CN106636307A - Application of miRNA-146a mutant in preparing test strip for early screening and rapid diagnosis of cervical cancer - Google Patents
Application of miRNA-146a mutant in preparing test strip for early screening and rapid diagnosis of cervical cancer Download PDFInfo
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Abstract
The invention relates to a reagent for early screening and diagnosis of cervical cancer, and in particular discloses an application of an miRNA-146a mutant in preparing a test strip for early screening and rapid diagnosis of the cervical cancer. With a nitrocellulose membrane serving as a solid-phase vector, the miRNA-146a (G/C) mutation rapid diagnosis test strip is prepared on the basis of the principle that a target mRNA, which is amplified by virtue of a nano-gold oligonucleotide probe and horse radish peroxidase (HRP), rapidly forms heteroduplex. In comparison with original conventional diagnosis methods, the technology provided by the invention has the outstanding advantages of being high in sensitivity, simple and rapid to operate and low in cost; an optimum platform is provided for the early diagnosis, prevention and treatment of the cervical cancer; and the platform is applicable to large-scale HPV and cervical cancer screening in low-income population and is capable of rapidly changing the current situation that the morbidity and mortality of the cervical cancer stay at a high level. The platform is of great significance to the epidemiological investigation, early diagnosis and treatment of HPV infection and the cervical cancer; and the platform has a broad application prospect.
Description
Technical field
The present invention relates to the early screening diagnostic reagent of cervical carcinoma, specifically, discloses miRNA-146a mutant in system
Application in standby cervical carcinoma early screening quick diagnosis test strips.
Background technology
Used as the second largest killer of global female malignant, its preventing and treating is all the time global public health to cervical carcinoma
One of major fields.Statistics shows that the whole world is annual newly to send out cervical carcinoma more than 50 ten thousand, wherein Chinese new cases account for 28.8%,
Up to 13.15 ten thousand.There are about 27.4 ten thousand women in world wide every year and die from cervical carcinoma, every year women dies from about more than 50,000 for China
Cervical carcinoma.Cervical intraepitheliaI neoplasia（cervical introepithelial neoplasia, CIN）It is precancerous lesion, according to
The shared scope in scaly epithelium of atypical hyperplasia cell is divided into CIN I（Slightly）、CIN II（Moderate）With CIN III（Weight
Degree）Three ranks.The World Health Organization（WHO）Description, CIN I levels and CIN II levels proceed to CIN III levels and about need 3-8
Year, it is 0.69%-6.2% that CIN I levels turn cancer rate, and it is 4.3%-13.3% that C1N II levels turn cancer rate, and CIN III levels turn cancer
Rate is then up to 12%-65%.Research shows that cervical cancer pathogenesis rate can be reduced 60-90% by effective examination.What clinic was commonly used
Detection methods have cervical exfoliated cell to check（Including traditional conventional smear and liquid based cytology technology）, vaginoscopy
And HPV-DNA technique of gene detection.
Screening method is visually observed including acetic acid dyeing and iodine staining experiment（VIA and VILI）.Due to fund and qualified
The shortage of the aspects such as cytology professional and technical personnel, VIA/VILI is that current WHO recommends to enter temporary dwelling palace in developing countries and regions
The alternative means of neck cancer examination.Both approaches are although simple to operate, low price, but sensitivity and specificity are relatively low（Take
It is magnificent, Cheng Yifan, Cheng Xiaodong etc..Accuracy evaluation of five kinds of detection methods in cervical carcinoma and its cercinoma prophase pathologic change examination. China
Medical journal .2011,91 (5)：309-312）, about 20-50%, and the case major part sifted out is that infiltrating carcinoma and early stage are former
Position cancer.Cervical cytological examination includes traditional conventional smear and liquid-based cytology.But the susceptibility of Pap smear
Relatively low, only 25-50% or so causes substantial amounts of false negative, thus clinical practice is restricted.U.S. FDA approval in 1996
Liquid-basedcytology technology (LBC) is used for cervical carcinoma screening, and LBC special flaking method makes abnormal cervical cell recall rate high
Up to more than 95%, susceptibility is higher by Pap smear 40% or so, and false negative rate reduces by 60%.At present in developed country, LBC by
Pap smear is gradually replaced to become recommended standard prescreening method（Sherman ME, Lorincz AT, Scott DR, et
al. Baseline cytology, human papillomavirus testing, and risk for cervical
neoplasia: a 10-year cohort analysis. J Natl Cancer Inst. 2003; 95:46-52）, but
Because its higher price and higher technical requirements are difficult to be promoted in developing countries and regions.Vagina sem observation is mainly with disease
The border motif of stove, color, the exception of four signs reflection focuses of blood vessel structure and Iod R.Determine if having abnormal position under mirror
Point takes biopsy specimen or scraping neck tube mucous membrane send pathologic finding.But because gynecatoptron operation is commonly present personal error in judgement, may
Diagnosis and the selection of biopsy site are had influence on, mistaken diagnosis occurs.
At present, the cervical carcinoma early screening scheme of world health organisation recommendations is the detection of HR-HPV associational cellses, but this
One scheme yet suffers from many limitations and deficiency.Novel tumor markers have specific expressed because of it in the different times of disease,
So will play a significant role in terms of the early detection of cervical carcinoma, treatment and Index for diagnosis.Such as HR-HPV E6/E7 mRNA inspections
Survey, the dyeing of P16INK4a/Ki-67 Double immunes, the method such as HPV L1 capsid proteins and the detection of hTERC gene associations may be into
For the secondary screening marker of HR-HPV DNA positive patients, with determine patient whether need biopsy under row gynecatoptron immediately to make a definite diagnosis or
Treatment.It is expected that either in developed country or developing country, with HR-HPV detections as primary dcreening operation, specific uterine neck
Carcinoma marker will play a significant role as the control prece of secondary examination in preventing and treating in cervical carcinoma.Cervical carcinoma specificity marker
The detection of thing will provide new thinking for the early diagnosis and therapy of clinical cervical carcinoma.
MicroRNA abnormal expressions and gene mutation have been confirmed in many tumours, and think to examine cancer early stage
Disconnected and prognosis evaluation have important value (Hasani S-S, Hashemi M, Eskandari-Nasab E, Naderi M,
Omrani M, Sheybani-Nasab M. A functional polymorphism in the miR-146a gene is
associated with the risk of childhood acute lymphoblastic leukemia: a
preliminary report. Tumor Biology. 2014;35:219-25).Research discovery, miR- in cervical cancer tissues
21,127,199a up-regulated and miR-146a, 214,218,34a expression is lowered, and points out miRNAs in cervical carcinogenesis mistake
Certain regulating and controlling effect has been played in journey（Lee JW, Choi CH, Choi JJ, et al. Altered MicroRNA
expression in cervical carcinomas[J]. Clin Cancer Res, 2008, 14(9):2535-
2542.）.Nearest research finds（Gadducci A,Guerrieri ME,Greco C.Tissue biomarkers as
prognostic variables of cervical cancer[J]. Crit Rev Oncol Hematol, 2013, 86
(2):104-129.）, miR-9,145,146a, 200a, 886-5p, 424 there is unconventionality expression in cervical carcinoma, due to these
The change of miRNA expression occurred in precancerous lesion early stage, therefore was expected to become the biomarker of cervical carcinoma early screening
(Srivastava K, Srivastava A. Comprehensive review of genetic association
studies and meta-analyses on miRNA polymorphisms and cancer risk. PloS one.
2012;7:e50966.).The research such as Wang finds（Wang X, Tang S, Le S Y, et al. Aberrant
expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is
required for cancer cell growth[J]. PloS one, 2008, 3(7): e2557.）miR-146a-5p
Raise in cervical cancer tissues, miR-146a-5p is transfected in Cervical Cancer HeLa Cells, it can be promoted to breed.miR-146a-
5p can activate NF- κ B by the expression of LMP1 (latent membrane protein 1)（nuclear factor kappa
B）, this shows that NF- κ B are one of target genes of miR-146a-5p regulation and control, the evolution of their common cervical carcinomas for participating in
(Cameron JE, Yin Q, Fewell C, Lacey M, McBride J, Wang X, et al. Epstein-Barr
virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a
modulator of lymphocyte signaling pathways[J]. Journal of virology. 2008,82
(4):1946-58)
Tumor necrosis factor α (Tumor Necrosis Factor, TNF- α) is a kind of with extensive biological effect
Cell factor.TNF- α belong to II type memebrane protein, are played a role in the form of tripolymer.The gene of TNF-α is about 3.6kb,
Positioned at human chromosome 6p21.1～p22, the kb of mRNA length 1.7.The TATA boxes of TNF- α genes are in transcription site upstream
At 20bp, at its upstream including at least 5 NF- κ B enhancers and the binding sites of c-jun/AP- 1 etc..Therefore study
It was found that inflammatory factor TNF-α can induce the expression of miR-146a by NF- κ B.Research above shows that some are specific
MicroRNAs such as miR-146a-5p can be used as Novel marker (Sandhu R, the Rein of cervical carcinoma screening and prognosis evaluation
J, D'Arcy M, Troester MA. Over-expression of miR-146a in basal-like breast
cancer cells confers enhanced tumorigenic potential in association with
altered p53 status. Cancer research. 2013;73:4196-.Palmieri A, Carinci F,
Martinelli M, Pezzetti F, Girardi A, Cura F, et al. Role of the MIR146A
polymorphism in the origin and progression of oral squamous cell carcinoma.
European journal of oral sciences. 2014;122:198-201.)。
The content of the invention
The present invention is directed to the deficiencies in the prior art, there is provided miRNA-146a mutant is fast in preparation cervical carcinoma early screening
Application in fast diagnosis test paper.
Another object of the present invention is to provide applications of the miRNA-146a in nm of gold oligonucleotide probe is prepared.
Another object of the present invention is to provide a kind of miRNA-146a cervical carcinomas early screening quick diagnosis test strips.
The above-mentioned purpose of the present invention is achieved by the following technical programs.
Application of the miRNA-146a mutant in cervical carcinoma early screening quick diagnosis test strips are prepared, miRNA-146a
The sequence of mutant such as SEQ ID NO:Shown in 1 ~ 2.The miRNA-146a-5p of miRNA-146a mutant sequences such as SEQ ID
NO:Shown in 1, the miRNA-146a-3p such as SEQ ID NO of miRNA-146a mutant sequences:Shown in 2.
Applications of the miRNA-146a in nm of gold oligonucleotide probe is prepared, using sequence such as SEQ ID NO:1 ~ 2 institute
The miRNA-146a mutant for showing and sequence such as SEQ ID NO:MiRNA-146a wild types design detection probe shown in 3 ~ 4,
The detection probe sequence such as SEQ ID NO:Shown in 5；The detection probe is carried out after sulfhydrylation modification, to be connected to nm of gold
Particle surface, the nanogold particle surface is also adsorbed with horseradish peroxidase HRP, obtains nm of gold oligonucleotide probe.
MiRNA-146a wild-type sequences miRNA-146a-5p such as SEQ ID NO:Shown in 3, miRNA-146a wild type sequences
The miRNA-146a-3p of row such as SEQ ID NO:Shown in 4.Detection probe sequence such as SEQ ID NO:Shown in 5.With reference to nm of gold
The sulfhydrylation detection probe sequence of particle is：5′-Thio/MC6-D/GGG GTT TCA CCG TGT TAG CCA GGA TGG
TCT- 3′.
A kind of miRNA-146a cervical carcinomas early screening quick diagnosis test strips, including following component：Receiving described in claim 2
The golden oligonucleotide probe of rice, respectively such as SEQ ID NO:Biotin capture probe A, B shown in 6 ~ 7, and the sample being sequentially connected
Product pad, gold standard pad, nitrocellulose membrane, absorption pad；The biotin capture spy as detection band is fixed with the nitrocellulose membrane
The pin A and biotin capture probe B as quality control band.
Biotin capture probe A such as SEQ ID NO:It is 5 '-biotin/CGC CCG GCT AAT TTT TTG shown in 6
TAT TTT TAG TAG AGA C - 3′；Biotin capture probe B such as SEQ ID NO:It is 5 '-biotin/ATG shown in 7
AGA CCA TCC TGG CTA ACA CGG TGA AAC CC - 3′。
The present invention adopts nitrocellulose membrane for solid phase carrier, using nm of gold oligonucleotide probe and horseradish peroxidase
（HRP）Target mRNA after dual amplification quickly forms the principle of heteroduplex, makes miRNA-146a (G/C) mutation and quickly examines
Disconnected test strips, realize cervical carcinoma early screening and quickly examine using the specific region of mutator specific primer identification target gene
It is disconnected.
Preferably, when using, sample drop is added on the sample-adding pad of test strips, after 15 minutes, on nitrocellulose filter
Detection zone show red for miRNA-146a mutant patients, be strong suspicion cervical cancer patient；Detection zone white shows
It is miRNA-146a wild types, shows that cervical tissue not yet occurs at present canceration.
By the sample of variable concentrations（Wild type, saltant type and non-specific nucleic acid）Drop is added in the sample-adding pad of test strips
On, after 15 minutes, detection zone on nitrocellulose filter shows red for miRNA-146a (G/C) mutant patient, is high
Degree suspects cervical cancer patient；Detection zone white is shown to be miRNA-146a wild types, shows that cervical tissue is not yet disliked at present
Become；Unnecessary nm of gold then can show red with the detection probe complete complementary of control area.MiRNA-146a wild types and prominent
Variant is detected in same test strips：Will be after amplification sample drop be added in disposable test paper slip upper 15 minute, celluloid
What detection zone showed red stripes on film shows sample for miRNA-146a (G/C) saltant type, and detection zone is still that white shows sample
Product are miRNA-146a wild types.
Compared with prior art, beneficial effect of the present invention is：What the technology had compared with conventional diagnostic method
Outstanding advantages are sensitive high, simple to operate（Any instrument is not required in addition to water bath, portable chemical light-emitting appearance）, it is quick（1 is little
When）, it is cheap（50-80 is first）, optimal platform is provided to early diagnose, preventing and treating cervical carcinoma, can be suitably used for owning
Country extensive HPV and cervical carcinoma screening especially in low-income groups of developing country, quickly changes cervical cancer pathogenesis
The high present situation of rate and the death rate.Epidemiology survey, early diagnosis and therapy to HPV infection and cervical carcinoma has non-
Often important meaning, and be with a wide range of applications.
Description of the drawings
Fig. 1 is to build miRNA-146a gene mutation quick diagnosis test strips schematic diagrames.
Fig. 2 is to build the few conjunction sweet acid probe schematic diagram of nm of gold.
Fig. 3 is miRNA-146a wild types and mutant gene structure.
Fig. 4 is the testing result of miRNA-146a quick diagnosis test strips condition optimizings and sample.
Specific embodiment
The present invention is described in further details with reference to Figure of description and specific embodiment, but embodiment is not right
The present invention is limited in any form.Unless stated otherwise, the reagent for adopting of the invention, method and apparatus are normal for the art
Rule reagent, method and apparatus.
Embodiment 1 builds quick diagnosis test strips
Build nm of gold oligonucleotide probe as shown in Figure 2：Using sequence such as SEQ ID NO:MiRNA-146a shown in 1 ~ 2 dashes forward
Variant and sequence such as SEQ ID NO:MiRNA-146a wild types design detection probe shown in 3 ~ 4, the detection probe sequence
Such as SEQ ID NO:Shown in 5；The detection probe is carried out after sulfhydrylation modification, nanogold particle surface is connected to, it is described to receive
Rice gold grain surface is also adsorbed with horseradish peroxidase HRP, obtains nm of gold oligonucleotide probe.Fig. 3 is miRNA-146a
Wild type and mutant gene structure.
Build HPV quick diagnosis test strips as shown in Figure 1：By nm of gold oligonucleotide probe obtained above, respectively such as
SEQ ID NO:Biotin capture probe A, B shown in 6 ~ 7, and be sequentially connected sample pad, gold standard pad, nitrocellulose membrane,
Absorption pad；The biotin capture probe A and the biotin as quality control band as detection band is fixed with the nitrocellulose membrane
Capture probe B.
Nanogold particle with probe is loaded and is connected on nitrocellulose membrane pad, and biotin capture probe A connects
The detection zone of nitrocellulose membrane is connected on, biotin capture probe B is connected to the check plot of nitrocellulose membrane.
Embodiment 2
The quick diagnosis test strips built using embodiment 1 are detected.Sample drop containing target dna is added in into pad
On, movement is started by siphonage, detection zone is first passed through, then to check plot.Target dna meeting and the nm of gold with probe
Grain specifically binds, and movement is may proceed to after combination, association reaction can occur with detection probe to after detection zone, with continuation
Mobile association reaction to occur with control probe again, the associated proteins in this twoth area can at this moment show redness, be HPV positive, inspection
Survey area remain white show that HPV is negative.
Target dna sequence such as SEQ ID NO:It is 5 '-AGA CCA TCC TGG CTA GTC TGT TGT shown in 8
CTC TAC TAA AAA TA- 3′。
The testing result of miRNA-146a quick diagnosis test strips condition optimizings and sample, is as a result shown in Fig. 4.
SEQUENCE LISTING
<110>Guangdong Medical College
<120>Application of the miRNA-146a mutant in cervical carcinoma early screening quick diagnosis test strips are prepared
<130>
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 99
<212> DNA
<213>The miRNA-146a-5p of miRNA-146a mutant sequences
<400> 1
ccgatgtgta tcctcagctt tgagaactga attccatggg ttgtgtcagt gtcagacctc 60
tgaaattcag ttcttcagct gggatatctc tgtcatcgt 99
<210> 2
<211> 99
<212> DNA
<213>The miRNA-146a-3p of miRNA-146a mutant sequences
<400> 2
tgaatatttt tgaaaaatta caaattaatt ttatgctact attaattatg attttatagc 60
gcgattaaat ttaagatagt ttcttttatg atacaaatg 99
<210> 3
<211> 99
<212> DNA
<213>MiRNA-146a wild-type sequence miRNA-146a-5p
<400> 3
ccgatgtgta tcctcagctt tgagaactga attccatggg ttgtgtcagt gtcagacctc 60
tgaaattcag ttcttcagct gggatatctc tgtcatcgt 99
<210> 4
<211> 99
<212> DNA
<213>The miRNA-146a-3p of miRNA-146a wild-type sequences
<400> 4
tgaatatttt tgaaaaatta caaattaatt ttatgctact attaattatg attttatagc 60
gccattaaat ttaagatagt ttcttttatg atacaaatg 99
<210> 5
<211> 30
<212> DNA
<213>Detection probe sequence
<400> 5
ggggtttcac cgtgttagcc aggatggtct 30
<210> 6
<211> 34
<212> DNA
<213>Biotin capture probe A
<400> 6
cgcccggcta attttttgta tttttagtag agac 34
<210> 7
<211> 32
<212> DNA
<213>Biotin capture probe B
<400> 7
atgagaccat cctggctaac acggtgaaac cc 32
<210> 8
<211> 38
<212> DNA
<213>Target dna sequence
<400> 8
agaccatcct ggctagtctg ttgtctctac taaaaata 38
Claims (4)
- Application of the 1.miRNA-146a mutant in cervical carcinoma early screening quick diagnosis test strips are prepared, it is characterised in that The sequence of miRNA-146a mutant such as SEQ ID NO:Shown in 1 ~ 2.
- Applications of the 2.miRNA-146a in nm of gold oligonucleotide probe is prepared, it is characterised in that using sequence such as SEQ ID NO:MiRNA-146a mutant and sequence such as SEQ ID NO shown in 1 ~ 2:MiRNA-146a wild types design inspection shown in 3 ~ 4 Probing pin, the detection probe sequence such as SEQ ID NO:Shown in 5；The detection probe is carried out after sulfhydrylation modification, is connected To nanogold particle surface, the nanogold particle surface is also adsorbed with horseradish peroxidase HRP, obtains nm of gold few nucleosides Acid probe.
- 3. a kind of miRNA-146a cervical carcinomas early screening quick diagnosis test strips, it is characterised in that including following component：Right The nm of gold oligonucleotide probe described in 2 is required, respectively such as SEQ ID NO:Biotin capture probe A, B shown in 6 ~ 7, and Sample pad, gold standard pad, nitrocellulose membrane, the absorption pad being sequentially connected；It is fixed with as detection band on the nitrocellulose membrane The biotin capture probe A and biotin capture probe B as quality control band.
- 4. quick diagnosis test strips according to claim 3, it is characterised in that when using, sample drop is added in into test paper On the sample-adding pad of bar, after 15 minutes, detection zone on nitrocellulose filter shows red for miRNA-146a mutant patients, For strong suspicion cervical cancer patient；Detection zone white is shown to be miRNA-146a wild types, shows that cervical tissue is not yet sent out at present Raw canceration.
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