CN108753983A - The diagnosis and treatment marker of cervical carcinoma - Google Patents
The diagnosis and treatment marker of cervical carcinoma Download PDFInfo
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Abstract
The invention discloses MNX1 genes can as the molecular marker of diagnosis of cervical cancer, the experiment proves that：Compared with normal control tissue, MNX1 genes expression quantity in cervical cancer tissues is high.The invention also discloses the drugs that MNX1 genes can be used for preparing treatment cervical carcinoma.The achievement in research of the present invention provides a kind of new cervical carcinoma methods for clinical diagnosis, while providing a kind of drug new target drone for treating cervical carcinoma.
Description
Technical field
The present invention relates to biomedical sectors, more particularly it relates to which MNX1 genes are preparing diagnosis and treatment cervical carcinoma
Application in product.
Background technology
Cervical carcinoma is most common gynecologic malignant tumor, the account for the first in tumors of female reproductive organ.According to world health
Statistical data in 2014 is organized, the year new cases of cervical carcinoma are about 52.8 ten thousand, and year death number of cases is about 26.6 ten thousand, wherein
85% patient is happened in developing country, and rural area is higher than city.China is the big country of cervical cancer pathogenesis, according to country
The newest cancer statistical data that tumor center announces, the cervical carcinoma new cases in China in 2015 are about 9.89 ten thousand, and year is dead
Number of cases is about 3.05 ten thousand, and the data present year by year rise and fall ill rejuvenation trend [Chen W, Zheng R,
Baade P D,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66
(2):115-132].The generation of cervical carcinoma and many factors are closely related, such as early marriage, early childbirth, fecund and sexual life disorder, but
Human papilloma virus (Human papilloma virus, HPV) infection is considered as the necessary condition of cervical cancer pathogenesis,
99.7% cervical cancer patient HPV screenings are positive, and the risk of HPV persistent infection patients' cervical carcinomas is HPV negative patients
More than 250 times [Wentzensen N, Arbyn M.HPV-based cervical cancer screening-facts,
fiction,and misperceptions[J].Prev Med,2017,98:33-35l；Torre L A,Islami F,
Siegel R L,et al.Global cancer in women:burden and trends[J].Cancer Epidemiol
Biomarkers Prev,2017].From the seventies and eighties in last century, clear HPV infection is the important origin cause of formation of cervical cancer pathogenesis for the first time
Afterwards, scholars induce the mechanism expansion further investigation of cervical cancer pathogenesis for HPV infection, and therefore have developed for cervical carcinoma
Diagnostic method and therapeutic strategy, including before sensitive special cancer diagnostic method, targeting immunoprophylaxis measure and treatment
Method greatly improves cervical cancer patient existence and quality of life.
With the development of modern biotechnology detection technique and the research that deepens continuously of Tumorigenesis, it is related to tumorigenic
Different kinds of molecules during various biological, as dissociated in nucleic acid, protein, carbohydrate, lipid, small molecule metabolites or even blood
Tumour cell, can be used as important tumor markers, specific foundation provided to clinical prevention, diagnosing and treating.In palace
In neck cancer, oneself is it is found that some tumor markers are used for the clinical preventions of cervical carcinoma at present.
Ki-67 and p16INK4a is two kinds and is commonly used in analysis tumor cell proliferation state and malignancy
Molecular marker.Wherein Ki-67 is not expressed in silent GO phases cell, and height is expressed in G1, S, G2 and M phase cells, therefore it can
The proliferation activity of analysis cell is widely used in assess tumor progression [Endl E, Gerdes J.The Ki-67protein:
fascinating forms and an unknown function[J].Exp Cell Res,2000,257(2):231-
237]；Simultaneously as it is expressed, pedigree is extensive, and the tumor markers as specificity still have certain defect, it is clinical it is still necessary to
Other molecular marker auxiliary diagnosis.P16INK4a specific can be combined as a kind of cycle regulating protein with CDK4 and CDK6
To inhibit the phosphorylation of its activity and downstream pRb, cell cycle progress and cell differentiation procedure are adjusted；Studies have found that
P16INK4a expression is proportionate with HR-HPV persistent infections and cervix neoplasms pathological grading, for the clear of the postoperative HPV of patient
It removes and persistent infection has certain guiding value [Koh J, Enders G H, Dynlacht B D, et al.Tumour-
derived p16alleles encoding proteins defective in cell-cycle inhibition[J]
.Nature,1995,375(6531):506-510]。
ProExC antibody (BD companies) can specificity the karyon albumen MCM2 that is induced by HPV infection of identification and topology
Isomerase TOP2A compounds.In cervix gland and squamous cell atypical hyperplasia, the cell S phase genes of E7 oncogenes induction
It can promote TOP2A and the MCM2 high expression in nucleus, while TOP2A can be combined to form stable structure with 6 MCM2 molecules
It is stranded in nucleus [Santin A D, Zhan F, Bignotti E, et al.Gene expression profiles of
primary HPV16-and HPV18-infected early stage cervical cancers and normal
cervical epithelium:identification of novel candidate molecular markers for
cervical cancer diagnosis and therapy[J].Virology,2005,331(2):269-291].Because its
It is not expressed in normal cervical epithelial cell, and the significantly high expression in the active squamous cell of the proliferation of HPV inductions, clinically may be used
For distinguishing, atypical hyperplasia and squamous cell ateliosis or atrophy etc. are similar to be changed.
Mainly a stable protection is collectively formed by capsid protein L 1 and L2 albumen in HPV DNA integrity and stabilities
Shell maintains.Wherein, L1 albumen can also promote virion to infect mucous membrane substrate by identifying the corresponding receptor on host cell
Film strips cell or cervical epithelial cells.HPV infect it is slight to moderate atypical hyperplasia during can be continuously detected L1 eggs
White expression, but as the be in progress expression of L1 albumen of uterine neck cancerization degree can fade away [Mcmurray H R, Mccance
D J.Human papillomavirus type 16E6activates TERT gene transcription through
induction of c-Myc and release of USF-mediated repression[J].J Virol,2003,77
(18):9852-9861].Therefore HPV-L1 expression disappear prompt viral genome oneself be incorporated into host genome, can be used for uterine neck
The diagnosis of cancer CIN3 phases.
Lanminin-5 is a kind of and the closely related tumor markers of tumor invasion, in a variety of different type malignant tumours
The expression of middle Lanminin-5 genes is often closely related with the progress of tumour.In the generating process of cervical carcinoma, Laminin-5 master
It expresses the early stage in tumour, especially at the skin lesion of microinvasion, therefore it can be used for Cervix Squamous Cell infiltration
The detection of early stage.MIB-I has the expression pattern similar with Ki-67, i.e., the proliferation shape of GINS each phases is expressed in significant height
The active tumour cell of state detects the periodic state that can be used for residing for assistant analysis tumour cell in conjunction with Ki-67.Therefore MIB-1
It is an important indicator of cell-proliferation activity during another important detection atypical hyperplasia.
Although in conclusion diagnosis of the tumor markers for clinical cervical carcinoma there are many oneself at present, oneself has tumour
Marker is horizontal because of constructive expression under normal circumstances or in non-malignant diseases also to be increased, and tumour-specific is lacked.
It would therefore be highly desirable to find tumour specific antigen as biological marker, early diagnosis and prognosis for clinical cervical carcinoma judge.
Invention content
One of the objects of the present invention is to provide one kind realizing diagnosis of cervical cancer by detecting MNX1 gene expression differences
Method.
The second object of the present invention is to provide a kind of method for treating cervical carcinoma by inhibiting MNX1 gene expressions.
The third object of the present invention is to provide a kind of method for screening treatment of human cervical cancer drug.
The fourth object of the present invention is to provide a kind of drug for treating cervical carcinoma.
To achieve the goals above, present invention employs following technical solutions：
The present invention provides application of the reagent of detection MNX1 in the tool for preparing diagnosing cervical.
Further, the reagent of the detection MNX1 includes the reagent for detecting MNX1 gene expression amounts.
Further, the reagent of the detection MNX1 includes that can quantify the reagent of MNX1 gene mRNAs, and/or can determine
Measure the reagent of MNX1 albumen.
The reagent of the quantitative MNX1 gene mRNAs of the present invention can play its work(based on the known method of nucleic acid molecules is used
Energy：Such as PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO
Method, high-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the reagent.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory
Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR methods etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR methods), PCR-RFLP methods, original position RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP methods, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR methods and PCR-SSCP (single-strand conformation polymorphism) methods detect.
The reagent that MNX1 gene mRNAs can be quantified can be the specific primer of MNX1 genes or transcript, also may be used
To be the specific recognition probe of MNX1 genes or transcript, or include primer and probe simultaneously.
The specific primer of MNX1 genes or transcript recited above includes the specific amplified used in real-time quantitative PCR
The primer of MNX1 genes.In the specific embodiment of the present invention, the primer sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2.
Primer can be prepared by chemical synthesis, by using those skilled in the art will know that method refer to known letter
Breath is prepared to be suitably designed by chemical synthesis.
Probe can be prepared by chemical synthesis, by using those skilled in the art will know that method refer to known letter
Breath appropriately designs, and is prepared by chemical synthesis, or can contain desired nucleic acid sequence by being prepared from biomaterial
Gene, and using it is expected designed for amplification nucleic acid sequence primer amplification it prepare.
The reagent of the quantitative MNX1 albumen of the present invention can play its function based on the known method of antibody is used：For example,
May include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The reagent of the quantitative MNX1 albumen of the present invention includes the antibody or its segment for specifically binding MNX1 albumen.It can make
With the antibody or its segment of any structure and size, immunoglobulin class, origin etc., as long as it combines target protein.This
The antibody or its segment that the detection product of invention includes can be monoclonals or polyclonal.Antibody fragment refers to reservation antibody
Peptide to the active antibody of the combination of antigen a part of (Partial Fragment) or containing an antibody part.Antibody fragment may include F
(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, the areas dimerization V it is (dual anti-
Body) or peptide containing CDR.The reagent of the quantitative MNX1 albumen of the present invention may include encoding antibody or Encoding Antibody Fragment
The nucleic acid of the separation of amino acid sequence, includes the carrier of the nucleic acid, and carries the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.Retain target all or in part for example, preparing
The polypeptide of protein integrates the mammalian cell expression vector for encoding their polynucleotides as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.The antibody of acquisition can finally be implemented by using MNX1 albumen for being used as antigen or part thereof
Antigentic specificity purifies to obtain the monoclonal antibody for MNX1 albumen.Polyclonal antibody can be prepared as follows：With with it is above
Identical antigen-immunized animal, collects blood sample from by immune animal, serum is isolated from blood, then uses upper
It states antigen and antigentic specificity purifying is implemented to serum.It can be by the antibody that is obtained with enzymatic treatment or by using the antibody of acquisition
Sequence information obtain antibody fragment.
The combination of marker and antibody or its segment can be implemented by method as commonly known in the art.For example, can
With following fluorescent marker protein or peptide：Clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, it is all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark
Remember kit-NH2, peroxidase labelling kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B- phycoerythrin labelling kit-NH2,
B- phycoerythrin labelling kits-SH, R-PE labelling kit-NH2, R-PE labelling kit SH
(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM)
555 labelling kit-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- marker protein marks
Remember kit (Funakoshi Corporation).For correct labeling, can be detected by label using suitable instrument
Antibody or its segment.
The acquisition of the sample for detecting the detection of MNX1 gene expression amounts of the present invention is the ordinary skill in the art, preferably
Noninvasive may be selected or the method with minimally-invasive property obtains.
The sample can be (but are not limited to)：Tissue, peripheral blood, marrow, lymph node, abdominal cavity cleaning solution, urine, sweat
Liquid.In specific embodiments of the present invention, tissue of the sample from subject.
The present invention also provides a kind of tool for diagnosing cervical, the tool can detect MNX1 gene expressions
Amount.
Further, the tool includes that can quantify the reagent of MNX1 gene mRNAs, and/or can quantify MNX1 albumen
Reagent.
In general, the reagent is present in container appropriate.Diluent can be used to draw as described in deionized water by each
Object or probe are adjusted to the concentration of at least one requirement, are sub-packed in container.
Further, the reagent that can quantify MNX1 gene mRNAs includes the specific amplified used in real-time quantitative PCR
The primer of MNX1 genes, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Further, the tool for diagnosing cervical includes but not limited to chip, kit, test paper or high throughput
Microarray dataset；High-flux sequence platform is a kind of special tool(s), with the development of high throughput sequencing technologies, to the gene of a people
The structure of express spectra, which will become, very easily to work.By comparing the gene expression profile of Disease and normal population, it is easy
The exception for analyzing which gene is related to disease.Therefore, the exception and cervical carcinoma of MNX1 genes are known in high-flux sequence
Generation correlation also belong to used the present invention MNX1 new application, equally within protection scope of the present invention.
It may also include the reagent for extracting nucleic acid in the kit of the present invention, the reagent of PCR be used for, for dyeing or showing
The reagent etc. of color.For example, these reagents include but not limited to：Extract, amplification liquid, hybridization solution, developing solution, washing lotion etc..
In addition, may also include the specification etc. of the method for description detection MNX1 gene expressions in the kit.
Kit of the present invention may include being suitable for a variety of different of practical (being such as directed to different detection methods)
Reagent, however it is not limited to cited reagent at present, as long as the detection based on MNX1 genes or transcript is come diagnosing cervical
Reagent is all contained in the scope of the invention.
The present invention also provides a kind of methods of diagnosing cervical, and described method includes following steps：
(1) sample of subject is obtained；
(2) MNX1 gene expression doses in Samples subjects are detected；
(3) the MNX1 gene expression doses measured are associated with the disease associated of subject.
(4) compared with normal control, MNX1 gene expression doses statistically increase, and show that subject is judged and suffer from
Cervical carcinoma or judge that risk of the subject with cervical carcinoma is high.
The present invention also provides a kind of therapies of cervical carcinoma, and the method includes inhibiting MNX1 gene expressions or suppression
The activity of MNX1 gene expression products processed.
The present invention also provides a kind of screening techniques of drug candidate that treating cervical carcinoma, can be by model cell
Some period after addition testing drug measures the expression of MNX1 genes or MNX1 albumen to improve to measure drug candidate
The effect of prognosis.More specifically, when the expression of MNX1 genes or MNX1 albumen is after adding or application testing drug
When reduction or when restoring normal level, the drug may be selected as the medicine for improving cervical carcinoma.
The present invention also provides a kind of drug for treating cervical carcinoma, the drug includes the reagent for inhibiting MNX1.
The reagent of the inhibition MNX1 of the present invention is unrestricted, as long as the reagent can inhibit MNX1 or be related to the upstreams MNX1
Or expression or the activity of the substance of downstream pathway, and for treating the effective drug of cervical carcinoma.
The present invention also provides the application of MNX1 genes or its expression product in the drug for preparing treatment cervical carcinoma.
Further, the drug includes the RNA interfering or negative regulation miRNA, negative regulation type for MNX1 gene expressions
Transcription regulatory factor or suppressive target micromolecular compound.
The drug of the present invention can be used by any of mode compounding pharmaceutical composition in this field.This combination
Object includes active constituent, in addition one or more pharmaceutically acceptable carriers, diluent, filler, bonding agent and other figurations
Agent, this depends on administering mode and designed dosage form.This field known treatment of branch art personnel is inert inorganic or has
The carrier of machine includes but is not limited to lactose, cornstarch or derivatives thereof, talcum, vegetable oil, wax, fat, polyhydroxy chemical combination
Object such as polyethylene glycol, water, sucrose, ethyl alcohol, glycerine, such, various preservatives, lubricant, dispersant, corrigent.It protects
It is wet cut to pieces, antioxidant, sweetener, colorant, stabilizer, salt, buffer solution is such can also be added thereto, these substances according to
It needs to be used to help the stability of formula or helps to improve activity or its biological effectiveness or generated in the case of oral
Acceptable mouthfeel or smell, the preparation that can be used in such a composition can be the form of its original chemical itself, or
The form of its pharmaceutically acceptable salt is optionally used, drug of the invention can be administered alone, or with various combination medicine-feedings,
And combining form is administered together with other healing potions.People in the art may be selected in the composition so prepared as needed
Any mode appropriate is administered drug known to member.It is by the present invention of safe and effective amount when using pharmaceutical composition
Medicament administration in people, specific dosage is also contemplated that the factors such as administration route, patient health situation, these are all skilled practitioners skills
Within energy range.
The drug of the present invention can be prepared into various dosage forms as needed.Including but not limited to, percutaneous, mucous membrane, nose, buccal,
Tablet, solution, granule, patch, paste, capsule, aerosol or suppository sublingual or orally use.
The administration method of the drug of the present invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e.
Can, including but not limited to intravenously, in peritonaeum, intraocular, intra-arterial, intrapulmonary takes orally, in vesicle, intramuscular, and tracheal strips, subcutaneously
, local by pleura by skin, sucking, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, rectum, vagina,
In skull, in urethra, in liver, in tumor.In some cases, it can systematically be administered.It is to be locally administered in some cases.
The dosage of the drug of the present invention is unrestricted, as long as obtaining desired therapeutic effect or preventive effect.This
The medicine of invention or the dosage of prophylactic agent can use therapeutic effect for example to disease or preventive effect as referring to
It marks to determine.
The present invention " MNX1 genes " (Chromosome 7, NC_000007.14 (157004853..157010653,
Complement)) sequence can be to be inquired in ncbi database.
In the context of the present invention, " diagnosis of cervical cancer " includes judging whether subject has suffered from cervical carcinoma, judged
Subject whether there is the risk with cervical carcinoma or judge that cervical cancer patient recurs.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with relevant disease or illness
Disease or morbid state, and include：
(1) prevent disease or morbid state occurs in mammals, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) inhibit disease or morbid state, that is, prevent its generation；Or
(3) alleviate disease or morbid state, even if disease or morbid state subside.
Term " treatment " is usually directed to treatment mankind or animal (for example, being applied by animal doctor), wherein can reach certain pre-
The therapeutic effect of phase, for example, inhibiting the development (including reduce development speed, development is made to stop) of illness, improving illness and healing
Illness.It further include the treatment as precautionary measures (such as prevention).To developing into illness not yet but developing into illness danger
The purposes of the patient of danger, is also included in term " treatment ".
The advantages of the present invention：
Present invention firstly discovers that and confirming the Close relation of MNX1 gene expressions and uterine neck carcinogenesis, the sample size of verification
It is more, as a result accurately.The it is proposed of the correlation provides new approach for the Clinics and Practices of cervical carcinoma.
Description of the drawings
Fig. 1 shows the system of the differential expression in cervical cancer tissues and normal control tissue using QPCR detection MNX1 genes
Meter figure；
Fig. 2 is shown using Western blot experiment detection MNX1 albumen in cervical cancer tissues and normal control tissue
The statistical chart of differential expression；
Fig. 3 shows the statistical chart using Western blot experiment detection MNX1 gene expression inhibition rates.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
Unconventionality expression gene in the cancerous tissue of the screening cervical cancer patient of embodiment 1
1, tissue collecting
The cervical cancer tissues 4 that obstetrics and gynecology hospital provides are collected, from postoperative through uterectomy (cone is cut or cut entirely)
Pathological diagnosis is the patient of cervical carcinoma, and wherein surrounding normal tissue is as a control group.
2, tissue RNA extractions
- 80 DEG C of cervical cancer patient tissues frozen and periphery normal cervical tissues about 50mg are taken, is put into liquid nitrogen and is ground
Mill is added 1m L Trizol and is used for Total RNAs extraction and real-time quantitative until being transferred to when without bulky grain tissue in 1.5mL EP pipes
PCR is analyzed, and detailed process is as follows：
1) 200 μ L chloroforms, manual fully shaking mixing rear chamber are added in above-mentioned containing the tissue suspension of 1mL Trizol
Temperature stands 10min；
2) 12000rpm, 4 DEG C of centrifugation 15min；
3) it waits after centrifuging, gentle aspiration EP pipes upper strata aqueous phase is secondary to new 1.5mL EP pipes, and 600 μ L isopropyls are added
Alcohol turns upside down and mixes well, and is placed at room temperature for 20min (or -20 DEG C of placement 2h, can increase the precipitation of RNA)；
4) 12000rpm, 4 DEG C of centrifugation 15min；
5) it discards supernatant, water-reducible 75% ethyl alcohol of DEPC is added, gently pressure-vaccum suspension precipitates with pipette tips；
6) 12000rpm, 4 DEG C of centrifugation 15min；
7) it discards supernatant, remaining liquid is drawn with pipette tips, hangs 5min at room temperature；
8) 20 μ L RNAase-free water dissolutions is taken to precipitate, it is quantitative after using or to freeze -80 DEG C of refrigerators for use.
3, quantitative RNA
It takes the 2 μ L of total serum IgE of said extracted to be diluted in 98 μ L DEPC water, wavelength is measured using ultraviolet specrophotometer
Absorbance value at 260nm and 280nm calculates sample according to the purity of this RNA of A260/A280 ratio in judgement according to A260 values
The concentration of RNA.
4, high-throughput transcript profile sequencing
(1) RNA-seq reads position
Low-quality read is removed to obtain cleaning read first, then utilize TopHat v1.3.1 will clean segment and
UCSC H.sapiens reference genes groups (hg19) are matched, the index of H.sapiens UCSC hg19 editions built in advance
It is downloaded from TopHat homepages, and as with reference to genome, when being matched with genome using TopHat, allows each read (acquiescence
To 20) having multiple matching sites, most 2 mispairing.TopHat establishes possible according to exon region and GT-AG shear signals
Shearing site library navigates to the read for not navigating to genome on genome according to these shearing site libraries.We use
The system default parameter of TopHat methods.
(2) transcript abundance is assessed
The read file matched is by Cufflinks v1.0.3 processing, and Cufflinks v1.0.3 are by RNA-seq pieces
Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to being matched in every 1,000,000 sequencing segment specific
The segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated values is calculated by Bayesian inference method.
The GTF comment files for the reference that Cufflinks is used download (Homo_ from Ensembl databases
sapiens.GRCh37.63.gtf)。
(3) detection of difference expression gene
It is transferred to Cuffdiff by the Ensembl GTF files of download and by the matched original documents of TopHat,
Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF files using original matching files, detects difference table
It reaches.The only q values < 0.01 in Cuffidff outputs, test display is considered as successfully more just differential expression.
4, result
For RNA-seq the results show that compared with normal control tissue, the gene of high expression is 351 in cervical cancer tissues, low
The gene of expression is 294, and difference all has statistical significance (P<0.05).
2 large sample of embodiment verifies the expression of difference expression gene
One, it is detected on transcriptional level
1, tissue collecting
According to the standard collection cervical cancer tissues and corresponding normal control tissue 40 of embodiment 1.
2, tissue RNA extractions and identification
Step is the same as embodiment 1.
3, the design and preparation of primer
Real-time quantitative PCR primer sequence used herein following (commission Sangon Biotech (Shanghai) Co., Ltd.) limited liability company
It designs and synthesizes)：
MNX1 gene primers：
Sense primer：5'-AGGTGAAGATTTGGTTCCA-3'(SEQ ID NO.1)；
Downstream primer 5 '-TTCTGTTTCTCCGCTTCC-3 ' (SEQ ID NO.2),
GAPDH gene primers：
Sense primer：5'-GACCTGACCTGCCGTCTA-3'(SEQ ID NO.3)；
Downstream primer：5'-AGGAGTGGGTGTCGCTGT-3'(SEQ ID NO.4).
4, reverse transcription PCR
Reverse transcription is carried out using Takara reverse transcription reagent box (going to dezymotize containing genomic DNA), specific reaction system is as follows：
Remove the reaction system of genomic DNA：
Table 1 removes the reaction system of genomic DNA
| Reagent | Volume |
| Total serum IgE | 1.0μg |
| gDNA Eraser | 1.0μL |
| 5*gDNA Eraser B buffer | 2.0μL |
| RNase Free water | It mends to 10 μ L |
5min is reacted at 42 DEG C.
Reverse transcription PCR reaction system：
2 reverse transcription PCR reaction system of table
| Reagent | Volume |
| RT Primer Mix | 1.0μL |
| PrimeScript RT Enzyme Mix I | 1.0μL |
| 5*Prime Script Buffer | 4.0μL |
| Remove the reaction system of genomic DNA | 10.0μL |
| RNase Free water | 4.0μL |
37 DEG C of reactions 15min, 85 DEG C of reaction 5s.
5, real-time quantitative PCR
Reaction system：
3 reverse transcription PCR reaction system of table
Reaction condition：95 DEG C of pre-degeneration 30s；
95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 20s, global cycle number are 40 times；
72 DEG C extend 5min (GAPDH is internal reference).
Using 2-△△CtRelative quantification method analyzes the expression of MNX1, and Ct is that thermal cycler detects glimmering in reaction system
The intensity value of optical signal.Computational methods are：Δ Δ Ct=(Ct target gene-Ct reference genes) cervical cancer tissues experimental group-(Ct
Target gene-Ct reference genes) normal control tissue group, 2-△△CtWhat is indicated is the expression of experimental group target gene relative to right
According to the variation multiple of group, analysis of experimental data is completed by Bio-RAD analysis softwares.
6, statistical analysis carries out data analysis using statistics software SPSS19.0, judges uterine neck using paired-samples T-test
The expression of cancerous tissue and MNX1 in normal control tissue sample whether there is the difference on statistical significance.Statistical check is all double
Side is examined, and P < 0.05 are that difference is statistically significant.
7, result
Compared with normal control tissue, in 40 cervical cancer tissues 38 MNX1 gene expressions significantly increase.Statistics knot
Fruit is as shown in Figure 1, compared with normal control tissue, and MNX1 genes significantly increase in cervical cancer tissues, and difference is anticipated with statistics
Adopted (P < 0.05).
Two, it is detected on protein level
1, the cervical cancer tissues total protein being collected into is extracted
(1) the cervical cancer patient tissue and periphery normal cervical tissues of preservation are taken out from liquid nitrogen, room temperature is after its defrosting
Cutting tissue about 100mg is transferred in tissue homogenizer and is shredded tissue using clean scissors；
(2) the about 300 μ L of RIPA lysates containing 1%PMSF are added, are then homogenized on ice；
(3) tissue homogenate is transferred in 1.5mL EP pipes, places 15min on ice；
(4) 12000rpm, 4 DEG C of centrifugation 15min；
(5) it shifts in supernatant to new 1.5mL EP pipes, takes 10 μ L to carry out BCA protein quantifications, remaining is set -20 DEG C and freezes.
2, electrophoresis
Using β-actin as internal reference.50 μ g total proteins are after SDS-PAGE points, electrotransfer to pvdf membrane, with containing 5% defatted milk
1 × TBST room temperature jogs of powder close 1h；It is separately added into MNX1 monoclonal antibodies and β-actin monoclonal antibodies, 4 DEG C overnight；1 × TBST washes film 4
It is secondary, secondary antibody is added, is incubated at room temperature 1h；After 1 × TBST washes film 4 times, it is placed in Super Signal chemical illuminating reagents and reacts
2min makes X-ray expose in darkroom, conventional method developing fixing, therefrom chooses photo expose and utilizes Image Pro softwares
Carry out gray analysis.
3, statistical procedures
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by MNX1 albumen
The gray value of band is normalized.Result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical softwares are next for statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
4, result
Compared with normal control tissue, 38 MNX1 protein levels significantly increase in 40 cervical cancer tissues, difference tool
It is statistically significant.Statistical result is as shown in Fig. 2, compared with normal control tissue, and MNX1 protein levels are aobvious in cervical cancer tissues
It writes and increases, difference has statistical significance (P < 0.05).
Embodiment 2 interferes MNX1 gene expressions
1, RNA interfering design synthesis
SiRNA is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd's design according to MNX1 gene orders.Shanghai Ji agate pharmacy
Technology Co., Ltd. provides and negative control siRNA (siRNA-NC) of the MNX1 genes without sequence homology simultaneously.
siRNA-MNX1：
Positive-sense strand is 5 '-ACUUGUUGAGCUUGAACUGGU-3 ' (SEQ ID NO.5)；
Antisense strand is 5 '-CAGUUCAAGCUCAACAAGUAC-3 ' (SEQ ID NO.6),
2, the culture of Hela cells
Cell is placed in 37 DEG C with containing dual anti-and 10% fetal calf serum 1640 culture mediums, 5%CO2Incubator in training
It supports, every changing 1 culture solution for 24 hours, 48h is passed on 1 time.The cell of logarithmic growth phase carries out subsequent experimental.
3, cell transient transfection
In day before transfection vitellophag, and by cell inoculation 6 orifice plates, per hole about 2x105A cell, overnight incubation are seen
It examines cell fusion degree (about 50-70%) and carries out cell siRNA transient transfections, detailed process is as follows：
1) according to the explanation on siRNA synthesis books, siRNA to 20 μM is diluted using RNase Free water；
2) two sterile EP tubes are taken to be respectively labeled as A and B, 125 μ L/ hole serum free mediums and 5 μ L are added in A pipes
125 μ L/ hole serum free mediums and 5 μ L Lipofectamine 3000 are added in siRNA, B pipe；
3) above-mentioned A is mixed well, the solution of B pipes is stored at room temperature 5min；
4) it is serum free medium to replace the culture medium in 6 orifice plates, per hole 2mL；
5) A of above-mentioned mixing, B mixed solutions is added, cross shake mixes well cell；
6) it is positioned over CO2In incubator, 37 DEG C of progress routine cultures；
7) after transfecting 48h, 1ml Trizol or 200 μ L RIPA lytic cells are separately added into, extract RNA and protein
Real-time quantitative PCR and Western blot detections are carried out, subsequent experimental is carried out after analyzing interference effect.
4, result
As shown in figure 3, compared with negative control group (transfection siRNA-NC), in experimental group (transfection siRNA-MNX1) cell
MNX1 expressing quantities significantly reduce, and difference has statistical significance (P<0.05).
Influence of the expression of 4 MNX1 genes of embodiment to Hela cell Proliferations
The detection of cell Proliferation uses CCK-8 methods
(1) it is transfected according to the method for embodiment 3；
(2) the Hela cells after transfection for 24 hours, suction are abandoned culture medium, with 0.25% trypsin digestion and cell, are contaminated with crystal violet
Liquid counts cell, and it is 2x 10 that cell, which is resuspended, with 1640 culture mediums containing 0.5%FBS and adjusts cell concentration4/ml；
(3) cell suspension 100 μ l, i.e. 2x10 is added under aseptic condition per hole into 96 new well culture plates3A cell, 37
DEG C, 5%CO2It stands for 24 hours；
(4) 10 μ l CCK solution are added into each hole respectively, cultivate 2h；
(5) 450nm wavelength, ultraviolet specrophotometer measure each experimental group light absorption value.
As a result：
The results show that negative control group (transfection siRNA-NC) cell OD values are 0.964 ± 0.108, experimental group (transfection
SiRNA-MNX1) cell OD values are 0.435 ± 0.064.The above results show inhibit MNX1 expression cervical cancer cell proliferation by
Inhibit, difference has statistical significance (P<0.05).
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection domain of the claims in the present invention.
Sequence table
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Claims (10)
1. detecting application of the reagent of MNX1 in the diagnostic tool for preparing cervical carcinoma.
2. application according to claim 1, which is characterized in that the reagent of the detection MNX1 includes detection MNX1 gene tables
Up to the reagent of amount.
3. application according to claim 1 or 2, which is characterized in that the reagent of the detection MNX1 includes that can quantify
The reagent of MNX1 gene mRNAs, and/or the reagent of MNX1 albumen can be quantified.
4. application according to claim 3, which is characterized in that the reagent that can quantify MNX1 gene mRNAs includes real
When quantitative PCR in the primer of specific amplified MNX1 genes that uses；The reagent that MNX1 albumen can be quantified includes specificity
In conjunction with the antibody of MNX1 albumen.
5. a kind of tool for diagnosis of cervical cancer, which is characterized in that the tool includes that can detect MNX1 gene expression amounts
Tool；Preferably, the tool includes that can quantify the reagent of MNX1 gene mRNAs, and/or can quantify MNX1 albumen
Reagent.
6. tool according to claim 5, which is characterized in that the reagent that can quantify MNX1 gene mRNAs includes real
When quantitative PCR in the primer of specific amplified MNX1 genes that uses, the primer sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2；The reagent that MNX1 albumen can be quantified includes the antibody for specifically binding MNX1 albumen.
7. tool according to claim 5 or 6, which is characterized in that the tool includes kit, chip, test paper, high pass
Measure microarray dataset.
8. a kind of drug for treating cervical carcinoma, which is characterized in that the drug includes the reagent for inhibiting MNX1.
9. drug according to claim 8, which is characterized in that the reagent includes that can inhibit MNX1 or be related on MNX1
The expression of the substance of trip or downstream pathway or active reagent.
The application of 10.MNX1 genes or its expression product in the drug for preparing treatment cervical carcinoma.
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| PCT/CN2019/098920 WO2020025029A1 (en) | 2018-08-02 | 2019-08-01 | Diagnostic marker for cervical cancer, and methods for diagnosing and treating cervical cancer |
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| WO2018035138A1 (en) * | 2016-08-15 | 2018-02-22 | Organovo, Inc. | Three dimensional bioprinted tumor models for drug testing |
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