the prior art, the purpose of the present invention is to provide a molecular marker that can be used for early diagnosis of osteoporosis (Osterarthritis, OA). The present invention proves through experiments that there are differences in the expression of CFAP20 gene in the blood of normal people and patients with osteoporosis, and thus it is believed that CFAP20 can be used as a marker for diagnosing osteoarthritis.

In order to achieve the above object, the present invention adopts the following technical solutions:

The invention provides an application of a product for detecting CFAP20 gene expression in preparing a tool for diagnosing osteoporosis.

Further, the above-mentioned products for detection of CFAP20 gene expression include: detection of CFAP20 gene expression by RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platform to diagnose osteoporosis .

Further, the product for diagnosing osteoporosis by RT-PCR includes at least one pair of primers for specific amplification of CFAP20 gene; the product for diagnosis of osteoporosis by real-time quantitative PCR includes at least one pair of primers for specific amplification of CFAP20 gene Primers; the product for diagnosing osteoporosis with immunoassay includes: an antibody that specifically binds to CFAP20 protein; the product for diagnosing osteoporosis with in situ hybridization includes: a probe that hybridizes with the nucleic acid sequence of CFAP20 gene The product for diagnosing osteoporosis with a chip includes: a protein chip and a gene chip; wherein, the protein chip includes an antibody that specifically binds to the CFAP20 protein, and the gene chip includes a probe that hybridizes with the nucleic acid sequence of the CFAP20 gene.

In a specific embodiment of the present invention, the product for diagnosing osteoporosis by real-time quantitative PCR at least includes the sequences of a pair of primers for specifically amplifying CFAP20 gene as shown in SEQ ID NO.3 and SEQ ID NO.4.

Preferably, the diagnostic tools include chips, kits, test strips or high-throughput sequencing platforms. Among them, the high-throughput sequencing platform is a special diagnostic tool, and products for detecting CFAP20 gene expression can be applied to this platform to detect the expression of CFAP20 gene. With the development of high-throughput sequencing technology, the construction of a person's gene expression profile will become a very convenient task. By comparing the gene expression profiles of disease patients and normal people, it is easy to analyze which gene abnormality is related to the disease. Therefore, it is known in high-throughput sequencing that the abnormality of the CFAP20 gene is related to osteoporosis, which also belongs to the use of the CFAP20 gene, and is also within the protection scope of the present invention.

The present invention also provides a tool for diagnosing osteoporosis, and the diagnosing tool includes a chip, a kit, a test strip, or a high-throughput sequencing platform.

Wherein, the chip includes a gene chip and a protein chip; the gene chip includes a solid phase carrier and an oligonucleotide probe immobilized on the solid phase carrier, and the oligonucleotide probe includes a gene chip for detecting CFAP20 gene transcription level. Oligonucleotide probes directed at the CFAP20 gene; the protein chip includes a solid phase carrier and a specific antibody to the CFAP20 protein immobilized on the solid phase carrier; the gene chip can be used to detect multiple genes including the CFAP20 gene (e.g., expression levels of multiple genes associated with osteoporosis). The protein chip can be used to detect the expression levels of multiple proteins including CFAP20 protein (for example, multiple proteins related to osteoporosis). By simultaneously detecting multiple markers of osteoporosis, the accuracy of osteoporosis diagnosis can be greatly improved.

Wherein, the kit includes a gene detection kit and a protein immune detection kit; the gene detection kit includes reagents for detecting the transcription level of CFAP20 gene; the protein immune detection kit includes a specific antibody for CFAP20 protein. Further, the reagents include reagents required in the process of detecting the expression level of CFAP20 gene by using RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization or chip method. Preferably, the reagents include primers and/or probes for the CFAP20 gene. Primers and probes that can be used to detect the expression level of CFAP20 gene can be easily designed according to the nucleotide sequence information of CFAP20 gene.

The probe hybridizing with the nucleic acid sequence of CFAP20 gene can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. The length of the probe is not limited, as long as it completes specific hybridization and specifically binds to the target nucleotide sequence, any length is acceptable. The probes can be as short as 25, 20, 15, 13 or 10 bases in length. Likewise, the probes can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and the longest is generally no more than 30 base pairs. The optimal length of complementarity is 15-25 base pairs. The self-complementary sequence of the probe is preferably less than 4 base pairs, so as not to affect the hybridization efficiency.

The high-throughput sequencing platform includes reagents for detecting the expression level of CFAP20 gene.

The test paper includes a test paper carrier and an oligonucleotide fixed on the test paper carrier, and the oligonucleotide can detect the transcription level of the CFAP20 gene.

Further, the specific antibody of CFAP20 protein includes monoclonal antibody and polyclonal antibody. The specific antibody of the CFAP20 protein includes the complete antibody molecule, any fragment or modification of the antibody (for example, chimeric antibody, scFv, Fab, F(ab') 2, Fv, etc. As long as the fragment can remain with CFAP20 Protein-binding ability is enough. Preparation of antibodies for protein level is well known to those skilled in the art, and any method can be used in the present invention to prepare the antibodies.

In a specific embodiment of the present invention, the primer sequence for the CFAP20 gene is as follows: the sequence of the forward primer is shown in SEQ ID NO.3, and the sequence of the reverse primer is shown in SEQ ID NO.4.

Sources of the CFAP20 gene and its expression products used for diagnosing osteoporosis include but are not limited to blood, tissue fluid, urine, saliva, spinal fluid, and other body fluids from which genomic DNA can be obtained. In a specific embodiment of the present invention, the source of the CFAP20 gene and its expression product used for diagnosing osteoporosis is blood.

In the context of the present invention, "CFAP20 gene" includes polynucleotides of the CFAP20 gene as well as any functional equivalents of the CFAP20 gene. The CFAP20 gene includes a DNA sequence that has more than 70% homology with the DNA sequence of the CFAP20 gene (NC_000016.10) in the current international public nucleic acid sequence database GeneBank, and encodes the same functional protein;

Preferably, the coding sequence of the CFAP20 gene includes any one of the following DNA molecules:

(1) the DNA sequence shown in SEQ ID NO.1 in the sequence listing;

(2) A DNA sequence that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes the same functional protein;

(3) A DNA molecule having 70%, preferably more than 90%, homology with the DNA sequence defined in (1) or (2), and encoding the same functional protein.

In a specific embodiment of the present invention, the coding sequence of the CFAP20 gene is the DNA sequence shown in SEQ ID NO.1.

In the context of the present invention, CFAP20 gene expression products include CFAP20 protein and partial peptides of CFAP20 protein. The partial peptide of the CFAP20 protein contains functional domains related to osteoporosis.

"CFAP20 protein" includes CFAP20 protein and any functional equivalents of CFAP20 protein. The functional equivalents include CFAP20 protein conservative variant proteins, or active fragments thereof, or active derivatives thereof, allelic variants, natural mutants, induced mutants, DNA capable of binding to CFAP20 under high or low stringent conditions The protein encoded by the hybridized DNA.

Preferably, the CFAP20 protein is a protein having the following amino acid sequence:

(1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

(2) The amino acid sequence shown in SEQ ID NO.2 is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as the amino acid sequence shown in SEQ ID NO.2. A protein derived from the amino acid sequence shown in ID NO.2. The number of substituted, deleted or added amino acids is usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10.

(3) having at least 80% homology (also known as sequence identity) with the amino acid sequence shown in SEQ ID NO.2, more preferably at least about 90% to the amino acid sequence shown in SEQ ID NO.2 95% homology, usually 96%, 97%, 98%, 99% homology of amino acid sequence polypeptide.

In a specific embodiment of the present invention, the CFAP20 protein is a protein having the amino acid sequence shown in SEQ ID NO.2.

In general, it is known that the modification of one or more amino acids in a protein does not affect the function of the protein. Those skilled in the art will recognize that changes to single amino acids or small percentages of amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications where changes to a protein result in a protein with similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.

An example of a protein modified by adding an amino acid or amino acid residues is a fusion protein of CFAP20 protein. There is no limitation on the peptide or protein fused with CFAP20 protein, as long as the resulting fusion protein retains the biological activity of CFAP20 protein.

The CFAP20 protein of the present invention also includes non-conservative modifications to the amino acid sequence shown in SEQ ID NO.2, as long as the modified protein can still retain the biological activity of the CFAP20 protein. The number of amino acids mutated in such modified proteins is usually 10 or less, such as 6 or less, such as 3 or less.

The present invention also provides a method for diagnosing osteoporosis, said method comprising the steps of:

(1) Obtain samples from subjects;

(2) Detect the expression level of CFAP20 gene or protein in the subject sample;

(3) Correlating the measured expression level of CFAP20 gene or protein with the subject's disease.

(4) If the expression level of CFAP20 gene or protein is increased compared with normal subjects, then the subject is judged to have a tendency to suffer from osteoporosis, or has already suffered from osteoporosis.

In the context of the present invention, "diagnosing osteoporosis" includes judging whether the subject already suffers from osteoporosis, and also includes judging whether the subject is at risk of suffering from osteoporosis.

Advantages and beneficial effects of the present invention:

The present invention discovers for the first time that the expression of CFAP20 gene is related to osteoporosis, and by detecting the expression of CFAP20 in the subject, it can be judged whether the subject suffers from osteoporosis, or whether the subject suffers from osteoporosis The risk of the disease, so as to guide the clinician to provide the subject with the preventive plan or the treatment plan.

The present invention discovers a new molecular marker-CFAP20 gene. Compared with traditional detection methods, gene diagnosis is more timely, specific and sensitive, and can realize early diagnosis of osteoporosis, thereby reducing the risk of osteoporosis. mortality rate.

Description of drawings

Figure 1 shows the difference in expression of CFAP20 gene detected by high-throughput sequencing between osteoporosis patients and normal people;

Figure 2 shows the difference in expression of CFAP20 gene detected by QPCR between osteoporosis patients and normal people.

specific implementation

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the embodiment, usually according to conventional conditions, such as people such as Sambrook, molecular cloning: the condition described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer's suggestion condition.

Example 1 Screening of genes differentially expressed in patients with osteoporosis and normal people

1. Research object:

Osteoporosis group: Randomly select 10 patients with primary osteoporosis from the orthopedics department of the hospital, aged 50-75 years.

Inclusion criteria: in line with the "Recommended Diagnostic Criteria for Osteoporosis in China" (Second Draft). No obvious heart, liver, kidney, pulmonary insufficiency, no various endocrine diseases causing secondary osteoporosis, no tumor, diabetes and other serious diseases that interfere with bone metabolism.

Normal group: 10 healthy volunteers aged 50-75 were selected.

There was no significant difference in age and gender between the two groups (P>0.10), which were comparable.

All research subjects were informed about this study and signed the informed consent.

2. Extraction of total RNA in blood

Total RNA in blood leukocytes was extracted using TRIzol according to conventional methods.

3. RNA concentration and purity determination

NanoDrop1000 spectrophotometer detects RNA samples, and the sample requirements for RNA-seq sequencing: OD260/OD280 is 1.8-2.2.

4. Quality analysis of RNA samples (Agilent Technologies 2100Bioanalyzer)

Agilent Technologies 2100Bioanalyzer detects the quality of RNA samples, observes that the main bands of 28S rRNA and 18SrRNA are obvious, there is no degradation, the RNA integrity index is qualified, and the concentration meets the requirements for sequencing cDNA library construction, which can be used for library construction and sequencing.

5. High-throughput transcriptome sequencing

(1) RNA-seq read mapping

First remove the low-quality reads to get clean reads, and then use TopHat v1.3.1 to match the clean fragments with the UCSC H. sapiens reference genome (hg19), the pre-built index of H. sapiens UCSC hg19 version is downloaded from the TopHat homepage , and as a reference genome, when using TopHat to match the genome, each read (default to 20) is allowed to have multiple matching sites and a maximum of 2 mismatches. TopHat builds a library of possible splicing sites based on exon regions and GT-AG splicing signals, and maps reads that have not mapped to the genome to the genome based on these splicing site libraries. We use the system default parameters of the TopHat method.

(2) Transcript abundance assessment

The matched read files were processed by Cufflinks v1.0.3, and Cufflinks v1.0.3 normalized the number of RNA-seq fragments to calculate the relative abundance of transcripts. The FPKM value refers to the number of fragments matching the 1 kb exon region of a specific gene per million sequenced fragments. Confidence intervals for FPKM estimates were computed by Bayesian inference methods. The reference GTF annotation file used by Cufflinks was downloaded from the Ensembl database (Homo_sapiens.GRCh37.63.gtf).

(3) Detection of differentially expressed genes

Transfer the downloaded Ensembl GTF file and the original file matched by TopHat to Cuffdiff, and Cuffdiff uses the original matched file to re-estimate the expression abundance of the transcripts listed in the GTF file and detect differential expression. Only q-values < 0.01 in the Cuffidff output where the test showed successful comparisons were considered differentially expressed.

6. Results

The results of RNA-seq showed (as shown in Figure 1) that compared with normal people, the mRNA level of CFAP20 gene in the blood of osteoporosis patients was significantly increased, and the difference was statistically significant (P<0.05).

Example 2 QPCR experiment verification of genes differentially expressed in patients with osteoporosis and normal people

1. Research object:

According to the method of Example 1, 50 cases of osteoporosis patients and 50 normal persons were collected.

2. Extraction of total RNA in blood

Method is with embodiment 1.

3. Reverse transcription

1 μg of total RNA was reverse-transcribed to synthesize cDNA using reverse transcription buffer. Use 25μl reaction system, take 1μg total RNA for each sample as template RNA, add the following components to the PCR tube respectively: DEPC water, 5× reverse transcription buffer, 10mmol/L dNTP, 0.1mmol/l DTT, 30μmmol/l Oligo dT, 200 U/μl M-MLV, template RNA. Incubate at 42°C for 1h, then centrifuge briefly at 72°C for 10min.

4. QPCR

(1) Primer design

QPCR amplification primers were designed according to the coding sequences of CFAP20 gene and GAPDH gene in Genbank, and synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The specific primer sequences are as follows:

CFAP20 gene:

The forward primer is 5'-ATACGGCACCAATTACAT-3' (SEQ ID NO.3);

The reverse primer is 5'-TAGAGTCTGTCTGAGAAGTA-3' (SEQ ID NO.4),

GAPDH gene:

The forward primer is 5'-TTTAACTCTGGTAAAGTGGATAT-3' (SEQ ID NO.5);

The reverse primer is 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO.6).

(2) Prepare the PCR reaction system according to Table 1:

Wherein, SYBR Green polymerase chain reaction system was purchased from Invitrogen Company.

Table 1 PCR reaction system

Reagent volume forward primer 1μl reverse primer 1μl SYBR Green polymerase chain reaction system 12.5μl template 2μl Deionized water Make up 25μl

(3) PCR reaction conditions: 95°C for 10 min, (95°C for 15 s, 60°C for 50 s)*45 cycles. Using SYBR Green as a fluorescent marker, the PCR reaction was carried out on a Light Cycler fluorescence quantitative PCR instrument, the target band was determined by melting curve analysis and electrophoresis, and the relative quantification was carried out by the ΔΔCT method.

5. Statistical methods

The results and data are expressed in the form of mean ± standard deviation, and SPSS13.0 statistical software is used for statistical analysis. The difference between the two is tested by t test, and it is considered statistically significant when P<0.05.

6. Results

The results are shown in Figure 2. Compared with normal people, the mRNA level of CFAP20 gene in the blood of osteoporosis patients was significantly increased, and the difference was statistically significant (P<0.05). The results were the same as the RNA-seq experiment.

The preparation of embodiment 3 osteoporosis diagnostic kits

According to the correlation between the CFAP20 gene and osteoporosis, the osteoporosis can be diagnosed by detecting the expression of the CFAP20 gene. Accordingly, the present invention provides a kit for diagnosing osteoporosis based on detecting the expression of the CFAP20 gene. The components in the diagnostic kit are as follows: a SYBR Green polymerase chain reaction system; a pair of primers for amplifying the CFAP20 gene and the GAPDH gene. The forward primer sequence for amplifying CFAP20 gene is 5'-ATACGGCACCAATTACAT-3', the reverse primer sequence is 5'-TAGAGTCTGTCTGAGAAGTA-3'; the forward primer sequence for amplifying GAPDH is 5'-TTTAACTCTGGTAAAGTGGATAT-3', reverse The primer sequence was 5'-GGTGGAATCATATTGGAACA-3'. SYBRGreen polymerase chain reaction system contains PCR buffer, dNTPs, SYBR Green fluorescent dye. The PCR buffer composition is: 25mM KCl, 2.5mM MgCl ₂ , 200mM (NH ₄ ) ₂ SO ₄ .

The description of the above embodiments is only for understanding the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

SEQUENCE LISTING

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Claims (9)

1. Application of the product for detecting the mRNA level of CFAP20 gene in blood in the preparation of a tool for diagnosing osteoporosis. 2. The application according to claim 1, characterized in that the product comprises: detecting the mRNA level of CFAP20 gene by RT-PCR, real-time quantitative PCR, in situ hybridization, or high-throughput sequencing platform to diagnose osteoporosis disease products. 3. application according to claim 2, is characterized in that, the described product that diagnoses osteoporosis with RT-PCR comprises at least a pair of primers of specific amplification CFAP20 gene; Described with real-time quantitative PCR diagnosis osteoporosis Syndrome products include at least one pair of primers for specifically amplifying the CFAP20 gene. 4. application according to claim 3, is characterized in that, the described product that diagnoses osteoporosis with real-time quantitative PCR comprises at least a pair of primers of specific amplification CFAP20 gene such as SEQ ID NO.3 and SEQ ID NO .4 shown. 5. A tool for diagnosing osteoporosis, characterized in that the tool can diagnose osteoporosis by detecting the mRNA level of CFAP20 gene in blood. 6. The tool according to claim 5, characterized in that the tool comprises a gene chip, a gene detection kit, a test strip or a high-throughput sequencing platform. 7. The tool according to claim 6, wherein the gene chip comprises a solid phase carrier and an oligonucleotide probe immobilized on the solid phase carrier, and the oligonucleotide probe comprises a solid phase carrier for detecting CFAP20 Oligonucleotide probes directed at the CFAP20 gene at the gene transcription level; the gene detection kit includes reagents for detecting the CFAP20 gene transcription level; the test paper includes reagents for detecting the CFAP20 gene transcription level; the Qualcomm The quantitative sequencing platform includes reagents for detecting the transcription level of CFAP20 gene. 8. The tool according to claim 7, wherein the reagent for detecting the transcription level of CFAP20 gene comprises primers and/or probes for CFAP20 gene. 9 . The tool according to claim 8 , wherein the primer sequences for the CFAP20 gene are as follows: the sequence of the forward primer is shown in SEQ ID NO.3, and the sequence of the reverse primer is shown in SEQ ID NO.4.