CN101182576A - Tiny RNA detecting probe used for stomach organization and detection method thereof - Google Patents
Tiny RNA detecting probe used for stomach organization and detection method thereof Download PDFInfo
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Abstract
The invention discloses a tiny RNA probe which is used for stomach tissue and a detection method hereof; the sequence of the probe consists of an oligonucleotide coding segment and an extended arm segment which are chemically modified; the oligonucleotide coding segment comprises the following tiny RNAs of hsa-miR-186, hsa-miR-20b, hsa-miR-18a, hsa-miR-181d, hsa-miR-424, hsa-miR-18b, hsa-miR-17, hsa-miR-20a, hsa-miR-106a, hsa-miR-374a, hsa-miR-224, hsa-miR-181, hsa-miR-25, hsa-miR-99a, hsa-miR-148b, hsa-miR-421, hsa-miR-199a-3p, hsa-miR-214, hsa-miR-92a and hsa-miR-574-5p; crossbreeding detection of the tiny RNA of the stomach tissue is implemented and the high-expression tiny RNA when the stomach tissue is cancerized can be detected; in this way, the high-expression tiny RNA is taken as a diagnosis mark of the cancerization of the stomach tissue; the tiny RNAs can be further designed to be the target points which orient the stomach cancer drug; therefore, the invention provides beneficial basis for judging stomach cancer and orienting the treatment.
Description
Technical field
The present invention relates to Microrna, be specifically related to a kind of Microrna probe and detection method thereof that is used to detect stomach-tissue.
Background technology
Microrna is that a class length is 19～24nt, and the strand microRNA of the not coded protein of relatively guarding on evolving is the newfound in recent years generegulation factor; In the animal and plant cell, Microrna is by bringing into play important regulatory role to the cutting of target mRNA or the inhibition of translation.
Microrna plays a part very important in the normal development of organism, found to have the Microrna about 250 in the human genome, some Microrna has tangible unconventionality expression in the unusual variation of different tissues cells physiological, tumour at different tissues takes place with the expression and the dysfunction of some Microrna confidential relation is arranged specifically, nearly 50% the Microrna that obtains explaining is positioned as the fragile site relevant with tumour on genome, therefore expression and the parafunctional research to concrete Microrna in the dissimilar tumor tissues is subject to people's attention.
As Calin equal reported first in 2002 in the chronic bone-marrow-derived lymphocyte leukemia disappearance of common 13q14 can cause not expressing of 2 kinds of Microrna genes (miR-15a and miR-16a), the prompting Microrna has and suppresses effect (the Proc Natl Acad Sci USA 2002 that leukemia takes place; 99:15524).Subsequently, by the analyses to 28 kinds of microrna expressions spectrum in the colorectal carcinoma, the down-regulated expression of discovery miR-143 such as Michael and miR-145 might promote generation (the Mol Cancer Res 2003 of tumour; 1:882).Further enlarge the disease kind number of observing,, it is found that Microrna (the Proc Natl Acad Sci USA 2006 of at least 21 kinds of unconventionality expressions in kinds of tumors by the difference of research tumour cell and Normocellular microrna expression spectrum; 103:2257).
China is the cancer of the stomach district occurred frequently, and the canceration that does not all relate to stomach-tissue in the existing technology is relevant with the Microrna of which high expression level.
Summary of the invention
Technical problem to be solved by this invention provides a kind of Microrna probe that is used to detect stomach-tissue Microrna high expression level when unusual, and the method for the microrna expression abnormalities that utilizes this probe in detecting stomach-tissue also is provided.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of Microrna probe that is used for stomach-tissue, the sequence of probe is made up of the oligonucleotide encode fragment and the extension arm fragment of chemically modified, and described oligonucleotide encode fragment comprises following small RNAs: hsa-miR-186
*, hsa-miR-20b, hsa-miR-18a, hsa-miR-181d, hsa-miR-424, hsa-miR-18b, hsa-miR-17, hsa-miR-20a, hsa-miR-106a, hsa-miR-374a, hsa-miR-224, hsa-miR-181b, hsa-miR-25, hsa-miR-99a, hsa-miR-148b, hsa-miR-421, hsa-miR-199a-3p, hsa-miR-214, hsa-miR-92a and hsa-miR-574-5p.
Described oligonucleotide encode fragment also comprises following small RNAs: hsa-miR-93, hsa-miR-92b, hsa-miR-100, hsa-miR-638, hsa-miR-106b, hsa-miR-923, hsa-miR-125a-5p, hsa-miR-23a, hsa-miR-199a-5p, hsa-miR-663, hsa-miR-99b, hsa-miR-125b, hsa-miR-23b, hsa-miR-181a and hsa-miR-10b.
Described oligonucleotide encode fragment also comprises following small RNAs: hsa-miR-21, hsa-miR-27a, hsa-miR-183, hsa-miR-483-5p, hsa-miR-361-5p, hsa-miR-19b, hsa-let-7i, hsa-miR-149
*, hsa-miR-191, hsa-miR-146b-5p, hsa-miR-27b, hsa-miR-451, hsa-miR-24, hsa-miR-151-5p and hsa-miR-26a.
A kind of method that detects the Microrna of stomach-tissue, comprise that successively total RNA extraction, total RNA mass analysis, little centrifuging obtain little RNA sample, add Poly (A) polysaccharase and with Cy3 and the little RNA sample of Cy5 specificity fluorescent mark, with above-mentioned probe fluorescently-labeled little RNA sample hybridized detection.
The oligonucleotide encode fragment of chemically modified can be hybridized with the target Microrna is complementary; The extension arm fragment can make the encode fragment that is connected with it from the sheet base certain distance be arranged, and reduces the hybridization steric restriction; The Tm value of probe hybridization is to come equilibrated by chemically modified.
Probe is to synthesize micro-fluid chip by the photosensitive reagents original position.
Above-mentioned Microrna can both find in the rna gene database, and the extension arm fragment can be with reducing any extension arm fragment of hybridizing steric restriction on the molecular biology.
Compared with prior art, the invention has the advantages that the Microrna probe that is used for stomach-tissue, the sequence of probe is made up of the oligonucleotide encode fragment and the extension arm fragment of chemically modified, the oligonucleotide encode fragment comprises above-mentioned Microrna, probe can be hybridized detection to the Microrna of stomach-tissue, the Microrna of high expression level in the time of detecting stomach-tissue generation canceration, the amount of every kind of Microrna forming at the stomach-tissue middle probe of canceration is just high than the amount of every kind of Microrna of normal stomach-tissue, the unconventionality expression of Microrna when the Microrna probe can detect stomach-tissue generation canceration, thereby can be used as a kind of diagnostic markers of stomach-tissue generation canceration, Microrna further can be used as the target spot of the directed cancer of the stomach medicine of design, so the present invention provides favourable foundation for the diagnosis and the targeted therapy of cancer of the stomach.
Description of drawings
Fig. 1 is the probe in detecting photo of stomach organization Microrna;
Fig. 2 organizes Microrna probe in detecting photo for cancer of the stomach is other;
Fig. 3 is that the other Microrna probe in detecting diversity ratio of organizing of stomach organization and cancer of the stomach is than photo.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
A kind of Microrna probe that is used for stomach-tissue, the sequence of probe is made up of the oligonucleotide encode fragment and the extension arm fragment of chemically modified, and the oligonucleotide encode fragment comprises following small RNAs: hsa-miR-186
*Hsa-miR-20b, hsa-miR-18a, hsa-miR-181d, hsa-miR-424, hsa-miR-18b, hsa-miR-17, hsa-miR-20a, hsa-miR-106a, hsa-miR-374a, hsa-miR-224, hsa-miR-181b, hsa-miR-25, hsa-miR-99a, hsa-miR-148b, hsa-miR-421, hsa-miR-199a-3p, hsa-miR-214 and hsa-miR-92a, hsa-miR-574-5p, the oligonucleotide encode fragment can with the complementation of target Microrna, the extension arm fragment can make the encode fragment that is connected with it from the sheet base certain distance be arranged, reduce the hybridization steric restriction, the Tm value of probe hybridization is to come equilibrated by chemically modified, and probe is to synthesize micro-fluid chip by the photosensitive reagents original position.
Embodiment 2
A kind of Microrna probe that is used for stomach-tissue, substantially the same manner as Example 1, different just oligonucleotide encode fragments also comprise following small RNAs: hsa-miR-93, hsa-miR-92b, hsa-miR-100, hsa-miR-638, hsa-miR-106b, hsa-miR-923, hsa-miR-125a-5p, hsa-miR-23a, hsa-miR-199a-5p, hsa-miR-663, hsa-miR-99b, hsa-miR-125b, hsa-miR-23b, hsa-miR-181a and hsa-miR-10b.
Embodiment 3
A kind of Microrna probe that is used for stomach-tissue, substantially the same manner as Example 1, different just oligonucleotide encode fragments also comprise following small RNAs: hsa-miR-21, hsa-miR-27a, hsa-miR-183, hsa-miR-483-5p, hsa-miR-361-5p, hsa-miR-19b, hsa-let-7i, hsa-miR-149
*, hsa-miR-191, hsa-miR-146b-5p, hsa-miR-27b, hsa-miR-451, hsa-miR-24, hsa-miR-151-5p and hsa-miR-26a.
Embodiment 4
A kind of Microrna probe that is used for stomach-tissue, substantially the same manner as Example 2, different just oligonucleotide encode fragments also comprise following small RNAs: hsa-miR-21, hsa-miR-27a, hsa-miR-183, hsa-miR-483-5p, hsa-miR-361-5p, hsa-miR-19b, hsa-let-7i, hsa-miR-149
*, hsa-miR-191, hsa-miR-146b-5p, hsa-miR-27b, hsa-miR-451, hsa-miR-24, hsa-miR-151-5p and hsa-miR-26a.
Embodiment 5
A kind of method that detects the Microrna of stomach-tissue, comprise that successively total RNA extracts: take by weighing the other tissue of 100mg stomach organization and cancer of the stomach separately, join respectively be ground in the liquid nitrogen dry powder → add 1ml Trizol → continue be ground to homogenate → change in the 1.5ml centrifuge tube → centrifugal 10min of 12000 * g → collection supernatant to new centrifuge tube → Virahol →-20 a ℃ precipitation that room temperature is placed 5min → the addings 0.2ml chloroform → vortex 15-30s → centrifugal 15min of room temperature placement 3min → 12000 * g → collection supernatant → add twice supernatant volume spends the night → the centrifugal 15min of 12000 * g, go supernatant → add 1mL to contain the liquid to the greatest extent of DEPC water 75% ethanol (precooling) → centrifugal 5min of 7500 * g →, slightly centrifugal, remove pipe end raffinate → seasoning 3min → precipitation and heavily be dissolved in 50 μ l DEPCH
2O obtains total RNA separately; Total RNA mass analysis: detect by A230, A260, A280, and total RNA is separately adopted agarose gel electrophoresis, according to the total RNA quality of the ratio Analysis of 18S rRNA and 28S rRNA; Little centrifuging obtains little RNA sample: separately total RNA is filtered with little centrifuging post, obtain fragment respectively and organize little RNA sample less than the little RNA sample of stomach organization of 300nt and cancer of the stomach are other; Add Poly (A) polysaccharase in the little RNA sample respectively at little RNA sample of stomach organization and other the organizing of cancer of the stomach: make little RNA 3 ' end add poly (A) tail; The little RNA sample of mark stomach organization and cancer of the stomach are other respectively organizes little RNA sample with Cy3 and Cy5 specificity fluorescent mark: be connected with this poly (A) tail with an oligonucleotide earlier, add Cy3 and Cy5 specificity fluorescent mark then; With the probe of embodiment 4 respectively to the little RNA sample of fluorescently-labeled stomach organization with cancer of the stomach is other organizes little RNA sample to hybridize detection: separately little RNA sample is added to (0.90M NaCl, 60 mM Na in 100 μ l, the 6 * SSPE damping fluid that contains 25% methane amide
2HPO
4, 6 mM EDTA pH6.8), place then on the probe synthetic micro-fluid chip with embodiment 4 and hybridize detection, and the hybridization instrument is microcirculation pump hybridization instrument, and hybridization temperature is 34 ℃, and hybridization time was to gather the hybridization image after the night; Hybridization image laser scanner (GenePix 4000B, Molecular Device) gathers, obtain photo respectively as accompanying drawing 1 and accompanying drawing 2, two kinds of samples hybridization combination image diversity ratios are obtained photo as accompanying drawing 3, software is handled and analyze with Array-Pro (Media Cybernetics) to the hybridization view data, data processing and analysis at first are background corrections, calculating repeats a mean value and standard deviation, filter by LOWESS (Locally-WeightedRegression) then and carry out stdn, data processed result adopts Hierarchical clustering, K-means/mediansclustering or ANOVA methods such as (analysis of variance) obtains the result as table 1; From the amount of some Microrna of the stomach organization more as can be seen of accompanying drawing 1, accompanying drawing 2 and accompanying drawing 3 amount height than the Microrna of the other tissue of cancer of the stomach, the amount of Microrna that further illustrates stomach organization from table 1 data processed result is more much higher than the amount of the Microrna of the other tissue of cancer of the stomach, therefore the unusual raising from the amount of some Microrna of stomach-tissue just can draw the result of stomach-tissue generation canceration, the present invention just provides foundation for the diagnosis of cancer of the stomach, further can select for use these Micrornas as cancer of the stomach is carried out directed target treatment.
Table 1: the amount of the Microrna of the other tissue of stomach organization and cancer of the stomach and logarithmic value relatively mutually thereof
| Sequence number | The Microrna title | Stomach organization | Cancer beside organism | Log2 (cancer beside organism/cancerous tissue) |
| 1 | hsa-miR-186 * | 325.63 | 11.53 | -4.98 |
| 2 | hsa-miR-20b | 3,785.63 | 385.88 | -3.26 |
| 3 | hsa-miR-18a | 588.77 | 75.08 | -2.94 |
| 4 | hsa-miR-181d | 166.34 | 23.04 | -2.79 |
| 5 | hsa-miR-424 | 532.70 | 85.76 | -2.67 |
| 6 | hsa-miR-18b | 230.13 | 34.30 | -2.66 |
| 7 | hsa-miR-17 | 7,571.93 | 1,234.02 | -2.62 |
| 8 | hsa-miR-20a | 9,394.27 | 1,527.90 | -2.61 |
| 9 | hsa-miR-106a | 7,221.83 | 1,199.42 | -2.59 |
| 10 | hsa-miR-374a | 224.36 | 40.09 | -2.54 |
| 11 | hsa-miR-224 | 287.36 | 62.40 | -2.35 |
| 12 | hsa-miR-181b | 403.18 | 79.85 | -2.34 |
| 13 | hsa-miR-25 | 8,164.81 | 2,213.61 | -1.99 |
| 14 | hsa-miR-99a | 604.04 | 160.83 | -1.89 |
| 15 | hsa-miR-148b | 504.24 | 163.30 | -1.85 |
| 16 | hsa-miR-421 | 220.35 | 67.15 | -1.74 |
| 17 | hsa-miR-199a-3p | 14,961.85 | 4,512.30 | -1.71 |
| 18 | hsa-miR-214 | 3,639.84 | 1,129.12 | -1.68 |
| 19 | hsa-miR-92a | 13,074.26 | 4,185.65 | -1.66 |
| 20 | hsa-miR-574-5p | 11,773.16 | 4,002.98 | -1.58 |
| 21 | hsa-miR-93 | 1,298.26 | 503.74 | -1.37 |
| 22 | hsa-miR-92b | 4,078.95 | 1,592.77 | -1.35 |
| 23 | hsa-miR-100 | 1,021.65 | 416.46 | -1.34 |
| 24 | hsa-miR-638 | 11,102.60 | 4,475.45 | -1.30 |
| 25 | hsa-miR-106b | 1,746.08 | 672.32 | -1.29 |
| 26 | hsa-miR-923 | 13,772.99 | 5,487.30 | -1.28 |
| 27 | hsa-miR-125a-5p | 4,578.20 | 1,910.78 | -1.25 |
| 28 | hsa-miR-23a | 28,925.70 | 12,304.60 | -1.23 |
| 29 | hsa-miR-199a-5p | 681.46 | 302.77 | -1.17 |
| 30 | hsa-miR-663 | 796.69 | 374.16 | -1.14 |
| 31 | hsa-miR-99b | 725.56 | 347.17 | -1.06 |
| 32 | hsa-miR-125b | 9,713.41 | 4,614.08 | -1.06 |
| 33 | hsa-miR-23b | 30,664.58 | 14,728.74 | -1.05 |
| 34 | hsa-miR-181a | 1,240.15 | 601.31 | -1.04 |
| 35 | hsa-miR-10b | 3,057.67 | 1,534.59 | -1.01 |
| 36 | hsa-miR-21 | 64,263.30 | 35,000.06 | -0.88 |
| 37 | hsa-miR-27a | 7,689.06 | 4,562.03 | -0.78 |
| 38 | hsa-miR-183 | 862.78 | 500.38 | -0.77 |
| 39 | hsa-miR-483-5p | 4,059.44 | 2,274.58 | -0.76 |
| 40 | hsa-miR-361-5p | 2,207.46 | 1,382.70 | -0.65 |
| 41 | hsa-miR-19b | 1,248.32 | 774.11 | -0.65 |
| 42 | hsa-let-7i | 16,004.29 | 10,686.76 | -0.58 |
| 43 | hsa-miR-149 * | 1,892.51 | 1,202.82 | -0.58 |
| 44 | hsa-miR-191 | 6,221.49 | 4,296.31 | -0.56 |
| 45 | hsa-miR-146b-5p | 4,271.78 | 3,243.45 | -0.38 |
| 46 | hsa-miR-27b | 9,019.00 | 7,129.02 | -0.37 |
| 47 | hsa-miR-451 | 4,361.53 | 3,413.23 | -0.37 |
| 48 | hsa-miR-24 | 5,693.80 | 4,512.81 | -0.32 |
| 49 | hsa-miR-151-5p | 3,652.71 | 2,938.51 | -0.32 |
| 50 | hsa-miR-26a | 28,738.43 | 26,619.49 | -0.11 |
Claims (4)
1. Microrna probe that is used for stomach-tissue, the sequence of probe is made up of the oligonucleotide encode fragment and the extension arm fragment of chemically modified, it is characterized in that described oligonucleotide encode fragment comprises following small RNAs: hsa-miR-186
*, hsa-miR-20b, hsa-miR-18a, hsa-miR-181d, hsa-miR-424, hsa-miR-18b, hsa-miR-17, hsa-miR-20a, hsa-miR-106a, hsa-miR-374a, hsa-miR-224, hsa-miR-181b, hsa-miR-25, hsa-miR-99a, hsa-miR-148b, hsa-miR-421, hsa-miR-199a-3p, hsa-miR-214, hsa-miR-92a and hsa-miR-574-5p.
2. a kind of Microrna probe that is used for stomach-tissue as claimed in claim 1 is characterized in that described oligonucleotide encode fragment also comprises following small RNAs: hsa-miR-93, hsa-miR-92b, hsa-miR-100, hsa-miR-638, hsa-miR-106b, hsa-miR-923, hsa-miR-125a-5p, hsa-miR-23a, hsa-miR-199a-5p, hsa-miR-663, hsa-miR-99b, hsa-miR-125b, hsa-miR-23b, hsa-miR-181a and hsa-miR-10b.
3. a kind of Microrna probe that is used for stomach-tissue as claimed in claim 1 or 2 is characterized in that described oligonucleotide encode fragment also comprises following small RNAs: hsa-miR-21, hsa-miR-27a, hsa-miR-183, hsa-miR-483-5p, hsa-miR-361-5p, hsa-miR-19b, hsa-let-7i, hsa-miR-149
*, hsa-miR-191, hsa-miR-146b-5p, hsa-miR-27b, hsa-miR-451, hsa-miR-24, hsa-miR-151-5p and hsa-miR-26a.
4. method of utilizing the Microrna of claim 1 or 2 or 3 described probe in detecting stomach-tissues, comprise successively that total RNA extracts, total RNA mass analysis, little centrifuging obtain little RNA sample, add Poly (A) polysaccharase and with Cy3 and the little RNA sample of Cy5 specificity fluorescent mark step, it is characterized in that with described probe fluorescently-labeled little RNA sample being hybridized detection.
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