Exosomal circRNA as a molecular marker for diagnosing heart failure in patients with dilated cardiomyopathy and its application

Abstract

This invention discloses exosomal circRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, relating to the field of molecular diagnostic technology. The exosomal circRNA is any one or a combination of two or more of the following molecular markers: hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837. The invention also discloses the application of exosomal circRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure. The exosomal circRNA is used as a marker for preparing kits, microarrays, or biochips for diagnosing dilated cardiomyopathy complicated with heart failure. The molecular marker provided by this invention effectively improves the detection accuracy of dilated cardiomyopathy complicated with heart failure. The combination of this molecular marker has a P value of <0.05 and |log fold change (FC)|>1, showing significant differential expression compared to the healthy control group.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics, and more specifically to an exosomal circRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure and its application. Background Technology

[0002] Dilated cardiomyopathy (DCM) is one of the most common hereditary cardiomyopathies. It is a heterogeneous cardiomyopathy that can cause cardiac enlargement, decreased ventricular systolic function, heart failure, and various types of arrhythmias. It is the leading cause of heart failure and heart transplantation. DCM has a high incidence rate, with a 52-month follow-up mortality rate of 42.24%, placing a heavy burden on families and society (Clinical Cardiovascular Journal, 2018, 34(05):421-434). Circular RNA (circRNA) is a special type of non-coding RNA molecule. circRNA molecules are rich in microRNA (miRNA) binding sites, which can relieve the inhibitory effect of miRNA on its target genes and increase the expression level of target genes. Differential expression of circRNAs is associated with a variety of diseases. For example, Circ101237 promotes MAPK1 expression in non-small cell lung cancer (NSCLC), thereby inhibiting tumor progression (CircRNA_101237 promotes NSCLC progression via the miRNA-490-3p / MAPK1 axis. Sci Rep. 2020). The expression level of circNlgn is significantly altered in patients with myocardial fibrosis (A Neuroligin Isoform Translated by circNlgn Contributes to Cardiac Remodeling. Circ Res. 2021). SCAR can inhibit the release of mitochondrial ROS, and expressing SCAR using a mitochondrial targeted delivery system can inhibit the pro-inflammatory phenotype of non-alcoholic lipohistiocytic hepatitis (Targeting Mitochondria-Located circRNA SCAR Alleviates NASH via Reducing mROS Output. Cell. 2020).

[0003] The prior art patent application CN114908156A discloses an application of circRNA for diagnosing and / or prognostic assessment of vascular remodeling or related diseases. In this invention, compared with healthy individuals, individuals with vascular remodeling or related diseases have significantly upregulated hsa_circ 026271, hsa_circ 026272 and / or rno_circ_0017665 in peripheral blood and / or tissues. By detecting the expression level of circRNA, vascular remodeling or related diseases or cardiovascular diseases can be accurately diagnosed.

[0004] Although the aforementioned invention patent discloses a technical solution that can be used to diagnose vascular remodeling, its circRNA is derived from plasma rather than plasma exosomes and is not applicable to the diagnosis of dilated cardiomyopathy complicated with heart failure.

[0005] Therefore, it is necessary to propose an exosomal circRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure and to explore its application in order to solve the above problems. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing the application of exosomal circRNA molecular markers for diagnosing dilated cardiomyopathy complicated with heart failure and its kit, thus solving the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention specifically adopts the following technical solution:

[0008] Exosomal circRNAs are used as molecular markers for diagnosing dilated cardiomyopathy complicated with heart failure. The exosomal circRNAs are any one or a combination of two or more of the following molecular markers: hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837. The specific nucleic acid sequences of the exosomal circRNAs are shown in SEQ NO.1-9.

[0009] Furthermore, the exosomes are plasma exosomes.

[0010] The application of exosomal circRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, wherein the exosomal circRNA is used as a marker for preparing kits, microarrays or biochips for diagnosing dilated cardiomyopathy complicated with heart failure.

[0011] Furthermore, the substances used to detect the expression level of the exosome circRNA are primers or probes.

[0012] Furthermore, the primer combination for quantitative real-time detection of exosomal circRNA expression levels is as follows:

[0013] (1) Primers for hsa-circ-0026806: 5'-AATGGAAATTGTACTTAACCTATGTG-3';

[0014] (2) Primers for hsa-circ-0054400: 5'-TATTGAAGGTGCCAGGTGCTG-3';

[0015] (3) Primers for hsa-circ-0074153: 5'-CACAAATGGGGATACCTTCTTAG-3';

[0016] (4) Primers for hsa-circ-0047519: 5'-CATCACTGCCTACTTCAAAGAAAT-3';

[0017] (5) Primers for hsa-circ-0063766: 5'-CTGTGAACCAGAATTCAATCAGA-3';

[0018] (6) Primers for hsa-circ-0076771: 5'-AACGAGATGTAAAAGTGTCACTGTG-3';

[0019] (7) Primers for hsa-circ-0019699: 5'-CCTTATCATCTAACCTCACTGAACTCT-3';

[0020] (8) Primers for hsa-circ-0056066: 5'-TAAAAACCAATAACAGTCGT-3';

[0021] (9) Primer for hsa-circ-0075837: 5'-CAAGGCAAAGCTGTTACTAG-3'.

[0022] A kit for diagnosing dilated cardiomyopathy complicated with heart failure, the kit being used to detect plasma exosomal circRNA expression levels, wherein the exosomal circRNA is any one or a combination of two or more molecular markers selected from hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837.

[0023] I. Compared with the prior art, the application provided by this invention has the following beneficial effects:

[0024] This invention selects one or more molecular markers from hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837 as molecular markers for exosomal circRNA expression levels. This effectively improves the detection accuracy of dilated cardiomyopathy complicated with heart failure. The combination of these molecular markers has a P-value < 0.05 and a |log fold change (FC)| > 1, showing significant differential expression compared to the healthy control group.

[0025] This invention discloses molecular markers for diagnosing dilated cardiomyopathy complicated with heart failure based on plasma exosomal circRNAs and their applications. RT-qPCR results showed that nine human plasma exosomal circRNAs, including hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837, were differentially expressed between patients with dilated cardiomyopathy complicated with heart failure and healthy controls. The diagnostic efficacy of seven plasma exosomal circRNAs was analyzed using ROC curves. The results showed that the AUC values ​​of the above exosomal circRNAs were 0.914, 0.928, 0.920, 0.886, 0.942, 0.901, 0.918, 0.927, and 0.934, respectively.

[0026] This invention demonstrates that the above-mentioned nine plasma exosomal circRNAs can serve as molecular diagnostic biomarkers for dilated cardiomyopathy complicated with heart failure. These biomarkers can be formulated into diagnostic kits, microarrays, or biochips. By detecting the expression levels of exosomal circRNAs in the subject's plasma, rapid diagnosis and determination of dilated cardiomyopathy complicated with heart failure can be achieved. When the expression level of at least one circRNA among exosomal hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837 is upregulated or downregulated, it indicates that the subject has dilated cardiomyopathy complicated with heart failure, demonstrating significant clinical application value.

[0027] II. Compared with the prior art, the reagent kit provided by the present invention has the following beneficial effects:

[0028] The kit provided by this invention diagnoses dilated cardiomyopathy complicated with heart failure by detecting the expression level of exosomal circRNA in plasma. Combinations of one or more of the molecular markers selected from exosomal hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837 effectively improve the detection accuracy of dilated cardiomyopathy complicated with heart failure. The combination of these molecular markers shows a p-value < 0.05, |log fold change (FC)| > 1, and significant differential expression compared to the healthy control group. Attached Figure Description

[0029] Figure 1 TEM images of plasma exosomes from a healthy control group and patients with dilated cardiomyopathy and heart failure;

[0030] Figure 2 The results of plasma exosome diameter particle analysis were obtained from healthy controls and patients with dilated cardiomyopathy and heart failure.

[0031] Figure 3 The images show protein imprinting of exosome-specific markers. In the image, Nor-Exo represents healthy controls, and DCM-Exo represents the dilated cardiomyopathy group with heart failure.

[0032] Figure 4 To verify the differential expression of circRNA in patients with dilated cardiomyopathy and heart failure and healthy controls using RT-qPCR;

[0033] Figure 5 To assess the diagnostic efficacy of circRNA in dilated cardiomyopathy complicated with heart failure;

[0034] Figure 6 ROC curve analysis of the diagnostic efficacy of combinations of molecular markers hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837 for dilated cardiomyopathy complicated with heart failure. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Exosomal circRNAs are used as molecular markers for diagnosing dilated cardiomyopathy complicated with heart failure. The exosomal circRNAs are any one or a combination of two or more of the following molecular markers: hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837. The specific nucleic acid sequences of the exosomal circRNAs are shown in SEQ NO.1-9.

[0037] In this invention, the exosomes are plasma exosomes.

[0038] The application of exosomal circRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, wherein the exosomal circRNA is used as a marker for preparing kits, microarrays or biochips for diagnosing dilated cardiomyopathy complicated with heart failure.

[0039] In this invention, the substances used to detect the expression level of exosome circRNA are primers or probes.

[0040] In this invention, the primer combination used for quantitative fluorescence detection of exosomal circRNA expression levels is as follows:

[0041] (1) Primers for hsa-circ-0026806:

[0042] Forward primer: 5'-AATGGAAATTGTACTTAACCTATGTG-3';

[0043] Reverse primer: 3'-TCACGAGATCGATTCCTGACTAC-5';

[0044] The amplified fragment of the product is 106 bp in size;

[0045] (2) Primers for hsa-circ-0054400:

[0046] Forward primer: 5'-TATTGAAGGTGCCAGGTGCTG-3';

[0047] Reverse primer: 3'-GCCAGGAAATCAGTTGGTGAGA-5';

[0048] The amplified fragment of the product is 103 bp in size;

[0049] (3) Primers for hsa-circ-0074153:

[0050] Forward primer 5'-CACAAATGGGGATACCTTCTTAG-3';

[0051] Reverse primer: 3'-CCTTGATTGCTTCTGATCTCTCT-5';

[0052] The amplified fragment size of the product is 99 bp;

[0053] (4) Primers for hsa-circ-0047519:

[0054] Forward primer 5'-CATCACTGCCTACTTCAAAGAAAT-3';

[0055] Reverse primer: 3'-CACCACAGTATCACTCTCTAGCCA-5';

[0056] The amplified fragment of the product is 142 bp in size;

[0057] (5) Primers for hsa-circ-0063766:

[0058] Forward primer 5'-CTGTGAACCAGAATTCAATCAGA-3';

[0059] Reverse primer: 3'-ATCCAGAACTCTTTGGAAGATAGTC-5';

[0060] The amplified fragment of the product is 62 bp in size;

[0061] (6) Primers for hsa-circ-0076771:

[0062] Forward primer 5'-AACGAGATGTAAAAGTGTCACTGTG-3';

[0063] Reverse primer: 3'-GCCTTCACTGCAGCTGAAAAG-5';

[0064] The amplified fragment of the product is 172 bp in size;

[0065] (7) Primers for hsa-circ-0019699:

[0066] Forward primer 5'-CCTTATCATCTAACCTCACTGAACTCT-3';

[0067] Reverse primer: 3'-CCAGTGGTATCTTCAGAGCGAAT-5';

[0068] The amplified fragment of the product is 94 bp in size;

[0069] (8) Primers for hsa-circ-0056066:

[0070] Forward primer 5'-TAAAAACCAATAACAGTCGT-3';

[0071] Reverse primer: 3'-TCACGTGAGAAAAAGTCCCC-5';

[0072] The amplified fragment size of the product is 50 bp;

[0073] (9) Primers for hsa-circ-0075837:

[0074] Forward primer 5'-CAAGGCAAAGCTGTTACTAG-3';

[0075] Reverse primer: 3'-GCTGGAACGTAGTCAAGGAA-5';

[0076] The amplified fragment of the product is 103 bp in size;

[0077] A kit for diagnosing dilated cardiomyopathy complicated with heart failure, the kit for detecting plasma exosomal circRNA expression levels, wherein the exosomal circRNA is any one or a combination of two or more molecular markers selected from hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837.

[0078] Experimental verification

[0079] The present invention will be further described in detail below with reference to specific embodiments.

[0080] 1) Test reagents: Plasma samples from the healthy control group and patients with coronary heart disease in this invention were obtained from the First Affiliated Hospital of Zhengzhou University. Other reagents or consumables used were commercially available or could be obtained by those skilled in the art through public channels.

[0081] 2) Experimental Methods: Peripheral blood was collected from individuals using EDTA-anticoagulated blood collection tubes. The tubes were centrifuged at 2500g for 15 min, and the supernatant plasma was transferred to a 2ml sterile tube and stored at -80℃. After removing the plasma from the freezer, it was thawed in a 25℃ water bath, then transferred to a centrifuge tube and centrifuged at 3000g for 10 min at 4℃ to remove cell debris. The supernatant, after removing cell debris, was transferred to a new centrifuge tube and centrifuged at 10000g for 20 min at 4℃ to remove impurities. 1.6ml of the supernatant was added to 0.4ml of Exoquick reagent (System Biosciences) and incubated at 4℃ for 30 minutes. After centrifugation (3000g × 30 min), the supernatant was discarded. The same centrifugation was repeated (3000g × 5 min), and the supernatant was discarded again. The resulting precipitate was the exosome. Exosomes were extracted using the Exoquick kit (System Biosciences). Five μl of the obtained exosome suspension was added to a Formvar-carbon copper grid. After washing the grid with PBS, it was placed on 50 μl of 1% glutaraldehyde solution for 5 min, followed by washing with 100 μl of dH2O for 2 min. The grid was stained with uranyl oxalate and methylcellulose solution, excess liquid was blotted on filter paper, and the grid was air-dried for 5 min. The morphology of the obtained exosomes was identified using a JEOL-1230 transmission electron microscope. The particle size and specific markers of the obtained exosomes were analyzed and identified using a ZetaView PMX110 particle tracker and Western blotting.

[0082] 1) Analytical methods: Graphpad Prism 8 software was used for statistical analysis and graphing. Continuous variables conforming to a normal distribution were expressed as mean ± standard deviation (mean ± SD). Independent samples t-tests were used to compare data between two groups. SPSS 22.0 software was used to calculate the area under the curve and plot the results to evaluate the diagnostic efficacy of exosome circRNA. P < 0.05 was considered statistically significant.

[0083] Example

[0084] Plasma was collected from 12 healthy controls and 20 patients with dilated cardiomyopathy and heart failure, and stored promptly at -80°C. The study was approved by the ethics committee; all patients signed written informed consent forms.

[0085] Exosomes were extracted from the plasma samples using the Exoquick kit (System Biosciences). The extracted exosomes were immobilized on a copper grid, stained, and dried. Morphology of the exosomes was identified using a JEOL-1230 transmission electron microscope. Particle size and specific markers of the exosomes were analyzed and identified using a ZetaView PMX 110 particle tracker and Western blotting. TEM images are shown below. Figure 1 The results of nanoparticle tracking analysis (NTA) are shown below. Figure 2 The results of the protein blot are shown below. Figure 3 (Alix, CD 63 and Hsp 70). Figures 1 to 3 The results showed that the extracted vesicles conformed to the characteristics of exosomes in terms of morphology, particle size distribution, and specific marker labeling.

[0086] Previous studies have suggested an association between nine exosomal circRNAs and dilated cardiomyopathy complicated with heart failure, namely: hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837, with specific nucleic acid sequences shown in SEQ NO. 1–9. These were validated using qRT-PCR, as follows:

[0087] 1. Extraction of total RNA from plasma exosomes: Take an appropriate amount of exosomes into a 1.5 ml EP tube, add 800 μL Trizol reagent (Invitrogen), mix thoroughly, lyse for 10 min, centrifuge at 12000 rpm for 10 min, and collect the supernatant. After vortexing and mixing, incubate at 4°C for 10 min, then add 200 μL chloroform, vortex and mix, incubate at 4°C for 10 min, and centrifuge at 12000 rpm for 10 min; collect the supernatant, add an equal volume of isopropanol, add a certain volume of glycogen, incubate at -20°C overnight, centrifuge again at 12000 rpm at 4°C for 10 min, and discard the supernatant; add 1 ml of 70% ethanol, centrifuge again at 12000 rpm at 4°C; when the RNA just turns transparent, add an appropriate amount of nuclease-free water, incubate at 55°C for 5 min to completely dissolve the RNA, and determine the concentration of the extracted RNA by UV analysis.

[0088] 2. RNA Reverse Transcription to cDNA: RNA was reverse transcribed into cDNA using the TAKARA Reverse Transcription Kit (containing gDNA removal reagent for qPCR). First, prepare the reaction mixture on ice according to Table 1 to eliminate genomic DNA from the RNA. To ensure the accuracy of the reaction mixture preparation, prepare MasterMix by adding 2 to the reaction number before each reaction, then aliquot the mixture into each reaction tube, and finally add the RNA sample. Incubate at 42°C for 2 minutes and store at 4°C.

[0089] Table 1 Elimination of genomes in RNA

[0090]

[0091] The reverse transcription reaction system and reaction conditions are shown in Tables 2 and 3:

[0092] Table 2 Reverse Transcription Reaction System

[0093]

[0094] Table 3 Reverse transcription reaction conditions

[0095]

[0096] 3. qRT-PCR detection

[0097] Exosomal circRNAs were analyzed using the Stepone Plus real-time PCR instrument (Applied Biosystems), with each sample analyzed in triplicate. Table 4 shows the nucleic acid sequences of the nine circRNAs and their forward and reverse primer sequences. Primer pairs were designed and synthesized by Gemma Biosciences, and NCBI BLAST alignment analysis was used to explore the sensitivity and specificity of the primers. Tables 5 and 6 show the qRT-PCR reaction system and reaction conditions, with the qPCR kit purchased from Bimake. Actin was used as an external reference, and Log2(2-ΔΔCT) represents the relative expression level of the target gene.

[0098] Table 4. Exosomal circRNA nucleic acid sequences and their forward primer sequences.

[0099]

[0100]

[0101] Table 5. qRT-PCR detection reaction system

[0102]

[0103] Table 6. qRT-PCR detection reaction conditions

[0104]

[0105] Results Analysis

[0106] RT-qPCR results showed differential expression of hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837 in patients with dilated cardiomyopathy and heart failure: compared with the healthy group, all nine circRNAs were upregulated or downregulated in the disease group, and the differences were statistically significant. (See attached figures). Figure 4 .

[0107] Statistical analysis and graphing were performed using R and SPSS 22.0, and the area under the curves was calculated to evaluate the diagnostic efficacy of exosomal circRNAs. ROC curve analysis of the expression levels of nine plasma exosomal circRNAs is shown below. Figure 5The results showed that the AUC values ​​of the exosomes hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837 were 0.914, 0.928, 0.920, 0.886, 0.942, 0.901, 0.918, 0.927, and 0.934, respectively. See [see attached image]. Figure 5 .

[0108] Nine exosomes (hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837) were used as a combination of molecular markers. The results showed that the AUC value of the combination of molecular markers reached 0.973, which was higher than that of individual markers (P<0.0001), with a sensitivity of 97.8% and a specificity of 95.0%. This demonstrates that the exosomal circRNA combination has good diagnostic efficacy for dilated cardiomyopathy complicated with heart failure. See the results below. Figure 6 .

[0109] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the scope of protection of the present invention.

Claims

1. The use of reagents for detecting exosomal circRNA molecular markers in the preparation of kits, microarrays, or biochips for diagnosing dilated cardiomyopathy complicated with heart failure, wherein the exosomal circRNA molecular markers are combinations of hsa-circ-0026806, hsa-circ-0054400, hsa-circ-0074153, hsa-circ-0047519, hsa-circ-0063766, hsa-circ-0076771, hsa-circ-0019699, hsa-circ-0056066, and hsa-circ-0075837.

2. The use of the reagent for detecting exosomal circRNA molecular markers according to claim 1 in the preparation of reagent kits, microarrays, or biochips for diagnosing dilated cardiomyopathy complicated with heart failure, characterized in that: The reagents used to detect the expression level of exosomal circRNA are primers or probes.

3. The use of the reagent for detecting exosomal circRNA molecular markers according to claim 2 in the preparation of reagent kits, microarrays, or biochips for diagnosing dilated cardiomyopathy complicated with heart failure, characterized in that: The primer combination for quantitative real-time detection of exosomal circRNA expression is as follows: (1) Primers for hsa-circ-0026806: Forward primer: 5'-AATGGAAATTGTACTTAACCTATGTG-3'; Reverse primer: 3'-TCACGAGATCGATTCCTGACTAC-5'; (2) Primers for hsa-circ-0054400: Forward primer: 5'-TATTGAAGGTGCCAGGTGCTG-3'; Reverse primer: 3'-GCCAGGAAATCAGTTGGTGAGA-5'; (3) Primers for hsa-circ-0074153: Forward primer 5'-CACAAATGGGGATACCTTCTTAG-3'; Reverse primer: 3'-CCTTGATTGCTTCTGATCTCTCT-5'; (4) Primers for hsa-circ-0047519: Forward primer 5'-CATCACTGCCTACTTCAAAGAAAT-3'; Reverse primer: 3'-CACCACAGTATCACTCTCTAGCCA-5'; (5) Primers for hsa-circ-0063766: Forward primer 5'-CTGTGAACCAGAATTCAATCAGA-3'; Reverse primer: 3'-ATCCAGAACTCTTTGGAAGATAGTC-5'; (6) Primers for hsa-circ-0076771: Forward primer 5'-AACGAGATGTAAAAGTGTCACTGTG-3'; Reverse primer: 3'-GCCTTCACTGCAGCTGAAAAG-5'; (7) Primers for hsa-circ-0019699: Forward primer 5'-CCTTATCATCTAACCTCACTGAACTCT-3'; Reverse primer: 3'-CCAGTGGTATCTTCAGAGCGAAT-5'; (8) Primers for hsa-circ-0056066: Forward primer 5'-TAAAAACCAATAACAGTCGT-3'; Reverse primer: 3'-TCACGTGAGAAAAAGTCCCC-5'; (9) Primers for hsa-circ-0075837: Forward primer 5'-CAAGGCAAAGCTGTTACTAG-3'; Reverse primer: 3'-GCTGGAACGTAGTCAAGGAA-5'.

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