CN106367507A - Application of LOC283856 in preparation of prostatic cancer early treatment or diagnosis products - Google Patents
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Abstract
The invention discloses application of LOC283856 serving as a molecular marker for detecting prostatic cancer. It is further proved that the expression of LOC283856 is up-regulated in prostatic cancer tissue. The invention further discloses application of LOC283856 in preparation of prostatic cancer early treatment or diagnosis products. The products comprise preparations or diagnosis kits for treating the prostatic cancer. When the molecular marker is used for detecting the prostatic cancer, early detection can be fast and effectively achieved, and a therapeutic target and an important basis are provided for gene therapy, medicine treatment and other clinical application.
Description
Technical field
The present invention relates to biological technical field is and in particular to loc283856 is in preparation carcinoma of prostate early treatment or diagnosis
Application in product.
Background technology
Carcinoma of prostate is the malignant tumor betiding in human male prostate tissue, has become as male in the world
Two big class kinds of tumor, positioned at cancer related mortality rate the 6th.In developed countries and regions such as America and Europes, it is that male is most common
Malignant tumor, its mortality rate occupies the second of various cancers；In Asia, its sickness rate is less than western countries, but is in recent years
Rapid ascendant trend, and increase rapider than European and American developed countries.China's prostate-cancer incidence is also improving year by year, position
In the 3rd of male genitourinary system malignant tumor, it is only second to bladder cancer and renal carcinoma.Carcinoma of prostate is typically after 50 years old
Morbidity, 95% betides the elderly men of more than 60 years old, and incidence rate constantly increases with age.Carcinoma of prostate early stage
Many no any symptoms, even if uncomfortable, are also not enough to cause the attention of patient, when tumor increases urethra, and often
Obscure with prostatic hyperplasia phase.Find that metastasis focus just finds carcinoma of prostate first in the patient of China about 80%.Now,
Pathological changes have reached late period, prognosis malas.So, the early diagnosiss of carcinoma of prostate have earthshaking to the treatment of prostate cancer patient
Meaning.
The clinical diagnostic modalities of carcinoma of prostate mainly have prostate digital rectal examination (dre), serum prostate special at present at present
Specific Antigen (psa) detection, the detection of endorectal ultrasonography ripple, living tissue pathologic finding etc..Digital rectal examination is the warp of prostate cancer diagnosis
Allusion quotation technology, mainly touches prostate by the forefinger of doctor, to judge the size and shape of prostatic tubercle, thus judging whether
With carcinoma of prostate.But the limitation of digital rectal examination is also very strong: when (1) patient prostate lump is little, easy to cause missed diagnosis；(2) part
Patient's prostate cancer enlargement is inconspicuous, but is already belonging to late period；(3) patient's rectum is had and can not be detected using this during illness；(4) doctor
May have when lacking experience fail to pinpoint a disease in diagnosis or mistaken diagnosis possibility.Although prostate ultrasound examination simple and direct-viewing operation, not damaged,
The method is easily confused with prostatitis and prostate hyperplasia, has been no longer used to stages of prostate cancer diagnosis.Living tissue pathology is examined
Look into because it is traumatic, complexity cannot function as the means of primary dcreening operation, but its goldstandard that to be carcinoma of prostate make a definite diagnosis, general and its other party
Law technology is used in conjunction.At present the sieving and diagnosis of carcinoma of prostate are relied primarily on serum psa detection and prostate biopsy pathological biopsy phase
In conjunction with method.Psa in blood not high (not higher than 4ng/ml) under normal circumstances, when being in carcinoma of prostate and other prostate
During disease disease state, psa raise become the most sensitive tumor mark of current examination carcinoma of prostate, but psa increase also common with non-before
Row adenocarcinoma disease, diseases of urinary system as multiple in prostatitis, prostatic hyperplasia etc. is relevant, is therefore difficult to make a definite diagnosis, or even can
The delay of flase drop, mistaken diagnosis or the state of an illness of disease can be led to.Additionally, psa increases when making a definite diagnosis carcinoma of prostate, often patient has belonged to
In middle and late stage, do not reach the purpose of early diagnosiss.And the number of times of the recall rate of aspiration biopsy and living body puncture, position have larger
Relation, is not highly stable diagnosis scheme.Therefore, study more effective prostate cancer marker to the morning improving carcinoma of prostate
Phase diagnoses and formulates rational individualized treatment scheme for patient, reduces patients with prostate cancer mortality rate and quality of making the life better has
Important meaning, if a kind of detection kit can be had, can improve recall rate and the accuracy rate of carcinoma of prostate, for prostate
The clinical treatment of cancer has great meaning.
Loc283856 belongs to lncrna (longnon-codingrna, long-chain non-coding rna), is a class transcript length
More than non-coding rna of 200bp nucleotide, research in recent years finds that it is the rna that a class has important biomolecule function, participates in base
Because of multiple important regulation and control such as transport in the group marking, chromosome silence, chromatin modification, transcriptional activation, transcription interference, core
Journey, all plays important regulation and control in the vital movement such as cell differentiation and growth, genetic transcription and translation, heredity and epigenetic
Effect.In recent years, more and more authority's research confirms that lncrna plays suppression in tumor develops or promotes tumor
Effect, at aspects such as modulate tumor cell proliferation, apoptosis, cell cycle, invasive ability, be respectively provided with particularly significant work
With.Oneself has more lncrnas to be proved including breast carcinoma, prostate, melanoma, hepatocarcinoma, colon cancer, bladder cancer etc. at present
Interior mankind's kinds of tumors has differences and expresses and execute important adjusting function.The generation of early stage effective detection tumor,
The curative effect of development and raising cancer therapy drug is particularly important for the treatment of cancer, and invention novel tumor markers as diagnosis and are controlled
The target spot treated is always the focus of tumor research.
Recent studies indicate that, lncrna is closely related with carcinoma of prostate, they may participate in generation, the development of tumor
And transfer, so the pathogenesis of tumor, early diagnosiss, individualized treatment, the detection of transfer and prognosis etc. may be had corresponding
Effect.
Content of the invention
In order to realize the early discovery of carcinoma of prostate, early intervention, it is an object of the invention to provide a kind of and prostate
The related new molecular marked compound of cancer.
The a further object of the present invention is the application providing this molecular marked compound before detection in row adenocarcinoma.
For achieving the above object, present invention firstly provides loc283856 is as the molecular marked compound of detection carcinoma of prostate
Application.
Preferably, described molecular marked compound loc283856 comprises the specific nucleotide sequence as shown in seq id no:2.
The present invention, it has been investigated that loc283856 up-regulated in carcinoma of prostate biological sample, shows loc283856
There is close dependency with the tumorigenesis of carcinoma of prostate.
Further, the invention provides loc283856 preparation carcinoma of prostate early treatment or diagnostic products in should
With.
Preferably, described product includes preparation or the diagnostic kit treating carcinoma of prostate.
It is furthermore preferred that containing the reagent of suppression loc283856 expression in described preparation.
Further, the invention provides loc283856 is in preparation prevention, alleviation and/or the medicine treating carcinoma of prostate
In application.
Preferably, described medicine includes the sirna of loc283856.
Further, the invention provides a kind of prostatic cancer early diagnosis kit, described diagnostic kit contains
The primer pair of nucleotide sequence as shown in seq id no:2 for the amplification.
It is furthermore preferred that described prostatic cancer diagnostic reagent kit is it is characterised in that include quantitative pcr reaction system:
(1) nucleotide sequence as shown in seq id no:2 for the specific amplified and comparison gapdh two are to primer；
The primer sequence of nucleotide sequence as shown in seq id no:2 for the detection is as follows:
Upstream primer sequence: seq id no:3
Downstream primer sequence: seq id no:4
The primer sequence of comparison gapdh is as follows:
Upstream primer sequence: seq id no:5
Downstream primer sequence: seq id no:6
(2) sybr green polymerase chain reaction system, including pcr buffer, sybr green fluorescent dye,
Dntps etc..
Beneficial effects of the present invention are as follows:
The invention discloses a kind of molecular marked compound loc283856 of detection carcinoma of prostate, and it is further characterized by
Loc283856 raises in expression in prostate cancer.Can not only quickly be had using this molecular marker analyte detection carcinoma of prostate
Imitate accomplishes early detection, and provides therapy target and important evidence for the clinical practice such as gene therapy, Drug therapy.
Brief description
Loc283856 up-regulated in prostate cancer tissue sample in embodiment 2 in Fig. 1 present invention；
In embodiment 4 in Fig. 2 present invention, loc283856-sirna significantly inhibits prostate gland cancer cell propagation.
Specific embodiment
Following examples are used for the present invention is described, but are not limited to the scope of the present invention.If not specializing, embodiment
In the conventional meanses that are well known to those skilled in the art of technological means used, reagent used can be commercially available.
The present inventor carries out high-flux sequence to 10 prostate cancer tissue samples and corresponding cancer beside organism sample,
Carry out genescreen in conjunction with bioinformatics method, pick out candidate lncrnaloc283856, in existing research not
The loc283856 report related with carcinoma of prostate, further, inventor carried out molecular biology method checking it was confirmed
Loc283856 raises in expression in prostate cancer, and its related preparations can be used for treating carcinoma of prostate.
The loc283856 of the present invention is known lncrna before making the present invention, and its essential information is as follows:
Genbank accession number: gene id:283856, from human genome, its nucleotide sequence such as seq id
Shown in no.1.
The present invention adopts rt-pcr method to detect expression in patients with prostate cancer and normal population for the above-mentioned lncrna, and
Demonstrate this lncrna up-regulated in patients with prostate cancer.
Fluorescent quantitation pcr method is by fluorescent dye or fluorescently-labeled specific probe, and pcr product is marked
Follow the tracks of, real time and on line monitoring course of reaction, product can be analyzed in conjunction with corresponding software, calculate testing sample template
Initial concentration.The appearance of fluorescent quantitation pcr, greatly simplifies the process of detection by quantitative, and is truly realized absolute quantitation.
The appearance of multiple detecting systems, makes the selectivity of experiment higher.Automation mechanized operation improves work efficiency, and reaction is quick, repetition
Property is good, sensitivity is high, high specificity, result are clear.
Cck-8 method is a kind of method of new detection cell proliferation.From its Cleaning Principle unlike mtt colorimetry
For living cells mitochondria dehydrogenation enzymatic conversion yellow crystal be high water soluble it is not necessary to cell lysis, can directly carry out colorimetric.
Measure its absorbance value with enzyme-linked immunosorbent assay instrument at 450nm wavelength, reference wavelength is 600-650nm.The method is extensive
For cell proliferating determining, screening anti-tumor medicine, cell toxicity test and anticancer sensitivity sense test.Its feature is operation
It is not necessary to radiosiotope and organic solvent, detection sensitivity height, little to cytotoxicity, repeatability is better than mtt method to simplicity.
The nucleotide full length sequence of loc283856 of the present invention or its fragment generally can be with pcr TRAP, recombination method or people
The method of work synthesis obtains.For pcr TRAP, can be according to published relevant nucleotide sequence, especially open reading frame
Sequence is designing primer, and the cdna with commercially available cdna storehouse or as prepared by conventional method well known by persons skilled in the art makees
For template, expand and obtain relevant sequence.When sequence is longer it is often necessary to carry out twice or repeatedly expanding, then will be many again
The secondary fragment amplifying is stitched together by proper order.
Once obtaining relevant sequence it is possible to obtain relevant sequence in large quantity with recombination method.This typically will
It is cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by conventional method and obtains relevant sequence.
Additionally, also relevant sequence can be synthesized with the method for synthetic, when especially fragment length is shorter.Generally, by first synthesizing
Multiple small fragments, are then attached obtaining the very long fragment of sequence again.
Embodiment 1 high-flux sequence screens difference expression gene
1st, sample
Choose 10 fresh prostate cancer tissues and corresponding cancer beside organism, sampling derives from BJ Union Hospital 2012 10
The prostate cancer patient of row radical-ability prostate excision bilateral pelvic lymphadenectomy during the moon in December, 2015, operation
Take carcinoma of prostate and cancer beside organism to put into liquid nitrogen after excision prostate under the guidance of Pathologis immediately, number rearmounted -80
DEG C cryogenic refrigerator preserves.
2nd, tissue samples are carried out with total rna extraction
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation
Carry out by product description, concrete operations are as follows:
It is ground in frozen mortar tissue samples being put into pre-cooling after the liquid nitrogen, taking-up after collecting sample, treat group
Knit sample powdered rear:
1. 1mltrizol, room temperature preservation 5 minutes are entered；
2. 0.2ml imitated by chlorination, uses forced oscillation centrifuge tube, fully mixes, and places 5 minutes -10 minutes under room temperature；
3. 12000rpm high speed centrifugation is drawn upper strata aqueous phase (inhaling 70%) and in another new centrifuge tube pipe, is noted after 15 minutes
The protein substance between two-layer aqueous phase must not be drawn onto.Move into new pipe, add isopyknic -20 DEG C pre- cold isopropanols, fully reverse
Mix, be placed in 10 minutes on ice；
4. 12000rpm high speed carefully discarded supernatant 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, place 5 minutes under room temperature fully to dry precipitation, add the water dissolution that depc was processed to sink
Form sediment；
6. use nanodrop2000 ultraviolet spectrophotometer measurement rna purity and concentration, frozen in -80 DEG C.Rna mass is sentenced
Calibration is accurate: between the od260/od280 value of rna sample is for 1.7-2.2；Total rna electrophoresis pattern has clearly 28s, 18s band；
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.
3rd, the quality analysiss of rna sample
Rna extract after agarose gel electrophoresiies, from electrophoresis result can with preliminary judgement extract rna sample quality qualified with
No, if to can be used for further transcriptome analysis.And then rna sample is detected by nanodrop1000 spectrophotometer
Extraction situation, the sample requirement of rna-seq sequencing: od260/od280 is 1.8-2.2.
4th, high-flux sequence
Microarray dataset is the hiseq 2500 high-flux sequence platform of illumina company, carries out high flux transcript profile depth
Sequencing, after sequencing, we use fast-qc (http://www.bioinformatics.babraham.ac.uk/projects/
Fastqc/) software carries out total evaluation to the quality of sequencing data, and including the quality Distribution value of base, the position of mass value is divided
Cloth, gc content, pcr duplication content, frequency of kmer etc..In differential genes expression analysis, according to obtaining
Fpkm value, differential screening is carried out using internationally recognized algorithm ebseq.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 carries out functional annotation to difference expression gene, in view of the result of data above analysis, in conjunction with document we
Screen differential expression loc283856, loc283856 up-regulated in carcinoma of prostate sample tissue.
Loc283856 expression in embodiment 2 rt-pcr checking prostate cancer tissue
1st, material
Choose 10 patients with prostate cancer, sampling is during BJ Union Hospital's in October, 2012 in December, 2015
Row radical-ability prostate excision the prostate cancer patient of bilateral pelvic lymphadenectomy, in pathology after excision prostate
Carcinoma of prostate and cancer beside organism is taken to put into liquid nitrogen under the guidance of section doctor immediately, rearmounted -80 DEG C of cryogenic refrigerators of numbering preserve.
2nd, method
2.1 pairs of subjectss' tissue samples carry out total rna extraction, with the extracting method of embodiment 1.
2.2 reverse transcription synthesis cdna
UsingIii reverse transcriptase (invitrogen, article No. 18080-044)
Carry out cdna reverse transcription, experimental implementation is carried out by product description, and concrete operations are as follows:
Using Reverse Transcriptase kit, carry out converse record synthesis cdna with RT Buffer rna total to l μ g.Using 25 μ l
Reaction system, each sample takes the total rna of 1 μ g as template rna.It is standby that -20 DEG C of refrigerators are put in the cdna preservation obtaining.
2.3、real-time pcr
2.3.1 instrument and analysis method
With abi 7500 type fluorescent quantitation pcr instrument, adopt 2-△△ctMethod carries out the relative quantitative assay of data.
2.3.2 design of primers
Using online primer-design software, gene order is with reference to ncbi:nr_027078.1 (loc283856), interior participation in the election
Gapdh, is synthesized by invitrogen company after design of primers.Concrete primer sequence is as shown in table 1:
Table 1 primer sequence
Operating process is as follows:
(1) reaction system: use powerGreen pcr master mix (invitrogen, article No.
4367659) expanded, experimental implementation is carried out by product description.Amplification program is: 95 DEG C of 5min, (95 DEG C of 15sec, 61 DEG C
45sec, 72 DEG C of 35sec) × 40 circulations.
Table 2realtime reaction system
| Component | Addition |
| 2×mix | 10μl |
| Forward primer (10 μm) | 0.5μl |
| Downstream primer (10 μm) | 0.5μl |
| Template | 2μl |
| Add sterile purified water | To 25 μ l |
(2) primer screening
After each sample cdna is mixed, carry out 5 times of gradient dilutions as template, after dilution, sample respectively takes 2 μ l to make template,
Expanded with genes of interest primer and reference gene primer respectively, carried out melt curve analysis analysis at 60-95 DEG C, according to expansion simultaneously
Increasing Efficiency is high and the unimodal principle of solubility curve carries out primer screening.
(3) sample realtime-pcr detection
Take 2 μ l to make template by after the 10 times of dilutions of each sample cdna, respectively genes of interest primer and reference gene primer are entered
Row amplification.Carry out solubility curve analysis at 60-95 DEG C simultaneously.
(4) sepharose electrophoresis
After above-mentioned reaction terminates, the pcr product of 5 μ l is taken to carry out 2% sepharose electrophoresis, with quick glue reclaim test kit
(invitrogen company) fragment for 157bp carries out cutting glue reclaim by size, takes part to be sequenced, result is entered with blast software
Row homology analysis.
(5) experimental result
Real-time quantitative pcr amplification curve flex point understands, amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is put down and no raised up now, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve is all unimodal, illustrates that amplified production only has one, is specific amplification；Relative quantification formula according to qrt-pcr: 2-δδct× 100%, compare expression in prostate cancer tissue and cancer beside organism for the loc283856.Result shows: qrt-
Pcr stable amplification result, the wherein loc283856 expression in prostate cancer tissue is 4.5 times of control tissue, such as schemes
Shown in 1.Result above demonstrate high flux transcript profile express data confluence analysiss loc283856 in patients with prostate cancer table
Reach the result of rise.
After rt-pcr product is recovered, the full-automatic sequenator of abi3730 is sequenced, the nucleotide of the fragment of this 157bp
Sequence is as shown in seq id no.2.With vector nti advance 10 software (invitrogen company) by this sequence with
Loc283856 whole mrna sequence is compared, and comparison result shows, the nucleotides sequence as shown in seq id no:2 is classified as
A part of sequence of loc283856, coincidence rate is 100%.
Embodiment 3 rnai interference loc283856 expression
First, material
(1) cell derived
Commercially available carcinoma of prostate pc-3 cell line (purchasing in Shanghai Bo Yan bio tech ltd).
(2) sirna design and synthesis
According to Photographing On-line software sidirect version 2.0 (http://design.rnai.jp/), according to gene
Sequence, with reference to ncbi:nr_027078.1 (loc283856), designs corresponding sirna, and particular sequence is shown in Table 3.It is sent to after design
Synesis Company synthesizes.
Table 3 sirna sequence list
2nd, experimental technique
Cell packet and transfection
1. cell packet
C group: blank control group；C1 group: transfection liposome group；C2 group: transfect nonspecific sirna group；S1, s2, s3
Group: transfect specific sirna group.
2. transfect
According to lipofectaminetmThe step that 2000transfection reagent provides is carried out.
(1) carcinoma of prostate pc-3 cell line is seeded on 6 orifice plates, these are used for the cell quantity of initial vaccination, should be able to
Cell confluency was made to reach 70% in 24 hours；
(2) prepare sirna-lipofectaminetm2000 complex:
A. dilute the lipofectaminetm 2000 of 5 μ l with 250 μ l opti-mem, gently mix, under room temperature, be incubated 5
Minute.
B. experiment each group takes 5 μ l, 20 μm/l sirna to add in 250 μ lopti-mem respectively and is diluted, and gently shakes
Move and mixed；
C. incubation 5 minutes after, will dilution sirna and lipofectaminetmIt is incubated 20 at room temperature after 2000 mixing
Minute.
(3) it is uniformly added into cell, culture medium and sirna-lipofectamine in each hole in culture platetm2000
Complex.Then it is shaken gently for culture plate, so that them is sufficiently mixed；
(4) it is placed on 37 DEG C, co2Incubation 48 hours in incubator, observation of cell transfection quantity, inspection under fluorescence microscope
Survey transfection efficiency；
(5) transfection efficiency is the ratio of the cell quantity in cryptoscope and the light microscopic visual field, and the transfection efficiency of cell all reaches
More than 90%, just can carry out follow-up experiment.Computing formula is as follows:
Cell quantity × 100% under the quantity/same field of view of the transfection efficiency=cell that fluoresces
3. the change that before and after application real-time pcr method detection transfection, loc283856 expresses
(1) structure of standard curve: choose 1 bottle of 50ml pc-3 cell, extract rna, measure rna concentration and purity, carry out
Reverse transcription reaction, ten times of dilutions of dna template that reaction is generated, obtain being equivalent to 104-100The dna template of copies/ μ l, point
Not Jia Ru loc283856 gene primer and internal control primer, prepare 25 μ l reaction systems, using real-time pcr amplification instrument, enter
Row pcr amplified reaction.Obtain the standard curve of loc283856 and internal reference.
(2) change of loc283856 gene expression before and after the detection of real-time pcr method transfects: extract each group cell
Rna, measure rna concentration and purity, carry out reverse transcription reaction, every group of dna template carries out loc283856 and internal reference simultaneously
Real-time pcr reacts, and experiment is in triplicate.
(3) row agarose gel electrophoresis are entered to pcr product.
3rd, experimental result
The sirna of 3 loc283856 and comparison sirna transfection first generation prostate gland cancer cell respectively, result is shown in greatly
Find green fluorescence it was demonstrated that having been obtained for the transfection of sirna in prostate gland cancer cell, then glimmering in amount prostate gland cancer cell
Light microscopic and light Microscopic observation prostate gland cancer cell quantity, carry out the detection of transfection efficiency, and result display transfection efficiency all reaches 90%
More than.Real-time pcr result shows, transfects nonspecific sirna group to loc283856 in carcinoma of prostate pc-3 cell
Expression no obvious inhibiting effect, and blank control group no difference of science of statistics, 3 loc283856sirna groups of transfection are all to prostatitis
In adenocarcinoma cell loc283856 expression play certain inhibitory action suppression ratio be 42%, wherein loc283856-sirna2 and
Loc283856-sirna3 inhibition is the most obvious, and suppression ratio reaches 52% and 58%.
Embodiment 4 cck-8 method detects the proliferative conditions of prostate gland cancer cell
Sirna transfection () of cell taking six orifice plates as a example
Transfection method is with embodiment 3.
2nd, cell proliferation experiment
1. resuspended after cell dissociation, it is inoculated in 96 orifice plates, every hole adds about 1 × 104Cell.
2. according to experiment packet: it is thin that blank control group, prostate gland cancer cell add nonspecific sirna group, carcinoma of prostate
Born of the same parents add 3 groups of loc283856-sirna, every group 6 repetitions.
3. experiment condition is 37 DEG C, 5%co2And 100% humidity.
4., after inoculation 0h, 24h, 48h, every hole is separately added into 10ul ckk-8 reagent, and continues to incubate in cell culture incubator
Educate 2 hours.
5. microplate reader wavelength is set as 570nm, detects the absorbance value (od) in each hole, reference wavelength 630nm (600-
650nm).
7. the meansigma methodss of blank well absorbance value are deducted as actual light absorption value using each hole absorbance value, average,
Curve, the proliferative conditions of reflection cell are made with 0h, 24h, 48h time point absorbance value.
3rd, experimental result
As shown in Fig. 2 sirna-nc represents that prostate gland cancer cell adds the feelings of nonspecific sirna experimental group cell proliferation
Condition, si-loc283856 represents that prostate gland cancer cell adds the situation of loc283856-sirna3 experimental group cell proliferation, and both are right
Ratio, it can be seen that loc283856-sirna3 significantly inhibits the propagation of prostate gland cancer cell, can be used for the system of carcinoma of prostate medicine
Standby.
The preparation of embodiment 5 test kit
The primer sets being obtained based on embodiment 2, assemble the test kit for detecting carcinoma of prostate of the present invention, described
Test kit includes the primer pair of specific amplified loc283856 as shown in seq id no:3 and seq id no:4, and specific amplified
The primer pair of house-keeping gene (gapdh) is as shown in seq id no:5 and seq id no:6；Also include sybr green polymerase
Chain reaction system, such as pcr buffer, sybr green fluorescent dye, dntps.The composition of described pcr buffer is 25mm
Kcl, 2.5mm mgcl2, 200mm (nh4)2so4；Also include prostate normal structure cdna: as negative control and detection sample
Cdna quantitative pcr detection jointly, each reaction system uses and detection sample cdna equal amount.
By the optimization to primer concentration and annealing temperature, final determination reaction system is as shown in table 3:
Table 3 pcr reaction system
| Component | Addition |
| Sybr green polymerase chain reaction system | 12.5μl |
| Forward primer (10 μm) | 0.5μl |
| Downstream primer (10 μm) | 0.5μl |
| Template cdna | 2.0μl |
| Add sterile purified water | To 25 μ l |
Optimum reaction condition is:
95 DEG C of denaturations 5min, (95 DEG C of degeneration 15sec, 61 DEG C of annealing 45sec, 72 DEG C of extension 35sec) × 40 circulations,
72 DEG C of extension 15min.
The application of the diagnostic kit of 6 carcinoma of prostate of embodiment
1st, case
Choose 20 patients with prostate cancer treating examination, sampling derives from BJ Union Hospital's in October, 2012 to 2015
The patient of urology department treatment during December.Obtain the prostate cancer tissue sample of all object of study, number rearmounted -80 DEG C of low temperature
Refrigerator store.All clinical samples of this research, all patient being carried out knows the inside story informs and passes through through this Hospital Ethical Committee.
2nd, method
Extract rna using conventional method or using specific test kit, reverse transcription becomes cdna, with the reagent in embodiment 5
Box carries out detection reaction.Using in test kit, prostate normal structure cdna, as the comparison cdna in qpcr detection by quantitative, detects
Loc283856 relative prostate normal tissue expression amount change in tissue samples.
3rd, result
Purpose band is determined by solubility curve analysis and electrophoresis, δ δ ct method carries out relative quantification, between sample and comparison
Relatively adopt t inspection, p < 0.05 is significant difference.Result shows, treats in the tissue samples of 20 patients with prostate cancer of examination
There are the expression of loc283856 and carcinoma of prostate normal tissue expression amount no significant difference in 6 patient tissue samples；There are 14
In patient tissue sample, the expression of loc283856 is more than 1 times of carcinoma of prostate normal tissue expression amount, wherein has 11 trouble
In person's tissue samples, the expression of loc283856 is more than 3 times of carcinoma of prostate normal tissue expression amount.Examine further through clinic
Survey, in 20 patients treating examination, make a definite diagnosis 11 carcinoma of prostate, the test kit of this 11 patients with prostate cancer and present invention preparation
Testing result is consistent.Infer accordingly, the diagnostic kit of this carcinoma of prostate clearly can distinguish patients with prostate cancer, and with this
There is provided diagnostic clue for clinical.
Although, above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, 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 (10)
1.loc283856 the application of the molecular marked compound as detection carcinoma of prostate.
2. application as claimed in claim 1 is it is characterised in that described loc283856 comprises the spy as shown in seq id no:2
Heterologous nucleotide sequence.
3. application as claimed in claim 1 or 2 is it is characterised in that described loc283856 is on expression in prostate cancer
Adjust.
4.loc283856 the application in preparation carcinoma of prostate early treatment or diagnostic products.
5. application as claimed in claim 4 is it is characterised in that described product includes preparation or the diagnosis examination treating carcinoma of prostate
Agent box.
6. application as claimed in claim 5 is it is characterised in that contain the reagent of suppression loc283856 expression in described preparation.
Application in the medicine of preparation prevention, alleviation and/or treatment carcinoma of prostate for the 7.loc283856.
8. apply it is characterised in that described medicine includes the sirna of loc283856 as claimed in claim 7.
9. a kind of prostatic cancer early diagnosis kit is it is characterised in that described diagnostic kit contains amplification such as seq id
The primer pair of the nucleotide sequence shown in no:2.
10. diagnostic kit as claimed in claim 9 is it is characterised in that described diagnostic kit includes quantitative pcr reactant
System:
(1) two pairs of primers of nucleotide sequence as shown in seq id no:2 for the specific amplified and comparison gapdh；
The primer sequence of nucleotide sequence as shown in seq id no:2 for the detection is as follows:
Upstream primer sequence: seq id no:3
Downstream primer sequence: seq id no:4；
The primer sequence of comparison gapdh is as follows:
Upstream primer sequence: seq id no:5
Downstream primer sequence: seq id no:6；
(2) sybr green polymerase chain reaction system, including pcr buffer, sybr green fluorescent dye, dntps etc..
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610815275.XA CN106367507A (en) | 2016-09-09 | 2016-09-09 | Application of LOC283856 in preparation of prostatic cancer early treatment or diagnosis products |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610815275.XA CN106367507A (en) | 2016-09-09 | 2016-09-09 | Application of LOC283856 in preparation of prostatic cancer early treatment or diagnosis products |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106367507A true CN106367507A (en) | 2017-02-01 |
Family
ID=57899105
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610815275.XA Withdrawn CN106367507A (en) | 2016-09-09 | 2016-09-09 | Application of LOC283856 in preparation of prostatic cancer early treatment or diagnosis products |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106367507A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154448A (en) * | 2015-09-16 | 2015-12-16 | 复旦大学 | Prostatic cancer molecular target RP11-1023L17.1 and application thereof to diagnostic kit |
| CN105316340A (en) * | 2015-09-16 | 2016-02-10 | 复旦大学 | MIR27A-3P serving as prostate cancer molecular marker and application of same to diagnostic reagent kit |
-
2016
- 2016-09-09 CN CN201610815275.XA patent/CN106367507A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154448A (en) * | 2015-09-16 | 2015-12-16 | 复旦大学 | Prostatic cancer molecular target RP11-1023L17.1 and application thereof to diagnostic kit |
| CN105316340A (en) * | 2015-09-16 | 2016-02-10 | 复旦大学 | MIR27A-3P serving as prostate cancer molecular marker and application of same to diagnostic reagent kit |
Non-Patent Citations (1)
| Title |
|---|
| MYERS JS 等: ""Differentally expressed genes and signature pathways of human prostate cancer"", 《PLOS ONE》 * |
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