CN108165550A - A kind of long-chain non-coding RNA and its application and biological products - Google Patents
A kind of long-chain non-coding RNA and its application and biological products Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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Abstract
Embodiment of the disclosure is related to a kind of long-chain non-coding RNA and its application, biological products, modification sequence of the long-chain non-coding RNA with the transcription sequence shown in SEQ ID No.1 or the transcription sequence shown in the homologous sequence of the transcription sequence shown in SEQ ID No.1 or SEQ ID No.1.Applicant carry out it is experimentally confirmed that expression of the long-chain non-coding RNA of the embodiment of the present disclosure in Hormone refractory prostate cancer (CRPC) is apparently higher than normal prostate tissue cell and hormone non-resistant prostate gland cancer cell.Embodiment of the disclosure further relates to application of the long-chain non-coding RNA as molecular marker in diagnose and treat Hormone refractory prostate cancer (CRPC), such as application of the long-chain non-coding RNA as prediction CRPC progresses and its as target spot in CRPC treatments, the expression of long-chain non-coding RNA described in silence can significantly inhibit proliferation, migration and the diffusion of cancer cell of CRPC tumour cells.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of long-chain non-coding RNA and its in diagnose and treat
Application in Hormone refractory prostate cancer.
Background technology
Prostate cancer is a kind of male malignant tumour occurred frequently, and the death rate is only second to lung cancer.In recent years, as people live
The change of mode, the aging of population, in apparent ascendant trend, the statistical data in Shanghai City is shown the incidence of prostate cancer,
The incidence of prostate cancer rose to 12.6/10 ten thousand current males from 2.9/10 ten thousand males of 1991, became serious
Endanger one of important diseases of China's male body health.
Invention content
In one aspect, embodiment of the disclosure provides a kind of long-chain non-coding RNA, has shown in SEQ ID No.1
Transcription sequence or SEQ ID No.1 shown in the homologous sequence of transcription sequence or transcription sequence shown in SEQ ID No.1
Modification sequence.
In some embodiments of the present disclosure, the homology of the homologous sequence is not less than 90%.
In some embodiments of the present disclosure, the modification sequence is to be modified through thio-modification or/and methoxyl group.
On the other hand, embodiment of the disclosure provides the application of above-mentioned long-chain non-coding RNA, including as molecule mark
Application of the will object in diagnosing or treating the biological products of Hormone refractory prostate cancer.
It yet still another aspect, embodiment of the disclosure provides a kind of biological products for diagnosing Hormone refractory prostate cancer,
The biological products include reagent, and the reagent includes the reagent for detecting long-chain non-coding RNA expression quantity in biological sample,
The long-chain non-coding RNA is above-mentioned long-chain non-coding RNA.
In some embodiments of the present disclosure, the biological products are kit, and the kit includes the reagent.
In some embodiments of the present disclosure, the reagent includes the mark for having specificity to the long-chain non-coding RNA
Remember object.
In some embodiments of the present disclosure, the marker includes the primer sets or fluorescence of the long-chain non-coding RNA
The primer sets or the compound with reference to the long-chain non-coding RNA of the long-chain non-coding RNA of label.
In some embodiments of the present disclosure, the primer sets of the long-chain non-coding RNA are the sequences shown in SEQ ID No.2
The primer pair of row and the sequence composition shown in SEQ ID No.3；Or the sequence shown in SEQ ID No.6 and SEQ ID No.7 institutes
The primer pair of sequence composition shown.
In further aspect, embodiment of the disclosure provides a kind of biology system for being used to treat Hormone refractory prostate cancer
Product, the biological products are drugs, and the drug includes the ingredient for long-chain non-coding RNA expression described in inhibition or silence,
The long-chain non-coding RNA is above-mentioned long-chain non-coding RNA.
The other aspects and technical detail of the disclosure, the description with reference to specific embodiment part will be people in the art
Member is understood.
For ease of writing, in following, above-mentioned long-chain non-coding RNA is represented with LncRNA-LOC100.
Description of the drawings
Fig. 1 is the growth figure of the human prostate cancer cell line DU 145 of experimental example and control group after LncRNA-LOC100 silences,
Middle A, B, C are followed successively by the growing state figure of the LncRNA-LOC100 in control group, siRNA1, siRNA2, and E, F are respectively to compare
Group, RNA interfering 1, the cell survival rate of LncRNA-LOC100 in RNA interfering 2, the expression rate of LncRNA-LOC100 are illustrated
Figure.
Fig. 2 be LncRNA-LOC100 silences after the DU145 tumor cell migration compares figures of experimental example and control group, wherein A
It does not take measures for control group, B, C are to show cell migration using siRNA1, siRNA2 silence LncRNA-LOC100, D
The comparison of rate.
Fig. 3 is the DU145 apoptosis of tumor cells FCM analysis of experimental example and control group after LncRNA-LOC100 silences
As a result, wherein A does not take measures for control group, B, C is show using siRNA1, siRNA2 silence LncRNA-LOC100, D
The comparison of apoptosis cell.
Fig. 4 is the DU145 tumor cell growth rates of experimental example and control group after LncRNA-LOC100 silences,
Middle A, B, C be respectively control group, using siRNA1, siRNA2 silence LncRNA-LOC100 DU145 cell volume, cell
Volume is compareed with the variation of LncRNA-LOC100 in all numbers, DU145.
The DU145 tumour cells of tumor models of the Fig. 5 to establish tumor model and control group after LncRNA-LOC100 silences
Cell quantity, wherein A, B, C, D be respectively control group, thin using the DU145 of siRNA1, siRNA2 silence LncRNA-LOC100
The quantity variation control of born of the same parents is changed, using the quantity of the DU145 cells of siRNA1 silences LncRNA-LOC100 using siRNA2
The quantity variations of the DU145 cells of silence LncRNA-LOC100, control group, using siRNA1, siRNA2 silence LncRNA-
The inhibiting rate variation control of the DU145 cells of LOC100.
Fig. 6 is sliced HE colored graphs for spleen tissue.Wherein, A, B, C are followed successively by control group, are sunk using siRNA1, siRNA2
Group after silent LncRNA-LOC100.
Fig. 7 detects LncRNA-LOC100 in 33 prostate cancer patient's cancerous tissues and cancer beside organism for real-time quantitative PCR
Expression, wherein N refers to cancer beside organism, and C refers to prostate cancer tissue.
Specific embodiment
In technology known for inventor, DU145 is from prostate cancer brain metastes tumour as a kind of CRPC cancer cells
It separates, differentiation degree is low, is the prostate gland cancer cell of androgen independent, has powerful metastatic potential, in shortage
The expression of the androgen receptor of source property.DU145 has significant mesothelial cell's characteristic, is that research epithelial-mesenchymal transition causes
The ideal cellular resources that metastatic potential of tumor cell changes.Proliferation and the transfer of DU145 tumour cells are effectively inhibited, it can
To prostate cancer is inhibited to have positive effect.It is of great significance to the research of the Effective target site of DU145 inhibiting tumour cells.
Long-chain non-coding RNA (LncRNA) is a kind of general not encode albumen or encode the limited base sequence of albumen ability
Row are expressed in a variety of level controlling genes such as epigenetics, transcriptional control, post-transcriptional control, in various biological process
In play a significant role, adjusted including human gene, cell growth differentiation and generation, fat metabolism etc. of tumor disease.Portion
Divide the study found that the occurrence and development of some long-chain non-coding RNAs and cancer are there are close relationship, long-chain non-coding RNA is expressed
Difference or certain cancer types specificity in level is related, so if a kind of and Hormone refractory prostate cancer can be obtained
(CRPC) associated LncRNA, it will help develop new diagnosis or the method for treating CRPC.
In the following embodiments, involved experimental implementation can be according to《Molecular Cloning:A Laboratory guide (third edition)》
(Molecular Cloning:A Laboratory Manual, 3red Huang Peitangs etc. are translated, Science Press .2002.8) in institute
It states condition and method carries out.Other test methods not being described in detail are those skilled in the art institute unless otherwise specified
Well known conventional method.The operation of each correlation test is summarized as follows below：
Cell culture
DU145 tumour cells are purchased from ATCC companies, DMEM culture mediums used in cell culture and fetal calf serum and vitellophag
Trypsase used is Sigma Aldrich products.It is cultivated in DU145 cell DMEM culture mediums.It is cultivated in DMEM
The streptomysin of 10%FBS, 100U/ml penicillin and 100mg/ml are added in base, in 37 DEG C of constant incubators containing 5%CO2
Culture.Culture medium is replaced daily, is passed on when cell density covers with bottom of bottle.
The extraction of total serum IgE
DU145 tumour cells carry total serum IgE, and RNA, into after cDNA, is expanded through reverse transcription using PCR.Growth conditions are good
Good DU145 tumour cells are inoculated in by 2 × 105 cells/wells in 6 orifice plates, and 6 orifice plates are placed in incubator, are cultivated；
DU145 tumour cells from cell incubator are taken out, culture medium is sucked out, are washed twice with sterile PBS, PBS is all sucked out；It is past
Trizol reagents are added in Tissue Culture Dish, are blown and beaten with pipettor, cell is made all to crack；It is thin being cracked with Trizol reagents
Dysuria with lower abdominal colic enters in the EP pipes of no RNA enzyme.It is blown and beaten with pipettor, cell is made all to crack；The cell cracked with Trizol reagents is turned
In the EP pipes for entering no RNA enzyme, chloroform, concussion stands low-temperature centrifugation；It draws supernatant to be transferred in new EP pipes, isopropanol is quiet on ice
It puts；Centrifugation；It discards supernatant, obtained precipitation is RNA, and precipitation is washed with DEPC alcohol；Centrifugation；It discards supernatant, nature to be precipitated
After air-drying, add in DEPC water and dissolved；Measure the concentration of the total serum IgE of extraction.
PCR quantitative analyses
Total serum IgE reverse transcription is carried out using the mRNA reverse transcription reagent box of GeneCopoeia companies, is added in every 25uL systems
2ug total serum IgEs；The program that reverse transcription uses is 45 DEG C of hour, 85 DEG C, 5 minutes, 4 DEG C of preservations；The cDNA that reverse transcription is obtained
Dilute 5 times of templates as quantitative PCR；Primer is diluted to the concentration of 2uM；Using GeneCopoeia companies mRNA fluorescent quantitations
PCR kit simultaneously prepares system and setting program according to specification.Specific procedure is as follows：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation
30s, 64 DEG C of annealing 30s, 72 DEG C of extension 15s, 35 recycle；72 DEG C of extension 10min.Interpretation of result：Analyze the specificity of primer
And amplification efficiency, the atopic of primer is judged according to solubility curve.Ct values are obtained according to amplification curve, using relative measurement
The analysis of target gene relative expression quantity is carried out with internal reference beta-actin.Calculation formula is：2^(-ΔCt),ΔCt+Ct
gene-Ct control。
The preparation of slow virus
Transfection procedure is carried out according to 2000 transfection reagent operation instructions of Invitrogen Lipofectamine, specifically
Process is as follows：HEK-293T cells in exponential phase are subjected to pancreatin digestion, cell suspension is made；Cell suspension is connect
Kind is cultivated in 24 orifice plates；The plasmid of extraction and Lipofectamine 2000 are dissolved separately in opti-MEM, mixed
It is even, it is stored at room temperature；By 2000 mixing of plasmid and Lipofectamine, it is stored at room temperature.Plasmid and Lipofectamine
2000 mixed liquor is added in HEK-293T cells, is cultivated；Vial supernatant is collected after transfection 72h, is centrifuged；Filtering point
Dress, the slow virus recombinated, for transfecting DU145 tumour cells.
Cell transfecting
The good DU145 tumour cells of growth conditions are inoculated in by 2 × 105 cells/wells in 6 orifice plates, 6 orifice plates are put
In incubator, cultivated；Cell growth to be cultivated can start the transfection of siRNA to 60 80% density；According to
2000 transfection reagents of Lipofectamine illustrate to transfect LncRNA-LOC100siRNA (purchased from Thermo Fisher
Scientific Bioisystech Co., Ltd), transfection process is as follows：
The lipofectamine2000 added in sterile EP pipes mixings in serum free medium are stood；By slow virus
It adds in serum free medium；Then with the above-mentioned mild mixing of the serum free medium comprising lipofectamine2000, room temperature
Stand minute；Cell is washed with D Hank's liquid 3 times；Serum free medium (antibiotic-free), temperature will be added in said mixture
With 1 hole in 6 orifice plates is added in after mixing；6 orifice plates are placed in CO2 incubators, are cultivated, then abandon supernatant, have been added in
Full culture medium continues to cultivate.
Mtt assay detects inhibiting effect of the LncRNA-LOC100siRNA to tumour growth
LncRNA-LOC100siRNA transfection tumor cells DU145 first adds culture solution to be placed in 37 DEG C to 100mL per hole,
5%CO2 incubator cultures.Prepare transfection when DU145 cell densities reach 80-90%.Per hole 10ug LncRNA-
LOC100siRNA adds in mixing in 200ul culture mediums, stands 5min (1 liquid), and 3-5ul lipo2000 is taken to add in 200ul cultures
In base, mixing (2 liquid).Standing 25min after 2 liquid and 1 liquid mixing.Cell is cleaned twice with culture medium, adds in transfection mixture.
The MTT liquid reaction solution of 20ul is added in per hole after 12h, 37 DEG C are protected from light incubation 4h, abandon supernatant, add in 150ul dimethyl sulfoxide (DMSO)s
(DMSO), light absorption value is surveyed at 490mn wavelength.
Blank control wells, cell control well and medicine feeding hole are set.Blank control wells add culture solution, MTT liquid, dimethyl sulfoxide (DMSO)
And siRNA.Cell control well and medicine feeding hole will refinement born of the same parents, culture solution, MTT liquid and dimethyl sulfoxide (DMSO)s.
Counting method measures growth of tumour cell curve
Inoculating cell：With trypsin digestion transfect 72h after each group PC3 cells, with the culture solution containing 10% fetal calf serum
Single cell suspension is made into, is inoculated in 6 orifice plates (3.3 × 105/hole).Cultivate cell：Move into cell incubator, 37 DEG C, 5%
It is cultivated under the conditions of CO2 and saturated humidity.Cell plates count：Blank control group continuously detects seven days, LncRNA-LOC100siRNA
Processing group detects three days.Daily pancreatin digests three hole cell counts and records, and draws cell growth curve, and experiment repeats three
It is secondary.
Scratch experiment detects the transfer ability of each group human prostata cancer PC3 cells
It is crossed behind in six orifice plates with marking pen, every mono- diatoms of 1cm, per hole at least across three lines, vitellophag paving
Plate.When cell density grows to 80-90%, with the vertical six orifice plates behind horizontal line cut of 10 μ l pipette tips.Old culture solution is abandoned, PBS washes three
Time, it adds in 2% culture solution and drug, experiment packet is same as above.It is observed under inverted microscope, rear film recording is as a result, experiment weight for 24 hours
Again three times.
The foundation of mouse DU145 Transplanted tumor models
The DU145 cells in exponential phase are collected, after 0.25% trypsin digestion, supernatant is abandoned in centrifugation, with culture
2 × 107 cells/mL single cell suspensions are made in liquid resuspension.4~5 week old SCID mice 35 is chosen, standard animal chambers adapt to one
Zhou Hou, every mouse right lower extremity root skin preserved skin, preserved skin position inoculate 0.2mL (2.0 × 107 cell/mL) Lewis
Lung carcinoma cell suspension observes injection site whether there is red and swollen ulceration daily, touches injection site, checks for tumour and grow.
The measure of mouse weight and tumorous size
After mouse-borne tumor, mouse injection site is touched daily, and the tumor mass until touching grain of rice size starts to use vernier
Calliper tumor size, three times a week.Each mouse is marked, carries out the dynamic monitoring of weight and tumor size.With
Same position is measured during vernier caliper measurement every time, measures the maximum major diameter (A) of knurl, the wide diameter of the maximum vertical with major diameter
(B), formula is utilized：Volume (V)=AB2/2 calculates the average external volume of knurl, draws tumor growth curve.The measure of tumour inhibiting rate
Last dose for 24 hours after, weigh mouse weight, eyeball, which takes, puts to death mouse after blood, removes mouse tumor tissue, removal
Surrounding tissue and blood, Hanks liquid are rinsed rear filter paper well and are dried, and put electronic balance and weigh knurl quality, calculate tumour inhibiting rate：
Data statistics
All data are analyzed using SPSS20.0 statistical softwares, and group is carried out using one-way analysis of variance and t inspections
Between compare, represented with " mean ± standard deviation " (x ± s), inspection level α=0.05, P ＜ 0.05 represent difference have statistics meaning
Justice and for significant difference, P >=0.05 is not statistically significant.Inspection level α=0.01, P ＜ 0.01 represents that difference has statistics
Meaning and for pole significant difference.
In at least one embodiment of the disclosure, a kind of long-chain non-coding RNA is provided, applicant is named as
LncRNA-LOC100, have SEQ ID No.1 shown in transcription sequence or the transcription sequence shown in SEQ ID No.1 it is homologous
The modification sequence of sequence or the transcription sequence shown in SEQ ID No.1.
Optionally, the homology of the homologous sequence is not less than 90%, such as homology is not less than 95%, such as homology
Not less than 98%.
Optionally, the modification sequence is the LncRNA-LOC100 by base group modification, such as thio-modification, such as first
Oxygroup is modified.
Applicant is it is discovered by experiment that expression of the LncRNA-LOC100 in CRPC is apparently higher than normal prostate tissue
Cell and hormone non-resistant prostate gland cancer cell, so as to as prediction, the CRPC progresses of diagnosis CRPC and its conduct
Target treatment CRPC.
In at least one embodiment of the disclosure, a kind of biology system for diagnosing Hormone refractory prostate cancer is provided
Product, the biological products include reagent, and the reagent includes the examination for detecting long-chain non-coding RNA expression quantity in biological sample
Agent, the long-chain non-coding RNA are above-mentioned long-chain non-coding RNA.
Optionally, the biological products are kit, and the kit includes the reagent.
Optionally, the reagent includes the marker for having specificity to the long-chain non-coding RNA.
Optionally, the long-chain of primer sets or fluorescent marker of the marker including the long-chain non-coding RNA is non-
The primer sets of coding RNA.
Wherein, the reagent can show as biologically any acceptable product form, including but not limited to probe,
Genetic chip or PCR primer.
Wherein, the biological sample is in vitro biological sample, in vitro new including but not limited to from detected object
It is fresh tissue or cell, formalin are fixed or paraffin-embedded tissue or cell, blood or body fluid.
Wherein, the kit is sold and using the kit encapsulated as complete business, in order to improve the accurate of detection
Property, negative and positive control is convenient for, relevant positive control and negative control can be also contained in mentioned reagent box, can be with
Comprising the other auxiliary reagents used convenient for fast operating, such as DNA extracts reagents, PCR reaction reagents, electrophoresis reagents, these
Ingredient is common in PCR amplification detection, dosage and using also skillfully grasping for those skilled in the art, not superfluous herein
It states.
In some embodiments of the present disclosure, the primer sets of the long-chain non-coding RNA are the sequences shown in SEQ ID No.2
The primer pair of row and the sequence composition shown in SEQ ID No.3.
In some embodiments of the present disclosure, the primer sets of the long-chain non-coding RNA are the sequences shown in SEQ ID No.6
The primer pair of row and the sequence composition shown in SEQ ID No.7.
In at least one embodiment of the disclosure, a kind of biology for being used to treat Hormone refractory prostate cancer is provided
Product, the biological products are drugs, the drug include for inhibit or silence described in long-chain non-coding RNA expression into
Point, the long-chain non-coding RNA is above-mentioned long-chain non-coding RNA.For example, the drug includes LncRNA-LOC100's
siRNA。
Optionally, the siRNA can include the antisense strand of LncRNA-LOC100, such as the sequence shown in SEQ ID No.2
The primer pair of row and the sequence composition shown in SEQ ID No.3, such as sequence and SEQ ID No.7 shown in SEQ ID No.6
The primer pair of shown sequence composition.
Embodiment 1 detects expressions of the LncRNA-LOC100 in CRPC tumour cells
To detect expressions of the LncRNA-LOC100 in CRPC tumour cells, first according to LncRNA-LOC100's
Gene order, designs specific primer pair, and the nucleotides sequence of F chains is classified as SEQ ID No:Sequence corresponding to 2, the core of R chains
Nucleotide sequence is SEQ ID No:3 (the siRNA2 experimental groups shown in respective figure)；The nucleotides sequence of F chains is classified as SEQ ID No:
Sequence corresponding to 6, the nucleotides sequence of R chains are classified as SEQ ID No:7 (the siRNA1 experimental groups shown in respective figure).
DU145 tumour cells are purchased from ATCC companies.DMEM culture mediums used in cell culture and fetal calf serum and vitellophag
Trypsase used is Sigma Aldrich departments product.The good DU145 tumour cells of growth conditions are pressed 2 × 105
Cells/well is inoculated in 6 orifice plates, and 6 orifice plates are placed in 37 DEG C, in 5%CO2 incubators, are cultivated；By DU145 tumour cells
It is taken out from cell incubator, culture medium is sucked out, washed twice with sterile PBS, PBS is all sucked out；Add in toward Tissue Culture Dish
Enter Trizol reagents, about every 5 × 106 cells add in 1mL reagents, with pipettor piping and druming several times, cell are made all to crack；Use
The cell of Trizol reagents cracking is transferred in the EP pipes of no RNA enzyme, and label is performed on tube wall.
Preparatory work of experiment：
1. the plastic products such as centrifuge tube, pipette tips 0.1% pyrocarbonic acid diethyl ester (DEPC) aqueous solution soaking 12 hours or more
Inactivate exogenous rna enzyme.High pressure steam sterilization removes remaining DEPC in 30 minutes, passes through carboxymethylation pair to prevent DEPC
The purine bases of RNA are modified.
2.0.1%DEPC processing water configuration：200ml ultra-pure waters add in 200 μ lDEPC, and 4 hours of magnetic agitation are stayed overnight, high
Press 30 minutes or more the DEPC that sterilize and inactivate.
75% alcohol 0.1%DEPC handles water and is configured.
3. vial and 200 DEG C of the tweezers hour inactivation exogenous rna enzyme of baking 3.
Extraction RNA is carried out qualitative (nanodrop) and quantitative (agarose electrophoresis)
Reverse transcription reaction
(1) in super-clean bench, the 20 μ lPCR pipes that DEPC is handled is taken to put on ice, are sequentially added
Total serum IgE 500ng
Primer (0.5 μ g/ μ l) 1 μ l immediately
Without RNA enzyme water 9ul to 12 μ l of total volume
Mixing centrifuges 3000 revs/min 30 seconds with microcentrifuge.
(2) 70 DEG C are denaturalized for 5 minutes, and cooled on ice is put in taking-up.
(3) PCR pipe is put on ice, is sequentially added
5×RT Buffer 4μl
RNase inhibitor(20U/μl) 1μl
10mM dNTP Mix 2μl
(4) M-MuLV reverse transcriptases (200U/ μ l) 1 μ l to 20 μ l of final volume are added in.
Mixing centrifuges 3000 revs/min 30 seconds with microcentrifuge.
(5) water-bath is put to carry out by the following conditions
25 DEG C 10 minutes
42 DEG C 60 minutes
70 DEG C 10 minutes
Cooled on ice immediately, product cDNA for PCR or put -20 DEG C of refrigerators preservations.
PCR reacts
(1) 20 μ IPCR pipes is taken to sequentially add
3000 revs/min of 30 seconds mixings are centrifuged with microcentrifuge.
(2) PCR reactions are carried out by the following conditions：
Experiment contrast
1. positive control：Reverse transcription and PCR amplification are carried out using the total serum IgE of known high expressing cell.
2. negative control：The reaction system for replacing total serum IgE with sterile deionized water carries out reverse transcription and PCR amplification.
3. internal reference：While carrying out PCR reactions with primer, PCR reactions are carried out with GAPDH primers.
The silence of embodiment 2LncRNA-LOC100 is to the inhibiting effect of CRPC tumour growths
DU145 cells are cultivated in vitro.Lewis cell cryopreservation tubes are taken out from liquid nitrogen, are immersed in 37 DEG C of waters bath with thermostatic control, soon
Speed, which shakes 1~2min, makes it melt as early as possible, and alcohol disinfecting after defrosting is moved in superclean bench, and liquid-transfering gun quickly moves cell
Into 10mL centrifuge tubes, and mixing after the dilution of 8mL cell culture fluids being added in, centrifugation channel closure postposition enters centrifuge 800r/min,
It is taken out after centrifugation 4min, abandons supernatant, added in 8mL cell culture fluids and cell is resuspended, dispense cell suspension to 25m2's after mixing
It is put into 37 DEG C of incubators and cultivates in Tissue Culture Flask, after label, the adherent situation of cell is observed after 6h.Observation cell growth daily
Situation after cell is adherent, abandons the culture solution that supernatant more renews to maintain cell normal activity.Cell growth confluent cultures after 3 days
The 90% of bottle, carries out the passage of cell.Liquid in culture bottle is discarded, 0.2% pancreatin is added in after rinsing culture bottle with Hanks liquid,
It is placed in 2~3min in 37 DEG C of incubators and carries out cell dissociation.Cell suspension is moved in 10mL centrifuge tubes after cell dissociation, is added
Enter culture solution dilution mixing and be placed on centrifuge 800r/min, centrifuge 4min, abandon supernatant, add in cell culture fluid dilution, mixing
Cell suspension is dispensed into the Tissue Culture Flask of 75m2 afterwards, is put into 37 DEG C of incubators and cultivates after label, replace cell in time
Culture solution.
Cell culture the 3rd day, cell density 90% is digested with pancreatin, cell suspension is made after centrifugation, after dilution
Cell suspension is made into a concentration of 1 × 105 cell/mL.Blank control group, cell controls group are set, at LncRNA-LOC100
Totally 3 groups of reason group, every group sets 3 multiple holes.The cell suspension prepared is laid in 96 orifice plates, 100 μ L cell suspensions are added in per hole.First
After 96 orifice plates are moistened with cell culture fluid, liquid feeding is carried out along wooden partition, is gently shaken up and down so that cell is uniform after liquid feeding
Tiling, edge hole are filled with PBS buffer solution, 5%CO2, are cultivated in 37 DEG C of incubators, until cell monolayer is paved with bottom hole.
96 orifice plate culture DU145 cells for 24 hours after, abandon supernatant, the culture solution more renewed.Culture solution is added to be put to 100mL per hole
In 37 DEG C, 5%CO2 incubator cultures.Prepare transfection when DU145 cell densities reach 80-90%.Per hole 10ug LncRNA-
LOC100siRNA plasmids add in mixing in 200ul culture mediums, stand 5min (1 liquid), and 3-5ullipo2000 is taken to add in 200ul trainings
It supports in base, mixing (2 liquid).Standing 25min after 2 liquid and 1 liquid mixing.Cell is cleaned twice with culture medium, adds in transfection mixing
Object.Change 10% culture solution after 12h into.Fluorescence microscopy Microscopic observation cell fluorescence after 48h.
MTT powder 0.5g are weighed, are dissolved in 100mL PBS buffer solution, are made into a concentration of 5mg/mL, i.e. 0.5%MTT liquid.With
0.22 μm of membrane filtration removes the bacterium in solution, puts 4 DEG C and be kept in dark place.Cell dosing culture for 24 hours, after 48h, 72h, drug
Processing group is separately added into 20 μ L of 0.5%MTT liquid, continues carefully to suck culture solution in hole after cultivating 4h, 150 μ L diformazans are added in per hole
Base sulfoxide puts low-speed oscillation 10min on shaking table, after object to be crystallized fully dissolves, puts and each hole is measured at microplate reader OD490nm wavelength
Light absorption value (OD values), according to formula：Inhibiting rate=[1- (experimental group OD values-zeroing hole OD values)/control group OD values-zeroing hole
OD values)] × 100%.
Blank control wells, cell control well and medicine feeding hole are set.Blank control wells add culture solution, MTT liquid, dimethyl sulfoxide (DMSO)
And siRNA.Cell control well and medicine feeding hole will refinement born of the same parents, culture solution, MTT liquid and dimethyl sulfoxide (DMSO)s.
Scratch experiment detects the transfer ability of each CRPC tumour cells.It is crossed behind in six orifice plates with marking pen, every 1cm
One diatom, per hole at least across three lines, vitellophag bed board.
When cell density grows to 80-90%, with the vertical six orifice plates behind horizontal line cut of 10 μ l pipette tips.
Old culture solution is abandoned, PBS is washed three times, adds in 2% culture solution and drug, experiment packet are same as above.It is seen under inverted microscope
It examines, rear film recording is as a result, test in triplicate for 24 hours.
3 streaming AnnexinV-FITC/PI of embodiment detects Apoptosis situation.
Using the apoptosis situation of streaming method detection tumour cell, test experience is divided to two groups, i.e. control group and LncRNA-
LOC100siRNA processing groups.DU145 cells spread six orifice plates (1 × 104/hole), after adherent, add the LncRNA- of different content
LOC100siRNA, control group only add culture solution.In incubator cultivate cell for 24 hours, vitellophag, PBS wash 2 times (2000rpm,
5mim), it puts in EP pipes.
It adds in Binding Buffer (500 μ l) and cell is resuspended.5 μ lAnnexinV-EGFP mixings are added in, add 5 μ l
Propidium Iodide mixings.
Room temperature is protected from light 5-15min.
It is quantitatively detected with flow cytometer (time is no more than 1h), excitation wavelength, launch wavelength EX 488nm, En530nm,
The green fluorescence of AnnexinV-EGFP is detected by FITC channels (FL1), and PI red fluorescences are detected by PI channels (FL2).It is real
It tests in triplicate.
The influence that the inhibition of 4 LncRNA-LOC100 of embodiment grows and shifts in mouse tumor model to CRPC.
SCID mice tumor model (mouse is purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) is established, in mouse
Back both sides inoculated tumour, inoculated tumour is after 7 days, in the accessible grain of rice size tumor mass in injection site.Mouse is weighed, and will be passed through
The tumor cell inoculation mouse of LncRNA-LOC100 silences, and with the tumour ratio of control group, that is, unused LncRNA-LOC100 silences
Compared with observation mouse injection site situation, touches tumor mass, and measure tumor size daily.
Mouse is put to death to 1cm sizes, visually observes spleen variation by tumour length.After putting to death tumor-bearing mice, spleen is taken out,
Color and luster, the thickness of spleen coating are observed, whether there is exudate, fibrous adherence, recess, breach etc., and whether observe spleen edge sharp keen,
Whether there is neoplasm metastasis etc..And further prepare the histotomy of spleen：
(1) materials are with fixing：Spleen is taken out, the tissue that size is 1.5cm × 1.5cm × 0.3cm is cut into after rinsing well
Block is placed in 10% formalin solution and fixes 24~48h.
(2) it is dehydrated transparent：By the ethyl alcohol of low concentration to high concentration as dehydrating agent, tissue moisture content in the block is gradually sloughed.
Tissue block is placed in again and is not only dissolved in ethyl alcohol, but also is dissolved in transparent in the clarifier dimethylbenzene of paraffin, tissue block is replaced out with dimethylbenzene
In ethyl alcohol, so as to waxdip embed.
(3) waxdip embeds：The tissue block that transparent processing is crossed is placed in the paraffin dissolved, treats that paraffin is completely immersed in tissue
It is embedded after block.Container is first got out, pours into the paraffin dissolved, the tissue block that rapid gripping has been impregnated with paraffin is put into it
In, cooled and solidified is blocking.
(4) slice and patch：Embedded paraffin mass is fixed on slicer, is cut into the thin slice of 5~8um thickness.It cuts
Thin slice be put into the water of heating and plate, be attached on glass slide, put in 45 DEG C of insulating boxs and dry.
(5) dewaxing dyeing：The paraffin in slice is sloughed, then through high concentration to low-concentration ethanol aquation, distillation with dimethylbenzene
Water rinses, you can carries out HE dyeing, process is as follows：
5~20min of dyeing in hematoxylin aqueous solution is put into 1. being sliced after ethyl alcohol aquation, tap water rinses 3~5min.
2. each 5~30s of color separation in sour water and ammonium hydroxide, flowing water enters distilled water a moment after rinsing 1h.
3. enter in 70% and 90% ethyl alcohol and be dehydrated each 10min.
4. entering eosin stains liquid dyes 2~3min.
(6) it is dehydrated transparent：Slice after dyeing is dehydrated through straight alcohol, then makes slice transparent through dimethylbenzene.
(7) sealing：By the transparent upper natural gum of slice drop, covered sealing.
Experiment conclusion
As described in Example 1, using following primer pair as the amplimer of LncRNA-LOC100
F:(SEQ ID NO.2)：5 '-GAGATGACAGAGGCATGCTTTA-3 ',
R:(SEQ ID NO.3)：5′-CCGAGGAATCACACAGTTCTAC-3′；
F:(SEQ ID NO.6)：5 '-CCAAGGAAGAAGCAGCTAAGA-3 ',
R:(SEQ ID NO.7)：5′-AGGATGCCTTTCTCCACATAAG-3′；
Using as follows as the amplimer of internal reference product beta-actin
F：(SEQ ID NO.4):5′-GTTGCTGTTTGGTTAGGTATTGG-3′
R：(SEQ ID NO.5):5′-CCACTGAGCTACAAGCTCTTAAT-3′.
If embodiment 2,3 is described, refering to what is shown in Fig. 1, by MTT methods being used to detect after LncRNA-LOC100 silences
It was found that the growth of human prostate cancer cell line DU 145 is suppressed, significant difference compared with the control group.Refering to what is shown in Fig. 2, cell migration
Experiment significantly weakens after showing LncRNA-LOC100 silences with comparing DU145 tumor cell migrations ability.With reference to 3 institute of figure
Show, with comparing DU145 apoptosis of tumor cells cell number showed increaseds after FCM analysis LncRNA-LOC100 silences；
Refering to what is shown in Fig. 4, using DU145 tumor cell growth rates after cell counting detection LncRNA-LOC100 silences,
DU145 growths are significantly inhibited.
As described in Example 4, HE colored graphs are sliced with reference to spleen tissue shown in fig. 6.In Fig. 6-A as it can be seen that control
Group slice on visible spleen essence white pulp, red pulp structure, acini lienalis structure is imperfect, massive tumor tissue in be dispersed in, tufted or
Small bulk arrangement, size are more consistent.Fig. 6-B expand snius lienis visible after LncRNA-LOC100 silences, and desmacyte increases in sinus
It is raw, it is seen that a small amount of remaining lymph follicle, therebetween tumor tissues significantly reduce.
Apart from the above, applicant has also randomly selected 33 patients in CRPC PATIENT POPULATIONs and has been investigated.Experiment
In the primer sequence of real-time quantitative of detection LncRNA-LOC100 expression used be：LncRNA-LOC100Primer F:(SEQ
ID NO.2) and LncRNA-LOC100Primer R:The primer pair of (SEQ ID NO.3) composition, internal reference product beta-
The primer nucleotide sequences of actin are beta-actin Primer F (SEQ ID NO.4) and beta-actin Primer R
(SEQ ID NO.5)。
Refering to what is shown in Fig. 7, in 33 clinical samples, have in 20 pairs of samples by Real_time quantitative detection discovery
The expression of LncRNA-LOC100 is apparently higher than cancer beside organism, and up-regulation rate reaches 74.3%, and statistical analysis shows p<0.01, tool
It is significant.It follows that the LncRNA-LOC100 that the embodiment of the present disclosure provides can be used as marker for diagnostic uses with pre-
Survey CRPC progresses.
It can be seen from the above, can predict CRPC progresses by detecting the expression of LncRNA-LOC100, it can be by heavy
Expression that is silent, inhibiting LncRNA-LOC100 is so as to inhibit the proliferation of CRPC tumour cells, migration and cancer cell diffusion, so as to rise
To the effect for the treatment of CRPC.
Sequence table
<110>Zhengzhou University
<120>A kind of long-chain non-coding RNA and its application and biological products
<130> 2210
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2544
<212> DNA
<213>Human Prostate Cancer Cells (DU145)
<400> 1
cttaaagggg gcccataaag acgcactgcc aagggctccg tgtgccccgg ggccgcggga 60
atgcggatcg cagccggcag ggaggggccg cggcggagcc ttagacttgg gggagccaag 120
gaggccactc ccaggaggct gcggagcaaa tgactgcctc cccgagcccc tggctgcaaa 180
agcagaggtt gcccgtggct tttgcagctc tggacacgcg ggtgcagccc agggaacgtg 240
gacaagaggt gttgacggac cggaccgtgc atttgagttg gggcatatcg aagtggttta 300
attaaaagcc gcgagtgtgg ccccagatcc tggcgttatc aaagcaaatc aaagcctttc 360
tttccagccg agcgggccct gtggaaatga gatgtacatt tacccttgac gcaccgcaag 420
cctggccgag gctctcaggt taattgatat ttaatggaaa tcatgcccat gaatatagtt 480
tccatcattt cacccttgat agacgtcagc cctgggcgca gggggaggct gaagaatgca 540
ggagggggca ttaggcttgc tgagagagta aatagaagag cggacagaaa cgttctgagg 600
cgggaaaaag ggctcgcgct ggggagggtg acatttcctc gggcaggacg cggctcccgg 660
gtctcagccc cggcgcgcag cgaaccaggg ccggctgcgg ttcccggagg agtgcggagg 720
gcagacgacc gcaagttccc cccaggagcc tcccagccgg cgttcagatc gcgccggttc 780
caggcagctg caggcggccc atcttatgct aacgatccta cttcctccgc caagcctgtg 840
aaattactgc cgagagagcg cgcaggcctc aaagttacga atttttccca aagcagagaa 900
agagaaggct cagcctagtt tgtgagcaga gatgaagaaa agttgcccga gtcatgtggc 960
ccagtcgagt gtgaatcgac aaaatattcc cgattttagc agctctctcc aaagctgtct 1020
ggctttaccc taccaaccgc ctccccacac acttcctttc cctctctgct cccgtttcaa 1080
tgtccaataa atgcaatggt tctctgttgt gtgtgtgtgt tttcccctct cctgctcggc 1140
ctttctcgtg ggctctttgg gtgactgagc cccgtttcct tcgggatagg tacaatgtcg 1200
tgagggcgcc aggcccacag ccttggaaac agacctccaa ggcgcccctg ctcaagctaa 1260
acgcaccacg ccggctttcc acaccaaatg caaatttccg aaaaccccag acggcctcag 1320
agatgctgca ggctggcgag cgatttcagc tacccctagc tccttgtggg ttggcccagc 1380
ctgcgagcgc gatcctgact tgtgtccggg tggactgaaa tatcattttt acaaatggag 1440
aaaatgagat cgagatggac agacatagca aggtgataat gtccgactca attatttcta 1500
ctgggaatga ctgaaaggta aaggaatcgt taatgaatct ggagatgaca gaggcatgct 1560
ttatttaatt tttaaattag taaggagtgc agacttgggg tggtggagag caagggtttt 1620
tttgttgttg ttgctgtttg tttttgtggg gttttttttc tgctggttat tgccaagtat 1680
aatgactttc tttaaatccg aaggcgaacg aattgattct gacaattaag gatttgtcct 1740
tcaatcttgc aaatgcccaa ttaaaagatt attattgtca ataaagcctt ggagctacct 1800
ttgtaattgt gccccttttt ttcagtttgg cctgtgtgtg tgtgtgtgtg tgtgtgtgtg 1860
tgtgtgtttg aaccctggat aaatgagtgg attttggaat ggaatggggg gggggaatga 1920
aagagcctgg atctcaacaa taccaggcat cttgtagaac tgtgtgattc ctcgggggaa 1980
taaaagaaaa atgaacacta gaaaattcat ttatagacat tcaggccaaa tttagaaaac 2040
gttacaacat ttcctcttgg gacaaaacag tttttaacat ttatttaagc attccgttta 2100
taatatgagt agaaaaagag cgcgattaac ttagtaagaa aagatctatg ctttttacag 2160
ctttctgttt tgatataata cattaattta tcatgtttta tagtttcagc tgtgccacag 2220
tccaaagttt ctgaagccat gatcgagcca aaattttatg cccagcaaac acagaaatta 2280
atagtacacc ttgtttattt ttggttaaaa ttatcctaat ttcggattaa aaataagatt 2340
gttaatattt ttaatcaaaa cctttctccg ccgcaattcc agctttacga gatcagaaag 2400
tggagcaaca gcgctctcta gtggccaaat gcgacctcca gtccactgaa tttaattttt 2460
attcatctca aaccaattct aatctcattc ttcaatttgg ggcttggact gctgacacta 2520
ataatgcttc attcctgtga ctgt 2544
<210> 2
<211> 22
<212> DNA
<213>Primer (LncRNA-LOC100 Primer F)
<400> 2
gagatgacag aggcatgctt ta 22
<210> 3
<211> 22
<212> DNA
<213>Primer (LncRNA-LOC100 Primer R)
<400> 3
ccgaggaatc acacagttct ac 22
<210> 4
<211> 23
<212> DNA
<213>Primer (beta-actin Primer F)
<400> 4
gttgctgttt ggttaggtat tgg 23
<210> 5
<211> 23
<212> DNA
<213>Primer (beta-actin Primer R)
<400> 5
ccactgagct acaagctctt aat 23
<210> 6
<211> 21
<212> DNA
<213>Primer (Prime F)
<400> 6
ccaaggaaga agcagctaag a 21
<210> 7
<211> 22
<212> DNA
<213>Primer (Primer R)
<400> 7
aggatgcctt tctccacata ag 22
Claims (10)
1. a kind of long-chain non-coding RNA, which is characterized in that there is the transcription sequence or SEQ ID No.1 shown in SEQ ID No.1
The modification sequence of the homologous sequence of shown transcription sequence or the transcription sequence shown in SEQ ID No.1.
2. long-chain non-coding RNA according to claim 1, which is characterized in that the homology of the homologous sequence is not less than
90%.
3. long-chain non-coding RNA according to claim 1, which is characterized in that the modification sequence be through thio-modification or/
It is modified with methoxyl group.
4. the application of long-chain non-coding RNA described in claim 1, which is characterized in that including being diagnosed as molecular marker
Or the application in the biological products for the treatment of Hormone refractory prostate cancer.
5. a kind of biological products for diagnosing Hormone refractory prostate cancer, which is characterized in that the biological products include reagent, institute
It states reagent and includes the reagent for detecting long-chain non-coding RNA expression quantity in biological sample, the long-chain non-coding RNA is right
It is required that the long-chain non-coding RNA described in 1.
6. biological products according to claim 5, which is characterized in that the biological products be kit, the kit
Including the reagent.
7. biological products according to claim 5 or 6, which is characterized in that the reagent, comprising to the long-chain non-coding
RNA has the marker of specificity.
8. biological products according to claim 7, which is characterized in that the marker includes the long-chain non-coding RNA
Primer sets or fluorescent marker the long-chain non-coding RNA primer sets or the chemical combination with reference to the long-chain non-coding RNA
Object.
9. biological products according to claim 8, which is characterized in that the primer sets of the long-chain non-coding RNA are SEQ
The primer pair of sequence composition shown in sequence and SEQ ID No.3 shown in ID No.2；Or the sequence shown in SEQ ID No.6
With the primer pair of the sequence composition shown in SEQ ID No.7.
10. a kind of biological products for being used to treat Hormone refractory prostate cancer, which is characterized in that the biological products are medicines
Object, the drug include the ingredient for long-chain non-coding RNA expression described in inhibition or silence, and the long-chain non-coding RNA is
Long-chain non-coding RNA described in claim 1.
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| CN113549690A (en) * | 2020-12-02 | 2021-10-26 | 天津医科大学总医院 | Long-chain non-coding RNA and application thereof as molecular marker of multiple myeloma cell |
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