CN103882095A

CN103882095A - Application of small molecule RNA as tuberculosis marker

Info

Publication number: CN103882095A
Application number: CN201210559345.1A
Authority: CN
Inventors: 钟球; 周琳; 李海成; 陈涛
Original assignee: Guangdong Tuberculosis Control Central
Current assignee: Guangdong Tuberculosis Control Central
Priority date: 2012-12-20
Filing date: 2012-12-20
Publication date: 2014-06-25

Abstract

The invention discloses an application of small molecule RNA as a tuberculosis marker. Expression profiles of miRNAs in serum of tuberculosis patients are obtained through experiments. The 10 miRNAs that have the highest expression quantity and are most different from those of healthy people are obtained through information analysis. High expression quantity can guarantee the sensitivity of the detection method taking the miRNAs as the targets. At the same time the great difference between the selected miRNAs and the miRNAs of health people can guarantee the specificity of the detection method. Based on the selected 10 miRNAs, a kit is developed, and the kit takes a small molecule RNA as the marker and carries out diagnosis through a reverse transcription technology and a real-time fluorescent quantitative PCR technology. The detection result of the kit is accurate and reliable. The operation of the kit is convenient. The kit is suitable for promotion and application.

Description

MicroRNA is as the application of tuberculosis marker

Technical field

The invention belongs to molecular diagnosis field, be specifically related to the application of microRNA as tuberculosis marker.

Background technology

Mycobacterium tuberculosis causes infection and dead.The population in World Health Organization's estimation whole world 1/ 3 is subject to tubercle bacillus affection, its major cause is multiple-drug resistance tuberculosis bacterium (multidrug resistance tuberculosiscle bacillus, MDR-TB) popular and human immunodeficiency virus (human immune deficiency virus, HIV) immunity infringement host (immuno-compromised host due to infection, organ transplantation, immunosuppressor use etc., ICH) increase with advanced age population in the low inferior factor of immunity of organisms, make infection due to Mycobacterium tuberculosis be difficult to control.In order further to improve diagnosis lungy, treatment and prevention and control level, separate and identify Mycobacterium tuberculosis in the urgent need to more accurately diagnosing fast new technology to be used for clinical samples now.

At present clinical existing diagnosis of tuberculosis technology according to every kind of technology for target difference can be divided into three major types.Micro-biological process, serological method, molecular biology method.

Micro-biological process is taking smear and various cultural method as representative.Smear method is wherein rapider, within general 1 hour, can go out result, but positive rate only has 20% left and right.Roche culture method is at present as the gold standard of diagnosis of tuberculosis, and the required cycle is long, general 4-8 week, and the treatment of tuberculosis strengthening phase is 2 months.If out patient is being intervened etc. cultivation results, will delay treatment; If but not etc. cultivation results does not treat, again may be because mistaken diagnosis make patient bear the misery that antitubercular agent side effect brings.

And about serological method, WHO has given a public statement, applied serology diagnosis of tuberculosis method all can not realize satisfactory results in the market.

Molecular biology method is a recent development field more rapidly, and the whole bag of tricks is all taking the RNA of mycobacterium tuberculosis or DNA as target.But the problem that these class methods face is to fail to find a target that another people is satisfied.

Summary of the invention

For the existing problem of prior art, the object of the present invention is to provide one taking microRNA as marker, the test kit of tuberculosis being diagnosed by reverse transcription, Real-Time Fluorescent Quantitative PCR Technique.

The technical solution used in the present invention is:

A kind of diagnostic kit lungy, it comprises the specific PCR primer that detects one or more microRNAs, and described microRNA comprises hsa-miR-92a, hsa-miR-122, hsa-let-7a, hsa-miR-486-5p, hsa-let-7b, hsa-miR-451, hsa-miR-320b, hsa-miR-320a, hsa-miR-192 and hsa-miR-185.

Wherein, the primer of detection hsa-miR-92a is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGGC（SEQ ID NO:1）

realtime-F：GCGCGCTATTGCACTTGTCCCG（SEQ ID NO:2）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:3）

The primer that detects hsa-miR-122 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAAACA（SEQ ID NO:4）

realtime-F：GCGCGCTGGAGTGTGACAATGG（SEQ ID NO:5）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:6）

The primer that detects hsa-let-7a is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT（SEQ ID NO:7）

realtime-F：GCGCGCTGAGGTAGTAGGTTGT（SEQ ID NO:8）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:9）

The primer that detects hsa-miR-486-5p is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTCGGG（SEQ ID NO:10）

realtime-F：GCGCTCCTGTACTGAGCTGC（SEQ ID NO:11）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:12）

The primer that detects hsa-let-7b is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACCAC（SEQ ID NO:13）

realtime-F：GCGCGCTGAGGTAGTAGGTTGT（SEQ ID NO:14）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:15）

The primer that detects hsa-miR-451 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTCA（SEQ ID NO:16）

realtime-F：GCGCGCGCAAACCGTTACCATTAC（SEQ ID NO:17）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:18）

The primer that detects hsa-miR-320b is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTGCCC（SEQ ID NO:19）

realtime-F：GCGCGCAAAAGCTGGGTTGAGA（SEQ ID NO:20）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:21）

The primer that detects hsa-miR-320a is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCGCCC（SEQ ID NO:22）

realtime-F：GCGCGCAAAAGCTGGGTTGAGA（SEQ ID NO:23）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:24）

The primer that detects hsa-miR-192 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCTGT（SEQ ID NO:25）

realtime-F：GCGCGCGCCTGACCTATGAATTG（SEQ ID NO:26）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:27）

The primer that detects hsa-miR-185 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGGA（SEQ ID NO:28）

realtime-F：GCGCGCTGGAGAGAAAGGCAGT（SEQ ID NO:29）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:30）

The invention has the beneficial effects as follows:

The present invention obtains miRNA express spectra in tuberculosis patient serum by experiment, then by information analysis obtain miRNA expression amount in crowd's serum the highest and with 10 kinds of miRNA of non-tuberculosis people constellation variance maximum.High expression level amount has ensured the sensitivity as detection method with this target, has ensured the specificity of the method with non-tuberculosis people constellation variance maximum simultaneously.10 kinds of miRNA that obtain based on above-mentioned screening, the present invention has built a kind of taking microRNA as marker, the test kit of tuberculosis being diagnosed by reverse transcription, Real-Time Fluorescent Quantitative PCR Technique.The detected result of this test kit accurately and reliably, easy and simple to handle, is suitable for applying.

Brief description of the drawings

Fig. 1 is the structure schema of the microRNA differential expression spectrum between different samples;

Fig. 2 is the structure schema of the microRNA differential expression spectrum between different samples;

Fig. 3 is the PCR product electrophorogram that miRNA detects primer;

Fig. 4 is the solubility curve figure that miRNA detects the PCR product of primer.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated, but be not limited to this.

one, can be used as the screening of the microRNA of tuberculosis marker

The little RNA (sRNA) of Solexa degree of depth order-checking gained is almost contained all RNA, comprises miRNA, siRNA, piRNA, rRNA, tRNA, snRNA, snoRNA, repeat associate sRNA, exon or intron degradation fragment etc.By and given data storehouse compare, find between sample and database in methods such as the locational overlap of genome, sRNA is annotated.MicroRNA is the important particular molecule of a class in organism, induced gene silence, the regulation process of many vital movements such as participation Growth of Cells, growth, genetic transcription and translation.Based on the little RNA digital assay of Solexa high throughput sequencing technologies, adopt the order-checking (SBS-sequencing by synthesis) while synthesizing, can reduce the disappearance in one section of region of causing because of secondary structure.And it is few to have required sample size, high-throughput, high precision, have automatization platform simple to operation and the feature such as powerful, millions of microRNA sequences of disposable acquisition, can identify all sidedly fast the microRNA of these species under this state and find new microRNA, build the microRNA differential expression spectrum between sample, for microRNA functional study provides powerful.Its experiment flow is as Fig. 1 and Fig. 2.

Solexa order-checking gained 35nt sequence, by removing joint, go inferior quality, the process such as depollute completes data processing and obtains clean sequence, statistics and sample room common sequence statistics that it is carried out to sequence length distribution.By the clean sequence classification annotation after cleaning, can obtain the each component and the expression amount information that in sample, comprise.To the known miRNA statistics of expressing in two samples, judge whether the expression amount between two samples exists significant difference, and use respectively the relatively difference of the miRNA expression amount of both co expression of log2-ratio, Scatter plot figure.Concrete steps are as follows: (1) first normalizes to same magnitude by two samples (control and treatment).Formula: normalized expression amount=miRNA expression amount/sample total expression amount * normalizing magnitude (2) is then used result statistics fold_change and the P-value after stdn and is figure.Fold_change formula: Fold_change=log2(treatment/control).P-value formula:

After normalization method, if certain miRNA gene expression amount of two samples is zero, be revised as 0.01; If certain miRNA gene expression amount of two samples is all less than 1, because its expression amount is too low, do not participate in Differential expression analysis.

The present invention utilizes Solexa high throughput sequencing technologies, analyzes the difference of Small rna expression spectrum in non-tuberculosis group and tuberculosis group serum.First we,, respectively with miRNA expression amount descending sort in two groups of serum, obtain the highest front 20 kinds of miRNA of expression amount in two groups of serum.Then ask the common factor of high expression level miRNA kind in two groups of serum, obtain 14 kinds of miRNA that expression amount is the highest in two groups of samples and the differential expression of these 14 kinds of miRNA in two groups of samples has statistical significance.Then we are clipped to ascending order and descending sort with fold-change (log2 S2/S1) point, then get front 5 kinds of miRNA, we have just obtained 10 kinds of (hsa-miR-92a, hsa-miR-122, hsa-let-7a, hsa-miR-486-5p, hsa-let-7b, hsa-miR-451, hsa-miR-320b, hsa-miR-320a, hsa-miR-192, hsa-miR-185) expression amount in crowd's serum miRNA the highest and expression amount difference maximum in non-tuberculosis group and tuberculosis group serum for diagnosis of tuberculosis mark like this.

materials and methods

1.1 material

1.1.1 sample source

27 examples are coated with positive lunger and (call " tuberculosis group " in the following text, comprise: male 19 examples, female's 8 examples, age is between 22～87 years old, average 42 years old) serum provides by Guangzhou chest hospital, 25 routine healthy volunteer's serum are taken from the worker of our unit and (are called " non-tuberculosis group " in the following text, comprise: male 11 examples, female's 14 examples, the age between 25～50 years old, average 34 years old).

sample collection and preservation

Venous blood samples 4～5 ml are in the solidifying pipe of 2 promoting vacuum, and room temperature leaves standstill approximately 30 min, and centrifugal 5 min of 4000 rpm draw serum and divide equally to 22 ml cryogenic vials, and-70 DEG C of Refrigerator stores are for subsequent use.

reagent and instrument

The short solidifying pipe of disposal vacuum is purchased from Guangzhou Improve Medical Co., Ltd.; Total RNA Purification Kit(TRK-1001) be LC SCIENCES company of U.S. product; From total RNA, separate the reagent such as sRNA, RT-PCR amplification, cDNA preparation of samples, the amplification of DNA cluster and order-checking and use Illumina company auxiliary products; Sequencing application Illumina HiSeqTM 2000 sequenator systems.

method

1.2.1 the total RNA of serum extracts

Take out 1 of every example serum sample for subsequent use from-70 DEG C of refrigerators, put room temperature and treat its dissolving; From the sample of case group, control group, respectively get 1 ml serum and add to respectively in 2 50 ml centrifuge tubes by group, preparation case group, each 1 pipe of control group pooled serum; The strict LC Sciences RNA that presses extracts the total RNA of test kit specification sheets extraction serum.

serum Small RNA order-checking

Serum sRNA separation, purifying, cDNA library foundation, amplification and order-checking, bioinformatic analysis have been assisted by BGI-Shenzhen, and experimental implementation is carried out in strict accordance with reagent and instrument specification sheets.

Main experiment flow comprises: from total RNA, separate order-checking on Cluster Station cluster amplification → Illumina HiSeqTM 2000 sequenators of the detection → DNA in purifying → library that sRNA → 5 ' joint connects → 3 ' joint connections → RT-PCR amplification → sRNA library → clean sequence employing of 10～30 nt sRNA GenomeStudio software is carried out to bioinformatic analysis, obtain the express spectra of Small RNA in two kinds of crowd's serum.

First we,, respectively with miRNA expression amount descending sort in two groups of serum, obtain the highest front 20 kinds of miRNA of expression amount in two groups of serum.Then ask the common factor of high expression level miRNA kind in two groups of serum, obtain 14 kinds of miRNA that expression amount is the highest in two groups of samples and the differential expression of these 14 kinds of miRNA in two groups of samples has statistical significance.Then we are clipped to ascending order and descending sort with fold-change (log2 S2/S1) point, then get front 5 kinds of miRNA, we have just obtained 10 kinds of (hsa-miR-92a, hsa-miR-122, hsa-let-7a, hsa-miR-486-5p, hsa-let-7b, hsa-miR-451, hsa-miR-320b, hsa-miR-320a, hsa-miR-192, hsa-miR-185) expression amount in crowd's serum miRNA the highest and expression amount difference maximum in non-tuberculosis group and tuberculosis group serum for diagnosis of tuberculosis mark like this.

Then adopt the method for real-time fluorescence quantitative PCR to analyze relatively the expression amount of these 10 kinds of miRNA in different crowd serum, further verify its value as diagnosis of tuberculosis mark.

two, the realtime-PCR detection method of miRNA

MiRNA realtime-PCR primer design method:

1) stem-loop RT design of primers: based on general loop-stem structure, only need to be according to different 6 bases of miRNA sequence amendment least significant end.General loop-stem structure sequence is: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC(SEQ ID NO:31)

For example design miR-1 UGGAAUGUAAAGAAGUAUGUAU(SEQ ID NO:32) RT primer, only need be on general stem ring sequence after-frame the reverse complementary sequence of 6 bases of miRNA3 ' end,

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATACAT（SEQ ID NO:33）

2) realtime upstream primer design: miRNA sequence is removed the remainder of 6 bases of 3 ' end as upstream primer, if the upstream primer of miR-1 is (noting U to change T into): TGGAATGTAAAGAAGT(SEQ ID NO:34).The Tm value (general reference dna MAN) that checks primer, if Tm value is lower, adds GC at 5 ' end and makes Tm value approach 60 degree.Therefore the upstream primer of miR-1 can be designed to: GCGCTGGAATGTAAAGAAGT(SEQ ID NO:35), 61.4 degree.

3) downstream primer is general, and sequence is GTGCAGGGTCCGAGGT(SEQ ID NO:36).

4) design of primers good after, need to detect by trial test the specificity of primer.Generally need to do solubility curve and detect the specificity of primer; Preferably PCR product is carried out to electrophoresis detection product whether single (because product length is very little, needing more than 3% agarose gel) simultaneously.

By above method, screening obtains the Auele Specific Primer for detection of hsa-miR-92a, hsa-miR-122, hsa-let-7a, hsa-miR-486-5p, hsa-let-7b, hsa-miR-451, hsa-miR-320b, hsa-miR-320a, these 10 kinds of miRNA of hsa-miR-192, hsa-miR-185, and sequence is as follows:

The primer that detects hsa-miR-92a is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGGC（SEQ ID NO:1）

realtime-F：GCGCGCTATTGCACTTGTCCCG（SEQ ID NO:2）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:3）

The primer that detects hsa-miR-122 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAAACA（SEQ ID NO:4）

realtime-F：GCGCGCTGGAGTGTGACAATGG（SEQ ID NO:5）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:6）

The primer that detects hsa-let-7a is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT（SEQ ID NO:7）

realtime-F：GCGCGCTGAGGTAGTAGGTTGT（SEQ ID NO:8）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:9）

The primer that detects hsa-miR-486-5p is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTCGGG（SEQ ID NO:10）

realtime-F：GCGCTCCTGTACTGAGCTGC（SEQ ID NO:11）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:12）

The primer that detects hsa-let-7b is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACCAC（SEQ ID NO:13）

realtime-F：GCGCGCTGAGGTAGTAGGTTGT（SEQ ID NO:14）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:15）

The primer that detects hsa-miR-451 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTCA（SEQ ID NO:16）

realtime-F：GCGCGCGCAAACCGTTACCATTAC（SEQ ID NO:17）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:18）

The primer that detects hsa-miR-320b is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTGCCC（SEQ ID NO:19）

realtime-F：GCGCGCAAAAGCTGGGTTGAGA（SEQ ID NO:20）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:21）

The primer that detects hsa-miR-320a is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCGCCC（SEQ ID NO:22）

realtime-F：GCGCGCAAAAGCTGGGTTGAGA（SEQ ID NO:23）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:24）

The primer that detects hsa-miR-192 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCTGT（SEQ ID NO:25）

realtime-F：GCGCGCGCCTGACCTATGAATTG（SEQ ID NO:26）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:27）

The primer that detects hsa-miR-185 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGGA（SEQ ID NO:28）

realtime-F：GCGCGCTGGAGAGAAAGGCAGT（SEQ ID NO:29）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:30）

Taking serum and the total RNA of blood lymphocyte as template, utilize the primer of above-mentioned detection hsa-let-7a to carry out pcr amplification respectively, amplification is shown in Fig. 3.Wherein, 1-4 is the amplification that hsa-let-7a detects primer; 5 is marker; 6-9 is the amplification of oligodT reverse transcriptase primer; 1,2,6,7 taking the total RNA of serum as template; 3,4,8,9 taking the total RNA of blood lymphocyte as template.This group primer only expands the single band of corresponding miRNA in serum and blood lymphocyte, and specificity is high.

The solubility curve of PCR product is shown in fig. 4.

three, the normal distribution curve of 10 kinds of miRNA expression amount in tubercular and non-tubercular serum

detect respectively in the tubercular and non-tubercular serum and blood lymphocyte of some amountthe content of 10 kinds of miRNA such as hsa-miR-92a, hsa-miR-122, hsa-let-7a, hsa-miR-486-5p, hsa-let-7b, hsa-miR-451, hsa-miR-320b, hsa-miR-320a, hsa-miR-192, hsa-miR-185.Obtain the normal state of these 10 kinds of miRNA in tubercular and non-tubercular body curve respectively.

four, diagnostic method lungy

Utilize miRNA Auele Specific Primer of the present invention to detect one or more the expression level in hsa-miR-92a, hsa-miR-122 in clinical sample serum, hsa-let-7a, hsa-miR-486-5p, hsa-let-7b, hsa-miR-451, hsa-miR-320b, hsa-miR-320a, hsa-miR-192, hsa-miR-185, the normal distribution curve that detected result and step 3 are obtained compares, if detected result drops in non-ill level, interpret sample supplier does not suffer from tuberculosis; If detected result drops in ill level, interpret sample supplier has suffered from tuberculosis.

Tuberculosis control center of <110> Guangdong Province

<120> microRNA is as the application of tuberculosis marker

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Claims

1. a diagnostic kit lungy, it comprises the specific PCR primer that detects one or more microRNAs, it is characterized in that, described microRNA comprises hsa-miR-92a, hsa-miR-122, hsa-let-7a, hsa-miR-486-5p, hsa-let-7b, hsa-miR-451, hsa-miR-320b, hsa-miR-320a, hsa-miR-192 and hsa-miR-185.

2. diagnostic kit according to claim 1, is characterized in that:

The primer that detects hsa-miR-92a is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGGC（SEQ ID NO:1）

realtime-F：GCGCGCTATTGCACTTGTCCCG（SEQ ID NO:2）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:3）

The primer that detects hsa-miR-122 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAAACA（SEQ ID NO:4）

realtime-F：GCGCGCTGGAGTGTGACAATGG（SEQ ID NO:5）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:6）

The primer that detects hsa-let-7a is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT（SEQ ID NO:7）

realtime-F：GCGCGCTGAGGTAGTAGGTTGT（SEQ ID NO:8）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:9）

The primer that detects hsa-miR-486-5p is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTCGGG（SEQ ID NO:10）

realtime-F：GCGCTCCTGTACTGAGCTGC（SEQ ID NO:11）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:12）

The primer that detects hsa-let-7b is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACCAC（SEQ ID NO:13）

realtime-F：GCGCGCTGAGGTAGTAGGTTGT（SEQ ID NO:14）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:15）

The primer that detects hsa-miR-451 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTCA（SEQ ID NO:16）

realtime-F：GCGCGCGCAAACCGTTACCATTAC（SEQ ID NO:17）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:18）

The primer that detects hsa-miR-320b is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTGCCC（SEQ ID NO:19）

realtime-F：GCGCGCAAAAGCTGGGTTGAGA（SEQ ID NO:20）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:21）

The primer that detects hsa-miR-320a is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCGCCC（SEQ ID NO:22）

realtime-F：GCGCGCAAAAGCTGGGTTGAGA（SEQ ID NO:23）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:24）

The primer that detects hsa-miR-192 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCTGT（SEQ ID NO:25）

realtime-F：GCGCGCGCCTGACCTATGAATTG（SEQ ID NO:26）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:27）

The primer that detects hsa-miR-185 is:

RT-PCR primer:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGGA（SEQ ID NO:28）

realtime-F：GCGCGCTGGAGAGAAAGGCAGT（SEQ ID NO:29）

realtime-R：GTGCAGGGTCCGAGGT（SEQ ID NO:30）。