CN106282366A - A kind of molecular marked compound relevant to carcinoma of prostate and application thereof - Google Patents
A kind of molecular marked compound relevant to carcinoma of prostate and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of molecular marked compound relevant to carcinoma of prostate, described molecular marked compound is LOC100190940.The invention also discloses a kind of prostatic cancer diagnostic reagent kit, described diagnostic kit contains the molecular marked compound LOC100190940 relevant to carcinoma of prostate.The present invention further discloses described molecular marked compound to screen carcinoma of prostate high-risk group, diagnose, treat, monitor and application in prognosis.Utilize this molecular marked compound and the detection of the diagnostic kit containing this molecular marked compound carcinoma of prostate can not only accomplish fast and effectively to detect in early days, 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 biological technical field, be specifically related to a kind of molecular marked compound relevant to carcinoma of prostate and answer
With.
Background technology
Carcinoma of prostate is to betide the malignant tumor in human male prostate tissue, has become as male in the world
Two big class kinds of tumor, are positioned at cancer related mortality rate the 6th.At 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 cancer；In Asia, its sickness rate be less than western countries, but in recent years in
Ascendant trend rapidly, 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 increases the most with age.Carcinoma of prostate is in early days
Many without any symptom, even if uncomfortable, also it is not enough to cause the attention of patient, when tumor increases urethra, the most often
Obscure mutually with prostatic hyperplasia.First patient in China about 80% finds that metastasis focus just finds carcinoma of prostate.Now,
Pathological changes has reached late period, prognosis mala.So, the treatment of prostate cancer patient is had earthshaking by the early diagnosis of carcinoma of prostate
Meaning.
The clinical diagnostic modalities of carcinoma of prostate currently mainly has prostate digital rectal examination (DRE), serum prostate special 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, the main forefinger touch prostate by doctor, to judge the size and shape of prostatic tubercle, thus judge whether
Suffers from carcinoma of prostate.But the limitation of digital rectal examination is the strongest: 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 can not use this to detect when having illness；(4) doctor
May have when lacking experience and fail to pinpoint a disease in diagnosis or the possibility of mistaken diagnosis.Although prostate ultrasound examination simple and direct-viewing operation, not damaged, but
The method easily confuses 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.Sieving and diagnosis to carcinoma of prostate relies primarily on the detection of blood-serum P SA and prostate biopsy pathological biopsy phase at present
In conjunction with method.PSA in blood the highest (not higher than 4ng/ml) under normal circumstances, when being in carcinoma of prostate and other prostate
During disease disease state, PSA raises becomes the tumor mark that current examination carcinoma of prostate is most sensitive, but PSA increase the most common and 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, even may be used
The delay of the flase drop of disease, mistaken diagnosis or the state of an illness can be caused.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 diagnosis.And the recall rate of aspiration biopsy has bigger with the number of times of living body puncture, position
Relation, is not highly stable diagnosis scheme.Therefore, study more effective prostate cancer marker to improve carcinoma of prostate morning
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.
LOC100190940 belongs to lncRNA (longnon-codingRNA, long-chain non-coding RNA), is a class transcript
Length is more than the non-coding RNA of 200bp nucleotide, and research in recent years finds that it is the RNA that a class has important biomolecule function, ginseng
With genomic imprinting, chromosome silence, chromatin modification, transcriptional activation, transcribe the multiple important regulation and control such as transport in interference, core
Process, all plays important tune in the vital movements such as cell differentiation and growth, genetic transcription and translation, heredity and epigenetic
Control effect.In recent years, increasing authority's research confirms that lncRNA plays suppression in tumor develops or promotes swollen
The effect of tumor, at aspects such as modulate tumor cell proliferation, apoptosis, cell cycle, invasive ability, is respectively provided with particularly significant work
With.Oneself has more lncRNAs to be proved to include breast carcinoma, prostate, melanoma, hepatocarcinoma, colon cancer, bladder cancer etc. at present
In interior mankind's kinds of tumors, there are differences expression and perform important adjusting function.In early days effectively detection tumor generation,
The curative effect of development and raising cancer therapy drug is particularly important for the treatment of cancer, and invention novel tumor markers is as diagnosis and controls
The target spot treated is always the focus of tumor research.
Recent studies indicate that, lncRNA is closely related with carcinoma of prostate, and they may participate in the generation 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 discovery of carcinoma of prostate, early intervention, it is an object of the invention to provide one and prostate
Relevant new molecular marked compound.
The second object of the present invention is to provide a kind of prostatic cancer diagnostic reagent kit.
The third object of the present invention be to provide described molecular marked compound screen carcinoma of prostate high-risk group, diagnose, treat,
Application in monitoring and prognosis.
For achieving the above object, present invention firstly provides a kind of molecular marked compound first closed with carcinoma of prostate, described molecule
Label is LOC100190940, and its nucleotides sequence is classified as shown in SEQ ID NO:1.The present invention it has been investigated that
LOC100190940 is down-regulated expression in carcinoma of prostate biological sample, and display LOC100190940 sends out with the tumor of carcinoma of prostate
Raw, development also exists close dependency.
Further, the invention provides a kind of prostatic cancer diagnostic reagent kit, described diagnostic kit contains and prostatitis
The molecular marked compound LOC100190940 that adenocarcinoma is relevant.
Preferably, described prostatic cancer diagnostic reagent kit, it is characterised in that include quantitative PCR reaction system:
(1) specific amplified LOC100190940 and comparison GAPDH two is to primer；
The primer sequence of detection LOC100190940 is as follows:
LOC100190940 forward primer sequence: SEQ ID NO:2
LOC100190940 downstream primer sequence: SEQ ID NO:3；
The primer sequence of comparison GAPDH is as follows:
GAPDH forward primer sequence: SEQ ID NO:4
GAPDH downstream primer sequence: SEQ ID NO:5；
(2) SYBR Green polymerase chain reaction system, including PCR buffer, SYBR Green
Fluorescent dye, dNTPs etc..
Further, the invention provides this molecular marker or diagnostic kit carcinoma of prostate high-risk group screen,
Diagnose, treat, monitor and application in prognosis.
Preferably, described application is by the content of LOC100190940 in detection subjects's tissue samples, and with normally
The content of horizontal LOC100190940 compares, to carry out carcinoma of prostate high-risk group's screening, to diagnose, treat, monitor and prognosis.
Beneficial effects of the present invention is as follows:
The invention discloses a kind of molecular marked compound LOC100190940 relevant to carcinoma of prostate, and be further characterized by
LOC100190940 lowers at expression in prostate cancer.Utilize this molecular marker analyte detection carcinoma of prostate can not only be quick
Effectively accomplish to detect in early days, and provide therapy target and important evidence for the clinical practice such as gene therapy, Drug therapy.
Accompanying drawing explanation
LOC100190940 down-regulated expression in prostate cancer tissue sample in embodiment 2 in Fig. 1 present invention.
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 present inventor carries out high-flux sequence to 10 example prostate cancer tissue samples and corresponding cancer beside organism sample,
Carry out genescreen in conjunction with bioinformatics method, pick out candidate lncRNALOC100190940, in existing research not
The report that LOC100190940 is relevant with carcinoma of prostate, further, inventor carried out molecular biology method checking it was confirmed
LOC100190940 lowers at expression in prostate cancer, and its related preparations can be used for treating carcinoma of prostate.
The LOC100190940 of the present invention is known lncRNA before making the present invention, and its essential information is as follows:
Genbank accession number: Gene ID:100190940, derives from human genome.
The present invention uses RT-PCR method to detect above-mentioned lncRNA expression in patients with prostate cancer and normal population, and
Demonstrate this lncRNA down-regulated expression in patients with prostate cancer.
Fluorescence quantitative PCR method is by fluorescent dye or fluorescently-labeled specific probe, is marked PCR primer
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 quantitative fluorescent PCR, greatly simplifies the process of detection by quantitative, and is truly realized absolute quantitation.
The appearance of multiple detecting system, the selectivity making experiment is higher.Automation mechanized operation improves work efficiency, and reaction is quick, repetition
Property is good, highly sensitive, high specificity, result are clear.
The nucleotide full length sequence of LOC100190940 of the present invention or its fragment generally can use PCR TRAP, recombination method
Or the method for synthetic obtains.For PCR TRAP, can especially open read according to published relevant nucleotide sequence
Frame sequence designs primer, and with commercially available cDNA storehouse or as prepared by conventional method well known by persons skilled in the art
CDNA, as template, expands and obtains relevant sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly expand, then
Again the fragment repeatedly amplified is stitched together by proper order.
Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically will
It is cloned into carrier, then proceeds to cell, then by conventional method relevant sequence of isolated from the host cell after propagation.
Additionally, can also be used with the method for synthetic to synthesize relevant sequence, when especially fragment length is shorter.Generally, by first synthesizing
Multiple small fragments, are attached obtaining the fragment that sequence is the longest the most again.
Embodiment 1 high-flux sequence screening difference expression gene
1, sampling
Choosing the 10 fresh prostate cancer tissues of example and corresponding cancer beside organism, sampling derives from BJ Union Hospital 2012 10
Month to row radical-ability prostate excision the prostate cancer patient of bilateral pelvic lymphadenectomy during in December, 2015, operation
Under the guidance of Pathologis, take carcinoma of prostate immediately after excision prostate and cancer beside organism puts into liquid nitrogen, number rearmounted-80
DEG C cryogenic refrigerator preserves.
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 samples being put into pre-cooling after liquid nitrogen, taking-up is ground, treats group
Knit sample powdered after:
1. 1mLTrizol, room temperature preservation 5 minutes are entered；
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 LOC100190940, LOC100190940 is down-regulated expression in carcinoma of prostate sample tissue.
LOC100190940 expression in embodiment 2RT-PCR checking prostate cancer tissue
1, material
Choose 10 example patients with prostate cancer, during sampling derives from BJ Union Hospital's in December, 2015 in October, 2012 to
Row radical-ability prostate excision the prostate cancer patient of bilateral pelvic lymphadenectomy, at pathology after excision prostate
Take carcinoma of prostate immediately under the guidance of section doctor and cancer beside organism puts into liquid nitrogen, number rearmounted-80 DEG C of cryogenic refrigerators and preserve.
2, method
2.1 pairs of subjects's 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 standby that-20 DEG C of refrigerators are put in the cDNA preservation obtained.
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-△△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_023357.2, and interior participation in the election GAPDH, primer sets
Synthesized by invitrogen company after meter.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) expanding, 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
(2) primer screening
After being mixed by each sample cDNA, carrying out 5 times of gradient dilutions as template, after dilution, sample respectively takes 2 μ L and makees template,
Expand with genes of interest primer and reference gene primer respectively, carry 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 after each sample cDNA 10 times being diluted and make template, enter with genes of interest primer and reference gene primer respectively
Row amplification.Carry out solubility curve analysis at 60-95 DEG C simultaneously.
Two, 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%, (-Δ Ct is to compare LOC100190940 expression in prostate cancer tissue and corresponding cancer beside organism
The difference of target lncRNA and the Ct value compareed).Result shows: qRT-PCR stable amplification result, and wherein LOC100190940 is front
Expression in row adenocarcinoma tissue is only the 30% of control tissue, as shown in Figure 1.Result above demonstrates high flux transcript profile
Express confluence analysis LOC100190940 result of down-regulated expression in patients with prostate cancer of data.
The preparation of embodiment 3 test kit
The primer sets obtained based on embodiment 2, assembles the test kit for detecting carcinoma of prostate of the present invention, described
Test kit include the primer of specific amplified LOC100190940 to as shown in SEQ ID NO:2 and SEQ ID NO:3, and special expansion
Increase the primer of house-keeping gene (GAPDH) to as shown in SEQ ID NO:4 and SEQ ID NO:5；Also include that SYBR Green is polymerized
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
The common quantitative PCR detection of cDNA, each reaction system uses and detection sample cDNA equal amount.
By the optimization to primer concentration and annealing temperature, finally determine that reaction system is as shown in table 3:
Table 3PCR reaction system
| Component | Addition |
| SYBR Green 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.
The application of the diagnostic kit of 4 carcinoma of prostate of embodiment
1, case
Choosing 20 examples and treat the patients with prostate cancer of examination, sampling derives from BJ Union Hospital 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 carrying out patient knows the inside story informs and passes through through this Hospital Ethical Committee.
2, method
Using conventional method or use specific test kit to extract RNA, reverse transcription becomes cDNA, with the reagent in embodiment 3
Box carries out detection reaction.In test kit, prostate normal structure cDNA is as the comparison cDNA in QPCR detection by quantitative, detection
LOC100190940 relative prostate normal tissue expression amount change in tissue samples.
3, result
Determine that purpose band, Δ Δ Ct method carry out relative quantification, between sample and comparison by solubility curve analysis and electrophoresis
Relatively using t inspection, P < 0.05 is significant difference.Result shows, treats in the tissue samples of 20 example patients with prostate cancer of examination
There are the expression of LOC100190940 in 9 example patient tissue samples and carcinoma of prostate normal tissue expression amount without significant difference；Have
In 11 example patient tissue samples, the expression of LOC100190940 is less than the 65% of carcinoma of prostate normal tissue expression amount, wherein
Having the expression of LOC100190940 in 8 example patient tissue samples is less than the 30% of carcinoma of prostate normal tissue expression amount.Warp
Clinical detection further, 20 examples treat to make a definite diagnosis 9 example carcinoma of prostate in the patient of examination, this 9 example patients with prostate cancer and system of the present invention
Standby test kit testing result is consistent.Inferring accordingly, the diagnostic kit of this carcinoma of prostate can clearly distinguish carcinoma of prostate
Patient, and provide 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 (6)
1. a molecular marked compound relevant to carcinoma of prostate, it is characterised in that described molecular marked compound is LOC100190940,
Its nucleotides sequence is classified as shown in SEQ ID NO:1.
2. molecular marked compound as claimed in claim 1, it is characterised in that described LOC100190940 is in prostate cancer tissue
Down-regulated expression.
3. a prostatic cancer diagnostic reagent kit, it is characterised in that described diagnostic kit contains divide relevant to carcinoma of prostate
Sub-label LOC100190940.
4. prostatic cancer diagnostic reagent kit as claimed in claim 3, it is characterised in that described diagnostic kit includes quantitatively
PCR reaction system:
(1) specific amplified LOC100190940 and two pairs of primers of comparison GAPDH；
The primer sequence of detection LOC100190940 is as follows:
LOC100190940 forward primer sequence: SEQ ID NO:2,
LOC100190940 downstream primer sequence: SEQ ID NO:3；
The primer sequence of comparison GAPDH is as follows:
GAPDH forward primer sequence: SEQ ID NO:4,
GAPDH downstream primer sequence: SEQ ID NO:5；
(2) SYBR Green polymerase chain reaction system, including PCR buffer, SYBR Green fluorescent dye, dNTPs etc..
5. molecular marked compound as claimed in claim 1 or 2 screens carcinoma of prostate high-risk group, diagnoses, treats, monitors and in advance
Application in Hou.
Apply the most as claimed in claim 5, it is characterised in that by LOC100190940 in detection subjects's tissue samples
Content, and compared with the content of normal level LOC100190940, to carry out carcinoma of prostate high-risk group's screening, to diagnose, control
Treat, monitor and prognosis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022237855A1 (en) * | 2021-05-11 | 2022-11-17 | 深圳市竞渡医疗器械科技有限责任公司 | Endometrial cancer biomarker and application thereof |
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| CN105102636A (en) * | 2013-03-14 | 2015-11-25 | 尼欧基因组学实验室股份有限公司 | Compositions and methods for detecting and determining a prognosis for prostate cancer |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108165550A (en) * | 2017-12-27 | 2018-06-15 | 郑州大学 | A kind of long-chain non-coding RNA and its application and biological products |
| CN108165550B (en) * | 2017-12-27 | 2021-06-25 | 郑州大学 | Long-chain non-coding RNA and application and biological product thereof |
| WO2022237855A1 (en) * | 2021-05-11 | 2022-11-17 | 深圳市竞渡医疗器械科技有限责任公司 | Endometrial cancer biomarker and application thereof |
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Application publication date: 20170104 |