CN111621562A

CN111621562A - Application of non-coding RNA in preparation of osteoarthritis early diagnosis and detection product

Info

Publication number: CN111621562A
Application number: CN202010551605.5A
Authority: CN
Inventors: 李恒; 闵继康; 唐慧斌; 袁永健; 豆晓杰; 翁伟
Original assignee: First Peoples Hospital of Huzhou
Current assignee: First Peoples Hospital of Huzhou
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2020-09-04
Anticipated expiration: 2040-06-17
Also published as: CN111621562B

Abstract

The invention discloses application of non-coding RNA in preparation of an osteoarthritis early diagnosis detection product, and particularly relates to NONHSAT016936.2, DANCR and miR-146a-3p genes of the non-coding RNA.

Description

Application of non-coding RNA in preparation of osteoarthritis early diagnosis and detection product

Technical Field

The invention relates to the technical field of biological medicines, and relates to application of non-coding RNA in preparation of an osteoarthritis early diagnosis product, in particular to NONHSAT016936.2, DANCR or miR-146a-3p gene.

Background

Osteoarthritis is a degenerative disease, which is caused by age, obesity, strain, trauma and other factors, such as articular cartilage degeneration and injury, joint margin and subchondral bone reactive hyperplasia, also known as osteoarthropathy, degenerative arthritis and the like, and commonly occurs in middle-aged and elderly people. The main symptom of osteoarthritis is joint pain, which often occurs in the morning, and pain is reduced after activity, but may be exacerbated if there is excessive activity.

At present, the clinical diagnosis of osteoarthritis mainly depends on X-ray examination, but the detection of the irreversible structural damage of the osteoarthritis in the late stage is not suitable for the early diagnosis of the osteoarthritis.

Disclosure of Invention

The invention mainly aims to provide an application of an osteoarthritis early diagnosis and detection product, which has important significance in that patients with osteoarthritis early can be found in time and adopt corresponding treatment modes, and the course of disease of the patients can be slowed down.

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

the application of non-coding RNA in preparing osteoarthritis early diagnosis and detection products, wherein the non-coding RNA is NONHSAT016936.2, DANCR or miR-146a-3p gene.

Preferably, the detection product is used for detecting the expression level of NONHSAT016936.2, DANCR or miR-146a-3p gene in a sample by reverse transcription PCR, fluorescent quantitative PCR, in situ hybridization, a chip or a high-throughput sequencing platform.

Preferably, the test product comprises primers and/or probes capable of specifically amplifying the NONHSAT016936.2, DANCR or miR-146a-3p gene.

Preferably, the primer pair sequence for specifically amplifying the NONHSAT016936.2 gene is as follows:

SQ1：5'–CGCCCAGGAACATGGTAACAC-3'，

SQ2：5'-GCCAAGGCTGTGACACAAGAG-3'。

preferably, the primer pair sequence for specifically amplifying the DANCR gene is

SQ3：5'-AGCGCAGGTTGACAACTACAG-3'

Or the combination of 5'-AGCGCAGAGTTTCATCACCTC-3', or 5'-AGCGCAGAGTTTCATCACCTC-3',

：5'-CTGCAGCTTGGGTGTGTATTC-3'。

preferably, the sequence of the primer pair for specifically amplifying the miR-146a-3p gene is as follows:

SQ5：5'-GACGGGCCTCTGAAATTCAG-3'，SQ6：5'-CAGTGCAGGGTCCGAGGTAT-3'。

preferably, the sample is peripheral blood.

Preferably, the detection product comprises a chip, a kit, a test strip or a high-throughput sequencing platform.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses non-coding genes NONHSAT016936.2, DANCR or miR-146a-3p related to osteoarthritis, and finds that when the expression level of the NONHSAT016936.2 gene is high and the expression levels of the DANCR and miR-146a-3p gene are low, a sample has higher risk of suffering from osteoarthritis, the expression level of the NONHSAT016936.2 gene is gradually increased along with the increase of the inflammation degree of the osteoarthritis, and the expression levels of the DANCR and miR-146a-3p gene are obviously reduced, so that the invention has important significance for early diagnosis of the osteoarthritis.

The foregoing is only an overview of the technical solutions of the present invention, and in order to more clearly understand the technical solutions of the present invention, the present invention is further described below with reference to the accompanying drawings.

Drawings

FIG. 1 is a graph showing peripheral blood expression of NONHSAT016936.2, DANCR and miR-146a-3p genes in a healthy control group and an OA patient group.

Detailed Description

For understanding the present invention, the present invention will be further explained with reference to the drawings and examples. The experimental procedures, for which specific conditions are not indicated in the examples, are carried out according to conventional rules, such as those described in Sambrook et al, handbook of molecular cloning, or according to conditions recommended by the manufacturer of the reagents or products.

Example 1 clinical case selection

Putting into a group standard:

1: the age is 45-70 years old;

2: the diagnosis is that the knee osteoarthritis patients or the patients who complain knee pain for more than 3 months;

3: signing the informed consent.

Exclusion criteria:

1: during the treatment period, blood and joint fluid samples are not taken on time according to the requirements of the research;

2: those whose joint is affected by the complication;

3: patients with serious diseases such as heart, brain, liver, kidney and hematopoietic system;

4: pregnant, women in preparation for pregnancy or lactation, psychiatric patients, etc.; patients with scarred skin;

5: patients who are participating in other clinical trials;

6: suspected or confirmed history of alcohol, drug abuse;

7: according to the judgment of researchers, other diseases or conditions which reduce the possibility of groups or complicate the use groups, such as the condition that the working environment changes frequently and the living environment is unstable and is easy to cause missed visits, are provided.

Thirdly, grouping the selected cases according to diagnosis classification standards formulated by the international osteoarthritis society:

1: 36 cases of healthy control group;

2: 115 cases of patients with OA of knee joint were confirmed;

115 patients with knee joint OA confirmed by X-ray film;

3: high risk group 42 cases

Mainly complain of more than 3 months of knee pain without obvious change of imaging, and excludes OA patients with other related diseases such as rheumatism and rheumatoid disease.

EXAMPLE 2 preparation of RNA samples

Separating mononuclear cells from peripheral blood:

1ml of antecubital venous blood is extracted from 36 healthy control groups and 115 OA patient groups, placed in a heparin anticoagulation tube, shaken up, and diluted by 1-2 times by using PH7.2-7.6Hank's solution;

taking 3-4 ml of LTS1077 ficoll-diatrizoate layered liquid, and putting the layered liquid into a 15 x 150mm test tube;

sucking the diluted blood by a capillary tube, slowly adding the diluted blood along the wall of the test tube at a position 1cm away from the layering liquid to ensure that the diluted blood is overlapped on the layering liquid, wherein the volume ratio of the diluted blood to the layering liquid is about 2: 1;

centrifuging at 2000rpm for 30min with horizontal centrifuge, dividing the tube content into 3 layers, the upper layer is plasma and Hank's solution, the middle layer is stratified solution, and the bottom layer is red blood cells and polymorphonuclear leukocytes.

A milky turbid mononuclear cell layer (white cloud layer narrow band) mainly comprising mononuclear cells can be seen at the interface of the upper layer and the middle layer of liquid, capillary vessels are gently inserted into the cloud layer, the mononuclear cells at the interface layer are absorbed along the periphery of the test tube, and the test tube is placed into another test tube;

adding more than 5 times of Ca-free²⁺、Mg²⁺The Hank's solution is evenly mixed, the rpm is 1500rpm for 10min, the supernatant is discarded, and the cell is repeatedly washed twice;

after the final centrifugation, the supernatant is discarded, 0.2ml of Hank's solution containing 20% calf plasma is added into each ml of blood sample to resuspend the cells, a drop of cell suspension is taken and placed in a blood counting plate for counting, and then the cell concentration is adjusted to 2 × 10⁶/ml；

Extraction of Total RNA from peripheral blood mononuclear cells

Extracting total RNA of peripheral blood mononuclear cells by using Trizol reagent, and carrying out experimental operation according to a product specification, wherein the experimental operation comprises the following specific steps: adding lm1Trizol into the separated peripheral blood mononuclear cells, performing vortex oscillation, standing at room temperature for 15min, adding 0.2ml chloroform, oscillating for 15s, and standing at room temperature for 10 min. Then centrifuged at 12000rm for 15 min. After the supernatant was absorbed, an equal volume of isopropanol was added for precipitation for 10 min. Centrifuging at 12000rm for 10min, removing supernatant, washing precipitate with lm 175% ethanol, centrifuging at 7500rm for 10min, removing supernatant, standing, and adding RNase-free water to dissolve RNA completely.

Quality analysis of RNA samples

And (3) determining the RNA purity by adopting an ultraviolet spectrophotometer: 1ul of RNA stock solution is added into a centrifuge tube, and then 249ul of ddH2O is added into the centrifuge tube, and the mixture is uniformly mixed. Zero point of A260 and A280 was adjusted sequentially using ddH2O as a control. A260 and A280 were then measured. And calculating the ratio of the two, wherein the ratio is greater than or equal to 1.8 to meet the experimental requirement. RNA concentration (ng/u1) ═ a260 × 40 × dilution factor.

Example 3 fluorescent quantitative PCR

Total RNA of peripheral blood mononuclear cells prepared in example 2 was subjected to reverse transcription, and then fluorescence quantitative PCR was performed using primer pairs SQ1 and SQ2 for NONHSAT016936.2 gene, primer pairs SQ3 and SQ4 for DANCR gene, and primer pairs SQ5 and SQ6 for miR-146a-3p gene, to detect the gene expression of osteoarthritis patients and healthy control groups NONHSAT016936.2, DANCR and miR-146a-3p

The results of gene expression of NONHSAT016936.2, DANCR and miR-146a-3p in 36 healthy control groups and 115 OA patient groups are shown in Table 1, and the expression levels of NONHSAT016936.2, DANCR and miR-146a-3p in peripheral blood mononuclear cells of the healthy control groups and OA patients are significantly different.

TABLE 1

Example 4

Further 115 patients with confirmed diagnosis of OA of the knee joint were classified into grade I to IV based on their X-ray films, i.e. according to the Kellgren and Lawrence metrics, wherein,

(1) 28 patients of class I (the clearance of the joint cavity is more than or equal to 6mm and osteophyte is formed on the joint surface);

(2) 36 patients of II (osteophyte and joint cavity clearance between 3mm and 6 mm);

(3) 27 patients of grade III (osteophytes and joint cavity clearance less than or equal to 3 mm);

(4) grade IV patients, 24, had osteophytes and the joint space disappeared.

In 115 cases of OA patients, the expression level of NONHSAT016936.2 gene was gradually increased with the increase in OA grade, while the expression levels of DANCR and miR-146a-3p gene were decreased, and the results are shown in Table 2.

TABLE 2

Example 5 changes in expression levels of NONHSAT016936.2, DANCR and miR-146a-3p in the group of high risk group for OA before and after follow-up

In the group of high-risk people with OA who complain of more than 3 months of knee pain without obvious change of imaging and exclude other related diseases such as rheumatism and rheumatoid disease, only 42 patients successfully obtain follow-up data after 24-38 months (33.4 months on average), and take X-ray again for accurate diagnosis of OA. Follow-up tests demonstrated that 18 of the patients were diagnosed with grade OA I, 2 with grade OAII, 20 with grade P20, and the remaining 22 with grade OA as unidentified with grade N22. The collection of peripheral blood mononuclear cells from 42 patients, the extraction and isolation of total RNA from peripheral blood mononuclear cells, and the quantitative fluorescence PCR were performed in the same manner as described above, to obtain 42 changes in expression levels of NONHSAT016936.2, DANCR, and miR-146a-3p in the OA high-risk group before and after the follow-up visit, and the results are shown in Table 3.

TABLE 3

Among the 42 cases of OA high risk group, the average expression quantity of NONHSAT016936.2, DANCR and miR-146a-3p in the first test is 12.36, 14.73 and 15.09. The NONHSAT016936.2 and miR-146a-3p index were between the average expression levels of the healthy human group and the OA patient group in example 2, except for the average expression level of DANCR of 14.73 and OA class I J.

As shown in Table 3, before the follow-up visit, the content of NONHSAT016936.2 in the P20 group was higher than that in the N22 group, and the expression level of NONHSAT016936.2 in the P20 group was gradually increased with the lapse of time, but the expression level of NONHSAT016936.2 in the N22 group was not significantly changed. The expression level of NONHSAT016936.2 in the P20 group was significantly higher than that in the N22 group after the follow-up visit. In contrast, before follow-up, the expression levels of DANCR and miR-146a-3P in the P20 group were lower than those in the N22 group, and as time progressed, the expression levels of DANCR and miR-146a-3P in the P20 group gradually decreased, but the expression levels of DANCR and miR-146a-3P in the N22 group did not change significantly. 3 indexes are proved to have the diagnostic value of early prompting OA subclinical patients.

ROC analysis

Setting the group P20 sample as 1 and the group N22 sample as 0, and obtaining a logistic regression equation by a logistic regression analysis method: logit (p) -85.157 +7.820 (noshsat 016936.2) -11.140(DANCR) -10.603(miR-146a-3 p). The left constant term of the equation is rounded off and divided by the coefficient 7.820 of NONHSAT016936.2 to obtain the joint predictor NONHSAT016936.2-1.425(DANCR) -1.356(miR-146a-3 p). ROC curve analysis was performed with NONHSAT016936.2, DANCR, miR-146a-3p and Joint predictor (combination), respectively. The results are shown in Table 4.

TABLE 4 ROC Curve analysis

Analysis results show that COMP and non-coding RNA multi-index joint detection can improve the sensitivity and specificity of early screening of OA subclinical population by using a single index, and has good clinical application value.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The application of non-coding RNA in preparing osteoarthritis early diagnosis and detection products is characterized in that the non-coding RNA is NONHSAT016936.2, DANCR or miR-146a-3p gene.

2. The use of claim 1, wherein the test product is used to test the expression level of the NONHSAT016936.2, DANCR or miR-146a-3p gene in a sample by reverse transcription PCR, fluorescence quantitative PCR, in situ hybridization, a chip or a high throughput sequencing platform.

3. Use according to claim 2, wherein the test product comprises primers and/or probes capable of specifically amplifying the NONHSAT016936.2, DANCR or miR-146a-3p gene.

4. The use as claimed in claim 3, wherein the primer pair for specifically amplifying the NONHSAT016936.2 gene has the sequence:

SQ1：5'-CGCCCAGGAACATGGTAACAC-3'，

SQ2：5'-GCCAAGGCTGTGACACAAGAG-3'。

5. use according to claim 3, characterized in that the primer pair sequence for the specific amplification of the DANCR gene is SQ 3: 5'-AGCGCAGGTTGACAACTACAG-3'

SQ4：5'-CTGCAGCTTGGGTGTGTATTC-3'。

6. the use of claim 3, wherein the primer pair sequence for specific amplification of the miR-146a-3p gene is as follows: SQ 5: 5'-GACGGGCCTCTGAAATTCAG-3' the flow of the air in the air conditioner,

SQ6：5'-CAGTGCAGGGTCCGAGGTAT-3'。

7. the use of claim 2, wherein the sample is peripheral blood.

8. The use of claim 1, wherein the assay product comprises a chip, a kit, a strip or a high throughput sequencing platform.