CN109880898B - Application of hsa-miR-23b-5p as acute altitude reaction susceptible molecular marker and kit - Google Patents
Application of hsa-miR-23b-5p as acute altitude reaction susceptible molecular marker and kit Download PDFInfo
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Abstract
The invention relates to a microRNA marker for screening acute altitude reaction susceptible persons, and the application of the microRNA hsa-miR-23b-5p as a molecular marker of the acute altitude reaction susceptible persons can be applied to the preparation of a reagent or a kit for screening the acute altitude reaction susceptible persons. And further provides a kit for detecting hsa-miR-23b-5p molecules in human plain blood plasma by a real-time fluorescent quantitative PCR method, and the acute altitude reaction risk is predicted according to the expression level of the kit. The microRNA is used as a molecular marker, has the characteristics of strong specificity, high sensitivity, no wound, convenience and the like, and is suitable for screening of large-scale acute altitude reaction susceptible people in plains.
Description
Technical Field
The invention belongs to the field of molecular biotechnology and application thereof, and relates to application of hsa-miR-23b-5p as an acute altitude stress susceptible molecular marker and a kit, wherein the acute altitude stress attack risk is screened through the expression level of hsa-miR-23b-5p in plain plasma. Therefore, the method is used for evaluating the susceptibility of the human body to the acute altitude reaction and reducing the risk of the human body caused by the acute altitude reaction.
Background
Acute altitude sickness (AMS) occurs in plain people who live in low altitude areas for a long time, and occurs 1-3 days after the plain people rapidly enter the plateau without adapting to a new environment, and comprises a series of symptoms such as headache, dizziness, fatigue, insomnia, emotional restlessness, flatulence, diarrhea, vomiting and the like, wherein severe headache is a typical symptom of AMS. Geographically, a wide area with an altitude higher than 500m and a relatively flat or fluctuating terrain is called a plateau, and medically, a place with an altitude higher than 2500-3000m is called a plateau. AMS has high incidence rate, which can reach 50-85% according to The difference of ascending speed and specific altitude (Bartsch P. and Swenson E. R., Clinical practice: Acute high-availability illnessees, The New England and Journal of Medicine, 2013,368(24), 2294, 302.); it is not only highly dangerous and seriously affects the ability of an individual to rapidly advance to a High plateau, but, more seriously, if AMS is not effectively controlled and treated, it is likely to develop into High Altitude cerebral edema with High lethality (Boos C.J.et al, High Altitude and Acute Motor therapy and Changes in Circulating Endothelin-1, Interleukin-6, and Interleukin-17a. High Altitude Medicine & Biology, 2016,17(1), 25-31.).
AMS has obvious genetic tendency and individual susceptibility, and both environmental factors and genetic factors of high altitude hypoxia can influence the occurrence of AMS. For many years, AMS genetic predisposition and individual susceptibility have been the focus of attention of scholars both at home and abroad, and although it has been proposed to use SNP sites such as hypoxia-associated genes EGLN1, HIF-1AN, NOS3 to predict AMS susceptible populations, it is now apparent that these markers are not satisfactory in terms of accuracy and specificity (Luks A.M., Swenson E.R., Bartsch P., Acute high-availability site). And the plateau area of China is very large (about 1/5 of the area of the national land) and the altitude is higher (the average altitude of the Qinghai-Tibet plateau is more than 4000 meters). In recent years, with the vigorous development of the highland tourism industry and the economic construction of the plateaus at home and abroad, more and more plains enter the plateaus, and the high development of the AMS not only causes very serious influence on the life and work of the people, but also constructs heavy burden on the fragile sanitary machines in the plateau areas. Therefore, an effective method for predicting the susceptibility of AMS to pathogenesis in plain is urgently needed to be found.
MicroRNA is a non-coding RNA molecule which is widely existed in eukaryote and has the length of 18-24 nucleotides. MicroRNA inhibits the expression of target genes horizontally after transcription, participates in the regulation of cell differentiation, proliferation, apoptosis and other life activities, and plays an important role in various physiological and pathological processes such as embryonic development, organism metabolism, disease occurrence and development and the like. In recent years, researchers have proposed the concept of circulating microRNA by detecting microRNA in various body fluids such as blood, saliva, and urine. And the expression level of the microRNA is highly related to the difference of the genetic genes of the microRNA. It is important to note that in recent years, a large number of researches show that circulating microRNA has good specificity and sensitivity on advanced diagnosis and disease prediction of tumors, hypertension, stroke and a series of diseases. In addition, body fluid samples such as blood and the like are easy to obtain, strong in clinical operability and small in wound, and Circulating microRNA has good stability and is convenient to detect, so that the Circulating microRNA has the potential of serving as an AMS noninvasive biomarker and is suitable for AMS susceptibility population screening (Toffolo K., Wang J.et al., Circulating microRNAs as biomarkers in Circulating vaccine in 2018.). However, the correlation between circulating microRNA and AMS susceptibility is rarely reported at present.
Disclosure of Invention
In view of the above, the invention aims to provide an application of hsa-miR-23b-5p as an acute altitude reaction susceptible molecular marker, and also provides a kit for screening an acute altitude reaction susceptible test, and screening the acute altitude reaction risk through the expression level of the hsa-miR-23b-5p in the plain plasma. Therefore, the method is used for evaluating the susceptibility of the human body to the acute altitude reaction and reducing the risk of the human body caused by the acute altitude reaction.
In order to achieve the purpose, the invention provides the following technical scheme:
application of hsa-miR-23b-5p as a molecular marker of acute altitude reaction susceptible people.
Furthermore, the sequence of the hsa-miR-23b-5p is shown in SEQ ID NO: 4, respectively.
Further, the expression of the hsa-miR-23b-5p nucleic acid molecule in target plasma is down-regulated compared to the expression in control plasma.
Further, the application is that hsa-miR-23b-5p is applied to preparation of a reagent or a kit for screening acute altitude reaction susceptible persons as a microRNA molecular marker.
2. The kit for screening the acute plateau reaction susceptible person is prepared according to the application of the hsa-miR-23b-5p serving as the molecular marker of the acute plateau reaction susceptible person, and comprises primers which can be specifically combined with the reverse transcription products of the hsa-miR-23b-5p respectively and carry out PCR amplification.
Further, the primers comprise a forward primer and a reverse primer, wherein the forward primer is shown as SEQ ID No.19, and the reverse primer is shown as SEQ ID No. 20.
Furthermore, the sequence of the hsa-miR-23b-5p is shown in SEQ ID NO: 4, respectively.
Further, the expression of the hsa-miR-23b-5p nucleic acid molecule in target plasma is down-regulated compared to the expression in control plasma.
Further, the kit also comprises a miRNA RT reaction system, wherein the miRNA RT reaction system comprises, per 10 ul: reverse transcription primer 1ul, 5 × reverse transcription Buffer 2ul, RTase Mix 2ul, RNase-free H2O3ul, the remaining 2ul being test RNA.
Further, the reverse transcription primer is shown as SEQ ID No. 10.
Further, the kit RT reaction temperature parameters are as follows: 60min at 42 ℃ and 10min at 70 ℃.
Go toThe kit also comprises a miRNA qPCR reaction system, wherein the miRNA qPCR reaction system comprises 20 ul: SYBR Green Mix 10ul, miRNA Forward primer0.8ul, miRNA Reverse primer0.8ul, ddH2O6.4 ul, the rest is RT reaction product 2 ul.
Further, the kit miRNA qPCR reaction conditions are pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 2s, annealing at 60 ℃ for 20s, and extension at 70 ℃ for 10 s.
The invention has the beneficial effects that: according to the invention, through screening of chip spectrums of the plain plasma circulating microRNA of the plain life habitants, after people enter plateaus, AMS and healthy people are distinguished according to the Louis lake scoring diagnosis system of the AMS international universal diagnosis standard, and significant differences between the AMS susceptible people and the healthy people are found by combining the disease occurrence conditions of the AMS after the plateau is exposed to oxygen deficiency. And detecting the expression level of each microRNA in the plain plasma circulation of another independent population by a SYBR (SYBR Green dye, abbreviated as SYBR) real-time fluorescent quantitative PCR (polymerase chain reaction) method, and fully confirming that the correlation exists between the expression level of hsa-miR-23b-5p in the plain plasma circulation and the AMS susceptibility. The plasma sample cases verified by the invention are large in quantity and strong in reliability, and the reliable effect of the microRNA indicated by the invention on prevention and treatment of AMS can be fully proved. According to the invention, miRNA which is abnormally expressed in blood plasma of a susceptible person of AMS and has an obvious and constant low level is determined as a preliminary research object, miRNA which is closely related to the susceptible person of AMS is found out, the screened miRNA has good screening efficiency on AMS, the risk of the altitude reaction after the altitude is screened in the plain, the prevention and treatment of AMS are guided, and the health and life threat of the altitude reaction to people are reduced. The method provides a new effective and reliable supplement for the existing laboratory evidence of the microRNA as a potential diagnosis biomarker of acute altitude stress.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a comparison of the expression levels of hsa-miR-1183 in plasma of AMS-susceptible and healthy controls;
FIG. 2 is a graph of the working characteristics of plasma hsa-miR-1183 from AMS-susceptible individuals.
FIG. 3 is a comparison of the expression levels of hsa-miR-3654 in plasma of AMS-susceptible persons and healthy controls;
FIG. 4 is a graph of the working characteristics of plasma hsa-miR-3654 of AMS-susceptible individuals.
FIG. 5 is a comparison of the plasma expression levels of hsa-miR-134-3p in AMS-susceptible and healthy controls;
FIG. 6 is a graph of the working characteristics of plasma hsa-miR-134-3p from AMS-susceptible individuals.
FIG. 7 is a comparison of the plasma expression levels of hsa-miR-15b-5p in AMS-susceptible and healthy controls;
FIG. 8 is a graph of the working characteristics of plasma hsa-miR-15b-5p from AMS-susceptible individuals.
FIG. 9 is a comparison of the plasma expression levels of hsa-miR-23b-5p in AMS-susceptible and healthy controls;
FIG. 10 is a graph of the working characteristics of plasma hsa-miR-23b-5p from AMS-susceptible individuals.
FIG. 11 is a graph showing the working characteristics of the combination of plasma hsa-miR-1183, hsa-miR-15b-5p and hsa-miR-23b-5p of AMS susceptible patients.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.
Firstly, by carrying out comparative analysis on plasma microRNA expression profiles of 20 AMS patients and 15 healthy controls, and then comparing the plasma microRNA expression levels of 209 AMS patients and 203 healthy controls, the correlation between each microRNA and AMS is researched, and sensitive and credible AMS susceptibility biological genetic markers are searched. Collecting 2ml of peripheral blood of people who intend to rapidly enter a plateau from a plain by using an EDTA-Na anticoagulation tube, separating the peripheral blood at 3000 Xg and 25 ℃ for 10 minutes, extracting upper plasma, storing the upper plasma at-80 ℃ for later use, detecting the microRNA expression level in the plasma by using a microRNA expression profile chip (mircurytM LNA Array (v.18.0)), distinguishing AMS from healthy people (Maggiorini M.et., analysis of acid monomer nucleic acid in silicosis lake scoring diagnosis system) according to AMS international universal diagnosis standard Louis lake after the people enter the plateau, comparing AMS with healthy people (Maggiorini M.et., analysis of acid monomer in nucleic acid by nucleic acid Environ, 1998,69(12),1186-92.), comparing AMS with healthy people to screen AMS expression profiles, screening a large amount of microRNAs related to susceptibility, and finding a-miR-3, miR-3654, 118hsp-134-1183-134, The expression of hsa-miR-15b-5p and hsa-miR-23b-5p is remarkably different in AMS susceptible people and healthy people.
Expression profiles of hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3p, hsa-miR-15b-5p, and hsa-miR-23b-5p in AMS susceptible (209 cases) and healthy control (203 cases) were tested in another independent population using the qPCR technique. In the whole process, Plasma RNA is extracted by a Plasma microRNA column extraction Kit (miRNeasy Serum/Plasma Kit, cat # 217184) of Kanji technology Co., Ltd, Germany, and then real-time fluorescent quantitative PCR (Bulge-Loop) is adoptedTMmiRNA qRT-PCR Starter Kit, cat No.: r10039.2) is carried out to amplify each microRNA and the external reference cel-miR-39; the normalized expression level of each microRNA of each sample to cel-miR-39 is respectively calculated, the result is tested by SPSS 19.0, and the expression levels of plasma hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3P, hsa-miR-15b-5P and hsa-miR-23b-5P of the AMS susceptible group (209 cases) and the healthy group (203 cases) are found to have significant difference by taking P < 0.05 as a significance test standard.
The invention aims to solve the technical problem that AMS susceptible persons and healthy persons can be screened by finding one or more than one kind of plain plasma microRNA markers. The method is characterized in that related microRNA markers in human plain plasma are detected, AMS susceptible persons and healthy persons are distinguished through the expression level, and then the risk of AMS after the AMS enters the plateau is screened, so that more risks caused by the occurrence of diseases are avoided.
The technical scheme for solving the problems is as follows: and detecting the content of one or more of the hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3p, hsa-miR-15b-5p and hsa-miR-23b-5p of the plain human plasma to distinguish AMS susceptible persons from healthy persons.
The invention also comprises application of plasma microRNA (one or more of hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3p, hsa-miR-15b-5p and hsa-miR-23b-5p) in preparation of a kit for screening AMS (AMS) morbidity risk.
Example 1 correlation study of MicroRNA expression from plasma samples with AMS risk
Description of Material and specimen Collection
AMS susceptible plasma specimens were collected from AMS patients in the population of the rapidly advanced plateau, totaling 209 cases before they entered the plateau. The plasma specimens of the normal population are collected from the normal healthy population in the fast-advancing plateau population, and 203 cases are counted before the normal healthy population enters the plateau. Diagnosis of AMS was confirmed by the International Universal diagnostic Standard-Louis lake score. All people did not take any preventive medication before blood was drawn. Each sample was pooled into 2ml of blood using an EDTA-Na anticoagulation tube. The clinical case characteristics of the patients are shown in table 1. The study was approved by the medical ethics committee of the army medical university (third military medical science), and all specimens were collected with informed consent from the patients.
TABLE 1 clinical data on plasma specimens
Second, sample treatment and RNA extraction
After centrifugation at 3000 Xg for 10 minutes at 25 ℃ within 10min after venous blood collection, the supernatant plasma was taken with RNAse-free and bacteria-free tips and stored at-80 ℃ in RNAse-free and bacteria-free EP tubes for later use. RNA in Plasma was extracted and purified by a Plasma microRNA column extraction Kit (miRNeasy Serum/Plasma Kit, cat # 217184) from Qiagen technologies, Germany, according to the procedure described in the specification.
1. The concentration of plasma microRNA is low, about 25-40 ng/ul;
a260/280 is 1.4-1.6;
3, cDNA synthesis adopts microRNA fluorescent quantitative PCR reverse transcription kit (Bulge-Loop)TMmiRNA qRT-PCR Starter Kit, cat No.: r10039.2): 10ul reverse transcription reaction system: 2ul of RNA stock solution, 1ul (5uM) of reverse transcription primer, 2ul of 5x reverse transcription Buffer, 2ul of RTase Mix, Nase-free H2O3 ul; reverse transcription primer information is shown in table 2;
RT reaction temperature parameters: 60min at 42 ℃ and 10min at 70 ℃.
TABLE 2 reverse transcription primer information
| miRNA | miRNA RT primer | SEQ ID |
| hsa-miR-134-3p | 5'---gtcgtatccagtgcgtgtcgtggagtcggcaattgcactggatacgacttggtga---3' | SEQ ID No.6 |
| hsa-miR-15b-5p | 5'---gtcgtatccagtgcgtgtcgtggagtcggcaattgcactggatacgactgtaaac---3' | SEQ ID No.7 |
| hsa-miR-1183 | 5'---gtcgtatccagtgcgtgtcgtggagtcggcaattgcactggatacgactgcccac---3' | SEQ ID No.8 |
| hsa-miR-3654 | 5'---gtcgtatccagtgcgtgtcgtggagtcggcaattgcactggatacgacttcctca---3' | SEQ ID No.9 |
| hsa-miR-23b-5p | 5'---gtcgtatccagtgcgtgtcgtggagtcggcaattgcactggatacgacaaatcag---3' | SEQ ID No.10 |
Three, real-time fluorescent quantitative PCR (SYBR dye method)
MicroRNA real-time fluorescence quantitative PCR kit (Bulge-Loop) using Chinese Guangzhou Ruibo Biotechnology LtdTMmiRNA qRT-PCR Starter Kit, cat No.: r10039.2) respectively amplifying hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3p, hsa-miR-15b-5p, hsa-miR-23b-5p (target microRNA) and exogenous cel-miR-39; ct values (cycle threshold) were obtained, respectively, by the formula: and (3) respectively calculating the expression level of microRNA of each sample when the expression quantity is 2Ct (cel-miR-39) -Ct (target microRNA), wherein the specific operation process is shown in the kit instruction. Three replicates per sample were performed. The basic information of hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3p, hsa-miR-15b-5p and hsa-miR-23b-5p is shown in Table 1.
1. An amplification reaction system: (20ul system): RT reaction product 2ul, SYBR Green Mix 10ul, miRNA Forward primer (5uM)0.8ul, miRNA Reverse primer (5uM)0.8ul, ddH2O 6.4.4 ul; the forward and reverse primer information is shown in table 3;
2. reaction conditions are as follows: pre-denaturation at 95 deg.C for 10min, denaturation at 95 deg.C for 2s, annealing at 60 deg.C for 20s, and extension at 70 deg.C for 10 s;
3. the Realtime PCR amplification curves of the miRNA to be detected and the exogenous cel-miR-39 are S-shaped;
and 4, melting curves of PCR products are all unimodal, which shows that the amplified target gene has good specificity and reliable results.
TABLE 1 basic MicroRNA information
TABLE 3 PCR Forward and reverse primer information
| miRNA | Forward and reverse directions | primer | SEQ ID |
| hsa-miR-134-3p | Forward primer | 5'---cctgtgggccacctagtc---3' | SEQ ID No.11 |
| hsa-miR-134-3p | Reverse primer | 5'---cagtgcgtgtcgtggagt---3' | SEQ ID No.12 |
| hsa-miR-15b-5p | Forward primer | 5'---tagcagcacatcatggt---3' | SEQ ID No.13 |
| hsa-miR-15b-5p | Reverse primer | 5'---cagtgcgtgtcgtggagt---3' | SEQ ID No.14 |
| hsa-miR-1183 | Forward primer | 5'---cactgtaggtgatggtgagagt---3' | SEQ ID No.15 |
| hsa-miR-1183 | Reverse primer | 5'---cagtgcgtgtcgtggagt---3' | SEQ ID No.16 |
| hsa-miR-3654 | Forward primer | 5'---gactggacaagctg---3' | SEQ ID No.17 |
| hsa-miR-3654 | Reverse primer | 5'---cagtgcgtgtcgtggagt---3' | SEQ ID No.18 |
| hsa-miR-23b-5p | Forward primer | 5'---tgggttcctggcatgct---3' | SEQ ID No.19 |
| hsa-miR-23b-5p | Reverse primer | 5'---cagtgcgtgtcgtggagt---3' | SEQ ID No.20 |
Fourth, statistical analysis method
Statistics were performed using statistical software SPSS 19.0. The positive Test adopts a Shapiro-Wilk method, and the significant difference is evaluated by using a Mann-Whitney Test (Mann-Whitney Test), a working characteristic curve (ROC curve for short) and an area under line (AUC) to evaluate the screening efficiency of each microRNA (hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3p, hsa-miR-15b-5p and hsa-miR-23b-5 p). Statistical differences were considered when P < 0.05.
Fifth, result analysis
1. FIG. 1 is a comparison of the expression levels of hsa-miR-1183 in plasma of AMS-susceptible and healthy controls; as shown in FIG. 1, the expression level of plasma hsa-miR-1183 between AMS susceptible persons and healthy controls is very significantly statistically different, with P < 0.001. Wherein AMS is an AMS susceptible subject and health is a Healthy control. When P is less than 0.05, the statistical difference is significant, when P is less than 0.01, the statistical difference is very significant, and when P is less than 0.001, the statistical difference is very significant.
2. FIG. 2 is a graph of the working characteristics of plasma hsa-miR-1183 from AMS-susceptible individuals. As shown in FIG. 2, the screening efficiency of plasma hsa-miR-1183 on AMS-susceptible persons and healthy controls can be known through an ROC curve, and the screening efficiency of the hsa-miR-1183 on AMS-susceptible persons and healthy controls is very good, wherein the AUC is 0.828 (95% CI, 0.788-0.863). The ROC curve, area under the curve (AUC), sensitivity, specificity (specificity) of AMS susceptibilities are well shown in this figure, where AUC reflects the screening efficacy. It is generally accepted that the highest AUC tested, the better the diagnostic value. Fully indicates that the hsa-miR-1183 in the plasma has certain accuracy and feasibility when being used as a biomarker for screening AMS susceptible persons independently. Table 4 shows the data of the working characteristic curves of various microRNAs (hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3p, hsa-miR-15b-5p and hsa-miR-23b-5p) in the blood plasma of healthy people and AMS susceptible people.
Table 4 operating characteristic curve data
3. FIG. 3 is a comparison of the expression levels of hsa-miR-3654 in plasma of AMS-susceptible persons and healthy controls; as shown in FIG. 3, there was a statistically significant difference between AMS-susceptible and healthy controls in the expression level of plasma hsa-miR-3654, with P < 0.001.
4. FIG. 4 is a graph of the working characteristics of plasma hsa-miR-3654 of AMS-susceptible individuals. As shown in FIG. 4, the screening efficiency of plasma hsa-miR-3654 on AMS-susceptible persons and healthy controls can be known through an ROC curve that the screening efficiency of hsa-miR-3654 on AMS-susceptible persons and healthy controls is very good, wherein AUC is 0.697 (95% CI, 0.650-0.741).
5. FIG. 5 is a comparison of the plasma expression levels of hsa-miR-134-3p in AMS-susceptible and healthy controls; as shown in FIG. 5, there was a statistically significant difference between AMS-susceptible and healthy controls in the expression level of plasma hsa-miR-134-3P, wherein P is less than 0.001.
6. FIG. 6 is a graph of the working characteristics of plasma hsa-miR-134-3p from AMS-susceptible individuals. As shown in FIG. 6, the ROC curve shows that the screening efficiency of plasma hsa-miR-134-3p on AMS susceptible persons and healthy controls is good, wherein the AUC is 0.745 (95% CI, 0.700-0.786).
7. FIG. 7 is a comparison of the plasma expression levels of hsa-miR-15b-5p in AMS-susceptible and healthy controls; as shown in FIG. 7, there was a statistically significant difference between AMS-susceptible and healthy controls in the expression level of plasma hsa-miR-15b-5P, wherein P is less than 0.001.
8. FIG. 8 is a graph of the working characteristics of plasma hsa-miR-15b-5p from AMS-susceptible individuals. As shown in FIG. 8, the ROC curve shows that the screening efficiency of plasma hsa-miR-15b-5p between AMS-susceptible persons and healthy controls is good, wherein the AUC is 0.808 (95% CI, 0.766-0.845).
9. FIG. 9 is a comparison of the plasma expression levels of hsa-miR-23b-5p in AMS-susceptible and healthy controls; as shown in FIG. 9, there was a statistically significant difference between AMS-susceptible subjects and healthy controls in which P was < 0.001 compared to the expression level of hsa-miR-23b-5P in plasma.
10. FIG. 10 is a graph of the working characteristics of plasma hsa-miR-23b-5p from AMS-susceptible individuals. As shown in FIG. 10, the ROC curve shows that the screening efficiency of plasma hsa-miR-23b-5p between AMS-susceptible persons and healthy controls is good, wherein the AUC is 0.769 (95% CI, 0.725-0.808).
FIG. 11 is a combined working characteristic curve of three microRNAs of plasma hsa-miR-1183, hsa-miR-15b-5p and hsa-miR-23b-5 p. The screening efficiency of three microRNA molecules alone between AMS susceptible persons and healthy controls is better, and the AUC is respectively: 0.828 (95% CI,0.788-0.863), 0.808 (95% CI,0.766-0.845), 0.769 (95% CI, 0.725-0.808). From the ROC curve for screening efficacy of the three micrornas in combination between AMS susceptible and healthy controls in fig. 11, it can be seen that the screening efficacy of the combination of the three plasma micrornas between AMS susceptible and healthy controls is better than that of the combination alone, with an AUC of 0.872 (95% CI, 0.836-0.903).
The inventor firstly carries out comparative analysis on plasma microRNA expression profiles of 20 AMS patients and 15 healthy controls, screens microRNA related to AMS susceptibility, further analyzes the expression quantity of each microRNA in 209 AMS susceptible plasma samples and 203 normal population plasma samples through a large amount of creative work, finds that hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3p, hsa-miR-15b-5p and hsa-miR-23b-5p in plasma have good screening efficiency on AMS susceptible persons and healthy controls, and jointly determines the morbid risk of AMS screening by three microRNAs of hsa-miR-1183, hsa-miR-15b-5p and hsa-miR-23b-5p more accurately. The inventor also carried out some studies on the relevance of microRNA to the incidence of mountain sickness at the previous stage, but no experimental data show that hsa-miR-1183, hsa-miR-3654, hsa-miR-134-3p, hsa-miR-15b-5p and hsa-miR-23b-5p are related to AMS susceptibility.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
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<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cactgtaggt gatggtgaga gt 22
<210> 16
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cagtgcgtgt cgtggagt 18
<210> 17
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gactggacaa gctg 14
<210> 18
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cagtgcgtgt cgtggagt 18
<210> 19
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tgggttcctg gcatgct 17
<210> 20
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cagtgcgtgt cgtggagt 18
Claims (5)
- The application of hsa-miR-23b-5p as a microRNA molecular marker in the preparation of a reagent or a kit for screening acute altitude reaction susceptible persons, wherein the sequence of the hsa-miR-23b-5p is shown in SEQ ID NO: 4, respectively.
- 2. The use of claim 1, wherein the kit comprises primers capable of specifically binding to the reverse transcription product of hsa-miR-23b-5p and performing PCR amplification.
- 3. The use of claim 2, wherein the primers comprise a forward primer and a reverse primer, wherein the forward primer is shown as SEQ ID No.19 and the reverse primer is shown as SEQ ID No. 20.
- 4. The use of claim 2, wherein the kit further comprises a miRNA RT reaction comprising, per 10 μ L: reverse transcription primer 1. mu.L, 5 × reverse transcription Buffer 2. mu.L, RTase Mix 2. mu.L, RNase-free H2O3. mu.L, the remaining 2. mu.L are test RNAs.
- 5. The use of claim 2, wherein the kit further comprises a miRNA qPCR reaction system comprising per 20 μ L: SYBR Green Mix 10. mu.L, miRNA forward primer 0.8. mu.L, miRNA reverse primer 0.8. mu.L, ddH2O6.4. mu.L, the remainder being 2. mu.L of RT reaction product.
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|---|---|---|---|---|
| CN107058472A (en) * | 2016-11-03 | 2017-08-18 | 中国人民解放军第三军医大学 | A kind of diagnostic kit by four kinds of blood plasma microRNA Combining diagnosis acute mountain sicknesses |
| JP2018502567A (en) * | 2015-01-12 | 2018-02-01 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | Diagnosis method of pancreatic cancer |
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| JP2018502567A (en) * | 2015-01-12 | 2018-02-01 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | Diagnosis method of pancreatic cancer |
| CN107058472A (en) * | 2016-11-03 | 2017-08-18 | 中国人民解放军第三军医大学 | A kind of diagnostic kit by four kinds of blood plasma microRNA Combining diagnosis acute mountain sicknesses |
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| Title |
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| A Signature of Circulating microRNAs Predicts the Susceptibility of Acute Mountain Sickness;Bao Liu等;《Front Physiol》;20170208;第8卷;Article 55 * |
| 急性高山病和高原红细胞增多症的microRNA表达特征及其病理生理学意义研究;黄河;《中国博士学位论文全文数据库 (医药卫生科技辑)》;20210715(第7期);E065-2 * |
| 高原习服及习服不良过程中基因表达特征及其病理生理学意义研究;刘宝;《中国博士学位论文全文数据库 (医药卫生科技辑)》;20170715(第11期);E065-86 * |
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