CN106148561A - The diagnosis and treatment label of carcinoma of prostate - Google Patents
The diagnosis and treatment label of carcinoma of prostate Download PDFInfo
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Abstract
The invention discloses a kind of label detecting carcinoma of prostate, described label is NCRNA00087, and is further characterized by NCRNA00087 in expression in prostate cancer rise.The present invention further discloses a kind of diagnostic kit for detecting carcinoma of prostate, described test kit includes that specific amplification carcinoma of prostate is correlated with the primer of lncRNA and description, and the described carcinoma of prostate lncRNA that is correlated with is NCRNA00087.The invention also discloses a kind of NCRNA00087 inhibitor, described inhibitor includes the siRNA of NCRNA00087.Utilize NCRNA00087 detection carcinoma of prostate can not only accomplish fast and effectively in early days to detect, 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 biomedicine field, relate to the diagnosis and treatment label of carcinoma of prostate, specifically this label is
NCRNA00087。
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) may have during doctors experience deficiency fail to pinpoint a disease in diagnosis or mistaken diagnosis can
Energy.
Currently for Late-stage Prostate Cancer patient, the weak effect of operative treatment, great majority use Drug therapy.According to
The different state of an illness can use chemotherapy, fluconazole ear drops, radionuclide internal radiotherapy and various therapy
Integrated application etc..But, radiotherapy and chemotherapy medicine acts not only on tumor, it is also possible to act on the tissue of tumor adjacent healthy, thus
While killing tumor, also bring the biggest side effect to body, finally affect the therapeutic effect to tumor.
Substantial amounts of high flux genome platform data shows that the subgenomic transcription product more than 98% is not encoding proteins
Non-coding RNA, wherein long non-coding RNA (long non-coding RNA, lncRNA) is that a class length is more than the non-of 200nt
Coding RNA.Increasing evidence shows that lncRNAs plays a significant role in multiple biological processes and progression of disease, such as
Immunoreation, cell differentiation, metabolism, tumor occur and development.LncRNAs manifests carcinogenic because of it in tumor suppression approach
Latent effect and become the new focus of tumor research.
Therefore, this area is in the urgent need to developing the Specific marker of carly fruit drop carcinoma of prostate, and develops carcinoma of prostate
Targeted drug.
Summary of the invention
In order to realize the early discovery of carcinoma of prostate, early intervention, an object of the present invention is to provide a kind of new
The diagnosis and treatment label NCRNA00087 of carcinoma of prostate.
The two of the purpose of the present invention are to provide a kind of diagnostic kit for detecting carcinoma of prostate.
The three of the purpose of the present invention are to provide a kind of NCRNA00087 inhibitor.
The four of the purpose of the present invention are to provide a kind of medicine treating carcinoma of prostate.
For achieving the above object, present invention firstly provides a kind of label detecting carcinoma of prostate, described label is
NCRNA00087。
Preferably, described NCRNA00087 raises at expression in prostate cancer；Preferably, detection NCRNA00087 exists
The method that expression in prostate cancer raises includes nucleic acid chip detection method or fluorescence quantitative PCR method.
Preferably, described NCRNA00087 includes the specific nucleotide sequences as shown in SEQ ID NO:1.
Further, the invention provides a kind of diagnostic kit for detecting carcinoma of prostate, described test kit includes
Specific amplification carcinoma of prostate is correlated with the primer of lncRNA and description, and the described carcinoma of prostate lncRNA that is correlated with is
NCRNA00087.Preferably, described primer has the primer shown in SEQ ID NO:2 and SEQ ID NO:3.
Preferably, dated herein below in described description:
When in the prostate gland cancer cell or tissue of detection object NCRNA00087 expression E1 and normal prostate cell or
Ratio >=2 of the NCRNA00087 expression E2 of tissue, then point out the probability of this detection object carcinoma of prostate higher than general population.Institute
The E1 stated is prostate gland cancer cell or the NCRNA00087 expression of tissue of detection object；Described E2 be normal population just
Often prostatic cell or the NCRNA00087 expression of tissue.Described normal prostate cell or tissue include the prostatitis that cancer is other
Glandular cell or tissue.Described expression is the relative expression quantity relative to crt gene (such as GAPDH).
Preferably, described test kit also includes 10 × Buffer, dNTP, MgCl2, Taq enzyme and SYBR Green fluorescence dye
Material.
Further, the invention provides a kind of NCRNA00087 inhibitor, described inhibitor includes NCRNA00087's
siRNA.Preferably, described NCRNA00087 inhibitor can suppress the expression of NCRNA00087 or can suppress
The function of NCRNA00087.
Preferably, the inhibitor of described treatment carcinoma of prostate contain following in one group or several groups of siRNA:SEQ ID
NO.6 and SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9.It is furthermore preferred that the inhibitor of described treatment carcinoma of prostate
It is SEQ ID NO.8 and SEQ ID NO.9 containing siRNA sequence.
Further, above-mentioned inhibitor answering in preparation prevents, alleviates and/or treat the biological preparation of carcinoma of prostate
With.
Further, the invention provides a kind of medicine treating carcinoma of prostate, described medicine includes above-mentioned inhibitor.
Preferably, described medicine also includes pharmaceutically acceptable carrier.Described carrier includes but not limited to: diluent, buffer agent,
Suspensoid, Emulsion, granule, encapsulation agents, excipient, filler, binding agent, spray, cutaneous permeable agent, wetting agent, disintegrate
Agent, absorption enhancer, surfactant, coloring agent, correctives or absorption carrier.
Preferably, described medicine is by being administered selected from the application method of lower group: oral, intravenous injection, muscle note
Penetrate, subcutaneous injection, sublingual administration, rectal perfusion, nasal spray, mouthspray, local skin or whole body transdermal administration.Described
The preparation of medicine is selected from lower group: tablet, capsule, injection, granule, spray.Described NCRNA00087 inhibitor with
The dosage of 0.01-20mg/kg body weight is applied to mammal at (each or every day).Described mammal includes people, mice, big
Mus, more preferably, for people.
The medicine of the present invention can be single preparations of ephedrine, it is also possible to be compound preparation.In compound preparation, except containing
Outside NCRNA00087 inhibitor, also can comprise other antitumoral compounds, such as chemotherapeutics.Representational chemotherapeutics, including
But it is not limited to, alkylating agent, antimetabolite, folacin, pyrimidine analogue, purine analogue and related inhibitors, length
Spring flower bases, antibiotic, L-Radix Asparagi phthalein amine enzyme, topoisomerase enzyme inhibitor, interferon, platinum coordination complex, rheum emodin replace
Urea, methyl hydrazine derivatives, adrenal cortex inhibitor, adrenocortical steroid, progestogen, estrogen, estrogen antagonist, hero
Hormone, androgen antagonist and promoting sexual gland hormone-releasing hormone analog.Preferably chemotherapeutics includes: 5-fluorouracil (5-FU),
Formyl tetrahydrofolic acid, irinotecan, oxaliplatin, capecitabine, paclitaxel and Docetaxel.
Beneficial effects of the present invention is as follows:
The invention discloses a kind of NCRNA00087 relevant to carcinoma of prostate, and be further characterized by this NCRNA00087 and exist
Expression in prostate cancer raises.Utilize this lncRNA detection carcinoma of prostate can not only accomplish fast and effectively in early days to examine
Survey, and provide therapy target and important evidence for the clinical practice such as gene therapy, Drug therapy.
Accompanying drawing explanation
Fig. 1 NCRNA00087 expression in prostate cancer tissue sample and cancer beside organism's sample.
Fig. 2 NCRNA00087-siRNA significantly inhibits prostate gland cancer cell propagation.
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 order-checking to prostate cancer tissue sample and cancer beside organism's sample, logical
Cross bioinformatics method and carry out genescreen, pick out candidate lncRNA NCRNA00087, in existing research not
The report that NCRNA00087 is relevant with carcinoma of prostate, further, inventor carried out molecular biology method checking it was confirmed
NCRNA00087 is up-regulated in prostate gland cancer cell.
The NCRNA00087 of the present invention is known lncRNA before making the present invention, and its essential information is as follows:
Genbank accession number: NCBI reference sequences: NR_024493.2, derives from human genome.
The present invention also uses RT-PCR method to detect above-mentioned lncRNA at prostate cancer tissue and the table of Carcinoma side normal tissue
Reach, and demonstrate this lncRNA up-regulated in carcinoma of prostate.
Terms used herein " up-regulated " 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 increases.According to the present invention,
" up-regulated " refer to by the inventive method intensity for hybridization measure at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10% or higher express and increase, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more
High or higher than 1 times, it is up to 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or higher.
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, experiment behaviour
Making to carry 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 is carried out functional annotation, the result analyzed in view of data above, in conjunction with document we
Having screened differential expression NCRNA00087, this lncRNA is up-regulated in prostate cancer tissue sample.
Embodiment 2RT-PCR checking prostate cancer tissue and cancer beside organism's NCRNA00087 expression
1, material
20 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.
2.3 Real-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:NR_024493.2 (NCRNA00087), interior participation in the election
GAPDH, is synthesized by invitrogen company after design of primers.Concrete primer sequence is as follows:
Table 1 primer sequence
Operating process is as follows:
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 MasterMix (invitrogen, article No. 4367659) is respectively to purpose
Gene primer and reference gene primer expand.Experimental implementation is carried out by product description.Amplification program is: 95 DEG C of 5min,
(95 DEG C of 15sec, 60 DEG C of 45sec, 72 DEG C of 35sec) × 40 circulations.Carry out solubility curve analysis at 60-95 DEG C simultaneously.Reaction
After end, the PCR primer taking 5ul carries out 2% sepharose electrophoresis, and reclaiming test kit (invitrogen company) with quick glue will be big
The little fragment for 313bp carries out cutting glue and reclaims and check order, and result blast software carries out homology analysis.
3, 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 flat and nothing raises up now, and exponent phase slope is relatively big, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve is all unimodal, illustrates that amplified production only has one, for specific amplification；Relative quantification formula according to qRT-PCR: 2-ΔΔCt× 100%, compare NCRNA00087 expression in prostate cancer tissue and cancer beside organism.Result shows: qRT-
PCR stable amplification result, wherein NCRNA00087 suffers from expression is cancer beside organism 3 times in organizing in carcinoma of prostate, with
Upper result verification high flux transcript profile expresses confluence analysis NCRNA00087 up-regulated in patients with prostate cancer of data
Result；After RT-PCR product is recovered, the full-automatic sequenator of ABI3730 checks order, the nucleotides sequence of the fragment of this 313bp
Row are as shown in SEQ ID NO.1.With VectorNTI advance 10 software (Invitrogen company) by this sequence with
NCRNA00087 whole mRNA sequence is compared, and comparison result shows, the nucleotides sequence as shown in SEQ ID NO:1 is classified as
A part of sequence of NCRNA00087 gene, coincidence rate is 100%.
The interference NCRNA00087 expression of embodiment 3RNAi and the impact on prostate gland cancer cell
One, material
1, cell derived
Human Prostate Cancer PC-3 Cell Line strain is purchased from ATCC company of the U.S.
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_024493.2 (NCRNA00087), 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
Two, experimental technique
1, cell packet
C group: blank group；C1 group: transfection liposome group；C2 group: transfect nonspecific siRNA-NC group；S1, S2
Group: transfect specific siRNA group.
2, transfection
According to LipofectamineTMThe step that 2000Transfection Reagent provides is carried out.
(1) PC-3 cell line is seeded on 6 orifice plates so that 24 hours inner cells converge and reach 70%；
(2) siRNA-Lipofectamine is preparedTM2000 complex:
A. dilute 5 μ L LipofectamineTM 2000 with 250 μ L Opti-MEM, mix gently, incubated at room temperature 5 points
Clock.
B. test each group to take 7.5 μ L siRNA respectively and add in 250 μ L Opti-MEMI and be diluted, and be shaken gently for by
Its mixing；
C. after hatching 5 minutes, by siRNA and Lipofectamine of dilutionTM20 are at room temperature hatched after 2000 mixing
Minute.
(3) each hole in culture plate adds cell, culture medium and siRNA-LipofectamineTM2000 is multiple
Compound.Then it is shaken gently for culture plate, makes them be sufficiently mixed；
(4) 37 DEG C it are placed on, CO2Hatch in incubator 48 hours, observation of cell transfection quantity, inspection under fluorescence microscope
Survey transfection efficiency；
(5) transfection efficiency is the ratio of cryptoscope and the cell quantity in the light microscopic visual field, and the transfection efficiency of cell all reaches
More than 90%, side 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, NCRNA00087 expresses
(1) structure of standard curve: be chosen in 50mL culture bottle the prostate gland cancer cell 1 bottle of normal cultivation, extract
RNA, measures RNA concentration and purity, carries out reverse transcription reaction, and DNA profiling ten times dilution reaction generated is equivalent to
104-101The DNA profiling of copies/ μ L, is separately added into NCRNA00087 primer and internal reference primer, prepares 25 μ L reaction systems, makes
Use Real-time PCR amplification instrument, carry out pcr amplification reaction.Obtain the standard curve of NCRNA00087 and internal reference.
(2) change that before and after Real-time PCR method detection transfection, NCRNA00087 expresses: extract each group of cell
RNA, measures RNA concentration and purity, carries out reverse transcription reaction, and often group DNA profiling enters the Real-of NCRNA00087 and internal reference simultaneously
Time PCR reacts, and experiment is in triplicate.
(3) PCR primer is carried out agarose gel electrophoresis.
Three, experimental result
Transfecting first generation prostate gland cancer cell with the siRNA and comparison siRNA of 2 NCRNA00087 respectively, result shows
Green fluorescence is found, it was demonstrated that have been obtained for the transfection of siRNA in prostate gland cancer cell, then in a large amount of prostate gland cancer cells
In cryptoscope and light Microscopic observation prostate gland cancer cell quantity, carrying out the detection of transfection efficiency, result display transfection efficiency all reaches
More than 90%.Real-time PCR result shows, transfects nonspecific siRNA-NC group in prostate gland cancer cell
NCRNA00087 expresses without obvious inhibiting effect, and blank group no difference of science of statistics, 2 NCRNA00087-of transfection
NCRNA00087 in prostate gland cancer cell is all expressed and plays certain inhibitory action by siRNA group, NCRNA00087-siRNA1 and
NCRNA00087-siRNA2 suppression ratio is respectively 60% and 69%.
The proliferative conditions of embodiment 4MTT method detection prostate gland cancer cell
One, mtt assay experimental procedure
1, in 96 orifice plates, about 1x10 is added according to every hole4Cell, then at 37 DEG C of 5%CO2Under conditions of cultivate 24 little
Time；
2, (with untransfected normal cell as matched group, non-specific siRNA-NC group, transfection are transfected according to experiment packet
NCRNA00087-siRNA2 group is experimental group, with only add culture medium be not added with cell blank control wells zeroing, often group 6 repetitions)
Continue to cultivate until being suitable for detection；
3, the siRNA transfection of cell, transfection method is with embodiment 3.
4, then at 37 DEG C, 5%CO2And 100% continue to hatch appropriate time under conditions of humidity；
5, with Dilution Buffer, 5xMTT is diluted to 1xMTT simultaneously；
6, after inoculation 0h, 24h, 48h, every hole adds 50 μ L 1xMTT, and under the conditions of 37 DEG C, hatches 4 hours；
7, after sucking-off supernatant, in every hole, also need to add 150 μ L DMSO, and place it in and carry out on plate shaker
Shake up；
8, microplate reader wavelength is set as 570nm, detects the optical density (OD value) in each hole.
9, deduct the meansigma methods of blank well absorbance value as actual light absorption value using each hole absorbance value, average,
Curve, the proliferative conditions of reflection cell is made with 0h, 24h, 48h time point absorbance value.
Two, experimental result
Compared with untransfected matched group, transfection siRNA-NC cell proliferation has no significant effect, and transfects NCRNA00087-
SiRNA2 can substantially suppress prostate gland cancer cell to breed, and can be used for the preparation of carcinoma of prostate medicine, suppression ratio prolonging in time
Growing and increase, result is as shown in Figure 2.
Embodiment 5 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 include the primer of specific amplified NCRNA00087 to as shown in SEQ ID NO:2 and SEQ ID NO:3, and specific amplified internal reference
The primer of 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.The composition of described PCR buffer is 25mM KCL,
2.5mM MgCL2, 200mM (NH4)2SO4。
By the optimization to primer concentration and annealing temperature, determine that optimal reaction system is as shown in table 4:
Table 4 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, 60 DEG C of annealing 45sec) × 40 circulations,
72 DEG C extend 15min.
Taking a small amount of prostatic cell of 30 example prostate cancer patient to be detected, prostate cancer patient to be checked is Beijing consonance doctor
Institute's urology department is gone to a doctor patients with prostate cancer.All clinical samples of this research, all carrying out patient knows the inside story informs and through this hospital
Ethics Committee passes through.Use conventional method (or using specific test kit) to extract RNA from prostatic cell, use reagent
Reagent in box, carries out PCR reaction according to optimal reaction system and condition, uses normal cancer beside organism cDNA conduct in test kit
Comparison cDNA in Real-Time PCR detection by quantitative, the NCRNA00087 of detection tissue samples relatively normal cancer beside organism table
The amount of reaching changes, and analyzes testing result, compares employing t inspection between sample and comparison, and P < 0.05 is significant difference, is judged to detect sample
Positive.
Testing result shows, in 30 example patients to be detected, has the NCRNA00087 of 24 example patients at prostatic cell table
The level of reaching is 3-5 times in cancer beside organism.Detecting further through clinic, this 24 example patient determines suffer from carcinoma of prostate, other 6 examples
Not suffering from carcinoma of prostate, the test kit testing result that Clinical detection result is prepared with the present invention is consistent.Infer accordingly, this prostatitis
The diagnostic kit of adenocarcinoma can clearly distinguish patients with prostate cancer, 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 (10)
1. the label detecting carcinoma of prostate, it is characterised in that described label is NCRNA00087.
2. mark as claimed in claim 1, it is characterised in that described NCRNA00087 is at expression in prostate cancer
Raise.
3. mark as claimed in claim 1 or 2, it is characterised in that described mark includes as shown in SEQ ID NO:1
Specific nucleotide sequences.
4. the diagnostic kit being used for detecting carcinoma of prostate, it is characterised in that before described test kit includes specific amplification
Row adenocarcinoma is correlated with the primer of lncRNA and description, and the described carcinoma of prostate lncRNA that is correlated with is NCRNA00087.
5. test kit as claimed in claim 4, it is characterised in that described primer has SEQ ID NO:2 and SEQ ID NO:3
Shown primer.
6. test kit as claimed in claim 5, it is characterised in that described test kit also include 10 × Buffer, dNTP,
MgCl2, Taq enzyme and SYBR Green fluorescent dye.
7. a NCRNA00087 inhibitor, it is characterised in that described inhibitor includes the siRNA of NCRNA00087.
8. inhibitor as claimed in claim 7, it is characterised in that described siRNA sequence is: SEQ ID NO.6 and SEQ ID
NO.7, SEQ ID NO.8 and SEQ ID NO.9.
9. inhibitor as claimed in claim 7 or 8 is in preparation prevents, alleviates and/or treat the biological preparation of carcinoma of prostate
Application.
10. the medicine treating carcinoma of prostate, it is characterised in that described medicine includes the suppression described in claim 7 or 8
Agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610862414.4A CN106148561A (en) | 2016-09-28 | 2016-09-28 | The diagnosis and treatment label of carcinoma of prostate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610862414.4A CN106148561A (en) | 2016-09-28 | 2016-09-28 | The diagnosis and treatment label of carcinoma of prostate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106148561A true CN106148561A (en) | 2016-11-23 |
Family
ID=57341558
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| CN201610862414.4A Withdrawn CN106148561A (en) | 2016-09-28 | 2016-09-28 | The diagnosis and treatment label of carcinoma of prostate |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018219264A1 (en) * | 2017-06-01 | 2018-12-06 | 上海长海医院 | Use of long-chain non-coding rna as prostatic cancer molecule marker |
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| CN104004840A (en) * | 2014-05-26 | 2014-08-27 | 高新 | Kit for early screening and diagnosis of prostate cancer |
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2016
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|---|---|---|---|---|
| CN104004840A (en) * | 2014-05-26 | 2014-08-27 | 高新 | Kit for early screening and diagnosis of prostate cancer |
| 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 |
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| MYERS JS等: "Differentially expressed genes and signature pathways of human prostate cancer", 《PLOS ONE》 * |
| SHUJIE SONG等: "Gene Silencing Associated with SWI/SNF Complex Loss during NSCLC Development", 《MOL CANCER RES》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018219264A1 (en) * | 2017-06-01 | 2018-12-06 | 上海长海医院 | Use of long-chain non-coding rna as prostatic cancer molecule marker |
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Application publication date: 20161123 |