CN106222270A - A kind of breast carcinoma occurs or the diagnostic kit of transfer - Google Patents
A kind of breast carcinoma occurs or the diagnostic kit of transfer Download PDFInfo
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- CN106222270A CN106222270A CN201610626354.6A CN201610626354A CN106222270A CN 106222270 A CN106222270 A CN 106222270A CN 201610626354 A CN201610626354 A CN 201610626354A CN 106222270 A CN106222270 A CN 106222270A
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Abstract
The invention belongs to biotechnology and medicine technology field, it is provided that a kind of miR 182, the let 7c application in the diagnostic kit of preparation breast carcinoma generation or transfer, test kit contains the quantitative RT PCR primer of miR 182.The test kit that the present invention provides, it is provided that a kind of serum microRNA newly extracts reagent, by detection miR 182, the expression of let 7c, can carry out early diagnosis to breast carcinoma, provides new thinking for the diagnosis of tumor.
Description
Technical field
The invention belongs to molecular biology and gene diagnosis field, be specifically related to serum let-7c and miR-182 to breast
Adenocarcinoma occurs or the application of transfer diagnosis.
Background technology
Breast carcinoma is modal familial neoplasms, is one of reason of causing women all over the world compared with high mortality.
In China, the patient dying from breast carcinoma increases year by year.Breast carcinoma usually originates with milk duct or lobule mammary gland tissue, accounts for all female
The 22.9% of property cancer (not including nonmelanoma skin cancer).But, the danger of patient with breast cancer's morbidity of about 50%-80%
Factor is indefinite, therefore finds risk factor or the diagnostic marker of effective pathogenesis of breast carcinoma, is beneficial to breast cancer diagnosis
And treatment.
Microrna (microRNA, miRNA) is small molecule RNA, and length is about the non-coding RNA of 22bp nucleotide.
MiRNA, by the expression at post-transcriptional level controlling gene, participates in different cell processes, including apoptosis, hematopoietic cell
The process such as differentiation, substance metabolism, neural growth and cell migration.Many miRNA take part in mankind's many tumors
There is evolution.Up to the present, there is many research reports miRNA in breast carcinoma cell strain and primary breast cancer tissue
Expression.Such as express miR-191/425 gene cluster at breast cancer cell camber, the gene table of breast cancer cell can be changed
Reach spectrum, participate in the invasiveness of breast carcinoma, affect the progress of breast carcinoma.The expression of the breast cancer tissue miR-21 by analyzing,
Highly express patient's prognosis survival rate patient with breast cancer significantly lower than low expression miR-21 of miR-21, show that miR-21 participates in
The morbidity progression of breast carcinoma；Process LAN miR-21 can also strengthen the growth of breast cancer cell MCF-7 cell, migrate and invade
Attack ability, increase self renewal and clonality.Process LAN miR-200a can stop breast cancer cell generation apoptosis, and promotees
Entering Nasopharyngeal neoplasms, suppression miR-200a expresses and can induce breast cancer cell generation apoptosis simultaneously.Reduce the table of miR-200f
Reaching the expression that can increase EMT correlation factor, miR-200f can be as the biomarker of breast carcinoma EMT process.Swell as one
Tumor suppressor gene, the signal that LET-7 is alpha mediated by regulating estrogen receptor (ER), can suppress breast carcinoma stem cell positive for ER
Growth and propagation.These researchs show, miRNA take part in the generation of breast carcinoma, migrates and the process such as infiltration.
The miRNAs that it is internal can be drained into blood circulation by apoptosis and downright bad primary tumo(u)r, and this kind of miRNA is referred to as
Circulation miRNA.Therefore, the miRNA in blood is from the cell (including tumor cell) of many tissues, and this makes us to analyze
MiRNA in blood, analyzes Different Organs, tissue or the feature of cell.Live additionally, the miRNA of circulation also has anti-RNase
Property, therefore highly stable in serum and blood plasma.Owing to circulation miRNA stablizes in individual human serum so that serum miRNA conduct
Attractive biomarker, its serum levels can be used to assisted diagnosis breast carcinoma.To so far, relevant circulation miRNA
Expression study at Peripheral Blood In Patients With Breast Cancer is reported few.In order to find the biomarker of blood serum of patients with human breast carcinoma miRNA,
We have inquired into serum miR-182 level value in patient with breast cancer diagnoses.
Before making the present invention, the relation between miRNA and human breast cancer is widely studied.The expression change of miRNA
Participate in generation and the progression of breast carcinoma.Study portion miRNA is at patient with breast cancer and normal healthy controls serum or blood plasma
Express, point out its value in breast cancer diagnosis.But the method for detection miRNA is complicated, the test kit that institute needs is expensive.
It is contemplated that found a kind of new serum miRNA diagnostic method, the diagnosis examination of design specific diagnosis miR-182, let-7c
Agent, it was demonstrated that they value in breast cancer diagnosis, for the thinking that the diagnosis offer of tumor is new.
Summary of the invention
The invention provides a kind of miR-182, let-7c to occur or in the diagnostic kit of transfer in preparation breast carcinoma
Application, also provides for a kind of breast carcinoma and occurs or transfering reagent box, and test kit contains the quantitation RT-PCR primer of miR-182, described
Primer is respectively as follows:
Forward primer sequence: GGCAATGGTAGAACTCACAC；Downstream primer sequence:
ACATGTACAGTCCATGGATG.Test kit contains the quantitation RT-PCR primer of let-7c, and described primer is respectively as follows:
Forward primer sequence: GTTGAGGTAGTAGGTTGTATGG；Downstream primer sequence: AACATGTACAGTCCATGGATG.The present invention
The test kit provided, by detecting the expression of miR-182, let-7c, can carry out early diagnosis, examining for tumor to breast carcinoma
The thinking that disconnected offer is new.The serum microRNA extracting method that the present invention has also provided for a kind of newly.
The present invention discloses a kind of breast carcinoma and occurs or the diagnostic kit of transfer, and test kit contains miR-182, let-7c
Specific diagnosis reagent.
Described test kit contains the quantitation RT-PCR primer of miR-182, and primer is respectively as follows:
Forward primer sequence: GGCAATGGTAGAACTCACAC
Downstream primer sequence: ACATGTACAGTCCATGGATG
Described test kit contains the quantitation RT-PCR primer of let-7c, and described primer is respectively as follows:
Forward primer sequence: GTTGAGGTAGTAGGTTGTATGG
Downstream primer sequence: AACATGTACAGTCCATGGATG
The expression of quantitative RT-PCR detection serum miR-182, let-7c is carried out in test kit.
Described quantitative RT-PCR adds serum microRNA when detecting the expression of serum miR-182, let-7c.
Described serum microRNA is extracted by following steps and detects:
(1) the conventional preparation of serum
Patient with breast cancer's blood and comparison blood are contained in centrifuge tube, are statically placed in 37 DEG C of environment solidification, treat blood coagulation
After, centrifugal after being balanced, the supernatant obtained is serum, sucts clear, and subpackage is standby；
(2) the extraction of serum microRNA
Take 450-550 μ L serum in 1.5mL EP pipe, add equal-volume phenol mixing 2min, stand on ice rapidly
2min, 13000rpm are centrifuged 3min.Collect supernatant to manage to new EP, add and the isopyknic phenol of supernatant, repeat this operation two
Secondary.Collect supernatant manage in new EP, add with the isopyknic chloroform isoamyl alcohol of supernatant (24:1), concussion mixing 2min, rapidly in
Stand 1min on ice, centrifugal 13000rpm, 3min, repeat this operation once.Suct clear layer in new EP pipe, add 1/10 body
Long-pending 3mol/L, pH=5.2 sodium acetate, equal-volume 100% ice ethanol, reverse mixing, place more than 20min, by this mixing for-20 DEG C
Liquid 13000rpm, centrifugal 3min, remove supernatant, add 750 μ L 0.1% DEPC.By above-mentioned mixed liquor 13000rpm, centrifugal
3min, removes supernatant, and appropriateness is drying precipitated, and the DEPC with the 0.1% of 15 μ L dissolves RNA precipitate, it is thus achieved that RNA sample；
(3) the detection of serum microRNA
Take the 2 above-mentioned RNA sample of μ L, add 98 μ L ultra-pure waters and be diluted to 100 μ L, mixing.Return to zero with ultra-pure water, diluted
RNA moves into cuvette, measures at ultraviolet spectrophotometer, according to OD260/OD280 ratio in judgement RNA mass, calculates RNA dense
Degree.Take 2 μ LRNA samples, add 1 μ L 6 × loading buffer mixing, the agarose gel electrophoresis detection of 1%, 80v electrophoresis
10min。
Described test kit also includes that quantitative RT-PCR reacts conventional enzyme and reagent.
Accompanying drawing explanation
Fig. 1: serum microRNA electrophoretogram；
Fig. 2: patient with breast cancer and the detection of expression of control serum miR-182；
Fig. 3: ROC curve figure.
With detailed description of the invention, technical scheme is further described in detail below in conjunction with the accompanying drawings.
Detailed description of the invention
Below as a example by serum miR-182 detects, it was demonstrated that its value in breast cancer diagnosis, make into one for the present invention
The elaboration of step.These examples are aimed at description of the present invention rather than limitation of the present invention.
In the present invention, in the above and below example, the technology used, prepare including serum, the carrying of microRNA
Take, PCR expands and detection equimolecular biology techniques, and statistical analysis etc., unless stated otherwise, the skill being in this area
Routine techniques known to art personnel；The instrument and equipment that used, reagent, software etc., only this specification is the most dated, is
The research of general this area and technical staff can be obtained by public approach.
The conventional preparation of embodiment 1 serum
Obtain patient and the non-anticoagulated blood 2mL of comparison, be contained in the vessel that centrifuge tube maybe can be centrifuged, stand or put 37
DEG C environment promotees its solidification, after blood coagulation, centrifugal after balance (generally 3000rpm is centrifuged 5-10min), obtain
Supernatant be serum, can be carefully by supernatant sucking-off (sucking-off cell component be sure not in attention), subpackage is standby.
The extraction of embodiment 2 serum microRNA and detection
1, take 450-550uL serum in 1.5mL EP pipe, add equal-volume reagent A mixing 2min, rapidly in the most quiet
Put 2min, centrifugal 13000rpm, 3min.
2, collect supernatant to manage to new EP, add and supernatant equal-volume reagent A, repeat step 1 two times.
3, collecting supernatant to manage in new EP, add and supernatant equal-volume reagent B, concussion mixing 2min, rapidly in the most quiet
Put 1min, centrifugal 13000rpm, 3min, repeat step 2 once.
4, suct clear layer in new EP pipe, add 1/10 volume reagent C, equal-volume reagent D, reverse mixing, put for-20 DEG C
Put more than 20min.
5, centrifugal 13000rpm, 3min, remove supernatant, add the reagent E of 750 μ L.
6, centrifugal 13000rpm, 3min, removes supernatant, and appropriateness is drying precipitated.
7, RNA precipitate is dissolved with the reagent F of 15 μ L.
8, RNA quality testing is extracted: take 2 μ L RNA sample, add 98 μ L ultra-pure waters and be diluted to 100 μ L, mixing.Zeroing is with super
Pure water (blank group), the RNA diluted moves into cuvette, measures at ultraviolet spectrophotometer UD-640, according to OD260/OD280
Ratio in judgement RNA mass (should be about 2.0), calculates RNA concentration.Take RNA sample 2 μ L, add 1 μ L 6 × loading
Buffer mixes, the agarose gel electrophoresis of 1% detection, electrophoresis 80v, 10min, find the RNA extracted be mainly microRNA (see
Fig. 1).
Reagent A: phenol
Reagent B: chloroform isoamyl alcohol (24:1)
Reagent C: 3mol/L, pH=5.2 sodium acetate
Reagent D: 100% ice ethanol
Reagent E: 75% ice ethanol
The DEPC (volume/volume) of reagent F:0.1%
The expression of embodiment 3 quantitative RT-PCR detection serum miR-182, let-7c
1, miRNAs add end reaction, with Poly A Ploymerase (Ambion company) tailing, concrete reaction system is such as
Under (25 μ L):
37 DEG C, 1h；65 DEG C, 10min.Product is in-20 DEG C of preservations.
2, reverse transcription reaction (reverse transcription, RT), concrete reaction system is as follows:
70 DEG C, 5min；2min on ice, is rapidly added afterwards.
5 × RT buffer:4.0 μ L
DNTPmix:4.0 μ L
Reverse Transcriptase:0.9 μ L
(Promega company)
42 DEG C of reactions 1h, the cDNA of synthesis and-20 DEG C of preservations.
3, PCR reaction: standard substance 5SrRNA is that this research department stores, and standard substance aquesterilisa does 10 times of gradient dilutions, 4
Individual gradient, concentration be respectively 1 × 109copies/ μ L, 1 × 108copies/ μ L, 1 × 107copies/ μ L, 1 ×
105copies/ μ L, with 5S rRNA as internal reference.The primer sequence of quantitative PCR is shown in Table 1 as follows:
PCR reaction system (Takara company) is following (20 μ L):
Reaction condition: 95 DEG C of degeneration 5min；95 DEG C of degeneration 20s；52 DEG C, 20s, 72 DEG C, 30s.Repeat 40 circulations, in 72
DEG C detection fluorescence, 5S rRNA is as internal reference.Draw solubility curve for 70 DEG C to 95 DEG C.
Embodiment 4 miR-182 value in breast cancer diagnosis
1, the basic condition of object of study
Object of study comes from Yantai, Shandong Province Laiyang central hospital, total new breast cancer patient 46 example and corresponding made a definite diagnosis
Comparison crowd 58 example, all patients all make a definite diagnosis through pathological diagnosis.Patient with breast cancer and the basic condition of matched group and clinic are special
Levy and be shown in Table 2.Case group and matched group are in terms of age, height and body weight, and through statistical test not statistically significant, two groups without bright
Significant difference is different, and equilibrium comparability is preferable.
2, the expression of serum miR-182
We, by miR-182 level in real-time quantitative PCR detection serum, study miR-182 diagnosis in breast carcinoma
Effect.It was found that the level of the miR-182 in blood serum of patients with human breast carcinoma is respectively 7.07 × 103 copy/ml (n=46),
The significant serum miR-182 level higher than normal healthy controls person (0.003 × 103 copy/ml, P < 0.01, n=58, see Fig. 2).Grind
Studying carefully result to show, in serum, the content of patient with breast cancer miR-182 is higher, shows that miR-182 is in the pathogenesis of breast carcinoma
Play a significant role.
For inquiring into miR-182 value in patient with breast cancer diagnoses, with ROC curve, we determine that miR-182 is distinguishing
Patients with lung cancer is 0.712 × 103copies/ml with the diagnosis dividing value of normal healthy controls, and sensitivity reaches 95.7%, and specificity reaches
75.9%, area under curve AUC=0.810,95% credibility interval is: 0.717-0.903 (P < 0.001) (Fig. 3).
Embodiment 5 Let-7c value in breast cancer diagnosis
Its value research method in breast cancer diagnosis of Let-7c is shown in example 1,2,3.Concrete detection primer is shown in, finds breast
Serum of adenocarcinoma patients's let-7c level is the lowest with matched group serum levels, it was demonstrated that its value in breast cancer diagnosis.
The primer table for detection of table 1 patent autonomous Design
2, table research detection patient with breast cancer and the basic clinical information of comparison
It should be noted last that, above detailed description of the invention only in order to technical scheme to be described and unrestricted,
Although the present invention being described in detail with reference to preferred embodiment, it will be understood by those within the art that, can be right
Technical scheme is modified or equivalent, and without deviating from the spirit and scope of technical solution of the present invention, it is equal
Should contain in the middle of scope of the presently claimed invention.
Claims (6)
1. a breast carcinoma occurs or the diagnostic kit of transfer, it is characterised in that test kit contains miR-182, let-7c
Specific diagnosis reagent.
A kind of breast carcinoma the most according to claim 1 occurs or the diagnostic kit of transfer, it is characterised in that test kit contains
The quantitation RT-PCR primer of miR-182, described primer is had to be respectively as follows:
Forward primer sequence: GGCAATGGTAGAACTCACAC,
Downstream primer sequence: ACATGTACAGTCCATGGATG.
A kind of breast carcinoma the most according to claim 1 occurs or the diagnostic kit of transfer, it is characterised in that test kit contains
The quantitation RT-PCR primer of let-7c, described primer is had to be respectively as follows:
Forward primer sequence: GTTGAGGTAGTAGGTTGTATGG,
Downstream primer sequence: AACATGTACAGTCCATGGATG.
A kind of breast carcinoma the most according to claim 1 occurs or the diagnostic kit of transfer, it is characterised in that at test kit
In carry out quantitative RT-PCR detection serum miR-182, let-7c expression.
A kind of breast carcinoma the most according to claim 4 occurs or the diagnostic kit of transfer, it is characterised in that described serum
MicroRNA is extracted by following reagent and step and is detected:
(1) the conventional preparation of serum
Patient with breast cancer's blood and comparison blood are contained in centrifuge tube, are statically placed in 37 DEG C of environment solidification, after blood coagulation, will
Being centrifuged after its balance, the supernatant obtained is serum, sucts clear, and subpackage is standby；
(2) extraction of serum microRNA
Take 450-550 μ L serum in 1.5mL EP pipe, add equal-volume phenol mixing 2min, stand rapidly 2min on ice,
13000rpm is centrifuged 3min, collects supernatant and manages to new EP, add and the isopyknic phenol of supernatant, repeat this operation secondary, receives
Collecting supernatant to manage in new EP, add and the isopyknic chloroform isoamyl alcohol of supernatant (24:1), concussion mixing 2min, rapidly in the most quiet
Put 1min, centrifugal 13000rpm, 3min, repeat this operation once；Suct clear layer in new EP pipe, add 1/10 volume
3mol/L, pH=5.2 sodium acetate, equal-volume 100% ice ethanol, reverse mixing, place more than 20min, by this mixed liquor for-20 DEG C
13000rpm, centrifugal 3min, remove supernatant, add 750 μ L 0.1% DEPC；By above-mentioned mixed liquor 13000rpm, centrifugal
3min, removes supernatant, and appropriateness is drying precipitated, and the DEPC with the 0.1% of 15 μ L dissolves RNA precipitate, it is thus achieved that RNA sample；
(3) the detection of serum microRNA
Take the 2 above-mentioned RNA sample of μ L, add 98 μ L ultra-pure waters and be diluted to 100 μ L, mixing；Returning to zero with ultra-pure water, the RNA diluted moves
Enter cuvette, measure at ultraviolet spectrophotometer, according to OD260/OD280 ratio in judgement RNA mass, calculate RNA concentration；Take 2 μ
L RNA sample, adds 1 μ L 6 × loading buffer mixing, the agarose gel electrophoresis detection of 1%, 80v electrophoresis 10min.
A kind of breast carcinoma the most according to claim 1 occurs or the diagnostic kit of transfer, it is characterised in that this test kit
Also include the reagent that quantitative RT-PCR reaction is conventional.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107699619A (en) * | 2017-11-17 | 2018-02-16 | 李宜健 | LncRNA compositions and the purposes for preparing diagnosis indication Luminal B type Bone of Breast Cancer transfering reagent boxes |
| CN108690861A (en) * | 2017-04-10 | 2018-10-23 | 肖晓莺 | MiRNA for the diagnosis of early-stage breast cancer disease |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108690861A (en) * | 2017-04-10 | 2018-10-23 | 肖晓莺 | MiRNA for the diagnosis of early-stage breast cancer disease |
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