CN106148554A - A kind of mark for carcinoma of prostate detection and the application in test kit thereof - Google Patents
A kind of mark for carcinoma of prostate detection and the application in test kit thereof Download PDFInfo
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Abstract
The invention discloses a kind of mark for carcinoma of prostate detection, described molecular marked compound is expressing protein and fragment, the sum analogous to general Dedekind sum of CCDC42B or this mRNA.The present invention further discloses a kind of individuality that detects and suffer from the test kit of carcinoma of prostate probability, described test kit contains the nucleotide sequence as shown in SEQ ID NO:1.The invention also discloses this marker detection carcinoma of prostate to be correlated with the method whether mrna expression level lower.The CCDC42B of the present invention, as the molecular marked compound of detection carcinoma of prostate, makes prostate cancer diagnosis, more accurately, quickly.And provide therapy target and important evidence for the clinical practice such as gene therapy, Drug therapy.
Description
Technical field
The present invention relates to genetic engineering field, particularly relate to mRNA relevant to carcinoma of prostate as detection carcinoma of prostate
Mark and application in test kit.
Background technology
Carcinoma of prostate is one of common male reproductive system malignant tumor.In world wide, prostate-cancer incidence exists
Occupying second in all malignant tumor of male, the sickness rate in U.S.'s carcinoma of prostate alreadys more than pulmonary carcinoma, becomes first harm
The tumor of men's health.The sickness rate of Asia carcinoma of prostate is well below American-European countries, but presents ascendant trend in recent years, and increases
Long rapider than European and American developed countries.Patients with prostate cancer is mainly elderly men, typically at 50 years old with sequela, and 95%
Being born in the elderly men of more than 60 years old, incidence rate increases the most with age.Carcinoma of prostate is the most without any disease
Shape, even if uncomfortable, is also not enough to cause the attention of patient, when tumor increases urethra, the most often increases with prostate
Life is obscured mutually.First patient in China about 80% finds that metastasis focus just finds carcinoma of prostate.Now, pathological changes has reached late
Phase, prognosis mala.
The clinical diagnostic modalities of carcinoma of prostate currently mainly has serum PSA (PSA) detection, rectum to refer to
Inspection, the detection of endorectal ultrasonography ripple, living tissue pathologic finding etc..PSA (Prostate Specific Antigen) is prostata tissue
Specific antigen, is understood PSA by prostatic anatomical structure and enters in blood and urine by prostate duct, some cases or
Under physiological conditions, PSA can enter in blood, such as prostatitis, urine retention, prostate infection, prostatic hyperplasia and prostate
By detection Serum PSA level, cancers etc., therefore predict that carcinoma of prostate has certain false positive rate.From the beginning of 1991, inspection
Surveying PSA content in serum and be used for predicting carcinoma of prostate, it is positive that content is considered carcinoma of prostate higher than 4ng/mL, sensitivity 79%,
Specificity 20%-59%, average sensitivity about 33%, in concentration 4-10ng/mL gray area part, specificity is minimum, at this moment
Generally require and be determined by invasive aspiration biopsy, bring considerable distress and spirit and financial burden to patient.So
PSA can not be preferably as the mark of diagnosis of prostate cancer.Digital rectal examination is the method for practicality the simplest, most economical, main
The forefinger of doctor to be passed through touches prostate, in order to find a lot of asymptomatic patients with prostate cancer, it is possible to obtains and examines in early days
Chance that is disconnected and that effect a radical cure.But all there is limitation in above method.The limitation of such as digital rectal examination is mainly at 4 aspects: (1)
When patient's prostate lump is little, easy to cause missed diagnosis；(2) enlargement of some patients carcinoma of prostate is inconspicuous, but is already belonging to late period, is difficult to root
Control；(3) patient's rectum can not use this to detect when having illness；(4) have during doctors experience deficiency and fail to pinpoint a disease in diagnosis or the possibility of mistaken diagnosis.
Recent studies indicate that, mRNA is closely related with carcinoma of prostate, they may participate in generations of tumor, development and
Transfer, so pathogenesis, early diagnosis, individualized treatment, the detection of transfer and prognosis etc. to tumor may have accordingly
Effect.
Summary of the invention
In order to realize the early diagnosis of carcinoma of prostate, individualized treatment, it is an object of the invention to provide a kind of new before
Row adenocarcinoma mRNA, and the expressing protein of this mRNA and fragment, sum analogous to general Dedekind sum.
It is another object of the present invention to the purposes that carcinoma of prostate is correlated with in mRNA row adenocarcinoma before detection.
For achieving the above object, present invention firstly provides a kind of mRNA mark for carcinoma of prostate detection, described mark
Will thing is expressing protein and fragment, the sum analogous to general Dedekind sum of CCDC42B or this mRNA；Described CCDC42B or this mRNA
Expressing protein and fragment, sum analogous to general Dedekind sum down-regulated expression in carcinoma of prostate biological sample.
Preferably, described mark includes the nucleotide sequence as shown in SEQ ID NO:1, and this nucleotides sequence is classified as
A part of sequence of CCDC42B gene.
Further, the invention provides a kind of individuality that detects and suffer from the test kit of carcinoma of prostate probability, described examination
Agent box contains the nucleotide sequence as shown in SEQ ID NO:1.
Preferably, described test kit includes the quantitative detecting reagent of the nucleotide sequence shown in SEQ ID NO:1；Described core
Nucleotide sequence quantitative detecting reagent includes the primer shown in SEQ IDNO.2 and SEQ ID NO.3.
Preferably, described test kit includes 10 × Buffer, dNTP, MgCl2, Taq enzyme, reverse transcriptase and RNase suppression
Agent.Preferably, described test kit also includes the normal prostata tissue of people or the total serum IgE of cell or cDNA.
Further, the invention provides a kind of carcinoma of prostate that detects to be correlated with the method whether mrna expression level lower.
(1) in detection testing sample, carcinoma of prostate is correlated with the expression of mRNA, and wherein said carcinoma of prostate is correlated with mRNA
It is CCDC42B；
(2) expression of mRNA of carcinoma of prostate in cell to be measured being correlated with is compared with a control, so that it is determined that prostate
Whether cancer expression is lowered.
Preferably, described testing sample is cell tissue sample.
Preferably, described cell tissue sample includes prostate cancer tissue and cancer beside organism.
Preferably, described cancer beside organism is normal structure.
Preferably, described detection is by genechip detection method, quantitative fluorescent PCR, protein chip detection method, immunity
Method or Ag-Ab detection method are carried out.
Preferably, described immunization method is ELISA detection and/or gold colloidal detection.
Preferably, described ELISA method is for using ELISA detection kit, and this test kit includes: be coated CCDC42B Dan Ke
The solid phase carrier of grand antibody, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, cleaning mixture, enzyme reaction
Stop buffer etc..Described colloidal gold method is for using detection kit, and described antibody can use commercially available CCDC42B monoclonal antibody
Or polyclonal antibody.It is highly preferred that described gold-immunochromatographyreagent reagent for assay box is to use colloidal gold immunochromatographimethod technology or gold colloidal
Percolation.It is highly preferred that detection zone (T) specking on described gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-
CCDC42B antibody, quality control region (C) specking have immunoglobulin IgG.
Beneficial effects of the present invention is as follows:
The invention discloses a kind of CCDC42B relevant to carcinoma of prostate, and be further characterized by this CCDC42B or this mRNA
Expressing protein and fragment, sum analogous to general Dedekind sum expression in prostate cancer lower.This mRNA is utilized to detect prostatitis
Adenocarcinoma can not only accomplish early diagnosis fast and effectively, and provide for the clinical practice such as gene therapy, Drug therapy and control
Treat target spot and important evidence.
Accompanying drawing explanation
Fig. 1 is prostate cancer tissue and cancer beside organism's CCDC42B expression.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
The conventional means that technological means used by is well known to those skilled in the art, reagent used can be commercially available.
The experimental technique of unreceipted actual conditions, usually this area conventional method in embodiment, as according to normal condition
Such as Sambrook et al., molecular cloning, the described condition in laboratory manual (third edition) (Science Press, 2002), or according to
Condition proposed by reagent manufacturing firm.
The present inventor carries out high flux transcript profile to the mRNA of prostate cancer tissue sample and cancer beside organism's sample
Order-checking, carries out genescreen by bioinformatics method, picks out candidate mRNACCDC42B, in existing research not
The report that CCDC42B is relevant with carcinoma of prostate, further, inventor carried out molecular biology method checking it was confirmed
CCDC42B is down-regulated expression in prostate gland cancer cell.
The CCDC42B of the present invention is known mRNA before making the present invention, and its essential information is as follows: Genbank accession number:
NCBI reference sequences: NM_001144872.1, derives from human genome.
The present invention also uses RT-PCR method and method for gene chip to detect above-mentioned mRNA by prostate cancer tissue and cancer
The expression of normal structure, and demonstrate this mRNA down-regulated expression in carcinoma of prostate.
Terms used herein " down-regulated expression " refers to the sequence corresponding to expressed gene, wherein the measurement card of sequence amount
Bright, and from normal individual or from being determined by stages of prostate cancer the individuality identifying morbid state different with carcinoma of prostate
Same gene in the biological sample separated is compared, and described gene is from suffering from carcinoma of prostate or being determined by stages of prostate cancer
Carcinoma of prostate identified that the expression in the biological sample separated in the individuality of morbid state reduces.According to the present invention,
" down-regulated expression " refer to by the inventive method intensity for hybridization measure at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10% or more express reduction, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more
Low.
Embodiment 1 high-flux sequence screening difference expression gene
1, sampling
In BJ Union Hospital's urological surgery, tissue specimen is obtained during taking in December, 2015 in October, 2012 to
27 examples, all specimen all confirm through pathological examination, and wherein cancer beside organism's sample 8 example, carcinoma of prostate specimen 19 example, after numbering
Put-80 DEG C of cryogenic refrigerators to preserve.
2, tissue samples is carried out Total RNAs extraction
UseReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation
Carrying out by product description, concrete operations are as follows:
After collecting sample, frozen mortar tissue being put into pre-cooling after liquid nitrogen, taking-up is ground, sample to be organized
After this is powdered:
1. Trizol, room temperature preservation 5 minutes are added；
2. adding chloroform 0.2mL, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5 minutes-10 minutes；
3. 12000rpm high speed centrifugation draws upper strata aqueous phase (inhale 70%) in another new centrifuge tube pipe after 15 minutes, notes
It is not drawn onto the protein substance between two-layer aqueous phase.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, the most reverse
Mixing, is placed in 10 minutes on ice；
4. 12000rpm carefully discarded supernatant at a high speed after 15 minutes, added 75% in the ratio of 1mL/mL Trizol
Paint precipitation (4 DEG C of preservations) washed by DEPC ethanol, washes paint precipitate, vibration mixing, 12000rpm high speed centrifugation 5 minutes at 4 DEG C；
5. discard ethanol liquid, ambient temperatare put 5 minutes fully to dry precipitation, add the water dissolution that processed of DEPC and sink
Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-80 DEG C.RNA mass is sentenced
Calibration standard: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophoresis pattern has 28S, 18S band clearly；
70 DEG C of water bath heat preservation electrophoresis patterns after 1 hour and the collection of illustrative plates no significant difference before water bath heat preservation.
3, the quality analysis of RNA sample
RNA extract after agarose gel electrophoresis, from electrophoresis result can with preliminary judgement extract RNA sample up-to-standard with
No, if to may be used for further transcriptome analysis.And then by NanoDrop1000 spectrophotometer detection RNA sample
Extraction situation, the sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.
4, high-flux sequence
Order-checking platform is the HiSeq 2500 high-flux sequence platform of Illumina company, carries out the high flux transcript profile degree of depth
Order-checking, after order-checking, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/
Fastqc/) software carries out total evaluation to the quality of sequencing data, and the mass value including base is distributed, and the position of mass value is divided
Cloth, G/C content, PCR duplication content, the frequency etc. of kmer.When differential genes expression analysis, according to obtaining
FPKM value, use internationally recognized algorithm EBSeq to carry out differential screening.Wherein, during screening, LOG2FC>1 or<-1, FDR<
0.05.In order to be better understood from the function of difference expression gene, we difference expression gene has been carried out Gene Ontology and
Signal path is analyzed, and difference expression gene carries out functional annotation and protein interaction analysis of network, in view of above number
According to the result analyzed, in conjunction with document, we have screened differential expression CCDC42B, and this mRNA expresses in prostate cancer tissue sample
Lower.
Embodiment 2RT-PCR checking prostate cancer tissue and cancer beside organism's CCDC42B expression
1, material
34 example prostate cancer tissue samples and 8 example cancer beside organism samples are taken from BJ Union Hospital and are secreted during 2012 to 2015 years
Carcinoma of prostate sample in urine surgical operation, is grouped it and numbers.All sample standard deviations confirm through pathological examination.
2, method
2.1 pairs of tissue samples carry out Total RNAs extraction, with the extracting method of embodiment 1.
2.2 reverse transcription synthesis cDNA
UseIII Reverse Transcriptase (invitrogen, article No. 18080-044)
Carrying out cDNA reverse transcription, experimental implementation is carried out by product description, and concrete operations are as follows:
Use Reverse Transcriptase kit, with RT Buffer, l μ g total serum IgE is carried out converse record and synthesize cDNA.Use 25 μ L
Reaction system, each sample takes 1 μ g total serum IgE as template ribonucleic acid.It is saved in-20 DEG C after the cDNA diluted sample 10 times of acquisition
Refrigerator is standby.
2.3Real-Time PCR
2.3.1 instrument and the method for analysis
With ABI 7500 type quantitative real time PCR Instrument, use 2-ΔΔCtMethod carries out data relative quantitative assay.
2.3.2 design of primers
Using online primer-design software, gene order is with reference to NCBI:NM_001144872.1
(CCDC42B), interior participation in the election GAPDH, synthesized by invitrogen company after design of primers.Concrete primer sequence is such as
Under:
Table 1 primer sequence
2.3.3 fluorescent quantitative PCR
25 μ L reaction systems are sequentially added into following component by table 2:
Table 2 Real Time reaction system
| Component | Addition |
| 2×mix | 10μL |
| Forward primer (10uM) | 0.5μL |
| Downstream primer (10uM) | 0.5μL |
| Template | 2μL |
| Add sterile purified water | To 25 μ L |
Use PowerGreen PCR Master Mix (invitrogen, article No. 4367659) is respectively to mesh
Gene primer and reference gene primer expand.Experimental implementation is carried out by product description.Amplification program is: 95 DEG C
5min, (95 DEG C of 15sec, 61 DEG C of 45sec) × 35 circulations.Carry out solubility curve analysis at 60-95 DEG C simultaneously.Reaction terminates
After, the PCR primer taking 5ul carries out 2% sepharose electrophoresis, reclaims test kit (invitrogen company) with quick glue and by size is
The fragment of 307bp carries out cutting glue and reclaims and check order, and result blast software carries out homology analysis.
Experimental result shows, real-time quantitative PCR amplification curve flex point understands, amplification curve entirety collimation is good, shows each
The amplification efficiency of reaction tube is close, and the limit is flat and nothing raises up now, and exponent phase slope is relatively big, illustrates that amplification efficiency is higher；
Sample amplified production solubility curve is all unimodal, illustrates that amplified production only has one, for specific amplification；According to qRT-PCR's
Relative quantification formula: 2-ΔΔCt× 100%, compare CCDC42B expression in prostate cancer tissue and cancer beside organism.Knot
Fruit such as Fig. 1 shows: compared with cancer beside organism, and CCDC42B expression in carcinoma of prostate suffers from tissue is only in cancer beside organism
48%, it was demonstrated that this mrna expression lower；After RT-PCR product is recovered, the full-automatic sequenator of ABI3730 checks order, should
The nucleotide sequence of the fragment of 307bp is as shown in SEQ ID NO.1.With VectorNTI advance 10 software
Whole with CCDC42B for this sequence mRNA sequence is compared by (Invitrogen company), and comparison result shows, such as SEQ ID
Nucleotides sequence shown in NO:1 is classified as a part of sequence of CCDC42B gene, and coincidence rate is 100%.
Embodiment 3 gene chip checking prostate cancer tissue and cancer beside organism's sample CCDC42B expression
1, the acquirement of material, with embodiment 2.
2, the extraction of total serum IgE, with the method for embodiment 1.
3, gene chip checking
After the linearized amplification of total serum IgE, cy3-UTP labelling, the cRNAs after fluorescent labeling uses RNEASY Mini Kit
Purification, carries out fragmentation process with the RNA Fragmentation Reagents of Amhion to the cRNAs that labelling is good.Use U.S.
People's full genome chip of expression spectrum (4x 44K gene) of Agilent company of state, 65 DEG C of hybridization 17h in chip hybridization stove, then
Eluting, dyeing, finally by Agilent DNA MicroarrayScanner scanner scanning.
Chip after hybridization, after chip scanner reads data point, imports data to analyze software, for two groups of ratios
Natural logrithm absolute value more than 2.0 or less than 0.5 mRNA as differential expression mRNA.
Use SPSS 13.0 statistical software carry out data analysis, group difference compare employing one factor analysis of variance method, P <
0.05 difference significance.Result shows, compared with cancer beside organism sample, and CCDC42B in prostate cancer tissue sample
MRNA level in-site is only 50%, and difference has statistical significance (P < 0.05).
Embodiment 4 carcinoma of prostate detection kit
The primer sets obtained based on embodiment 2, assembles the test kit for carcinoma of prostate of the present invention, described reagent
Box includes that the primer of specific amplified nucleotide sequence as shown in SEQ ID NO.1 is to such as SEQID NO:2 and SEQ ID NO:3
Shown in, and the primer of specific amplified reference gene (GAPDH) is to as shown in SEQ ID NO:4 and SEQ ID NO:5；Also include
SYBR Green polymerase chain reaction system, such as PCR buffer, SYBR Green fluorescent dye, dNTPs.Described PCR buffers
The composition of liquid is 25mM KCL, 2.5mM MgCL2, 200mM (NH4)2SO4。
By the optimization to primer concentration and annealing temperature, finally determine that reaction system is as shown in table 3:
Table 3 PCR reaction system
| Component | Addition |
| SYBRGreen polymerase chain reaction system | 12.5μL |
| Forward primer (10 μMs) | 0.5μL |
| Downstream primer (10 μMs) | 0.5μL |
| Template cDNA | 2.0μL |
| Add sterile purified water | To 25 μ L |
Optimum reaction condition is:
95 DEG C of denaturations 5min, (72 DEG C extend 35sec for 95 DEG C of degeneration 15sec, 61 DEG C of annealing 45sec) × 40 circulations,
72 DEG C extend 15min.
Take a small amount of prostatic cell of patient Bai to be detected, use conventional method (or using specific test kit) from
Prostatic cell extracts RNA, uses seminal plasma fructose detection kit, carry out PCR reaction according to optimal reaction system and condition, use examination
In agent box, normal cancer beside organism cDNA is as the comparison cDNA in Real-TimePCR detection by quantitative, detects tissue samples such as SEQ
The expression change of nucleotide sequence shown in ID NO.1 relatively normal cancer beside organism.
Testing result, compared with normal cancer beside organism, the nucleotides sequence as shown in SEQ ID NO.1 is listed in this detection patient
Prostatic cell expression is only in cancer beside organism 48%.Detecting further through clinic, this example patient determines suffer from prostatitis
Adenocarcinoma, the test kit testing result that Clinical detection result is prepared with the present invention is consistent.Infer accordingly, the diagnosis examination of this carcinoma of prostate
Patients with prostate cancer can clearly be distinguished by agent box, and provides diagnostic clue as clinic.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (9)
1. the mark for carcinoma of prostate detection, it is characterised in that described mark is the table of CCDC42B or this mRNA
Reach albumen and fragment, sum analogous to general Dedekind sum.
2. mark as claimed in claim 1, it is characterised in that described CCDC42B or the expression product of this mRNA are front
Down-regulated expression in row adenocarcinoma tissue.
3. mark as claimed in claim 1 or 2, it is characterised in that described mark includes as shown in SEQ ID NO:1
Nucleotide sequence.
4. one kind is detected individuality and suffers from the test kit of carcinoma of prostate probability, it is characterised in that described test kit contains such as SEQ
Nucleotide sequence shown in ID NO:1.
5. test kit as claimed in claim 4, it is characterised in that described test kit includes the nucleoside shown in SEQ ID NO:1
The quantitative detecting reagent of acid sequence；Described quantitative detecting reagent includes the primer shown in SEQ IDNO.2 and SEQ ID NO.3.
6. test kit as claimed in claim 4, it is characterised in that described test kit also include 10 × Buffer, dNTP,
MgCl2, Taq enzyme, reverse transcriptase and RNase inhibitor.
7. one kind is detected carcinoma of prostate and is correlated with the method whether mrna expression level lower, it is characterised in that it includes walking as follows
Rapid:
(1) in detection testing sample, carcinoma of prostate is correlated with the expression of mRNA, and the wherein said carcinoma of prostate mRNA that is correlated with is
CCDC42B；
(2) expression of mRNA of carcinoma of prostate in cell to be measured being correlated with is compared with a control, so that it is determined that carcinoma of prostate table
Reach whether level is lowered.
8. method as claimed in claim 7, it is characterised in that described testing sample is cell tissue sample.
9. method as claimed in claim 7, it is characterised in that described detection is by genechip detection method, fluorescent quantitation
PCR, protein chip detection method, immunization method or Ag-Ab detection method are carried out.
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| CN107043811A (en) * | 2017-02-08 | 2017-08-15 | 泰山医学院 | Application of the CFAP20 genes in diagnosis of osteoporosis |
| CN111679074A (en) * | 2020-07-11 | 2020-09-18 | 成都益安博生物技术有限公司 | Prostate cancer peripheral blood TCR marker and detection kit and application thereof |
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Application publication date: 20161123 |