CN110205386A - One kind miRNA molecule miR-33a-5p relevant to carcinoma of endometrium and its application - Google Patents
One kind miRNA molecule miR-33a-5p relevant to carcinoma of endometrium and its application Download PDFInfo
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Abstract
The present invention provides a kind of miRNA molecule miR-33a-5p relevant to carcinoma of endometrium and its applications, belong to biological gene therapy field.Expression rate in miRNA molecule miR-33a-5p endometrial tissues is significantly lower than normal endometrial tissue, can be used for preparing in carcinoma of endometrium auxiliary diagnosis or carcinoma of endometrium adjuvant treatment/prognosis evaluation reagent or kit.
Description
Technical field
The invention belongs to molecular biology and oncology, and in particular to a kind of miRNA relevant to carcinoma of endometrium
Molecule miR-33a-5p and its application.
Background technique
Carcinoma of endometrium is one group of epithelial malignancy for betiding endometrium, also known as carcinoma of uterine body, is that women is raw
Grow one of three big common cancers.It mostly occurs in climacteric and postmenopausal women.With the increasing of population average life span
It is subject to and the change of living habit, rises and rejuvenation trend in lasting within the disease incidence of carcinoma of endometrium nearly 20 years.In west state
Family, it is the first that carcinoma of endometrium already takes up female reproductive system Cancer Mortality, in China, as after cervical carcinoma the
Two common gynecologic malignant tumors, account for about the 20%~30% of gynecologic malignant tumor.The carcinoma of endometrium in the flourishing city in part
Disease incidence has reached gynecologic malignant tumor first.Under existing medical technique level, carcinoma of endometrium early detection and trouble
The treatment of the assessment of person's prognosis and advanced stage or recurrent endometrial carcinoma is still a kind of problem.
Microrna (microRNA, miRNA) be the small-sized non-coding of a kind of endogenous be about 18-22 nucleotide at
The RNA of ripe form, adjustable one group of target gene simultaneously lead to mRNA degradation or Translational repression.MiRNAs cancer starting and
It plays an important role in progress, in cell growth and cell differentiation, apoptosis, proliferation, embryonic development and stem cell update etc. are each
Kind bioprocess.MiRNAs is to tumour either cancer suppressing action, can also play tumor promotion, existing research report
Imbalance of expression in miRNA Endometrial Carcinomas prompts to play an important role in the occurrence and development of miRNA Endometrial Carcinomas,
Existing research shows exception table in miR-200a, miR-200b, miR-200c, miR-141 and miR-429 Endometrial Carcinomas
It reaches.But there is no the relevant reports about expression in miR-33a-5p endometrial tissues.
Summary of the invention
In view of the above-mentioned problems, the present invention provides one kind miRNA molecule miR-33a-5p relevant to carcinoma of endometrium and answers
With.MiRNA molecule miR-33a-5p can be used as the diagnostic markers of carcinoma of endometrium, provide molecule for the treatment of carcinoma of endometrium
Target spot can be applied to the preparation of anti-tumor drug for the mimics of its design.
To achieve the above object, the present invention provides a kind of miRNA molecule miR-33a-5p relevant to carcinoma of endometrium,
Its nucleotide sequence is as shown in SEQ ID NO.1.
The miRNA molecule miR-33a-5p Endometrial Carcinomas auxiliary diagnosis or Treatment of Endometrial Neoplasms/prognosis evaluation
In application.
The miRNA molecule miR-33a-5p preparation carcinoma of endometrium auxiliary diagnosis or carcinoma of endometrium adjuvant treatment/
Application in prognosis evaluation reagent or kit.
For detecting the reagent of miRNA molecule miR-33a-5p in preparation carcinoma of endometrium auxiliary diagnosis or carcinoma of endometrium
Application in treatment/prognosis evaluation reagent or kit.
It is described for detect the reagent of miRNA molecule miR-33a-5p to include for expanding the miRNA molecule miR-
The specific primer of 33a-5p, the primer sequence are as follows:
F:5 '-GAC TTA AAC GTG GAT GTA CTT GC-3 ' (SEQ ID NO.2).
For detecting the kit of miRNA molecule miR-33a-5p relevant to carcinoma of endometrium in preparation carcinoma of endometrium
Application in auxiliary detection or auxiliary diagnostic tool.
For detecting the kit of miRNA molecule miR-33a-5p relevant to carcinoma of endometrium in preparation carcinoma of endometrium
Application in treatment/prognosis evaluation tool.
It is a kind of for carcinoma of endometrium auxiliary diagnosis or the kit of prognosis evaluation, including:
(1) for expanding the specific primer of miR-33a-5p;
(2) standard DNA template;
(3) PCR reaction solution.
A method of detection miRNA includes the following steps:
(1) sample total serum IgE is extracted;
(2) sample cdna is prepared;
(3) quantitative amplification miR-33a-5p.
Its mimics for being overexpressed miR-33a-5p is designed by Suzhou GenePharma Co., Ltd..Obtaining its tool
After the sequence of body, and designs mimics sequence and transfected;Endometrial carcinoma cell line is overexpressed using the segment
Ishikawa has found the migration invasive ability decline of tumour cell.Therefore the function of the molecule may be invaded with the migration of cancer cell
Attack ability correlation.
The present invention proposes the miRNA molecule of low expression in carcinoma of endometrium, can be used as carcinoma of endometrium molecular marker
Or target spot, it is applied to the early diagnosis of carcinoma of endometrium clinic, Index for diagnosis or targeted therapy, and be conducive to further elucidate uterus
The occurrence and development mechanism of endometrial carcinomas, signal path etc., great application prospect and theoretical value.
Detailed description of the invention
Fig. 1 is the corresponding solubility curve of miR-33a-5p and diffusion profile figure in real-time-PCR, and wherein Fig. 1-1 is
Corresponding solubility curve, Fig. 1-2 are corresponding amplification curve diagram.
Fig. 2 be real-time-PCR in miR-33a-5p cancer group and non-cancer group relative expression's spirogram.
Fig. 3 is the ROC curve analysis chart of miR-33a-5p in endometrial.
Fig. 4 is that PCR detects mimics miR-33a-5p transfection efficiency figure in Ishikawa cell.
Fig. 5 is to detect miR-33a-5p using scratch experiment method to be overexpressed the influence diagram for migrating endometrial carcinoma cell.
Fig. 6 is to detect miR-33a-5p using transwell experimental method to be overexpressed the shadow for invading endometrial carcinoma cell
Ring figure.
Specific embodiment
Invention is further explained combined with specific embodiments below.
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
Material, reagent etc., are commercially available unless otherwise specified.
As known to those skilled in the art, primer sequence of the invention can be made suitable according to the sequence of Research of predicting markers
When adjustment and modification, these modified primer sequences still can be used for detecting the marker.The present invention also includes these
Equivalent technical solution.
Expression in 1 miR-33a-5p Endometrial Carcinomas of embodiment and normal endometrial tissue.
1. collection of specimens.
Sample collection collection attached Shengjing city hospital, Chinese Medical Sciences University factor endometrial carcinoma, He Gong during 2011 to 2017 years
The normal-sub of III grade of neck intraepithelial neoplasia (cin) (cervicalintraepithelialneoplasia III, CIN III) row operation excision
In utero membrane tissue.Accordingly it is divided into carcinoma of endometrium group (n=80), Normal group (n=40).Patient is preoperative not to receive putting
Treatment and hormone therapy, and definitive pathological diagnosis.Carcinoma of endometrium group operation clinical stages and pathological grading (FIGO 2009): early
Phase: I phase added II phase 60, advanced stage: III phase and the IV phase 20;Histological grade (WHO standard): differentiated carcinoma of endometrium 33
Example, middle differentiation 32, low differentiation carcinoma of endometrium 15;Lymphatic metastasis is 11 positive, no lymphatic metastasis 69;Infiltrate muscle layer
<1/2 totally 44, infiltrate muscle layer>=1/2 totally 36.This research has passed through attached Shengjing city Hospital Ethical Committee, Chinese Medical Sciences University
Approval.
2. design of primers.
According to the sequence of hsa-miR-33a-5p, design primer.Particular sequence is as follows:
Reverse transcription primer sequence SEQ ID the No.2:F:5 '-GAC TTA AAC GTG GAT of hsa-miR-33a-5p
GTA CTT GC-3';
The forward primer sequence SEQ ID No.3:GGAACGATACAGAGAAGATTAGC of U6;
The reverse primer sequences SEQ ID No.4:TGGAACGCTTCACGAATTTGCG of U6.
Above-mentioned primer is synthesized by Shanghai Sheng Gong Biotechnology Co., Ltd, and wherein U6 is internal reference calibrating cdna.
3, miR-33a-5p endometrial tissues, normal endometrium group are detected using Real-time-PCR method
The expression quantity knitted.
3.1, the tissue specimen Total RNAs extraction collected.
(1) by the carcinoma of endometrium sample fetched (1ml trizol lysate has been added), 5min is stood on ice.
(2) 200 μ l chloroforms are added, oscillation is placed at room temperature for 2-3min after mixing.4 DEG C of 12,000g are centrifuged 15min.
(3) upper strata aqueous phase is drawn, until in another centrifuge tube.Note: intermediate interface must not be drawn.
(4) isopropanol that equivalent is added mixes, -20 DEG C of precipitatings.
(5) 4 DEG C of 12,000g are centrifuged 10min, abandon supernatant, and RNA is sunken to tube bottom.
(6) 75% ethyl alcohol of 1ml is added, suspend precipitating.
(7) 4 DEG C of 12000g are centrifuged 5min, as far as possible abandoning supernatant.
(8) 5-10min is dried or be dried in vacuo to room temperature.Note: RNA sample not dried excessively, otherwise be difficult to dissolve.
(9) RNA sample is dissolved with 20 μ l DEPC H2O.
(10) measurement of RNA purity and RNA take 2 μ l RNA solutions in enzyme quantitatively with DEPC H2O to compare (Blank)
It marks and detects concentration of specimens and quality on instrument.
3.2, reverse transcription synthesizes.
(1) reverse transcription reaction.
Firstly, carrying out the reverse transcription initial denaturation of miRNA, initial denaturation reaction solution is configured, configuration method is as shown in table 1.
1 miRNA initial denaturation reaction solution of table
Reagent | Usage amount |
mRQ buffer(2×) | 5.OuL |
RNA sample(0.25-8ug) | 3.751ul |
Mrq enzyme 1.25uL | 1.25uL |
37 DEG C of 1h, 85 DEG C of 5min, it is 100ul, -20 DEG C of preservations that 90ul DEPC water to total volume is added in each pipe backward.
3.3、PCR。
Fluorescent quantitative PCR, such as table 2.
2 PCR reaction system of table
Reagent | Usage amount |
SYBR Premix Ex Taq II(2X) | 10.0μL |
RNase Free dH2O | 6.4μL |
PCR Forward Primer(10μM) | 0.8μL |
PCR Reverse Primer(10μM) | 0.8μL |
DNA profiling | 2.0μL |
Total volume | 20.0μL |
Wherein, Forward primer is the forward primer for hsa-miR-33a-5p, nucleotide sequence such as SEQ2.
Wherein, the nucleotide sequence of the forward primer of internal reference U6 is as shown in SEQ ID No.3, the nucleotides sequence of reverse primer
Column are as shown in SEQ ID No.4.
Statistical analysis
All results are presented in the form of mean value ± SEM value, and table is made.The software that data statistic analysis uses is
SPSS22.0, mapping software are Graphpad prism 5 (Graphpad Software, San Diego, CA), P < 0.05 and P
< 0.01 is the screening criteria of significant difference and extremely significant sex differernce.
Interpretation of result.
Data analysis
Quantitative PCR data is handled with the 7500Fast Software v2.3 software that amplification instrument configures, establishes each gene
Threshold value and baseline, dissolve amplification curve by Software Create, such as Fig. 1, show that amplification is good.Obtain each sample reaction pair
The CT value answered.In order to ensure the reliability of result data, it is believed that endogenous control gene CT value greater than 30 sample RNA mass compared with
It is low, and it is excluded from analysis, the relative expression quantity in sample uses 2-ΔΔctAlgorithm.
Expression in endometrial tissues and normal endometrial tissue utilizes Real-time PCR method,
Such as result in Fig. 2, the results showed that the expression in miR-33a-5p endometrial tissues is substantially less than in normal endometrium
In expression (P < 0.05), carry out ROC curve analysis, have checked the sensibility and specificity of miR-33a-5p.And use curve
Under relevant range (AUC) confirm diagnosis effect of miR-33a-5p.As shown in figure 3, the AUC of miR-33a-5p reaches
0.9166, (95%CI:0.865to 0.9682).These are the result shows that miR-33a-5p can distinguish carcinoma of endometrium and normal
Endometrial tissue.
By the expression in miR-33a-5p Endometrial Carcinomas and the relationship between clinical pathological factors such as 4 institute of table
Show, wherein patient in group has 80 people altogether, it is age-based, clinical stages, differentiation degree, invasive depth and lymphatic metastasis
Situation is grouped.The result shows that early stage endometrial carcinomas (I~II phase) miR-33a-5p expression is apparently higher than in advanced stage endometrial carcinomas
The expression of (III~IV phase), difference are statistically significant (P < 0.05).Clinical and Pathological Analysis the result shows that, miR-33a-5p
Expression it is related with the clinical stages of endometrial carcinoma.
Expression and the relationship of its clinicopathologic features of 4 miR-33a-5p of table in endometrial tissues
Note:
P=0.2036, High differentiation vs.Middle differentiation;
P=0.1507, High differentiation vs.Low differentiation.
2 In vitro cell experiment of embodiment.
Noble cells system is purchased from Chinese Academy of Sciences's Shanghai cell bank respectively in 1.Ishikawa people's carcinoma of endometrium.
Cell culture medium RPMI1640 and fetal calf serum are purchased from Gibico company, are overexpressed mimics synthesis and are purchased from Suzhou
Ji Ma Technology Co., Ltd. is turned using 6 well culture plate culture Ishikawa cells when cell growth reaches 80% or so
The miR-33a-5p of dye, overexpression is mimics-33a-5p, and miR-NC is control group, Ishikawa cell is blank control group,
Lip3000 is transfected as transfection reagent.Cell RNA is extracted after transfection 48h, RNA extraction agent RNAiso Plus is purchased from
Precious bioengineering Co., Ltd, reverse transcription quantification kit are purchased from precious biotech firm.Carry out detection transfection efficiency.Such as Fig. 4, as a result
Show successfully to be overexpressed miR-33a-5p in Ishikawa cell.
2. the transfer ability of cell is detected with scratch experiment, the cell culture in 6 orifice plates is in good condition, pollution-free, carefully
Born of the same parents cultivate to degrees of fusion be 90 percent, dynamics uniformly use in pipette tips metal marker straight line, continue to train with serum free medium
Cell is supported, during this period, every 12h, for 24 hours, 48,60h shoot the photo of scratch healing under the microscope, detect cell migration energy
Power.The result shows that the Ishikawa cell migration ability of miR-33a-5p overexpression group significantly reduces compared with miR-NC group
(Fig. 5) illustrates that the transfer ability of endometrial carcinoma cell can be inhibited by being overexpressed miR-33a-5p.
3. the matrigel by specification of kit is diluted, in the matrigel of 20 μ L of cell upper berth diluted, cell is set
In 700 μ L complete mediums on 24 orifice plates, are added under cell.Microscopically observation cell state is good, is disappeared with 0.25% pancreatin
Change liquid cell, mixed liquor is placed in centrifuge tube, 1000rpm is centrifuged 5 minutes;Supernatant is removed, is washed with the culture solution without serum
Cell, cell count under microscope, is put into 200 μ L cell suspensions, cell number about 5*10 for the cell transwell upper chamber4It is a thin
Born of the same parents.Cell is worn 16 hours in being incubated for incubator, rear to stop to take out the cell transwell, is gently washed with PBS 2 times, more than 4%
Polyformaldehyde fixes cell, and every hole is added 700ul cell fixer, fixes 60min in fixer, PBS cleans cell, dries small
Room dyes 50min in haematoxylin, cell is put into dyeing, rear to use PBS wash cell 3 times, is being inverted 20 times of microscopic observations, with
Machine takes 3 visuals field, counts number of cells, detects the invasive ability of cell.Transwell the result shows that, compared with control group
Compared with the invasive ability of the Ishikawa cell of miR-33a-5p overexpression group significantly reduces, and illustrates that overexpression miR-33a-5p can
To inhibit the invasive ability (Fig. 6) of endometrial carcinoma cell.
The preferred embodiment of the present invention has been described in detail above, it should be understood that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
According to present inventive concept by logic analysis, reasoning or according to the limited available technology of experiment in prior art basis
Scheme, should be among the protection scope determined by the claims.
Sequence table
<110>attached Shengjing city hospital, Chinese Medical Sciences University
<120>a kind of miRNA molecule miR-33a-5p relevant to carcinoma of endometrium and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213>mankind (Homo sapiens)
<400> 1
GUGCAUUGUA GUUGCAUUGC A 21
<210> 2
<211> 23
<212> DNA
<213>artificial sequence
<400> 2
GACTTAAACG TGGATGTACT TGC 23
<210> 3
<211> 23
<212> DNA
<213>artificial sequence
<400> 3
GGAACGATACAGAGAAGATTAGC 23
<210> 4
<211> 22
<212> DNA
<213>artificial sequence
<400> 4
TGGAACGCTTCACGAATTTGCG 22
Claims (8)
1. a kind of miRNA molecule miR-33a-5p relevant to carcinoma of endometrium, nucleotide sequence such as SEQ ID NO .1 institute
Show.
Answering in 2.miRNA molecule miR-33a-5p Endometrial Carcinomas auxiliary diagnosis or Treatment of Endometrial Neoplasms/prognosis evaluation
With.
3.miRNA molecule miR-33a-5p is commented in preparation carcinoma of endometrium auxiliary diagnosis or carcinoma of endometrium adjuvant treatment/prognosis
Estimate the application in reagent or kit.
4. the reagent for detecting miRNA molecule miR-33a-5p is controlled in preparation carcinoma of endometrium auxiliary diagnosis or carcinoma of endometrium
Application in treatment/prognosis evaluation reagent or kit.
5. including for expanding the miRNA molecule miR-33a-5p for detecting the reagent of miRNA molecule miR-33a-5p
Specific primer, the primer sequence are as follows:
F:5 '-GAC TTA AAC GTG GAT GTA CTT GC-3 ' (SEQ ID NO .2).
6. the kit for detecting miRNA molecule miR-33a-5p relevant to carcinoma of endometrium is auxiliary in preparation carcinoma of endometrium
Help the application in detection or auxiliary diagnostic tool.
7. the kit for detecting miRNA molecule miR-33a-5p relevant to carcinoma of endometrium is controlled in preparation carcinoma of endometrium
Application in treatment/prognosis evaluation tool.
8. a kind of for carcinoma of endometrium auxiliary diagnosis or the kit of prognosis evaluation characterized by comprising
(1) for expanding the specific primer pair of miR-33a-5p;
(2) standard DNA template;
(3) PCR reaction solution.
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CN110628909A (en) * | 2019-10-09 | 2019-12-31 | 辽宁省肿瘤医院 | miRNA molecule miR-4500 related to endometrial cancer and application thereof |
CN113444803A (en) * | 2021-07-14 | 2021-09-28 | 武汉大学中南医院 | Cervical cancer prognosis marker microorganism and application thereof in preparation of cervical cancer prognosis prediction diagnosis product |
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