CN107058468B - Kit for predicting acute mountain sickness incidence risk through circulating microRNA-369-3p expression level - Google Patents

Abstract

The invention relates to a microRNA marker for detecting a person susceptible to acute mountain sickness and application of the microRNA marker in preparation of a kit for predicting the onset risk of the acute mountain sickness. The microRNA is used as a molecular marker, has the characteristics of strong specificity, high sensitivity, no invasion, convenience and the like, and is particularly suitable for screening of large-scale acute mountain sickness susceptible persons in the plain.

Description

Kit for predicting acute mountain sickness incidence risk through circulating microRNA-369-3p expression level

Technical Field

The invention relates to a kit for detection, in particular to a method for predicting the risk of acute mountain sickness by the expression level of plain plasma circulating microRNA-369-3p, so as to evaluate the susceptibility of the acute mountain sickness of a human body.

Background

Acute Mountain Sickness (AMS), also known as acute mild altitude sickness, occurs in people who live in low altitude areas for a long time and rapidly enter into plateaus of over 2500m without adaptation to new environments for 1-3 days, and includes a series of symptoms such as insomnia, headache, dizziness, anorexia, emotional restlessness, vomiting, and the like, wherein severe headache is a typical symptom of AMS. AMS has a disease rate of 50-85% according to the difference of ascending speed and specific altitude (Bartsch P and Swenson E R (2013), Clinical practice: Acute high-altitudins, N Engl J Med (368 (24), 2294-. More seriously, if AMS is not effectively controlled and treated, it is likely to develop into High Altitude Cerebral Edema (HACE) with High lethality rate (Boos C J et al (2016), High Altitude Albuted and Acute Motor Package and Change in circulating Endothelin-1, Interleukin-6, and Interleukin-17a, High Altitude Albuted Medicine & Biology, 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, the genetic tendency and individual susceptibility of AMS have been the focus of attention of researchers at home and abroad, and although some people have proposed using SNP sites of hypoxia-sensitive genes EGLN1and HIF-1AN to predict AMS susceptible population, these markers are not satisfactory in accuracy and specificity (Zhang E, Zhang J, Jin J, Qin J, Li H, Huang L: Variants of the low oxygen sensors EGLN1and HIF-1AN assisted with access mountain facility, 2014, 15 (12): 21777 21787.) since International patent office of molecular sciences, 2014, 15 (1/5) of our country, the average of the Tibetan plateau is above 4000 meters, the national mountain height of the plateau has been increased with the development of economic tourism, and many more International nationals have entered the national world, the occurrence of AMS has seriously affected people's life and work, so an effective method for predicting the disease susceptibility of AMS in plains is urgently needed to be found.

MicroRNA is a kind of endogenous small molecular non-coding RNA widely existing in eukaryote, and the length of the MicroRNA is 18-24 nucleotides. MicroRNA inhibits the expression of target genes horizontally after transcription, regulates and controls the vital activities of cell differentiation, proliferation, apoptosis and the like, and plays an important role in various physiological and pathological processes such as embryonic development, body 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. In addition, the expression level of the microRNA is highly related to the difference of the genetic genes of the microRNA, and a large number of researches show that the circulating microRNA has good specificity and sensitivity on the advanced diagnosis and disease prediction of tumors, hypertension, stroke and a series of diseases in recent years. 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 is good in stability and 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 (Ghai V and Wang K (2016), Recent progress aware of circulating microRNAs as clinical biomarkers, Arch toxin).

However, no report has been made on the correlation between circulating microRNA and AMS susceptibility.

No report for detecting the expression level of microRNA-369-3p in blood to predict the risk of AMS is found.

Through screening of chip spectrums of plain plasma circulating microRNA of the plain life habitants, and combining with the AMS morbidity of the plain life habitants after high altitude hypoxia exposure, the difference of the plain plasma circulating microRNA-369-3p between AMS susceptible persons and AMS tolerant persons is found. And detecting the expression level of the plain plasma circulating microRNA-369-3p by a SYBR (SYBR Green dye, abbreviated as SYBR) real-time fluorescence quantitative PCR (polymerase chain reaction) method, and confirming that the correlation exists between the expression level of the plain plasma circulating microRNA-369-3p and the AMS susceptibility.

Disclosure of Invention

The invention aims to search novel biological markers of the plain plasma related to AMS, and provides application of the plain plasma microRNA-369-3p in preparation of a kit for predicting the incidence risk of AMS by the plain plasma. The kit can be used for screening AMS susceptible persons before plateau entrance of plateau persons, guiding AMS prevention and reducing AMS threat.

The inventor researches the correlation between microRNA-369-3p and AMS by comparing the expression profiles of plasma microRNA of 13 AMS patients and 9 healthy controls and then compares the expression levels of plasma microRNA-369-3p of 41 AMS patients and 46 healthy controls, and finds out a sensitive and credible biological genetic marker of AMS. Collecting 22 peripheral blood 2ml of population which is expected to rapidly enter a plateau from a plain by using an EDTA-Na anticoagulation tube, separating at 3000 Xg and 25 ℃ for 10 minutes, extracting upper plasma, storing at-80 ℃ for later use, detecting the microRNA expression level in the plasma by using a microRNA expression profile chip (mircurytMLNA Array (v.18.0)), distinguishing AMS from healthy population (Maggiorini M et al (1998)) according to an AMS international universal diagnostic standard Lewis lake scoring diagnostic system after the population enters the plateau, comparing AMS with the healthy population (Maggiorini M et al, 1998) Assessment of acute mountain sickness and diabetes mellitus plasma Environ 69, 12), comparing AMS with the healthy population microRNA expression profile, screening related microRNA, and finding that the difference of the microRNA-3 p in the AMS susceptible population and the healthy population exists.

The expression profile of microRNA-369-3p in AMS susceptible (41 cases) and healthy control (46 cases) was examined in another independent population using the qPCR technique. In the whole process, Plasma RNA is extracted by adopting a Plasma microRNA column extraction Kit (miRNeasy serum/Plasma Kit, the product number is 217184) of Germany Qiagen technology limited company, and then the amplification of microRNA-369-3p and external reference cel-miR-39 is carried out by adopting a real-time fluorescence quantitative PCR (Hairpin-itTMmiRNAs RT-PCR quantification Kit, the product number is E01008); the normalized expression level of microRNA-369-3P relative to cel-miR-39 of each sample is respectively calculated, the result is tested by SPSS 19.0, and the expression level of plasma microRNA-369-3P of AMS susceptible group samples (41 samples) and normal population (46 samples) is found to have significant difference by taking P < 0.05 as a significance test standard (Table 1)

The invention aims to solve the technical problem that an AMS susceptible person and an AMS tolerant person can be screened by finding a plain plasma microRNA marker. By detecting the content of microRNA-369-3p in human plain plasma and by the expression level, AMS susceptible persons and AMS tolerant persons are distinguished, and the disease risk of AMS after the AMS enters the plateau is predicted.

The technical scheme for solving the problems is as follows: and detecting the content of microRNA-369-3p in the plasma of the plain human to distinguish AMS susceptible persons from AMS tolerant persons, wherein the specific information of the microRNA-369-3p is shown in a table 2.

The benefits of the invention are: the screened microRNA has good prediction efficiency on AMS, can predict the morbidity risk of AMS after the AMS reaches the plateau in the plain, guides the prevention and treatment of the AMS, and lightens the threat of the AMS.

Besides the technical scheme, the invention also makes the following improvements.

The invention also comprises the function of the plasma microRNA (microRNA-369-3p) in the preparation of the kit for predicting the risk of AMS.

Drawings

FIG. 1. MicroRNA-369-3p expression levels (i.e., normalized levels of microRNA-369-3p compared to cel-miR-39) in plasma of AMS susceptible and tolerant subjects. Wherein AMS is the patient with AMS and non-AMS is the healthy control. *: p < 0.05, i.e. there was a significant statistical difference, x: p < 0.01, i.e. there were very significant statistical differences, x: p is less than 0.001, namely, the statistical difference is very significant.

FIG. 2 is a characteristic curve (ROC curve for short) of AMS human plasma microRNA-369-3p, which shows the ROC curve, area under the curve (AUC), sensitivity (sensitivity) and specificity (specificity) of AMS human plasma, wherein the AUC reflects the predicted efficiency (AUC is 0.5, no predicted efficiency; AUC < 0.5 is very small predicted value; AUC < 0.7 < 0.9 is very accurate predicted value; 0.9 < AUC < 1 is very accurate predicted value).

Detailed Description

The present invention will now be described with reference to the accompanying drawings, which illustrate examples only for the purpose of illustrating the present invention and do not limit the scope of the present invention.

Example 1 correlation study of MicroRNA-369-3p expression from plasma specimens with risk of AMS onset

Description of Material and specimen Collection

AMS susceptible plasma specimens were collected from AMS patients in the population of rapidly advancing plateaus, and the total number was 41. The plasma specimens of the normal population are collected from the normal healthy population in the fast-advancing plateau population before entering the plateau, and the total number is 46. 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.

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.

Three, real-time fluorescent quantitative PCR (SYBR dye method)

Amplifying microRNA-369-3p and exogenous cel-miR-39 by using a microRNA real-time fluorescent quantitative PCR Kit (Hairpin-itTMmiRNAs RT-PCR quantification Kit, the product number is E01008) of Shanghai Jima pharmaceutical technology Limited company in China; ct values (cycle threshold) were obtained, respectively, by the formula: and (3) respectively calculating the expression level of 2Ct (cel-miR-39) -Ct (microRNA-369-3p) in each sample, wherein the specific operation process is shown in the specification. Three replicates per sample were performed. The sequences of the microRNA-369-3p and cel-miR-39 primers are shown in Table 3.

And fourthly, a statistical analysis method.

Statistics were performed using statistical software SPSS 19.0. The Shapiro-Wilk method is adopted in the normality Test, and the Mann-Whitney Test (Mann-Whitney Test), a working characteristic curve (ROC curve for short) and an area under the line (AUC) are used for evaluating the prediction efficiency of the microRNA-369-3 p. Statistical differences were considered when p < 0.05.

Sixthly, result analysis

Compared with the expression level of plasma microRNA-369-3p of an AMS susceptible person and an AMS tolerant person, p is less than 0.001, and the statistical difference is very significant.

2. Prediction efficacy of plasma microRNA-369-3p on AMS susceptible and AMS tolerant subjects it was shown by the ROC curve that microRNA-369-3p is very good in prediction efficacy on AMS susceptible and AMS tolerant subjects, AUC is 0.859 (95% CI, 0.783-0.985).

Seven, conclusion

The microRNA-369-3p in blood has good prediction efficiency on AMS susceptible persons and AMS tolerant persons, and can predict the morbidity risk of AMS.

TABLE 1 expression levels of plasma microRNA-369-3p for AMS susceptible and AMS tolerant groups

AMS susceptible group vs. AMS tolerant group: p < 0.001, data expressed as median (25% -75% quantile)

TABLE 2 basic information of microRNA-369-3p

TABLE 3 primer information of microRNA-369-3p and cel-miR-39

Claims (1)

1. The application of the primer for detecting the expression level of the microRNA-369-3p in preparing a pre-test kit for a person susceptible to acute mountain sickness is characterized in that the sequence of the microRNA-369-3p is SEQ ID NO. 1.

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