CN112410417A - Annular circRNA AFF1 and application thereof - Google Patents
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Abstract
The invention belongs to the field of biomedical inspection, and particularly relates to a circular circRNA AFF1 for diagnosing subarachnoid hemorrhage (SAH), application of the circular circRNA AFF1 in diagnosing subarachnoid hemorrhage (SAH) and a related kit. The nucleotide sequence of the circular RNA circRNA AFF1 is shown as SEQ ID No. 1. According to the invention, after RNA in peripheral blood plasma is extracted, circRNA AFF1 is found to be used as an important biological detection index and can be applied to quantitative detection, so that whether a patient has SAH risk or not is judged, early diagnosis, early prevention and early treatment of the SAH patient are realized, and the method plays an important role in early diagnosis and differential diagnosis of subarachnoid hemorrhage (SAH).
Description
Technical Field
The invention belongs to the field of biomedical inspection, and particularly relates to a circular circRNA AFF1 for diagnosing subarachnoid hemorrhage (SAH), application of the circular circRNA AFF1 in diagnosing subarachnoid hemorrhage (SAH) and a related kit.
Background
Subarachnoid hemorrhage (SAH) is a neurological emergency with a mortality rate of 50%. Except for rupture of aneurysm, the rest causes are unclear, and the risk of bleeding after disease is particularly high. The reasons are closely related to the abnormal function of blood vessels. SAH causes not only devastating attacks on the central nervous system but also severe effects on many other organs, the most common complications being loss of neural function due to cerebral ischemia, hypoxia, and permanent neurological dysfunction.
SAH is a sudden and high-mortality neurological disease, and its diagnosis has long been mainly based on imaging means such as CT, and there is no effective diagnostic means for early diagnosis of the disease and for secondary bleeding caused by recurrence. Therefore, the occurrence and development process of SAH is deeply researched, key molecular markers and drug targets are searched, and the method has important scientific significance and clinical value for developing effective diagnosis and treatment measures.
Circular RNA (circRNA) is a non-coding RNA which is widely found in mammals in recent years, is rich in expression and is formed by performing variable shearing on special precursor mRNA with reverse complementary ends. The circRNA is connected end to form a closed circular structure, so that the closed circular structure is more stable than linear RNA and can resist the degradation of exonuclease. Meanwhile, the expression of the circRNA has the specificity of tissues and cell types, and the abundant circRNA can be detected in blood, saliva, urine or exosomes. These features of circRNA suggest its potential as a diagnostic marker for disease.
SAH is closely related to vascular dysfunction and vascular stress injury caused by hypoxia, and the abnormal activation of the CircRNA AFF1 in vascular endothelial cells under the hypoxia state causes the up-regulation of the phosphorylation level of a final effector YAP in a Hippo/YAP signal pathway, and finally causes partial loss of the functions of vascular regeneration and repair. Through high-throughput circular RNA sequencing analysis of patient samples, the screen discovers that circAFF1 is abnormally increased in hypoxic vascular injury and can diffuse into peripheral blood before imaging performance, and the method has certain clinical significance of early warning.
Disclosure of Invention
Based on the above problems, the present invention aims to overcome the disadvantages of the prior art and provide a rapid, sensitive and specific diagnostic indicator for subarachnoid hemorrhage (SAH) and a corresponding diagnostic kit.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following aspects:
in a first aspect, the invention provides a circular RNA circRNA AFF1, the nucleotide sequence of circular RNA circRNA AFF1 being shown in SEQ ID No. 1.
In a second aspect, the invention provides a molecular marker for subarachnoid hemorrhage, wherein the molecular marker is circular circRNA AFF 1.
According to the invention, the circAFF1 in the subarachnoid hemorrhage patient is found to be up-regulated, so that the circAFF1 can be used as an important biological detection index of the subarachnoid hemorrhage, can be applied to quantitative detection, can judge whether the patient has SAH risk, realizes early diagnosis, early prevention and early treatment of the SAH patient, and has an important role in early diagnosis and differential diagnosis of the subarachnoid hemorrhage (SAH).
In a third aspect, the invention provides the use of a circular circRNA AFF1 quantitative detection agent in the preparation of a diagnostic kit for subarachnoid hemorrhage.
The quantitative detection reagent is a qRT-PCR primer capable of amplifying circular circRNA AFF1, the sequence of an upstream primer of the qRT-PCR primer is shown as SEQ ID No.2, and the sequence of a downstream primer is shown as SEQ ID No. 3.
In a fourth aspect, the present invention provides a diagnostic kit for subarachnoid hemorrhage, which contains a reagent for detecting the content of cyclic circRNA AFF1 in vascular endothelial cells.
Preferably, the kit contains a qRT-PCR primer capable of amplifying circular circRNA AFF1, wherein the sequence of an upstream primer of the qRT-PCR primer is shown as SEQ ID NO.2, and the sequence of a downstream primer is shown as SEQ ID NO. 3.
Further, the kit also comprises an internal reference primer pair.
Preferably, the reference primer pair comprises one or more of a U6 amplification primer pair and a GAPDH amplification primer pair.
Specifically, the nucleotide sequences of the U6 amplification primer pair are respectively shown as SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequences of the GAPDH amplification primer pair are respectively shown as SEQ ID NO.6 and SEQ ID NO. 7.
Preferably, the kit further comprises at least one of an RNA extraction reagent, a PCR reaction buffer, dNTPs, and a DNA polymerase.
Compared with the prior art, the invention has the following advantages:
according to the invention, after RNA in peripheral blood plasma is extracted, circRNA AFF1 is found to be used as an important biological detection index and can be applied to quantitative detection, so that whether a patient has SAH risk or not is judged, early diagnosis, early prevention and early treatment of the SAH patient are realized, and the method plays an important role in early diagnosis and differential diagnosis of subarachnoid hemorrhage (SAH).
Drawings
FIG. 1-A shows a reaction system in which CoCl is present2Relative expression of circAFF1 in HUV-EC-C and HBEC-5i cells under treatment;
FIG. 1-B is reverse splicing of CircAFF1 derived from exons 3 and 4 of the AFF1 gene;
FIG. 1-C is a qRT-PCR analysis of circAFF1 and AFF1mRNA after treatment with or without RNase R in HUV-EC-C and HBEC-5i cells;
FIGS. 1-D are qRT-PCR analyses of circAFF1 in HUV-EC-C and HBEC-5i nuclei and cytoplasm;
FIG. 1-E shows FISH that circAFF1 is mainly localized in the cytoplasm, where nuclei were stained with DAPI and circAFF1 was labeled with Cy 3;
FIG. 2-A is a qRT-PCR analysis of circAFF1 in HUV-EC-C and HBEC-5i cells after stable transfection of circAFF1 or vector;
FIG. 2-B is a qRT-PCR analysis of AFF1mRNA in HUV-EC-C and HBEC-5i cells after stable transfection of circAFF1 or the vector;
FIG. 2-C is a diagram of the evaluation of cell viability of HUV-EC-C and HBEC-5i cells with circAFF1 or transfected control vectors by the CCK-8 assay;
FIG. 2-D is a graph evaluating tube-forming ability of HUV-EC-C and HBEC-5i cells transfected with circAFF1 or vector;
FIG. 2-E is a graph evaluating the migration capacity of HUV-EC-C and HBEC-5i cells transfected with circAFF1 or vectors by the round Healing assay;
FIG. 2-F is a graph of the ability of cells to invade by transwell;
FIG. 2-G is a flow cytometry assessment of the apoptotic capacity of HUV-EC-C cells transfected with circAFF1 or vector;
FIG. 2-H is a graph evaluating the apoptotic capacity of HBEC-5i cells transfected with circAFF1 or vector by flow cytometry;
FIG. 3-A is CoCl2After treatment, the level of CircAFF1 in exosomes from HUV-EC-C and HBEC-5i cells;
FIG. 3-B is the expression of cirAFF1 in patients of different ages;
FIG. 3-C is the expression of cirAFF1 in different Hunt-Hess grade patients;
FIG. 3-D is the expression of cirAFF1 in patients of different sex;
fig. 4 is the mechanism of vascular endothelial cell dysfunction caused by hypoxia-induced circAFF 1.
Detailed Description
In order to facilitate an understanding of the invention, the invention is further described below, and preferred embodiments of the invention are given. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The term "quantitative detector for circular RNA circRNA AFF 1" in the present invention shall not be understood as a mere detector for circular RNA circRNA AFF1, but shall include the remaining detection reagents known to those skilled in the art to reflect the expression level of circular RNA circRNA AFF 1. For example, the expression level of circular RNA circRNA AFF1 can be indirectly detected by quantitatively detecting cDNA reverse transcribed from circular RNA circRNA AFF 1.
Example 1
In CoCl2Under the condition of induced cell hypoxia, the expression of the CircRNA AFF1 in two vascular endothelial cells of HUV-EC-C and HEBC-5i is abnormally increased, which indicates that a certain relation exists between the CircRNA AFF1 and the hypoxic stress injury of blood vessels. The specific operation is as follows: genomic DNA was isolated using MiniBEST Universal genomic DNA extraction kit Ver.5.0 (Japan, Takara) and total RNA was extracted from cells using TRIzol reagent (USA, Invitrogen) according to the manufacturer's instructions. For circRNA detection, RNA was split into two equal parts prior to RNase R treatment. One was treated with RNase R to obtain circRNA and the other was used to detect GAPDH expression. RNase R (Epicentre Technologies, USA) was used to remove linear RNA. Mu.g of total RNA was treated with or without RNase R (3U/. mu.g) at 37 ℃ for 30 min and then the treated RNA was purified with the RNeasy MinElute Cleanup Kit (Qiagen, Germany). According to manufacturer's specificationsFractions were extracted using the PARISTM kit (Life Technologies, USA). Complementary DNA (cDNA) was synthesized from total RNA using PrimeScript RT kit (Tiangen) and used in
QPCR was performed on a sequence detector (Roche, Switzerland) and Fast SYBR Green Master Mix kit (Tiangen), according to the manufacturer's recommendations. GAPDH and U6 were used as internal controls. Then the results were analyzed by 2- Δ Δ Ct relative expression method. All primer sequences are as follows:
| SEQ ID NO.2 | circAFF1-F | 5'-CTTGAAAGTGCCTGCCAAAG-3' |
| SEQ ID NO.3 | circAFF1-R | 5'-GGCTTCCTGGTTGCGTCT-3' |
| SEQ ID NO.4 | U6-F | 5'-TGCGGGTGCTCGCTTCGGCAGC-3' |
| SEQ ID NO.5 | U6-R | 5'-CCAGTGCAGGGTCCGAGGT-3' |
| SEQ ID NO.6 | GAPDH-F | 5'-GACCTGACCTGCCGTCTA-3' |
| SEQ ID NO.7 | GAPDH-R | 5'-AGGAGTGGGTGTCGCTGT-3' |
as shown in fig. 1-a through 1-E. Wherein, FIG. 1-A is in CoCl2Relative expression of circAFF1 in HUV-EC-C and HBEC-5i cells under treatment. FIG. 1-B shows reverse splicing of CircAFF1 derived from exons 3 and 4 of the AFF1 gene. FIG. 1-C is a qRT-PCR analysis of circAFF1 and AFF1mRNA after treatment with or without RNase R in HUV-EC-C and HBEC-5i cells. FIGS. 1-D show the qRT-PCR analysis of circAFF1 in the HUV-EC-C and HBEC-5i nuclei and cytoplasm. FIG. 1-E shows FISH that circAFF1 is mainly localized in the cytoplasm, with nuclei stained with DAPI and circAFF1 labeled with Cy 3.
Subsequently, the CircRNA AFF1 is artificially overexpressed in vascular endothelial cells, and the detection shows that the overexpression of the CircRNA AFF1 can really inhibit the angiogenesis capacity, the proliferation capacity and the migration capacity of the vascular endothelial cells and promote the apoptosis of the cells. To further verify that hypoxia causes cell damage to depend on CircRNA AFF1, it was found that a callback to CircRNA AFF1 could indeed restore functional damage to vascular endothelial cells caused by hypoxia after knocking down the expression of CircRNA AFF1 in hypoxic treated cells. As shown in fig. 2-a through 2-H. Wherein FIG. 2-A is a qRT-PCR analysis of circAFF1 in HUV-EC-C and HBEC-5i cells after stable transfection of circAFF1 or vector; FIG. 2-B is a qRT-PCR analysis of AFF1mRNA in HUV-EC-C and HBEC-5i cells after stable transfection of circAFF1 or the vector; FIG. 2-C is a diagram of the evaluation of cell viability of HUV-EC-C and HBEC-5i cells with circAFF1 or transfected control vectors by the CCK-8 assay; FIG. 2-D is a graph evaluating tube-forming ability of HUV-EC-C and HBEC-5i cells transfected with circAFF1 or vector; FIG. 2-E is a graph evaluating the migration capacity of HUV-EC-C and HBEC-5i cells transfected with circAFF1 or vectors by the round Healing assay; FIG. 2-F is a graph of the ability of cells to invade by transwell; FIG. 2-G is a flow cytometry assessment of the apoptotic capacity of HUV-EC-C cells transfected with circAFF1 or vector; FIG. 2-H is a graph evaluating the apoptotic capacity of HBEC-5i cells transfected with circAFF1 or vector by flow cytometry.
Example 2
After informed consent, 235 SAH patients' peripheral blood were correlated with circAFF1 for SAH pathology. Clinical data for 235 SAH patients are shown in table 1:
TABLE 1, 235 clinical data of SAH patients
| 0 | I | II | III | IV | |
| Age (age) | 54.3±13.4 | 64.2±8.4 | 56.6±11.9 | 55.9±11.4 | 56±10.9 |
| Male, n (%) | 10(35.7) | 2(40) | 58(36.7) | 12(37.5) | 4(36.4) |
Peripheral blood was collected by centrifugation for 2 hours and 1200g for 10 minutes, and the upper plasma was separated. TRZOL is added into plasma to extract RNA, then reverse transcription is carried out, and finally real-time quantitative PCR is carried out. RNA extraction and RT-PCR protocol refer to example 1. The results show that the expression of CircRNA AFF1 is significantly elevated in SAH patients compared to the expression of CircRNA AFF1 in the blood of the normal population. As shown in fig. 3-a through 3-E. Wherein FIG. 3-A is CoCl2After treatment, the level of CircAFF1 in exosomes from HUV-EC-C and HBEC-5i cells. FIG. 3-B is the expression of cirAFF1 in patients of different ages. FIG. 3-C is the expression of cirAFF1 in different Hunt-Hess grade patients; FIG. 3-D is the expression of cirAFF1 in patients of different sex.
Pathological vascular endothelial injury caused by hypoxia is the basis of many vascular-related diseases. However, the role of circular RNA in hypoxic vascular injury is still poorly understood. Hypoxia-induced AFF1 circular RNA (circAFF1) can activate SAV1/YAP1 and cause vascular endothelial cell dysfunction. Aberrant expression of circAFF1 inhibited proliferation, tube formation and migration of vascular endothelial cells. The role of circAFF1 is achieved by the release of SAV1 by the adsorption of miR-516b, SAV1 in turn causing phosphorylation of YAP 1. In addition, the present invention found upregulation of circAFF1 in 235 cases of subarachnoid hemorrhage patients. As shown in fig. 4, the figure reveals the mechanism of vascular endothelial cell dysfunction caused by hypoxia-induced circAFF 1. In conclusion, the invention clarifies the function of circAFF1/miR-516b/SAV1/YAP1 axis in vascular endothelial dysfunction and the potential early diagnosis value of the vascular endothelial dysfunction on diseases caused by hypoxic injury, and can be used for preparing a diagnosis kit for subarachnoid hemorrhage.
Claims (10)
1. The circular RNA circRNA AFF1 is characterized in that the nucleotide sequence of the circular RNA circRNA AFF1 is shown as SEQ ID No. 1.
2. A molecular marker for subarachnoid hemorrhage, wherein the molecular marker is the circular circRNA AFF1 of claim 1.
3. Application of a circular circRNA AFF1 quantitative detection agent in preparation of a diagnostic kit for subarachnoid hemorrhage.
4. The use according to claim 3, wherein the quantitative detection agent is a qRT-PCR primer capable of amplifying circular circRNA AFF1, the sequence of the upstream primer of the qRT-PCR primer is shown as SEQ ID No.2, and the sequence of the downstream primer is shown as SEQ ID No. 3.
5. A diagnostic kit for subarachnoid hemorrhage is characterized by comprising a reagent for detecting the content of circular circRNA AFF1 in vascular endothelial cells.
6. The diagnostic kit of claim 5, wherein the kit comprises a qRT-PCR primer capable of amplifying circular circRNA AFF1, wherein the sequence of the upstream primer of the qRT-PCR primer is shown as SEQ ID No.2, and the sequence of the downstream primer is shown as SEQ ID No. 3.
7. The diagnostic kit of claim 6, further comprising an internal reference primer pair.
8. The diagnostic kit of claim 7, wherein the reference primer pair comprises one or more of a U6 amplification primer pair and a GAPDH amplification primer pair.
9. The diagnostic kit of claim 8, wherein the nucleotide sequences of said U6 amplification primer pair are shown as SEQ ID No.4 and SEQ ID No.5, respectively, and the nucleotide sequences of said GAPDH amplification primer pair are shown as SEQ ID No.6 and SEQ ID No.7, respectively.
10. The diagnostic kit of claim 9, wherein the kit further comprises at least one of RNA extraction reagents, PCR reaction buffers, dNTPs, and DNA polymerase.
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Citations (3)
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|---|---|---|---|---|
| WO2017046203A1 (en) * | 2015-09-15 | 2017-03-23 | Luxembourg Institute Of Health (Lih) | Biomarkers for heart failure |
| CN109868315A (en) * | 2019-01-28 | 2019-06-11 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | For early detection cerebral aneurysm subarachnoid hemorrhage severity and the in-vitro method of prognosis |
| CN111926074A (en) * | 2020-09-03 | 2020-11-13 | 南京医科大学眼科医院 | Biomarker for detecting ischemic retinopathy, detection kit and application |
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2020
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| WO2017046203A1 (en) * | 2015-09-15 | 2017-03-23 | Luxembourg Institute Of Health (Lih) | Biomarkers for heart failure |
| CN109868315A (en) * | 2019-01-28 | 2019-06-11 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | For early detection cerebral aneurysm subarachnoid hemorrhage severity and the in-vitro method of prognosis |
| CN111926074A (en) * | 2020-09-03 | 2020-11-13 | 南京医科大学眼科医院 | Biomarker for detecting ischemic retinopathy, detection kit and application |
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