Application of specific primer in aortic valve calcification disease detection kit

Abstract

The invention discloses application of a specific primer in an aortic valve calcification disease detection kit. The specific primer comprises an upstream primer and a downstream primer, wherein the upstream primer and the downstream primer are F1 (SEQ ID NO: 1): 5 '-GAACAATGGTCGCACACCAC-3'; the invention relates to a kit for detecting CAVD by using Hsacirc0064557, which is characterized in that the Hsacirc0064557 is used as a biomarker for CAVD diagnosis, R1 (SEQ ID NO: 2): 5 '-GTGGACCCTTCGGATCACCT-3', the kit also comprises primers F2 (SEQ ID NO: 3): 5 '-AAGGTGAAGGTCGGAGTCAA-3', and R2 (SEQ ID NO: 4): 5 '-AATGAAGGGGTCATGATGG-3'. The Hsacirc0064557, which is remarkably and highly expressed in plasma and tissue samples of a CAVD patient, is used as the biomarker for CAVD diagnosis, and is an effective tool for CAVD detection, diagnosis, progress condition and treatment effect judgment.

Description

Technical Field

[0001] The invention relates to the technical field of kits, in particular to the application of a specific primer in a kit for detecting aortic valve calcification disease. Background Art

[0002] Valvular heart disease (VHD) is a common cardiovascular disease in my country. Elderly VHD and valvular lesions caused by coronary heart disease and myocardial infarction are becoming more and more common. Without intervention, they will eventually lead to heart failure [Godfrey S, Kirkpatrick JN, Kramer DB, Sulistio MS. Expanding the Paradigm for Cardiovascular Palliative Care. Circulation, 2023, 148(13): 1039-1052]. Most valvular calcification is degenerative VHD in the elderly. Valvular calcification is mainly aortic valve calcification (CAVD), followed by mitral valve calcification [Zhang Shuai, Lu Wenyuan, Tang Hanwei, Lin Hongyuan, Gong Jiamiao, Wu Yongjian, Zheng Zhe, Hou Jianfeng. Clinical characteristics and current status of surgical treatment of elderly patients with valvular heart disease and heart failure in China. Chinese Journal of Circulation, 2023, 38(05): 520-525]. About one-third of the elderly people who were diagnosed with calcific aortic valve sclerosis by echocardiography or radiology examination accounted for calcific aortic valve sclerosis, which is also an early and subclinical form of degenerative CAVD. Early diagnosis and treatment are crucial for CAVD patients, but the current early diagnosis and detection methods based only on cardiac ultrasound are far from sufficient for the needs of CAVD patients. At the same time, for the treatment of CAVD, surgical thoracotomy and conventional valve replacement with extracorporeal circulation will cause serious damage to multiple organs such as the heart, lung, brain, liver, and kidney in some patients. For elderly patients with degenerative CAVD, especially those with underlying diseases, they cannot tolerate extracorporeal circulation. Therefore, it is urgent to further explore the influencing factors of the occurrence and development of CAVD in order to provide strong evidence for the diagnosis and treatment of CAVD. hsa_circ_0064557 is a circular RNA, which is located on the genome at: chr3:18456602-18462483, and the corresponding mRNA is SATB1. The specific primers designed based on it are effectively applied to the aortic valve calcification disease detection kit, which will bring new solutions to the diagnosis of CAVD. Summary of the invention

[0003] The present invention aims to at least partially overcome the above and / or other potential problems existing in the prior art: to provide an application of a specific primer in a kit for detecting aortic valve calcification disease.

[0004] The technical solution of the present invention is as follows: an application of a specific primer in an aortic valve calcification disease detection kit, wherein the specific primer is specifically a specific RT-qPCR upstream and downstream primer of hsa_circ_0064557:

[0005] F1 (SEQ ID NO: 1): 5'-GAACAATGGTCGCACACCAC-3';

[0006] R1 (SEQ ID NO:2): 5'-GTGGACCCTTCGGATCACTC-3'.

[0007] Furthermore, the kit also includes specific RT-qPCR upstream and downstream primers for external reference GAPDH:

[0008] F2 (SEQ ID NO:3): 5'-AAGGTGAAGGTCGGAGTCAA-3';

[0009] R2 (SEQ ID NO:4): 5'-AATGAAGGGGTCATTGATGG-3'.

[0010] Specifically, the method for detecting circRNA biomarkers comprises the following steps:

[0011] 1) Collect plasma and tissue samples from CAVD patients and extract total RNA from plasma and tissues;

[0012] 2) Reverse transcription of total RNA into cDNA;

[0013] 3) Design specific primers spanning the circular site of hsa_circ_0064557 as described above;

[0014] 4) The cDNA was amplified using the RT-qPCR fluorescent dye method, and after amplification, the Cq values ​​of hsa_circ_0064557 and the external reference gene GAPDH in the sample were detected;

[0015] 5) According to the Cq value, the expression level of hsa_circ_0064557 was normalized by the expression level of the external reference gene GAPDH, and the relative expression level of hsa_circ_0064557 was calculated and used as the -ΔCq The RT-qPCR relative quantitative value of hsa_circ_0064557 was calculated by the formula, where ΔCq = Cq (hsa_circ_0064557) -Cq (GAPDH) ;

[0016] 6) When the PCR relative quantitative ΔCq value of the biomarker hsa_circ_0064557 in the plasma sample is less than 3.650, it can be diagnosed as CAVD; when it is greater than or equal to 3.650, it can be diagnosed as a non-CAVD sample. When the PCR relative quantitative ΔCq value of the biomarker hsa_circ_0064557 in the tissue sample is less than 10.72, it can be diagnosed as CAVD; when it is greater than or equal to 10.72, it can be diagnosed as a non-CAVD sample.

[0017] Furthermore, the process of extracting total RNA from plasma and tissue specimens in the above step 1) is as follows:

[0018] Step a, collection of plasma samples: about 5 mL of whole blood was collected from patients and normal controls through the median cubital vein. The collected blood samples were placed in EDTA anticoagulant tubes. The blood samples were centrifuged at 3000 rpm within 10 minutes to extract the upper pale yellow plasma, which was added to 2 mL enzyme-free tubes and each sample was numbered and labeled. All plasma samples were stored in a -80°C refrigerator for later use.

[0019] Step b, Trizol LS reagent lysis: Use a pipette to add 300 μL of plasma sample to an enzyme-free tube, then add 900 μL of Trizol LS reagent, vortex mix for 15 seconds, and let stand at room temperature for 5 minutes.

[0020] Step c, phase separation: Centrifuge the above mixture at 4°C, 12000rpm for 10 minutes. The centrifuge tube is divided into upper and lower layers. The upper layer is a homogenized pink liquid, and the lower layer is a darker viscous impurity. Pipette about 1mL of the upper layer into a new 1.5mL enzyme-free tube.

[0021] Step d, chloroform extraction: add 0.2 mL of chloroform, cover and vortex for about 15 seconds, let stand at room temperature of 20°C for 5 minutes, and then centrifuge at 4°C, 12000 rpm for 15 minutes; slowly draw about 0.5 mL to 1 mL of the upper aqueous phase into an enzyme-free centrifuge tube.

[0022] Step e, isopropanol precipitation: add isopropanol of the same volume as the upper water layer in step d, mix well and let stand on ice for 10 minutes, then centrifuge at 4°C and 12,000 rpm for 10 minutes.

[0023] Step f, ethanol washing and precipitation: pour off the supernatant and add 1 mL of 75% ethanol to wash the precipitate. Mix the washed precipitate in the enzyme-free tube on an oscillator and centrifuge at 4°C, 8000 rpm for 5 minutes. Discard the supernatant again and carefully use a pipette to thoroughly remove the residual liquid. Dry the white precipitate at room temperature, which is the total RNA in the plasma, and store it at -80°C for later use.

[0024] The total RNA extraction method for tissue samples is similar to that for plasma. First, take out about 20 mg of tissue from a -80°C refrigerator and homogenize the tissue with a handheld automatic homogenizer until there are no obvious tissue particles. Then add 1 mL of Trizol reagent and blow it thoroughly with a pipette. After standing at room temperature for 10 minutes, add 200 μL of chloroform, and the subsequent operation method is the same as the extraction method in plasma.

[0025] Furthermore, the RT-qPCR reaction conditions in step 4) are as follows: pre-denaturation at 95°C for 5 minutes; then denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and 45 cycles; finally denaturation at 95°C for 1 minute, annealing at 55°C for 30 seconds, enzyme denaturation at 95°C for 30 seconds, and insulation at 4°C.

[0026] The present invention also provides an aortic valve calcification disease detection kit, which also includes Taq enzyme for PCR reaction, dNTP mixed solution, fluorescent reagent, PCR buffer, diethyl pyrocarbonate (DEPC), and water.

[0027] The method for detecting the biological function of circRNA includes the following steps:

[0028] 1) CCK-8 assay to detect the proliferation ability of hsa_circ_0064557 in CAVD: transfected human valvular interstitial cells (hVICs) (5×10 3 , 100 μL) were inoculated in a 96-well plate and cultured for 1, 2, 3, 4, and 5 days. 10 μL CCK-8 reagent was added and incubated for 3 hours. The absorbance value was measured and recorded using an enzyme labeling instrument.

[0029] 2) Transwell assay to detect the migration ability of hsa_circ_0064557 in CAVD: transfected hVICs cells (8×10 4 , 200μL) was added to the middle chamber of the Transwell plate, and 500μL of cell culture medium containing 20% ​​fetal bovine serum was added to the lower chamber. After incubation for 24h, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.

[0030] 3) Flow cytometry was used to detect the changes in the apoptotic ability of hsa_circ_0064557 in CAVD: After cell transfection, the cells were digested with EDTA-free trypsin and resuspended in buffer. After standing at room temperature, they were stained with Annexin V-FITC / PI apoptosis kit and protected from light for 15 minutes. Finally, cell apoptosis was detected by flow cytometry. All steps were performed according to the manufacturer's instructions.

[0031] The beneficial effects of the present invention are as follows: compared with the prior art, the advantage of the present invention is that hsa_circ_0064557, which is significantly highly expressed in the plasma and tissue samples of CAVD patients, is used as a biomarker for the diagnosis of CAVD. hsa_circ_0064557 is a circular RNA, and its location on the genome is: chr3:18456602-18462483. The corresponding mRNA is SATB1. A detection kit is made by using its specific primers. RNA with good quality and concentration is extracted by using RNA extraction reagents, mixing, centrifugation and other methods. Only about 300 μL of CAVD plasma or about 20 mg of tissue specimens need to be collected to detect the expression of hsa_circ_0064557, which becomes an effective tool for CAVD detection, diagnosis, progression, and treatment efficacy judgment, and has certain clinical application value. The RT-qPCR instrument used is a common amplification instrument, and SYBR The Green fluorescent dye method does not require the design of additional probes to simultaneously detect the expression of the target hsa_circ_0064557 and the external reference gene, which is economical and convenient. Simultaneous detection of the biological function of hsa_circ_0064557 in cells can provide direction for the application of targeted treatment of CAVD. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram showing the detection results of circRNA gene chip technology in CAVD plasma specimens in Example 1 of the present invention.

[0033] Figure 2 It is an amplification curve diagram of the external reference gene GAPDH in three plasmas and two tissue specimens in Example 2 of the present invention (sample 1 is a normal control plasma specimen, sample 2 is a non-CAVD plasma specimen, sample 3 is a CAVD plasma specimen; sample 4 is a non-CAVD tissue specimen, and sample 5 is a CAVD tissue specimen).

[0034] Figure 3 This is the amplification curve of hsa_circ_0064557 in three plasmas and two tissue specimens in Example 2 of the present invention (sample 1 is a normal control plasma specimen, sample 2 is a non-CAVD plasma specimen, sample 3 is a CAVD plasma specimen; sample 4 is a non-CAVD tissue specimen, and sample 5 is a CAVD tissue specimen).

[0035] Figure 4 This is a schematic diagram of the comparison of the expression levels of hsa_circ_0064557 in plasma and tissues in Example 3 of the present invention.

[0036] Figure 5 This is the ROC curve diagram of hsa_circ_0064557 in Example 3 of the present invention.

[0037] Figure 6 This is the relevant biological function of hsa_circ_0064557 in CAVD in Example 5 of the present invention. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below with specific examples, but the present invention is not limited to the following specific examples.

[0039] Example 1

[0040] Detection of hsa_circ_0064557 expression in CAVD plasma and tissue specimens:

[0041] 1. CircRNA gene chip detection: The circRNA gene chip technology of Arraystar Company in the United States was used to detect the level of circRNA in CAVD tissue specimens and non-CAVD control tissue specimens, such as Figure 1 shown.

[0042] 2. Result analysis: The fold change difference of hsa_circ_0064557 in CAVD tissues and control specimens was 5.558 times, suggesting that hsa_circ_0064557 may play a promoting role in the occurrence and development of CAVD.

[0043] Example 2

[0044] The plasma samples were divided into three groups: normal control group, non-CAVD group, and CAVD group. The expression level of hsa_circ_0064557 was detected according to the following steps, including the following steps:

[0045] Step a: Collect CAVD plasma samples: Take about 5 mL of venous whole blood, extract the upper light yellow plasma after centrifugation, and store it in a 2 mL enzyme-free tube.

[0046] Step b, Trizol LS reagent lysis: Use a pipette to add 300 μL of plasma sample to an enzyme-free tube, then add 900 μL of Trizol LS reagent, vortex mix for 15 seconds, and let stand at room temperature for 5 minutes.

[0047] Step c, phase separation: Centrifuge the above mixture at 4°C, 12000rpm for 10 minutes. The centrifuge tube is divided into upper and lower layers. The upper layer is a homogenized pink liquid, and the lower layer is a darker viscous impurity. Pipette about 1mL of the upper layer into a new 1.5mL enzyme-free tube.

[0048] Step d, chloroform extraction: add 0.2 mL of chloroform, cover and vortex for 15 seconds, then let stand at room temperature of 20° C. for 5 minutes, and then centrifuge at 4° C. and 12,000 rpm for 15 minutes; slowly draw about 0.5 mL to 1 mL of the upper aqueous phase into an enzyme-free centrifuge tube.

[0049] Step e, isopropanol precipitation: add isopropanol of the same volume as the upper water layer in step d, mix well and let stand on ice for 10 minutes, then centrifuge at 4°C and 12,000 rpm for 10 minutes.

[0050] Step f, ethanol washing and precipitation: pour off the supernatant and add 1 mL of 75% ethanol to wash the precipitate. Mix the washed precipitate in an enzyme-free tube on an oscillator and centrifuge at 8000 rpm for 5 minutes at 4°C. Discard the supernatant again and carefully use a pipette to thoroughly remove the residual liquid. Dry the white precipitate at room temperature, which is the total RNA in the plasma, and store it at -80°C for later use.

[0051] Tissue specimens were divided into two groups: non-CAVD group and CAVD group. For CAVD tissue samples, about 20 mg of tissue was first taken and homogenized with a handheld automatic homogenizer until no obvious tissue particles were present. Then 1 mL of Trizol reagent was added and blown evenly with a pipette. After standing at room temperature for 10 min, 200 μL of chloroform was added, and the subsequent operation method was the same as the total RNA extraction method from plasma.

[0052] Step g, reverse transcription: using a reverse transcription kit to reverse transcribe the total RNA extracted in step f above to obtain cDNA, which is stored at -20°C for future use;

[0053] Step h, SYBR Green fluorescent dye method RT-qPCR detection: the cDNA obtained by reverse transcription in the above step g is prepared according to the reaction system ratio in Table 1, and the relevant RT-qPCR parameters are set according to the temperature control program in Table 2;

[0054] The specific upstream and downstream primers for amplification of the external reference gene GAPDH used are:

[0055] F2: 5'-AAGGTGAAGGTCGGAGTCAA-3';

[0056] R2: 5'-AATGAAGGGGTCATTGATGG-3'.

[0057] The specific upstream and downstream primers for amplification of hsa_circ_0064557 used are:

[0058] F1: 5'-GAACAATGGTCGCACACCAC-3';

[0059] R1: 5'-GTGGACCCTTCGGATCACTC-3'.

[0060] Table 1. RT-qPCR amplification reaction system

[0061]

[0062] Table 2. RT-qPCR amplification reaction parameters

[0063]

[0064]

[0065] from Figure 2 and Figure 3 It can be seen that hsa_circ_0064557 and GAPDH in healthy plasma and tissue specimens were effectively amplified, and the amplification curves of hsa_circ_0064557 and GAPDH RT-qPCR in plasma and tissue specimens can be obtained. Among them, from the amplification curve of GAPDH, the Cq values ​​of the plasma specimens of the healthy control group, non-CAVD group and CAVD group detected were 29.35, 29.58 and 30.07, respectively, and the Cq values ​​of the tissue specimens of the non-CAVD group and CAVD group were 16.68 and 17.23, respectively. Figure 2 Similarly, from the amplification curve of hsa_circ_0064557, the Cq values ​​of the plasma samples of the healthy control group, non-CAVD group and CAVD group were 34.51, 34.08 and 33.62, respectively, and the Cq values ​​of the tissue samples of the non-CAVD group and CAVD group were 31.39 and 30.57, respectively. Figure 3 shown; and from Figure 2 and Figure 3 It can be concluded that the expression levels of hsa_circ_0064557 in plasma and tissue CAVD specimens are consistent with the results of the circRNA gene chip in Example 1.

[0066] The Cq values ​​of hsa_circ_0064557 and GAPDH in the same sample were used at the same time, and the calculation formula was ΔCq = Cq (hsa_circ_0064557) -Cq (GAPDH) The ΔCq value of hsa_circ_0064557 was calculated and then according to 2 -ΔCq The value can be used to determine the relative expression level of hsa_circ_0064557; 2 -ΔCq The lower the value, the lower the expression level of hsa_circ_0064557. -ΔCq The higher the value, the higher the corresponding expression level of hsa_circ_0064557;

[0067] from Figure 2 and Figure 3 The 2 -ΔCq =0.028; 2 -ΔCq =0.044, CAVD group plasma 2 -ΔCq =0.085; the plasma level of the CAVD group was significantly higher than that of the control group and the non-CAVD group. -ΔCq The value indicated that hsa_circ_0064557 was highly expressed in the plasma of CAVD patients. -ΔCq =3.731E-05, CAVD group organized 2 -ΔCq =9.644E-05; the CAVD group tissue was significantly larger than the non-CAVD control group tissue 2 -ΔCq The value indicated that hsa_circ_0064557 was highly expressed in CAVD patient tissues.

[0068] Example 3

[0069] Method for CAVD detection using hsa_circ_0064557 as a biomarker

[0070] The steps include:

[0071] 1. Collect CAVD plasma and tissue samples: Take about 5 mL of venous whole blood, extract the upper light yellow plasma after centrifugation and store it in a 2 mL enzyme-free tube; take about 20 mg of tissue and use a handheld automatic homogenizer to homogenize the tissue until there are no obvious tissue particles.

[0072] 2. Extraction of RNA from CAVD plasma and tissue samples (extraction method is the same as in Example 2).

[0073] 3. Reverse transcription and SYBR Green fluorescent dye method RT-qPCR detection The same operation as in “Reverse transcription and SYBR Green fluorescent dye method RT-qPCR detection” in Example 2 was performed.

[0074] 4. hsa_circ_0064557 was used as a biomarker for the detection of plasma and tissue samples of CAVD, and the expression level of hsa_circ_0064557 in 24 plasma samples of the healthy control group, non-CAVD group and CAVD group was analyzed. -ΔCq It was significantly higher than that in the healthy control group, P < 0.001, which proved that the expression level of hsa_circ_0064557 in the plasma of CAVD patients was significantly higher than that in the healthy control group. Figure 4As shown. The cutoff value of hsa_circ_0064557 as a CAVD biomarker is 3.650, which means that when the RT-qPCR relative quantitative value ΔCq of the hsa_circ_0064557 biomarker in the sample is less than 3.650, it is considered a CAVD plasma sample; when it is greater than or equal to 3.650, it is considered a non-CAVD control plasma sample. At the same time, the expression level of hsa_circ_0064557 in 27 and 11 tissue specimens of the non-CAVD group and CAVD group were analyzed. The 2 -ΔCq It was significantly higher than that in the non-CAVD group, P < 0.001, which proved that the expression level of hsa_circ_0064557 in the plasma of CAVD patients was significantly higher than that in the non-CAVD group. Figure 4 As shown in A. The cutoff value of hsa_circ_0064557 as a CAVD biomarker is 10.72, which means that when the RT-qPCR relative quantitative value ΔCq of the hsa_circ_0064557 biomarker in the sample is less than 10.72, it is considered to be a CAVD tissue sample; when it is greater than or equal to 10.72, it is considered to be a non-CAVD tissue sample, such as Figure 4 As shown in B.

[0075] After detecting the expression level of hsa_circ_0064557 in the plasma specimens of 24 non-CAVD control group and CAVD group, the ROC curve was drawn to further explore the diagnostic value of hsa_circ_0064557 in CAVD. Figure 5 As shown in A, the ROC curve shows that the AUC value is 0.6710, P < 0.05. It can be further concluded that the sensitivity of hsa_circ_0064557 as a biomarker for CAVD plasma samples is 0.4167 and the specificity is 0.9167. After detecting the expression level of hsa_circ_0064557 in the plasma samples of 24 healthy control groups and CAVD groups, in order to further explore the diagnostic value of hsa_circ_0064557 in CAVD, the ROC curve was drawn, as shown in Figure 1. Figure 5 As shown in Figure 2, the ROC curve shows that the AUC value is 0.7101, P < 0.05. It can be further concluded that the sensitivity of hsa_circ_0064557 as a biomarker for CAVD plasma samples is 0.6667, and the specificity is 0.7500. After simultaneously detecting the expression level of hsa_circ_0064557 in 27 non-CAVD control group and 11 CAVD group tissue specimens, in order to further explore the diagnostic value of hsa_circ_0064557 in CAVD, the ROC curve was drawn, as shown in Figure 2. Figure 5As shown in C, the ROC curve shows that the AUC value is 0.7424, P < 0.05. It can be further concluded that the sensitivity of hsa_circ_0064557 as a biomarker for CAVD tissue samples is 0.9091 and the specificity is 0.5926. Figure 5 As shown in Figure D, the combined ROC curve analysis of hsa_circ_0064557 in plasma and tissues showed an AUC value of 0.803, P < 0.05. It can be further concluded that the sensitivity of hsa_circ_0064557 as a biomarker for CAVD plasma samples is 0.667 and the specificity is 0.7500.

[0076] Example 4

[0077] Detection kits and applications

[0078] The hsa_circ_0064557 molecular marker detection kit for CAVD detection in this embodiment includes, in addition to real-time RT-qPCR reagents, hsa_circ_0064557 primers and external reference GAPDH primers. The real-time RT-qPCR reagents include Taq enzyme, dNTP reagent, PCR buffer, DEPC treated water (RNase free water), and fluorescent reagent.

[0079] When the above detection kit is used for detection, the specific operation method can refer to the operation method of Example 2 for sample detection and CAVD diagnosis.

[0080] The detection kit in this example can be used to simply, quickly and conveniently detect the hsa_circ_0064557 molecular marker to determine the progression of CAVD, facilitating diagnosis and treatment.

[0081] The above are only examples of the features of the present invention and do not constitute any limitation on the protection scope of the present invention. Any technical solution formed by equivalent exchange or equivalent replacement falls within the protection scope of the present invention.

[0082] Example 5

[0083] 1. CCK-8 assay to detect the proliferation of hsa_circ_0064557 in CAVD cells: transfected hVICs (5×10 3 , 100 μL) were inoculated into 96-well plates, and 10 μL CCK-8 reagent was added after 1, 2, 3, 4, and 5 days of culture, and incubated in an incubator for 3 hours. Finally, the absorbance value was measured and recorded by an enzyme-labeled instrument.

[0084] 2. Transwell assay to detect the migration ability of hsa_circ_0064557 in CAVD cells: transfected hVICs cells (8×10 4 , 200μL) was added to the upper chamber of the Transwell plate, and 500μL of culture medium containing 20% ​​fetal bovine serum was added to the lower chamber of the Transwell plate. After incubation for 24h, the cells were fixed with 4% paraformaldehyde and finally stained with 0.1% crystal violet to observe the number of cell migration.

[0085] 3. Flow cytometry detection of hsa_circ_0064557 apoptosis in CAVD cells: Digest the transfected cells with EDTA-free trypsin and resuspend the cells in buffer. After standing at room temperature, stain with Annexin V-FITC / PI apoptosis kit and keep in the dark for 15 minutes. Detect changes in cell apoptosis rate on a flow cytometer.

[0086] Finally, it was found that the proliferation ability of hVICs cells was decreased and increased after silencing and overexpressing hsa_circ_0064557 levels, respectively ( Figure 6 A), the migration ability decreased and increased respectively ( Figure 6 B), the apoptosis rate increased and decreased respectively ( Figure 6 C).

[0087] The above are only examples of the features of the present invention and do not constitute any limitation on the protection scope of the present invention. Any technical solution formed by equivalent exchange or equivalent replacement falls within the protection scope of the present invention.

Claims

1. Use of a specific primer in a kit for detecting aortic valve calcification disease, characterized in that: The specific primers are specifically the specific RT-qPCR upstream and downstream primers of hsa_circ_0064557: F1 (SEQ ID NO: 1): 5'-GAACAATGGTCGCACACCAC-3'; R1 (SEQ ID NO: 2): 5'-GTGGACCCTTCGGATCACTC-3'.

2. The use of the specific primers according to claim 1 in a kit for detecting aortic valve calcification disease, characterized in that: The kit includes specific RT-qPCR upstream and downstream primers for the external reference GAPDH: F2 (SEQ ID NO: 3): 5'-AAGGTGAAGGTCGGAGTCAA-3'; R2 (SEQ ID NO: 4): 5'-AATGAAGGGGTCATTGATGG-3'.

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