CN112322732A - Application, expression method and kit of biological marker for detecting intracranial aneurysm - Google Patents
Application, expression method and kit of biological marker for detecting intracranial aneurysm Download PDFInfo
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Abstract
The invention belongs to the technical field of biological markers, and discloses a biological marker application, an expression method and a kit for detecting intracranial aneurysm, wherein the biological marker for detecting intracranial aneurysm is CD11 a; the biological marker CD11a is a protein encoded by a parent gene ITGAL corresponding to hsa _ circ _ 0000690; the expression method for detecting the biological marker of the intracranial aneurysm comprises the following steps: collecting a specimen; extracting total RNA; detecting the expression level of mRNA of hsa _ circ _0000690, ITGAL and CD11a in ICA and normal human peripheral blood; finding biomarkers for diagnosing intracranial aneurysms; and (5) carrying out statistical analysis. The invention defines the expression difference of hsa _ circ _0000690 in ICA patients and normal human peripheral blood, explores the possibility of the hsa _ circ _0000690 as a noninvasive biological marker and has stronger innovation.
Description
Technical Field
The invention belongs to the technical field of biological markers, and particularly relates to application, an expression method and a kit of a biological marker for detecting intracranial aneurysm.
Background
At present, intracranial aneurysm (ICA) is characterized by saccular expansion protrusion of cerebrovascular pathological, and in cerebrovascular accidents, ICA is second to cerebral infarction and hypertensive cerebral hemorrhage and is third, but the risk of operation, postoperative disability rate and lethality rate are far higher than those of cerebral infarction and hypertensive cerebral hemorrhage. At present, ICA diagnosis, disease condition and prognosis evaluation mainly depend on imaging, hemodynamics and biological model construction. DSA is used as a gold standard for diagnosing ICA, is invasive examination, has high cost, needs hospitalization examination, has a complicated process, and is not suitable for wide screening of common people. The hemodynamic examination has complex process, low accuracy of result, special equipment and difficult wide development. Meanwhile, the death and disability rate after intracranial aneurysm is caused is high, and the current early diagnosis and screening methods are not ideal. Therefore, exploring the molecular mechanism of ICA formation, finding biomarkers for early diagnosis of ICA becomes an urgent task.
Circular RNA (Circular RNA) is a class of endogenous non-coding RNA molecules that do not have a 5 'end cap and a 3' end poly (A) tail and are covalently bonded to form a Circular structure. The circRNA is widely present in blood and is predicted to be a potential noninvasive candidate biomarker, van Rossum and the like find that some circRNA is specifically expressed in the brain, and a plurality of researches show that the circRNA is closely related to the occurrence and development of cerebrovascular diseases and is predicted to be a novel biomarker of ICA in the future.
hsa _ circ _0000690 is a CircRNA located on chromosome 16, the sequencing length is 284 pairs of base sequences, the corresponding parental gene is called integrin subbunit alpha L in English, and is called ITGAL for short, and alpha L subunit coding integrin lymphocyte function-associated antigen-1 (LFA-1), namely CD11 a. hsa _ circ _0000690 was derived from the exon-coding region of ITGAL, contains 2 exon regions, and was found to be present in reality in several reports, sample tissues for these experiments including neutrophils, mesencephalon tissue, and partial cell lines.
Integrins (ITGs) are a class of transmembrane glycoproteins that are heterodimers of two subunits, alpha and beta, linked by non-covalent bonds. Integrins are not only a class of adhesion molecules that mediate cell and extracellular matrix adhesion, but also are important signaling molecules that specifically bind to the corresponding receptors and activate subsequent integrin-mediated intracellular signaling pathways. Integrin is not only closely related to functions of cell proliferation, migration, apoptosis and the like, but also is suggested to participate in the processes of endothelial cell growth, survival and migration as a key molecule by a plurality of researches. It has also been found that ITG α v β 8 of glial cells is closely related to the development of cerebral blood vessels. In the research, the ITG alpha 4 beta 1 antagonist is administered to the mice planted with the mouse lung cancer cells, and the monocyte and the vascular progenitor cells are obviously inhibited, and the vascular density is obviously reduced. These studies also demonstrated that integrins are closely related to angiogenesis.
The integrin family is a general term for some integrins with similar structures. The integrin beta 2 family is a generic term for a class of integrins having the same beta-chain structure. This family is the most important adhesion molecule for which the combination of leukocytes with intercellular adhesion molecules (ICAMs) of vascular endothelial cells is currently known, and includes 4 members, ITGAL/Itgb2 (i.e., LFA-1), ITGAM/Itgb2, ITGAX/Itgb2 and ITGAD/Itgb 2.
LFA-1, one of the integrin beta 2 family, is also composed of two subunits, alpha and beta, the alpha subunit, also known as CD11a antigen, encoded by ITGAL, and the beta subunit, also known as CD18, encoded by ITGB 2. LFA-1 is expressed on the surface of all leukocytes such as lymphocytes, neutrophils, monocytes, macrophages and the like, plays a central role in the intercellular adhesion process of the leukocytes through the interaction with the ligands ICAMs1-3 thereof, and plays an important role in the lymphocyte activation and proliferation processes.
The alpha subunit of LFA-1 encoded by ITGAL of the parent gene of hsa _ circ _0000690, especially the site for binding with ICAM-1, can form stable immune synapse with ICAM-1, plays an important role in activation and proliferation of T cells, can mediate mutual binding between cells, can transmit costimulatory signals, and plays an important role in inflammation and immune response.
The formation mechanism of ICA is still unclear at present, and the research on the participation of the inflammatory reaction of arterial blood vessels in the formation, progression and rupture of ICA is concerned more and more. Meta-studies have shown that white blood cell counts are predictive of aneurysmal subarachnoid hemorrhage and are presumably associated with chronic inflammation. The aggregation and adhesion of leukocytes to inflamed tissue is the basis for any inflammatory response.
The nature of ICA is that vascular endothelial cells are damaged, locally thinned, leading to pathological cystocele, eventually ruptured. After vascular endothelial injury, macrophages are the first subset of leukocytes to initiate and regulate subsequent immune responses. There are studies demonstrating a significant decrease in the probability of developing aneurysms in macrophage depleted mice. Macrophages are classified into types I and II according to the inflammatory signaling pathway. Macrophage-type I is a proinflammatory cell that can lead to the production of aneurysms; macrophage-type II is associated with the elimination of inflammation and tissue repair.
In both the animal model and the cracked and non-cracked ICA specimen, there is a phenomenon that lymphocytes infiltrate the early ICA tumor wall, and T cells are the main, and B cells are rare. T lymphocytes also belong to a subset of leukocytes and are also involved in the inflammatory response of ICA formation. The detection of related cytokines expressed and secreted by T lymphocytes of ICA patients by Chalouhi et al finds that the content of gamma-interferon induced monokine in the plasma of femoral artery is obviously reduced compared with the content of monokine in the plasma of tumor-bearing artery, and suggests that infiltration of T lymphocytes into the wall of cerebral vessels is one of the important reasons for causing ICA. Piantino et al reported that the incidence of ICA was significantly increased in patients with immunodeficiency, suggesting that T lymphocyte immunodeficiency may directly or indirectly promote the occurrence and development of ICA. Zheng, S and the like find that proinflammatory factors such as TNF-alpha, IFN-gamma, IL6 and the like exist in the tumor wall of the early ICA, so that T lymphocytes are supposed to secrete the TNF-alpha, the IFN-gamma and the IL6 at the same time, induce subsequent inflammatory reactions and participate in the formation and development of the ICA.
Neutrophils are another subgroup belonging to leukocytes, also inflammatory cells of damaged, migrating, aggregated arterial walls. Similar to the first two classes of inflammatory cells, the next cytokines of action also include TNF- α.
TNF-alpha is taken as an inflammatory cytokine and is highly expressed in human ruptured ICA, and partial animal and human experiments find that the TNF-alpha has an important role in the formation and rupture process of ICA and is the cytokine secreted by vascular endothelial cells in the first batch after the damage of the artery wall. Starke, R.M., et al, demonstrated that TNF- α plays a critical role in the development and disruption of ICA by a rat model.
TNF- α has multiple roles in the initiation and disruption of ICA: TNF-alpha stimulates the production of Reactive Oxygen Species (ROS), which in turn leads to vascular endothelial cell dysfunction, stimulates inflammatory cytokine expression and macrophage-type I infiltration, promoting Vascular Smooth Muscle Cell (VSMC) phenotypic changes; 2. adhesion molecules such as ICAM-1, vascular cell adhesion molecule I (VCAM-1) and E-selectin are upregulated in vascular endothelial cells, fibroblasts and VSMCs. These adhesion molecules attract and promote the aggregation and migration of leukocytes on the ICA wall, promoting ICA formation.
ICAM-1 is an important adhesion molecule in ICA formation, and its main receptor is LFA-1 of the aforementioned integrin beta 2 family. LFA-1 and ICAM-1(CD54) mediate the adhesion of leukocytes to endothelial cells, their transvascular migration and the initiation of the entire process of infiltration into diseased tissue. Binding of LFA-1 to ICAM-1 expressed on vascular endothelial cells mediates migration, extravasation of T lymphocytes, especially LFA-1 mediated migration in cases of impaired blood brain barrier leading to infiltration and killing of T lymphocytes.
Because of the protective effect of the blood brain barrier on brain tissue, there is almost no peripheral blood lymphocyte accumulation in normal human brain tissue. The interaction between the adhesion molecule expressed on lymphocyte membrane and the corresponding ligand expressed by blood brain barrier endothelial cell plays an important role in the migration and infiltration of lymphocyte to blood vessel wall tissue. In particular, the adhesion between LFA-1/ICAM-1 is a key mechanism for the penetration of normal blood brain barrier by peripheral blood activated T lymphocytes.
In the study of colon epithelial cells of chronic inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, Chidlow, J.H., Jr. and the like find that deletion of ITGAL gene leads to obvious reduction of T lymphocyte adhesion mediated by TNF-alpha.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) DSA is invasive examination, high in cost, needs hospitalization examination, is complex in process, and is not suitable for wide screening of common people. The hemodynamic examination has complex process, low accuracy of result, special equipment and difficult wide development.
(2) The death and disability rate after intracranial aneurysm is caused is high, and the current early diagnosis and screening methods are not ideal.
(3) At present, no molecular marker which can quickly and conveniently detect intracranial aneurysm and has certain clinical value exists.
The significance of solving the problems and the defects is as follows:
at present, in order to achieve success through experimental detection, clinical research needs to be converted into clinical research, and a detection kit needs to be improved, so that the aims of rapidness, convenience, cost reduction and accuracy improvement are fulfilled.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an application, an expression method and a kit of a biological marker for detecting intracranial aneurysm.
The invention is realized by the fact that the biological marker for detecting the intracranial aneurysm is CD11 a.
Further, the CD11a is a protein encoded by the parental gene ITGAL corresponding to hsa _ circ _ 0000690.
CD11a (fragment number 1170) is a known protein, immunoprotein protein structure, FASTA format for the amino acid sequence of the protein as follows (query site)
https://www.ncbi.nlm.nih.gov/protein/NP_002200.2report＝fasta)
Related web sites alsohttps://www.ncbi.nlm.nih.gov/gene/3683。
https://www.ncbi.nlm.nih.gov/genome/gdv/browser/gene/？id＝3683。
Further, the CD11a is used as an alpha subunit of LFA-1 to be combined with ICAM-1, and is applied to the aggregation, migration and infiltration of leukocytes on the wall of cerebral blood vessels mediated by TNF-alpha.
Another object of the present invention is to provide a kit for detecting intracranial aneurysm, comprising the CD11a according to any one of claims 2 to 4.
Another object of the present invention is to provide a method for detecting expression of a biological marker of intracranial aneurysm, comprising the steps of:
step one, specimen collection: setting an experimental group and a control group, and adding EDTA-K2 anticoagulant for later use;
step two, extracting total RNA: extracting total RNA in peripheral blood, determining the concentration and purity of the extracted total RNA by using an ultramicro nucleic acid protein determinator, and judging the integrity of the RNA by using agarose gel electrophoresis;
step three, detecting the expression level of hsa _ circ _0000690 in ICA and peripheral blood;
detecting the expression level of the mRNA of the ITGAL in ICA and peripheral blood;
step five, detecting the content of CD11a in ICA and peripheral blood;
analyzing a proper biomarker of the intracranial aneurysm;
and seventhly, performing statistical analysis.
Further, in step three, the method for detecting the expression level of hsa _ circ _0000690 in ICA and peripheral blood comprises:
(1) reverse Transcription (RT)
Synthesis of cDNA Using High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (ABI,4374966) according to the instructions; 20 μ L reverse transcription System 10 × RT Buffer 4 μ L, Enzyme Mix1 μ L, Primer Mix1 μ L, 25 × dNTP Mix 0.8 μ L, 10 × RT Random Primers 2 μ L remaining in RNA template (0.5 μ g) plus RNase Free dH2O is balanced to 20 mu L; the method comprises the following steps: at 25 ℃ for 10 min; at 37 ℃ for 120 min; 5min at 85 ℃; 4 ℃;
(2) detection of hsa _ circ _0000690 by Real-Time PCR
The Real-Time PCR experiment was performed on an ABI PRISM7500 sequence detection System (PE Applied Biosystems, US) using a Power SYBR Green PCR Master Mix (ABI, US); the reaction system is 10 μ L, wherein: 2 × SYBR Green PCR buffer 5 μ L, F primer0.5 μ L, R primer0.5 μ L, cDNA 1 μ L, RNase-free water3 μ L; the PCR reaction condition is 50 ℃ and 2 min; at 95 ℃ for 10 min; 40cycles (95 ℃, 15 s; 60 ℃, 1 min);
the Ct value (threshold cycle) at which the result is obtained is defined as the number of cycles required to reach the detection threshold of the fluorescence signal; the real-time PCR of each sample was repeated 3 times, and the obtained data were used to calculate the relative expression of hsa _ circ _0000690 between the two groups by using the software provided by ABI as 2- Δ Ct.
In step four, the method for detecting the expression level of ITGAL mRNA in ICA and peripheral blood comprises:
1) reverse Transcription (RT)
Synthesis of cDNA Using Primescript RT reagent kit with gDNA Eraser (Takara, Japan), the procedure was as described; 20 μ L reverse transcription Primer Script RT Master Mix 4 μ L, Total RNA 2 μ g, remaining with RNase Free dH2O is balanced to 20 mu L; the method comprises the following steps: 15min at 37 ℃ and 5s at 85 ℃;
2) detection of ITGAL mRNA by Real-Time PCR method
The Real-Time PCR experiment was performed on ABI PRISM7500 sequence detection System (PE Applied Biosystems, US) using SYBR Premix ExTaq (TaKaRa, Japan); the reaction system is 20 μ L, wherein: SYBR premix Ex Taq 10. mu.L, F primer 0.4. mu.L, R primer 0.4. mu.L, cDNA 1. mu.L, RNase-free water 8.2. mu.L; the PCR reaction conditions were thermal denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 10s, then denaturation at 95 ℃ for 10s and denaturation at 95 ℃ for 10s, and the reaction was continued for 40 cycles;
the Ct value (threshold cycle) at which the result is obtained is defined as the number of cycles required to reach the detection threshold of the fluorescence signal; the real-time PCR of each sample was repeated 3 times, and the obtained data were used to calculate the relative expression amount of ITGAL mRNA between the two groups by the method of 2- Δ Ct using software provided by ABI.
In the fifth step, the method for detecting the content of CD11a in ICA and peripheral blood comprises the following steps:
1) firstly, labeling CD45 with Perdinin chlorophyll protein (PerCP) for later use;
2) putting 10 mu L of marked CD45 and 100 mu L of sample into the same test tube after collecting the peripheral blood within 6h, and adding a CD11a related monoclonal antibody (McAb) for treatment;
3) labeling 10 mu L of cell antigen by a direct immunofluorescence labeling method, adding the labeled cell antigen into the test tube, uniformly mixing, and incubating for 20-30 min at room temperature in a dark place;
3) adding 1ml of erythrocyte lysate into the test tube, incubating for 10min at room temperature in a dark place, centrifuging at 1000rpm for 5min, and removing supernatant; washing the precipitate with PBS for 2 times, and expanding the volume of the precipitate to 400-600 mu L; 10000 cells/tube are obtained by a flow cytometer;
4) performing data processing analysis by using Cell quest3.0 software;
in step six, the method for analyzing the appropriate biomarkers of the intracranial aneurysm comprises the following steps:
evaluating 3 indexes of hsa _ circ _0000690 expression level, ITGAL expression level and CD11a content by using a working characteristic curve;
in step seven, the method for performing statistical analysis includes:
processing experimental data by adopting SPSS19.0 statistical software;
quantitative data between the experimental group and the control group are tested by an independent sample t, all data are expressed by mean +/-standard deviation (mean +/-SD), the difference is considered to be significant when P is less than 0.05, and all test levels are that bilateral probability alpha is 0.05;
the intra-group correlation analysis used Spearman correlation analysis and partial correlation analysis.
The invention also aims to provide application of the biological marker for detecting the intracranial aneurysm in detection of peripheral blood circRNA, ITGAL expressed mRNA and CD11a of the intracranial aneurysm.
By combining all the technical schemes, the invention has the advantages and positive effects that: the biological marker and the expression method for detecting intracranial aneurysm, which are provided by the invention, combine the latest research results of inflammatory response in aspects of ICA generation, development and rupture at home and abroad and the suggestion that circRNA becomes a potential noninvasive candidate biomarker for various diseases, and combine the early-stage research results of the invention group to extract scientific problems and propose own hypothesis: the ITGAL of parent gene corresponding to hsa _ circ _0000690 encodes CD11a, and is combined with ICAM-1 as alpha subunit of LFA-1, thereby playing an important role in the aggregation, migration and infiltration of leukocytes on the wall of cerebral blood vessels mediated by TNF-alpha, and possibly being one of the mechanisms of ICA formation and rupture caused by inflammatory reaction. Therefore, the invention has theoretical innovation.
At present, no relevant research on hsa _ circ _0000690 is reported in the literature. The invention takes the preliminary experiment result and related documents as a basis for establishing that hsa _ circ _0000690 has expression difference in ICA patients and normal human peripheral blood, and explores the possibility of the hsa _ circ _0000690 as a noninvasive biological marker, so that the invention has stronger innovation, and the result of the invention can become the preliminary experiment foundation of natural invention in subsequent applications.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flow chart of a method for detecting expression of a biological marker of intracranial aneurysm according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of a technical route provided by an embodiment of the present invention.
Fig. 3 is a diagram of cluster analysis provided by an embodiment of the present invention.
FIG. 4 is a scatter diagram provided by an embodiment of the invention
Figure 5 is a volcano plot provided by embodiments of the present invention.
FIG. 6 is a signal transduction pathway diagram of the "mechanism of leukocyte migration across endothelium" provided by the embodiments of the present invention.
FIG. 7 is a diagram of the "NK cell mediated cytotoxicity response" signaling pathway provided by the present invention.
Fig. 8 is a diagram illustrating the result of the small sample size ttest provided by the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides the application, the expression method and the kit of the biological marker for detecting intracranial aneurysm, and the invention is described in detail with reference to the accompanying drawings.
The biological markers for detecting intracranial aneurysms provided by the embodiment of the invention are CD11a and hsa _ circ _ 0000690.
The biological marker CD11a provided by the embodiment of the invention is a protein encoded by a parental gene ITGAL corresponding to hsa _ circ _ 0000690.
As shown in fig. 1, the method for detecting expression of a biological marker of intracranial aneurysm provided by the embodiment of the present invention comprises the following steps:
s101, specimen collection: setting experimental group and control group, reserving 5ml of whole blood of each experimental group and control group, and adding EDTA-K2 anticoagulant for standby.
S102, extracting total RNA: extracting total RNA in peripheral blood, measuring the concentration and purity of the extracted total RNA by using an ultramicro nucleic acid protein tester, and judging the integrity of the RNA by using agarose gel electrophoresis.
S103, detecting the expression level of hsa _ circ _0000690 in ICA and normal human peripheral blood.
S104, detecting the expression level of ITGAL mRNA in ICA and normal human peripheral blood.
S105, detecting the content of CD11a in ICA and normal human peripheral blood.
S106, searching for a proper biomarker for diagnosing intracranial aneurysm.
And S107, performing statistical analysis.
The present invention will be further described with reference to the following examples.
Content (a)
1、
The death and disability rate after intracranial aneurysm is caused is high, and the current early diagnosis and screening methods are not ideal. The circRNA is widely present in blood and stable in property, and has potential value as a noninvasive biological marker. Previous experiments in the present invention found that hsa _ circ _0000690 may differ in peripheral blood between intracranial aneurysm patients and normal persons. The protein CD11a coded by the parent gene ITGAL plays a key role in the process of leukocyte aggregation and adhesion. The accumulation and adhesion of leukocytes are the initiating links in the development of any inflammation that plays an important role in the development, progression and rupture of aneurysms. The present invention therefore speculates that: the ITGAL of parent gene corresponding to hsa _ circ _0000690 encodes CD11a, and is used as alpha subunit of LFA-1 to be combined with ICAM-1, plays an important role in the aggregation and migration infiltration of leucocytes on the wall of cerebral blood vessels, and can be one of the mechanisms of intracranial aneurysm formation and rupture caused by inflammatory reaction. The invention aims to explore the expression difference of circRNA, ITGAL expressed mRNA and CD11a in peripheral blood between intracranial aneurysm patients and normal people, search a proper noninvasive biological marker and provide a prophase basis for later mechanism research.
According to the invention, the peripheral blood samples of 3 ICA patients and 3 normal persons are subjected to RNA sequencing experiments, and the experimental results are subjected to subsequent data processing such as clustering analysis of differential expression, GO functional significance enrichment analysis, KEGG Pathway significance enrichment analysis and the like, and are preliminarily verified to obtain the expression difference of hsa _ circ _0000690 in the peripheral blood of the ICA patients and the normal persons.
The invention speculates that the ITGAL of the parent gene corresponding to hsa _ circ _0000690 encodes CD11a, and is combined with ICAM-1 as an alpha subunit of LFA-1, thereby playing an important role in the aggregation and migration infiltration of leukocytes, especially T lymphocytes, on the wall of the cerebral blood vessel mediated by TNF-alpha and being one of the possible mechanisms of ICA formation and rupture caused by inflammatory reaction.
On the basis of previous work, the sample size is further expanded, whether the hsa _ circ _0000690 in the peripheral blood is different between ICA patients and normal people or not is researched, and whether the expression level of the ITGAL gene and the content of CD11a in the peripheral blood are different between the ICA patients and the normal people or not is also researched. Whether hsa _ circ _0000690, ITGAL and CD11a can be used as biomarkers for diagnosing ICA is judged through a receiver operator characteristic curve (ROC curve), a novel biomarker capable of simply, accurately and efficiently carrying out early screening or diagnosis of ICA is sought, and an early basis is provided for the next step of discussing a possible mechanism of formation and rupture of ICA caused by an inflammatory reaction. Therefore, the research of the invention has important potential scientific research and clinical value.
2. Summary of the invention
2.1 the invention will be from hsa _ circ _0000690 expression level, parental gene ITGAL mRNA expression level and corresponding coded protein CD11a content three different aspects, will clarify the three in ICA and normal people in the expression difference, explore the correlation between the three, and through the ROC curve verification three in diagnosis ICA clinical value.
2.1.1 clear that there is a difference in expression of hsa _ circ _0000690 in ICA and normal humans
40 ICA peripheral blood samples and 40 normal human peripheral blood samples were taken, the expression level of hsa _ circ _0000690 was determined by the quantitative RT-PCR method, and the presence or absence of expression difference between the two groups was counted by the t-test.
2.1.2 clear differences in the expression of ITGAL in ICA and Normal humans
40 ICA peripheral blood samples and 40 normal human peripheral blood samples were taken, and the expression level of mRNA of ITGAL gene was measured by quantitative RT-PCR method, and the presence or absence of expression difference between the two groups was counted by t-test.
2.1.3 clear that there is a difference in the content of CD11a between ICA and normal human
40 ICA peripheral blood samples and 40 normal human peripheral blood samples are taken, the content of CD11a in the peripheral blood is detected by a flow cytometer, and whether the content between two groups is different or not is counted by a t test.
2.1.4 unambiguous correlation between hsa _ circ _0000690, ITGAL and CD11a
Through bivariate correlation analysis, the correlation relationships of hsa _ circ _0000690 and ITGAL, hsa _ circ _0000690 and CD11a, ITGAL and CD11a are respectively defined and further verified through partial correlation analysis.
2.1.5 search for suitable ICA biomarkers
The clinical value of the 3 indexes hsa _ circ _0000690, ITGAL and CD11a for diagnosing ICA is clarified through ROC curve, and suitable biomarkers for screening or diagnosing ICA are searched.
2.2 target
2.2.1 defining the expression differences in ICA and normal humans of peripheral blood hsa _ circ _0000690, ITGAL and CD11 a;
2.2.2 exploring the relationship between peripheral blood hsa _ circ _0000690, ITGAL and CD11 a;
2.2.3 validation of peripheral blood hsa _ circ _0000690, ITGAL and CD11a could be biomarkers for diagnosis of ICA.
2.3 Key problems to solve
2.3.1 whether peripheral blood hsa _ circ _0000690 differs between ICA and normal? This is a key scientific problem to solve.
Although in the previous experiments, the invention preliminarily found that hsa _ circ _0000690 has difference between ICA and normal human peripheral blood, the sample size is small, and the sample size still needs to be expanded further, so that the difference exists clearly.
2.3.2 what relationship does the expression of hsa _ circ _0000690, ITGAL and CD11a in human peripheral blood? This is the second key scientific problem to solve.
It was first clarified that ITGAL and CD11a also had expression differences in ICA and normal human peripheral blood, and then the correlations of hsa _ circ _0000690 and ITGAL, hsa _ circ _0000690 and CD11a, ITGAL and CD11a were verified by statistical methods such as bivariate analysis and partial correlation analysis, respectively.
2.3.3 whether peripheral blood hsa _ circ _0000690, ITGAL and CD11a could be used as biomarkers for diagnosing ICA? This is the third key scientific problem to be solved.
The SPSS was used to separately evaluate ROC curves for hsa _ circ _0000690 expression level, ITGAL mRNA expression level, and CD11a content in the two groups of samples, and these 3 indices were evaluated for clinical value in diagnosing ICA.
3. The invention is further described below in connection with specific experiments.
3.1 schemes
Collecting a specimen:
40 patients with intracranial aneurysm were used as experimental group, 40 patients with health examinee were used as control group, and 5ml of whole blood was collected from each experimental group and control group, and EDTA-K2 anticoagulant was added for use.
Extracting total RNA:
total RNA in peripheral blood was extracted according to the product instructions of the miRNeasy Serum/Plasma Kit (Qiagen, Germany). The concentration and purity of the extracted total RNA are determined by a ultramicro nucleic acid protein determinator, and the integrity of the RNA is judged by agarose gel electrophoresis.
(1) Detection of expression level of hsa _ circ _0000690 in ICA and Normal human peripheral blood
1) Reverse Transcription (RT)
Synthesis of cDNA Using High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (ABI,4374966) the protocol was used. 20 μ L reverse transcription System 10 × RT Buffer 4 μ L, Enzyme Mix1 μ L, Primer Mix1 μ L, 25 × dNTP Mix 0.8 μ L, 10 × RT Random Primers 2 μ L remaining in RNA template (0.5 μ g) plus RNase Free dH2O was trimmed to 20. mu.L. The method comprises the following steps: at 25 ℃ for 10 min; at 37 ℃ for 120 min; 5min at 85 ℃; 4 ℃ is prepared.
2) Detection of hsa _ circ _0000690 by Real-Time PCR
The Real-Time PCR experiment was performed on an ABI PRISM7500 sequence detection System (PE Applied Biosystems, US) using a Power SYBR Green PCR Master Mix (ABI, US). The reaction system is 10 μ L, wherein: 2 × SYBR Green PCR buffer 5. mu.L, F primer 0.5. mu.L, R primer 0.5. mu.L, cDNA 1. mu.L, RNase-free water 3. mu.L. The PCR reaction condition is 50 ℃ and 2 min; at 95 ℃ for 10 min; (95 ℃, 15 s; 60 ℃, 1min)40 cycles.
The Ct value (threshold cycle) at which the result is obtained is defined as the number of cycles required to reach the detection threshold of the fluorescence signal. The real-time PCR of each sample was repeated 3 times, and the data obtained were used to calculate the relative expression of hsa _ circ _0000690 between the two groups by the method of 2- Δ Ct using software supplied by ABI (Applied Biosystems).
(2) Detection of expression level of ITGAL mRNA in ICA and Normal human peripheral blood
1) Reverse Transcription (RT)
Synthesis of cDNA Using Primescript RT reagent kit with gDNA Eraser (Takara, Japan), the procedure was as described. 20 μ L reverse transcription Primer Script RT Master Mix 4 μ L, Total RNA 2 μ g, remaining with RNase Free dH2O was trimmed to 20. mu.L. The method comprises the following steps: 15min at 37 ℃ and 5s at 85 ℃. .
2) Detection of ITGAL mRNA by Real-Time PCR method
The Real-Time PCR experiment was performed on ABI PRISM7500 sequence detection System (PE Applied Biosystems, US) using SYBR Premix ExTaq (TaKaRa, Japan). The reaction system is 20 μ L, wherein: SYBR premix Ex Taq 10. mu.L, F primer 0.4. mu.L, R primer 0.4. mu.L, cDNA 1. mu.L, RNase-free water 8.2. mu.L. The PCR reaction conditions were thermal denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 10s, then denaturation at 95 ℃ for 10s and then at 95 ℃ for 10s and 95 ℃ for 10s for 40 cycles.
The Ct value (threshold cycle) at which the result is obtained is defined as the number of cycles required to reach the detection threshold of the fluorescence signal. The real-time PCR of each sample was repeated 3 times, and the obtained data were used to calculate the relative expression amount of ITGAL mRNA between the two groups by a 2- Δ Ct method using software provided by ABI (Applied Biosystems).
(3) Detecting the content of CD11a in ICA and normal human peripheral blood
1) CD45 was first labeled with Perdinin chlorophyll protein (PerCP) and was ready for use.
2) 10 μ L of labeled CD45 and 100 μ L of the peripheral blood within 6 hours after collection were placed in the same tube, and then treated with the addition of a CD11 a-related monoclonal antibody (McAb).
3) And (3) marking 10 mu L of cell antigen by using a direct immunofluorescence marking method, adding the cell antigen into the test tube, uniformly mixing, and incubating for 20-30 min at room temperature in a dark place.
3) 1ml of erythrocyte lysate is added into the test tube, incubated for 10min at room temperature in the dark, centrifuged at 1000rpm for 5min, and the supernatant is removed. After washing the precipitate 2 times with PBS, the volume of the precipitate was increased to 400-600. mu.L. 10000 cells/tube were obtained with a flow cytometer.
4) And (4) performing data processing analysis by using Cell quest3.0 software.
(4) Finding suitable biomarkers for diagnosing intracranial aneurysms
Clinical value of hsa _ circ _0000690 expression level, ITGAL expression level, and CD11a level were assessed using receiver operating characteristic curves (ROC curve), respectively, for diagnosing ICA.
(5) Statistical analysis
Experimental data were processed using SPSS19.0 statistical software. Quantification data between experimental and control groups were tested using independent sample t-tests, all data are expressed as mean ± standard deviation (mean ± SD), differences were considered significant when P <0.05, and all test levels were bilateral with a probability α of 0.05. The intra-group correlation analysis used Spearman correlation analysis and partial correlation analysis.
3.2 technical route (as shown in FIG. 2)
3.3
(1) According to the invention, hsa _ circ _0000690 is selected as a research object through early RNA sequencing analysis and subsequent screening process, and the research object is revealed to be mainly enriched in multiple inflammatory signal pathways such as leukocyte migration and NK cell killing through KEGG Pathway. The role of inflammatory reaction in the development and progression of intracranial aneurysms is becoming more and more important. And the applicant preliminarily concluded, by small sample size experiments, that hsa _ circ _0000690 is differentially expressed in ICA patients and normal population. (see the work foundation in detail)
(2) The hospital central laboratory has the hardware equipment required by the invention
The large modern laboratory which is characterized by clinical immunology inspection, cytogenetics inspection and molecular biology gene diagnosis and provided by the invention has a fluorescence quantitative PCR instrument, a flow cytometer, a gene sequencer, a biological safety cabinet, a high-speed centrifuge, a fluorescence microscope, a micro oscillator, an inverted microscope, a Finland Leibo enzyme labeling instrument, a gel imaging analysis system, an ultrapure water manufacturing system and the like.
4. The features and innovation of the present invention
The invention combines the latest research results of inflammatory reaction in aspects of ICA generation, development and rupture at home and abroad and the suggestion that circRNA becomes a potential noninvasive candidate biomarker for various diseases, combines the earlier research results of the invention group, extracts scientific problems and proposes own hypothesis: the ITGAL of parent gene corresponding to hsa _ circ _0000690 encodes CD11a, and is combined with ICAM-1 as alpha subunit of LFA-1, thereby playing an important role in the aggregation, migration and infiltration of leukocytes on the wall of cerebral blood vessels mediated by TNF-alpha, and possibly being one of the mechanisms of ICA formation and rupture caused by inflammatory reaction. Therefore, the invention has theoretical innovation.
At present, no relevant research on hsa _ circ _0000690 is reported in the literature. The invention takes the preliminary experiment results and related documents as the basis for establishing that hsa _ circ _0000690 has expression difference in ICA patients and normal human peripheral blood, and explores the possibility of the hsa _ circ _0000690 as a noninvasive biological marker, thereby having stronger innovation. And the result of the invention can become the early-stage experimental basis of the natural invention in the subsequent application province.
5. Results
(1) hsa _ circ _0000690 differed in ICA and normal human peripheral blood with reduced expression levels in ICA patients;
(2) ITGAL and CD11a were different in ICA and normal human peripheral blood, and both ITGAL expression level and CD11a level were elevated in ICA patients;
(3) hsa _ circ _0000690 is negatively correlated with both ITGAL and CD11a, respectively;
(4) hsa _ circ _0000690 can be a biomarker for diagnosis or screening of ICA.
The invention is further described below in connection with the basis and operating conditions.
1. Work foundation
1.1 the invention takes 3 cases of ICA as an experimental group, 3 cases of patients have no obvious abnormality in physical examination, and the age, the past medical history and the experimental group can be matched with normal people to be taken as a control group, blood is extracted, and a CircRNA sequencing experiment is carried out, so that most of the CircRNA is found to be in a down-regulated state in ICA patients.
Some of the results are shown below:
1.1.1 Cluster analysis graph (as shown in FIG. 3)
Also known as Heatmap (Heatmap), suggests that most of the circrnas in the experimental group were down-regulated compared to the control group.
1.1.2 dot Plot (Scatter Plot) (as shown in FIG. 4)
The invention detects that 2566 sites of 3328 CircRNA sites are in a down-regulation state, 620 sites are in an up-regulation state, and the whole trend is more down-regulation sites which are consistent with the trend of a cluster analysis chart.
1.1.3 Volcano Plot (Volcano Plot) (as shown in FIG. 5)
3328 of the CircRNA sites were screened for statistically significant up-regulated 20 and down-regulated 38 of CircRNA by abscissa log2FC greater than 2 or less than-2, ordinate-log 10(P-value) greater than 1.3 and (-log10(0.05) equal to 1.3). The overall trend is still that there are more downregulation sites, consistent with the trends of the cluster analysis plots and scatter plots.
1.2 statistical 58 CircRNA sites were subjected to KEEG analysis, sites associated with inflammatory response were selected, and hsa _ circ _0000690 was found to have a possible difference in expression between ICA and normal population as verified by small sample size RT-PCR pre-stage verification of blood samples from 9 ICA and 10 normal individuals.
1.2.1 Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis
Through KEGG analysis, the gene ITGAL corresponding to hsa _ circ _0000690 is found to be involved in multiple inflammatory response pathways, including a leukocyte transendothelial migration mechanism and a Natural Killer (NK) cell-mediated cytotoxicity response.
The signal transduction pathway of the "mechanism of leukocyte migration across endothelium" is shown in FIG. 6, and the signal transduction pathway of the "NK cell mediated cytotoxicity response" is shown in FIG. 7.
1.2.2 Small sample size T test (as shown in FIG. 8)
According to the invention, the peripheral blood samples of 9 cases of ICA and 10 cases of normal people are subjected to RT-PCR, and by comparing 2-delta Ct values between the two groups, the invention discovers that the 2-delta Ct value of hsa _ circ _0000690 is 0.022 in the ICA group, 0.089 in the normal people group and 0.0005 in the P value, and the statistical difference exists. Thus, the present inventors have preliminarily recognized that hsa _ circ _0000690 may have differences in expression between ICA and the normal population.
The laboratory of the invention is an experimental center integrating clinic, scientific research and teaching. The laboratory is continuously introducing the most advanced experimental techniques and equipments, and the basic instruments and equipments include fluorescence quantitative PCR instrument (ABI7500, ABI of USA), flow cytometer (BD FACSCalibur), desk micro-freezing centrifuge (Microfuge 22R, BECKMAN of USA), gene sequencer (3500dx, ABI of USA), biosafety cabinet (BSC-1300 IIB 2), high speed centrifuge (Minispin plus, Germany eppendorf), fluorescence microscope (BX51, Japan Olympus), micro-oscillator (LAB DANCE S25, Germany IKA), inverted microscope (IX71, Japan Olympus), Finland Leber enzyme reader (MultisKan Ascent), gel imaging analysis system, and ultra-pure water manufacturing system, etc.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. A biological marker for detecting intracranial aneurysm, wherein the biological marker for detecting intracranial aneurysm is CD11 a.
2. The biological marker for detecting intracranial aneurysm as set forth in claim 1, wherein the CD11a is a protein encoded by the parental gene ITGAL corresponding to hsa _ circ _ 0000690.
3. The use of claim 1, wherein CD11a is used as an alpha subunit of LFA-1 in combination with ICAM-1 in TNF-alpha mediated aggregation, migration and infiltration of leukocytes into the walls of cerebral blood vessels.
4. A kit for detecting intracranial aneurysm, comprising the CD11a according to any one of claims 1 to 3.
5. A method of expressing a biological marker for detecting intracranial aneurysm as in any one of claims 1 to 4, comprising the steps of:
step one, specimen collection: setting an experimental group and a control group, and adding EDTA-K2 anticoagulant for later use;
step two, extracting total RNA: extracting total RNA in peripheral blood, determining the concentration and purity of the extracted total RNA by using an ultramicro nucleic acid protein determinator, and judging the integrity of the RNA by using agarose gel electrophoresis;
step three, detecting the expression level of hsa _ circ _0000690 in ICA and peripheral blood;
detecting the expression level of the mRNA of the ITGAL in ICA and peripheral blood;
step five, detecting the content of CD11a in ICA and peripheral blood;
analyzing a proper biomarker of the intracranial aneurysm;
and seventhly, performing statistical analysis.
6. The method of detecting expression of a biological marker of an intracranial aneurysm as in claim 5, wherein the method of detecting the expression level of hsa _ circ _0000690 in ICA and peripheral blood in step three comprises:
(1) reverse transcription
Synthesis of cDNA Using High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (ABI,4374966) according to the instructions; 20 μ L reverse transcription System 10 × RT Buffer 4 μ L, Enzyme Mix1 μ L, Primer Mix1 μ L, 25 × dNTP Mix 0.8 μ L, 10 × RT Random Primers 2 μ L remaining in RNA template (0.5 μ g) plus RNase Free dH2O is balanced to 20 mu L; the method comprises the following steps: at 25 ℃ for 10 min; at 37 ℃ for 120 min; 5min at 85 ℃; 4 ℃;
(2) detection of hsa _ circ _0000690 by Real-Time PCR
The Real-Time PCR experiment was performed on an ABI PRISM7500 sequence detection System (PE Applied Biosystems, US) using a Power SYBR Green PCR Master Mix (ABI, US); the reaction system is 10 μ L, wherein: 2 × SYBR Green PCR buffer 5 μ L, F primer0.5 μ L, R primer0.5 μ L, cDNA 1 μ L, RNase-free water3 μ L; the PCR reaction condition is 50 ℃ and 2 min; at 95 ℃ for 10 min; 40cycles (95 ℃, 15 s; 60 ℃, 1 min);
the Ct value at which the result is obtained is defined as the number of cycles required to reach the detection threshold of the fluorescence signal; the real-time PCR of each sample was repeated 3 times, and the obtained data were used to calculate the relative expression of hsa _ circ _0000690 between the two groups by using the software provided by ABI as 2- Δ Ct.
7. The method for detecting expression of a biological marker of intracranial aneurysm as in claim 5, wherein in step four, the method for detecting expression level of mRNA of ITGAL in ICA and peripheral blood comprises:
1) reverse Transcription (RT)
Synthesis of cDNA Using Primescript RT reagent kit with gDNA Eraser (Takara, Japan), the procedure was as described; 20 μ L reverse transcription Primer Script RT Master Mix 4 μ L, Total RNA 2 μ g, remaining with RNase Free dH2O is balanced to 20 mu L; the method comprises the following steps: 15min at 37 ℃ and 5s at 85 ℃;
2) detection of ITGAL mRNA by Real-Time PCR method
The Real-Time PCR experiment was performed on ABI PRISM7500 sequence detection System (PE Applied Biosystems, US) using SYBR Premix ExTaq; the reaction system is 20 μ L, wherein: SYBR premix Ex Taq 10. mu.L, F primer 0.4. mu.L, R primer 0.4. mu.L, cDNA 1. mu.L, RNase-free water 8.2. mu.L; the PCR reaction conditions were thermal denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 10s, then denaturation at 95 ℃ for 10s and denaturation at 95 ℃ for 10s, and the reaction was continued for 40 cycles;
the Ct value (threshold cycle) at which the result is obtained is defined as the number of cycles required to reach the detection threshold of the fluorescence signal; the real-time PCR of each sample was repeated 3 times, and the obtained data were used to calculate the relative expression amount of ITGAL mRNA between the two groups by the method of 2- Δ Ct using software provided by ABI.
8. The method for detecting expression of biological markers of intracranial aneurysms according to claim 5, wherein in step five, the method for detecting the content of CD11a in ICA and peripheral blood comprises:
1) firstly, labeling CD45 with Perdinin chlorophyll protein (PerCP) for later use;
2) putting 10 mu L of marked CD45 and 100 mu L of sample into the same test tube after collecting the peripheral blood within 6h, and adding a CD11a related monoclonal antibody (McAb) for treatment;
3) labeling 10 mu L of cell antigen by a direct immunofluorescence labeling method, adding the labeled cell antigen into the test tube, uniformly mixing, and incubating for 20-30 min at room temperature in a dark place;
3) adding 1ml of erythrocyte lysate into the test tube, incubating for 10min at room temperature in a dark place, centrifuging at 1000rpm for 5min, and removing supernatant; washing the precipitate with PBS for 2 times, and expanding the volume of the precipitate to 400-600 mu L; 10000 cells/tube are obtained by a flow cytometer;
4) performing data processing analysis by using Cell quest3.0 software;
in step six, the method for analyzing the appropriate biomarkers of the intracranial aneurysm comprises the following steps:
evaluating 3 indexes of hsa _ circ _0000690 expression level, ITGAL expression level and CD11a content by using a working characteristic curve;
in step seven, the method for performing statistical analysis includes:
processing experimental data by adopting SPSS19.0 statistical software;
quantitative data between the experimental group and the control group are tested by an independent sample t, all data are expressed by mean +/-standard deviation (mean +/-SD), the difference is considered to be significant when P is less than 0.05, and all test levels are that bilateral probability alpha is 0.05;
the intra-group correlation analysis used Spearman correlation analysis and partial correlation analysis.
9. Use of a biological marker for detecting intracranial aneurysm as defined in any one of claims 1 to 4 for detection in peripheral blood circRNA, ITGAL-expressed mRNA and CD11a of intracranial aneurysm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316417A (en) * | 2015-11-27 | 2016-02-10 | 北京泱深生物信息技术有限公司 | ZNRD1 gene and expression products thereof serving as diagnosis and treatment targets of intracranial aneurysm |
| CN105400885A (en) * | 2015-12-18 | 2016-03-16 | 四川省人民医院 | Applications of TFF2 gene as intracranial aneurysm diagnosis and treatment marker |
| CN109295218A (en) * | 2018-12-11 | 2019-02-01 | 南京医科大学 | Circular rna marker hsa_circ_0001788 and its application |
-
2020
- 2020-10-27 CN CN202011167210.1A patent/CN112322732A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316417A (en) * | 2015-11-27 | 2016-02-10 | 北京泱深生物信息技术有限公司 | ZNRD1 gene and expression products thereof serving as diagnosis and treatment targets of intracranial aneurysm |
| CN105400885A (en) * | 2015-12-18 | 2016-03-16 | 四川省人民医院 | Applications of TFF2 gene as intracranial aneurysm diagnosis and treatment marker |
| CN109295218A (en) * | 2018-12-11 | 2019-02-01 | 南京医科大学 | Circular rna marker hsa_circ_0001788 and its application |
Non-Patent Citations (4)
| Title |
|---|
| CAO H ET AL.: "Circular RNA expression profile in human primary multiple intracranial aneurysm", 《EXP THER MED》 * |
| FALIN CHEN ET AL.: "Circular RNAs expression profiles in plasma exosomes from early‐stage lung adenocarcinoma and the potential biomarkers", 《J CELL BIOCHEM》 * |
| YANMING HUANG ET AL.: "Circular RNA hsa_circ_0000690 as a potential biomarker for diagnosis and prognosis of intracranial aneurysm: Closely relating to the volume of hemorrhage", 《BRAIN AND BEHAVIOR》 * |
| 张明铭: "颅内动脉瘤的miRNA、mRNA表达谱及其分子网络调控研究", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
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