Human circular RNA and application thereof

Abstract

The invention relates to the technical field of detection markers in the field of research on human muscle aging, in particular to a human circular RNA (Ribonucleic Acid) and application thereof. The invention discloses a circular RNA marker for detecting human muscle senescence and application of the circular RNA marker, human source circular RNA circN6 is has-circN6, the nucleotide sequence of the human source circular RNA circN6 is shown as SEQ ID NO: 1, meanwhile, the invention provides detection primers of the human source circular RNA circN6, and the nucleotide sequences of the primers are shown as SEQ ID NO: 3-4. According to the present invention, the existence of the annular transcript has-circN6 of hsa-N6 is found, the full-length cloning is performed on the molecule, and the strong tolerance of the has-circN6 on the RNase R excision enzyme is detected; fluorescent quantitative PCR analysis finds that has-circN6 is highly expressed in muscular tissues of young people, is low in expression in serum of old people, and has translation potential.

Description

Technical Field

[0001] The present invention relates to the technical field of detecting markers in the field of studying human muscle aging, and in particular to a human circular RNA circN6 for detecting a serum marker of human muscle aging. Background Art

[0002] In today's society, with the increasing aging of the population, it is crucial to understand and monitor the human aging process. Muscle aging, as an important aspect of human aging, not only affects people's ability to carry out daily activities, but is also closely related to the occurrence and development of many chronic diseases. In recent years, circular RNA (circRNA), as a new type of biomarker, has attracted widespread attention in the study of human muscle aging.

[0003] Circular RNA is a type of non-coding RNA with a special circular structure. Unlike traditional linear RNA, it does not have a 5' cap and a 3' poly(A) tail, making it more stable. Circular RNA is widely present in eukaryotes and has tissue specificity and developmental stage specificity. Recent studies have shown that circular RNA plays an important role in a variety of physiological and pathological processes, including gene expression regulation, cell proliferation, differentiation and apoptosis.

[0004] In the study of muscle aging, circular RNA has the following advantages, making it a potential serum marker: The stability of circular RNA makes it easier to detect in body fluids such as serum. Compared with linear RNA, circular RNA is not easily degraded by nucleases and can exist stably in the blood for a long time. This makes it possible to reflect the state of muscle aging by detecting the level of circular RNA in serum.

[0005] At present, the research on circular RNA as a serum marker of human muscle aging is still in its infancy, but some preliminary results have been achieved. Through high-throughput sequencing technology and bioinformatics analysis, researchers have identified some circular RNAs that are differentially expressed during muscle aging. These circular RNAs may become potential serum markers of muscle aging, providing a new means for early diagnosis and monitoring of muscle aging.

[0006] Human circular RNA circN6 comes from human chromosome 1 and is formed by reverse splicing of exons 6, 7, 8, and 9 of its maternal gene (has-N6mRNA). Currently, no information about this circRNA is found on websites such as circBase, and there is no research on human muscle aging with this circRNA, so this circRNA is temporarily named circN6. In short, as a new type of biomarker, circular RNA has broad application prospects in the study of human muscle aging. With the continuous deepening of research, it is believed that circular RNA will provide a more accurate and reliable means for early diagnosis and monitoring of muscle aging, and contribute to improving the health status and quality of life of the elderly. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a detection primer for identifying the presence of human circular RNA circN6 in the human body. The present invention provides a full-length human circular RNA circN6, provides an application of a fluorescent quantitative PCR detection primer in detecting human circular RNA circN6, and provides a full-length amplification primer for human circular RNA circN6.

[0008] The present invention also discovered that human circular RNA circN6 is differentially expressed in the serum of young and elderly people, and therefore can be used as a serum marker detection kit for human muscle aging.

[0009] The present invention provides a vector pLO5-hsa-circN6+FLAG containing human circular RNA circN6 with a FLAG tag, and a vector pLO5-hsa-circN6+FLAG+ΔATG containing human circular RNA circN6 with a FLAG tag and a mutated start codon ATG, so as to verify the translation of circular RNA circN6.

[0010] Furthermore, the present invention verifies that the polypeptide translated by circular RNA circN6 has significant differences in the muscles of the elderly and young people, and therefore can be used as a detection kit for human muscle aging.

[0011] In order to achieve the above object, the present invention is implemented by the following scheme:

[0012] The present invention uses muscle tissue and serum of fracture patients of all ages taken from the hospital as research samples, and the informed consent of each research subject has been obtained before the study is carried out. The research subjects are divided into a young group (age ≤ 20 years old) and an elderly group (age ≥ 65 years old) according to their age. First, detection primers and full-length amplification primers are designed according to the sequence characteristics of the reverse splicing site (cross-splicing site) of circN6. After PCR cloning of the specific sequence and full-length sequence across the splicing site, second-generation sequencing is performed; then the designed detection primers are used to detect the relative expression of circular RNA circN6 in muscle samples and serum samples of the young group and the elderly group by fluorescent quantitative PCR; at the same time, circular RNA circN6 with a FLAG tag and a vector with a FLAG tag and a mutation of the start codon ATG are constructed respectively, and 293T cells are transfected, and cell extraction protein samples are collected 48h after transfection for Western-blot experiments to verify whether circular RNA circN6 has the ability to translate.

[0013] Therefore, the present invention claims the following:

[0014] A circular RNA circN6, wherein the circN6 nucleotide sequence is shown in SEQ ID NO:1.

[0015] SEQ ID NO: 1:

[0016] GAATGCCATCTCCAACCACACTGAAGAAGTCGGAGAAGTCTGGTTTCAGCAGCCCCTCCCCTTCACAGACCTCCTCCCTGGGAACGGCGTTCACACAGCATCACCGACCTGTCATTACAGGACCCAGAGCAAGTCCGCATGCAACACCATCGACTCTTCATTTCCCGACATCACCCATTATCCAGCAGCCTGGGCCTTACTTCTCACACCCAGCCATCCGCTATCACCCTCAGGAGACGCTGAAAGAATTTGTCCAACTTGTCTGCCCTGATGCTGGTCAGCAGGCTGGACAGGTGGGGTTCCTCAATCCCAATGGGAGCAGCCAAGGCAAGGTGCACAACCCATTCCTTCCCACCCCAATGTTGCCACCGCCACCGCCACCACCGATGGCCAGGCCTGTGCCTCTGCCGGTGCCAGACACAAAGCCTCCAACCACGTCAACAGAAGGAGGTGCAGCCTCCCCCACGTCACCAACCTACTCGACACCCAGCACCTCCCCCGCAAACCGATTCGTCAGTGTTGGACCACGGGATCCAAGCTTTGTAAATATCCCTCAACAGACACAG

[0017] The translated polypeptide amino acid sequence of a circular RNA circN6 is shown in SEQ ID NO:2.

[0018] SEQ ID NO:2:

[0019] MPSPTTLKKSEKSGFSSPSPSQTSSLGTAFTQHHRPVITGPRASPHATPSTLHFPTSPIIQQPGPYFSHPAIRYHPQETLKEFVQLVCPDAGQQAGQVGFLNPNGSSQGKVHNPFLPTPMLPPPPPPPMARPVPLPVPDTKPPTTSTEGGAASPTSPTYSTPSTSPANRFVSVGPRDPSFVNIPQQTQECHLQPH

[0020] The primers for fluorescence quantitative PCR detection of a circular RNA circN6 have nucleotide sequences shown in SEQ ID NO:3 - 4.

[0021] A full-length amplification primer for circular RNA circN6, whose nucleotide sequence is shown in SEQ ID NO:5-6.

[0022] Application of a circular RNA circN6 as a serum marker for human muscle aging.

[0023] A vector pLO5-hsa-circN6+FLAG containing circular RNA circN6 and carrying a FLAG tag is used to verify whether circular RNA circN6 has translation potential. Preferably, the vector is pLO5-ciR.

[0024] A vector pLO5-hsa-circN6+FLAG+ΔATG containing circular RNA circN6 and having a FLAG tag and a mutated start codon ATG is used to verify whether circular RNA circN6 has translation ability. Preferably, the vector is pLO5-ciR.

[0025] Application of a protein translated by circular RNA circN6 as a detection kit for human muscle aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0027] Figure 1 Determination of circular structure and full-length cloning of circular RNA circN6; A: amplification results of the backsplicing site of circN6; M: DL2000 maker, lane 1: the cross-splicing site amplified using the muscle cDNA of the Y3-18 sample as a template, i.e., the back splicing site; B: Full-length cloning results of circular RNA circN6, M: DL2000 maker, lane 1: the full-length circular RNA circN6 amplified using the muscle cDNA of the Y3-18 sample as a template; C: amplification and sequencing results of the cross-splicing site of circular RNA circN6, i.e., the circular position; D: Schematic diagram of the circularization of circular RNA circN6;

[0028] Figure 2Analysis of RNase R tolerance and reverse transcription of circular RNA circN6 and hsa-N6 mRNA; A: Effect of RNase R on the expression abundance of circular RNA circN6 and hsa-N6 mRNA; B: Effect of different reverse transcription primers on the expression of circular RNA circN6 and hsa-N6 mRNA; C: Expression of circular RNA circN6 and hsa-N6 mRNA in human primary myoblasts;

[0029] Figure 3 Relative expression levels of circular RNA circN6 in muscles and serum of young and old groups, respectively; A: expression level of circN6 in 10 muscle samples; B: relative expression level of circN6 in muscles of young and old groups; C: expression level of circN6 in 10 serum samples; D: relative expression level of circN6 in serum of young and old groups;

[0030] Figure 4 To construct the vector pLO5-hsa-circN6+FLAG containing circN6 with a FLAG tag, and to construct the vector pLO5-hsa-circN6+FLAG+ΔATG containing circN6 with a FLAG tag and with the start codon ATG mutated; A: Schematic diagram of vector construction; B: Sequencing results of the pLO5-hsa-circN6+FLAG vector construction; C: Sequencing results of the pLO5-hsa-circN6+FLAG+ΔATG vector construction; D: Western-blot results, the lanes on both sides are protein markers, lane 1 is the positive control, lane 2 is the protein sample of 293T cells transfected with the pLO5-hsa-circN6+FLAG vector, and lane 3 is the protein sample of 293T cells transfected with the pLO5-hsa-circN6+FLAG+ΔATG vector;

[0031] Figure 5 To detect the content of circN6-aa, a polypeptide translated from circN6, in serum and muscle samples of the young and elderly groups; A: western-blot results, the upper and lower groups represent the content of circN6-aa, a polypeptide translated from circN6, detected in serum samples of the young and elderly groups, respectively; B: western-blot results, the upper and lower groups represent the content of circN6-aa, a polypeptide translated from circN6, detected in muscle samples of the young and elderly groups, respectively. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] Example 1: Full-length cloning of circular RNA circN6

[0034] 1. Experimental Methods

[0035] Based on the previous sequencing study of human muscle samples, a circular RNA highly expressed in the muscles of young people was screened, namely circN6. Through the prediction and comparison information on the NCBI website, the full-length primers has-circN6-Full length-F / R for amplifying circN6 and the identification primers Q-hsa-circN6-F / R for circN6 were designed, and the internal reference gene was GAPDH. The primers were designed by Primer-BLAST (Primer designing tool (nih.gov)) and Primer3 (Primer3Input). Primer information is shown in Table 1.

[0036] The total RNA of muscle tissues of the young group (including 4 young samples, Y1-11, Y2-16, Y3-18, and Y4-20, where the first two digits represent the sample number and the last two digits represent the age) and the elderly group (including 6 elderly samples, O1-68, O2-65, O3-70, O4-71, O5-74, and O6-76) were extracted by Trizol method, and the total RNA was reverse transcribed into cDNA. The reverse transcription procedures were 25℃, 10min; 42℃, 30min; 80℃, 10min. The cDNA of Y3-18 was used as a template for full-length amplification of circN6 and amplification across splice junctions.

[0037] Table 1 Primer sequence information

[0038]

[0039]

[0040] 2. Experimental Results

[0041] Since circular transcripts are formed by reverse splicing, which connects the spliceosomes end to end to form a circular structure, primers were designed for the circN6 loop position sequence and the target sequence was amplified by PCR. Figure 1A is the amplification result of the back splicing site of circN6 using Q-has-circN6 primer. The electrophoresis results show that the target fragment was successfully amplified. The target fragment of the circN6 linker position exists at the 190bp position (such as Figure 1 A), which preliminarily indicates that circN6 really exists. The above PCR products were sent for sequencing, and the base sequence of the sequencing results was consistent with the base sequence of the back splicing site of circN6. circN6 is derived from exons 6, 7, 8, and 9 of its maternal gene (as shown in Figure 1 C). The full-length molecule was cloned using the hsa-circN6-Full length primer. The cloning results showed that the full length of circN6 was 566 nt, which was formed by reverse splicing of exons 6 to 9 of its maternal gene, and it was predicted to contain 2 open reading frames by the bioinformatics website Open Reading Frame Finder (ORFfinder Home-NCBI (nih.gov)).

[0042] Example 2: Circular structure and nucleocytoplasmic distribution of circN6

[0043] 1. Analysis of circN6 tolerance to RNase R and reverse transcription

[0044] 1. Experimental Methods

[0045] Using the RNA extracted from human muscle tissue in Example 1, the extracted total RNA was divided into a control group (RNaseR(-)) and an RNase R treatment group (RNase R(+)). The RNase R treatment group used RNase R enzyme for digestion at 37°C for 15 min, inactivated at 70°C for 10 min, and then reverse transcribed into cDNA; the control group used sterilized double distilled water to replace the RNase R enzyme, and the other reaction conditions were consistent with the RNase R treatment group, so as to detect the stability of circRNA.

[0046] Real-time fluorescence PCR was used to quantify the relative expression of circN6. Q-has-circN6 primers were used to detect the expression of circN6, and GAPDH was used as an internal reference gene. The fluorescence quantitative PCR method was as follows: cDNA generated by reverse transcription was used as a template for real-time fluorescence quantitative PCR. The PCR reaction system was: 2 μL of cDNA template, 0.3 μL of upstream and downstream primers, 5 μL of 2×SYBR Green qPCR Mix, 2.4 μL of double distilled water, and a total system of 10 μL. The qPCR reaction conditions were 95°C, 5 min; 95°C, 5 s; 60°C, 30 s; 72°C, 15 s. 35 cycles; the melting curve program was 95°C, 15 s; 60°C 1 min; 95°C, 1 s. The information of fluorescence quantitative primers is shown in Table 2.

[0047] Table 2 Primer sequence information

[0048]

[0049] In addition, the extracted total RNA was divided into two types: using random primers and using oligothymidine primers for reverse transcription. The system and reaction conditions are shown in Table 3:

[0050] Table 3 Reaction system

[0051]

[0052] The expression of circN6 was then quantitatively analyzed based on the above-mentioned fluorescence quantitative method.

[0053] 2. Experimental results

[0054] The results of RNase R tolerance showed that the relative expression of has-N6 mRNA in the RNase R-treated group decreased significantly, while the relative expression of circN6 did not change significantly (e.g. Figure 2 A). Random primers and oligothymidine primers were used for reverse transcription, and the RT-qPCR quantitative results showed that the expression level of has-N6 mRNA in the oligothymidine primer group and the random primer group did not change significantly, while the expression level of circN6 reverse transcribed using random primers was significantly higher than that in the oligothymidine primer group (as shown in Figure 2 B). It was further confirmed that circN6 has the characteristics of a circular molecule without a 3'-end Poly (A) tail.

[0055] 3. Distribution of circN6 and has-N6 mRNA in primary human myoblasts

[0056] 1. Experimental Methods

[0057] (1) Nuclear and cytoplasmic separation

[0058] ① Preparation of nuclear lysis buffer and cell preparation

[0059] Table 4 Preparation of cytosolic lysis buffer

[0060] Stock concentration Working concentration Dilution ratio Dosage (1mL) Hepes (pH = 7.9) 0.5M 10mM 1：50 20μL MgCl2 1M 1.5mM 1：667 1.5μL KCl 1M 10mM 1：100 10μL NP-40 10％ 0.1％ 1：100 10μL DTT 1M 0.5mM 1：2000 0.5μL cocktail 1：100 10μL RNase inhibitor 0.1U / μL ddH2O to 1mL

[0061] Table 5 Preparation of nuclear lysis buffer

[0062] Stock concentration Working concentration Dilution ratio Dosage (1mL) Tris-HCl (pH = 7.4) 1M 50mM 1：20 50μL NaCl 5M 450mM 1：11 91μL glycerin 100％ 20％ 1：5 200μL NP-40 10％ 1％ 1：10 100μL DTT 1M 2mM 1：500 2μL PMSF 100mM 1mM 1:100 10μL cocktail 10μL Na3VO4 0.2M 0.2mM 1:1000 1μL RNase inhibitor 0.1U / μL ddH2O to 1mL

[0063] a. Primary myoblasts were isolated from leg muscle samples of Y3-18 and cultured in complete medium (DMEM / HG + 20% fetal bovine serum (v / v) + 1% double antibody (v / v) + 0.01% ciprofloxacin (v / v)) at 37°C and 5% CO2 (v / v).

[0064] b. (In a 10 cm plate) First, aspirate the cell supernatant, then use PBS to wash the myoblasts 1 to 2 times, and finally blow off the cells with 1 mL of PBS, transfer them to a 1.5 mL tube, centrifuge at 1500 rpm at 4°C for 5 min to precipitate the cells, and remove the supernatant.

[0065] c. Extract cytoplasmic RNA: Add 3 times the volume of cytoplasmic lysis buffer (100-150 μL) to the precipitate, gently blow the cells, let stand on ice for 10 minutes, and then centrifuge at 4°C, 5000 rpm for 10 minutes.

[0066] d. Transfer the supernatant to a 1.5 mL tube without RNase. This is the cytoplasmic RNA. Be careful not to touch the cell pellet during the transfer of the supernatant. You can keep some supernatant appropriately.

[0067] e. Add cytoplasmic lysis buffer, blow the cells evenly, place on ice for 10 min, centrifuge at 4°C, 10,000 rpm for 10 min, and discard the supernatant. Repeat this step 4 times to thoroughly wash the cytoplasmic RNA.

[0068] f. Get nuclear RNA: Add 3 times the volume of nuclear lysis solution (100-150 μL) to the precipitate washed in the previous step, blow off the precipitate, shake vigorously for 30 seconds, let it stand on ice for 10 minutes, centrifuge at 4°C, 12000 rpm for 10 minutes, and collect the supernatant, which is the nuclear RNA.

[0069] (2) Nuclear and cytoplasmic quantification

[0070] The extracted cytoplasmic and nuclear RNA was reverse transcribed into cDNA. The reverse transcription method was based on Example 1. Real-time fluorescence PCR was used to quantify the relative expression levels of circN6 and has-N6 mRNA. The primer information was based on Table 2. Q-has-circN6 primer was used to detect the expression of circN6, and Q-has-N6 primer was used to detect the expression of has-N6. GAPDH was used as a cytoplasmic reference gene, and U6 was used as a nuclear reference gene.

[0071] 2. Experimental results

[0072] RT-qPCR quantitative results after nuclear-cytoplasmic separation showed that circN6 was generally localized in the cytoplasm of myoblasts, while the expression level of its linear transcript has-N6 in the nucleus was significantly higher than that in the cytoplasm (e.g. Figure 2 C).

[0073] Example 3: Application of circN6 as a human muscle aging serum marker kit

[0074] 1. Experimental Methods

[0075] As described in Example 1, total RNA was extracted from muscle and serum samples of all elderly and young groups and used as a template after reverse transcription. The designed circN6 identification primers Q-hsa-circN6-F / R were used to identify the relative expression levels of circN6 in muscle and serum samples of the elderly and young groups by using fluorescent quantitative PCR, and GAPDH was used as an internal reference gene.

[0076] 2. Experimental Results

[0077] Figure 3 A and B show the relative expression of circN6 in the muscle tissue of the young group and the old group. The results show that the relative expression level of circN6 in the muscle tissue of the young group is significantly higher than that of the old group. Figure 3 C and D show the relative expression of circN6 in the serum of the young and elderly groups. The results show that the relative expression level of circN6 in the muscle tissue of the young group is significantly lower than that of the elderly group. In summary, circN6 has the potential to be used as a serum marker kit for detecting human muscle aging.

[0078] Example 4: Verification of the ability of circN6 to translate and the application of the circN6-aa polypeptide translated from circN6 as a human muscle aging detection kit in serum and muscle

[0079] 1. Experimental Methods

[0080] The vector pLO5-hsa-circN6+FLAG containing circN6 and FLAG tag was constructed, and the vector pLO5-hsa-circN6+FLAG+ΔATG containing circN6 and FLAG tag with mutated start codon ATG was constructed. The two vectors were transfected into 293T cells (12-well plate) respectively, and the verified circRNA that can be translated was used as a positive control to eliminate system errors. PEI was used as a transfection reagent for transfection. 1μg of vector and 4μg of PEI were diluted with 50μL of blank DMEM / HG medium, respectively. After standing at room temperature for 5 minutes, PEI was carefully added to the vector, and it was allowed to stand at room temperature for 25min. Then, the PEI and vector mixture was added dropwise to 293T cells, and the DMEM / HG medium with 10% FBS was replaced after 4-6h. 48h after transfection, the cells were harvested and protein samples were extracted for Western-blot experiment. Then, serum and muscle proteins were extracted from three young patients, Y1-11, Y2-16, and Y3-18, and three elderly patients, O4-71, O5-74, and O6-76, for Western-blot experiments.

[0081] 2. Experimental Results

[0082] like Figure 4 As shown in B and C, the vector containing circN6 and the vector with the start codon ATG mutated were successfully constructed. Figure 4 D shows that circN6 (lane 2) has the potential for translation, with a light band near 25 kDa. After the start codon was mutated (lane 3), the light band disappeared, preliminarily proving that circN6 has the ability to translate. Figure 5 As shown in A, the content of circN6-aa, a polypeptide translated from circN6, in the serum of the young group was significantly lower than that in the serum of the elderly group. Figure 5 B shows that the content of circN6-aa, a polypeptide translated from circN6, in the muscles of the young group is significantly higher than that in the muscles of the elderly group, indicating that circN6-aa can be used as a serum detection kit for human muscle aging in serum and muscle.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A human circular RNA circN6, wherein the circN6 nucleotide sequence is as shown in SEQ ID NO:

1.

2. A polypeptide translated from human circular RNA circN6, whose amino acid sequence is shown in SEQ ID NO:

2.

3. A fluorescent quantitative PCR detection primer for human circular RNA circN6, whose nucleotide sequence is shown in SEQ ID NO:3-4.

4. Use of the fluorescent quantitative PCR detection primers according to claim 3 in detecting human circular RNA circN6.

5. Application of human circular RNA circN6 as a serum marker for human muscle aging in the preparation of detection kits.

6. An application to verify that circular RNA circN6 has translation ability.

7. Application of the polypeptide translated by circular RNA circN6 as a serum marker of human muscle aging in the preparation of detection kits.

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