CN106244686A - The new application of mir 3620 - Google Patents
The new application of mir 3620 Download PDFInfo
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- CN106244686A CN106244686A CN201610618150.8A CN201610618150A CN106244686A CN 106244686 A CN106244686 A CN 106244686A CN 201610618150 A CN201610618150 A CN 201610618150A CN 106244686 A CN106244686 A CN 106244686A
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Abstract
The present invention relates to the new application of mir 3620, be specifically related to the new opplication in mir 3620 and ripe miRNA diagnosis and treatment adenocarcinoma of colon thereof.Invent order-checking secondary to adenocarcinoma of colon tissue and cancer beside organism and obtain data, in combination with data base's data with existing, after confluence analysis, it is thus achieved that its miRNA expresses data, filter out the mir 3620 of candidate.Further clinical verification and the display miR 3620 3p low expression in cancerous tissue of cell experiment result, process LAN miR 3620 3p can suppress the in-vitro multiplication ability of colon cancer cell.The present invention provides new adenocarcinoma of colon diagnosis and treatment gene for clinic, and the research and development for next step related reagent lay the foundation.
Description
Technical field
The present invention relates to tumor cells diagnostic field, be specifically related to mir-3620 and the new application of ripe miRNA thereof, more
It is specifically related to mir-3620 and ripe miRNA new opplication in diagnosis and treatment adenocarcinoma of colon thereof.
Background technology
MiRNA is a kind of little molecule of endogenous non-coding, is carried out the expression of controlling gene by specific binding target gene.Single
Bar miRNA can be with up to a hundred target genes of targeting, and substantial amounts of miRNA and the signal relevant to cancer of interacting between target gene
Path serves vital regulating and controlling effect.MiRNA plays regulation feedback and the effect of buffering in normal cell physiology, as
Accurate adjustment target gene protein level, prevents it from exceeding the key factor of normal physiological category.Additionally, miRNA is also regulation difference point
Switch between subsignal path.MiRNA expression and activity exception very likely affect cell normal physiological function thus
Cause the generation of disease.Tested by substantial amounts of tumor tissues and cell strain miRNA chip detection, increasing miRNA quilt
Find to play in cancer beyond important function expected from people.In the experiment in vivo of transgenic mice, research worker finds
The unconventionality expression of miRNA would generally cause the generation of cancer.And some miRNA even play difference in different tumor cells
The most diametrically opposite effect.So, miRNA and the relation of cancer are that we must get clear during defeating this disease
Problem.
The present invention is based on high-flux sequence method, and cancerous tissue and cancer beside organism to 6 example adenocarcinoma of colon patients carry out secondary
Order-checking, it is thus achieved that the expression data of its miRNA, in conjunction with 337 example adenocarcinoma of colon patient's miRNA data in TCGA data base, enters meanwhile
Row Integrative Bioinformatics is analyzed, and picks out and several carry out molecular biology checking from the miRNA of candidate, and result show,
The mir-3620 that invention provides is closely related with adenocarcinoma of colon, can be used for clinical diagnosis and the prevention detection of adenocarcinoma of colon, for facing
On bed, the exploitation of dependent diagnostic reagent or chip etc. lays the foundation.
Summary of the invention
It is an object of the invention to provide the preparation of detection mir-3620 and ripe miRNA thereof in preparation diagnosing colon cancer examination
Application in agent.The sequence of mir-3620 is shown in that sequence table SEQ ID NO 1, miR-3620-5p sequence are shown in sequence table SEQ ID NO
2；MiR-3620-3p sequence is shown in sequence table SEQ ID NO 3.
Preferably colon cancer is adenocarcinoma of colon.
Further, diagnosis adenocarcinoma of colon reagent include based on high-flux sequence method and/or based on quantifying PCR method and/
Or based on the transcribing or based on immune detection of mir-3620 and ripe miRNA thereof in probing procedure detection adenocarcinoma of colon sample
The expression of the target gene of mir-3620 and ripe miRNA regulation and control thereof in method detection adenocarcinoma of colon sample, it is preferred to use
Northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on microsphere
In Flow cytometry adenocarcinoma of colon sample, mir-3620's and ripe miRNA thereof transcribes；Use ELISA and/or gold colloidal
The expression of the target gene of mir-3620 and ripe miRNA regulation and control thereof in ELISA test strip adenocarcinoma of colon sample.
Preferably, include specific amplification mir-3620 and the primer of ripe miRNA thereof based on quantifying PCR method, enter one
Preferably, the primer sequence of specific amplification miR-3620 is SEQ ID NO 4 to step；Include based on probing procedure and mir-
The probe of the nucleic acid array hybridizing of 3620 and ripe miRNA.
It is an object of the invention to provide and raise transcribing and/or promoting mir-3620 of mir-3620 and ripe miRNA thereof
And the reagent of the activity of ripe miRNA prevents and treats the application in colon cancer preparation in preparation.
Further, colon cancer is adenocarcinoma of colon.
Further, colon cancer preparation preventing and treating Colon Cancer Cells is prevented and treated.
Preferably, microRNA gain-of-function technology based on RNA and/or gene specific miR Mimics skill are used
Art rise mir-3620 and ripe miRNA thereof transcribes and/or promotes mir-3620 and the activity of ripe miRNA thereof.Preferably people
Work synthesizes short hairpin RNA or raises mir-3620 and ripe miRNA thereof by regulation and control promoter.
It is an object of the invention to provide the target gene of mir-3620 and ripe miRNA thereof in preparation diagnosis and/or preventing and treating
Application in adenocarcinoma of colon preparation.
It is an object of the invention to provide above-mentioned adenocarcinoma of colon diagnostic preparation answering in preparing adenocarcinoma of colon diagnostic tool
With.
It is an object of the invention to provide one and prevent and treat colon cancer reagent, described reagent comprises:
A () rise mir-3620 and ripe miRNA thereof transcribes and/or promotes mir-3620 and the work of ripe miRNA thereof
The reagent of property；
Receptible carrier on (b) pharmaceutics.
Preferably, microRNA gain-of-function technology based on RNA and/or gene specific miR Mimics skill are used
Art rise mir-3620 and ripe miRNA thereof transcribes and/or promotes mir-3620 and the activity of ripe miRNA thereof.Preferably people
Work synthesizes short hairpin RNA (short hairpin RNA, shRNA) or raises mir-3620 and maturation thereof by regulation and control promoter
The expression of miRNA.
Further, described colon cancer is adenocarcinoma of colon.
It is an object of the invention to provide a kind of adenocarcinoma of colon diagnostic reagent, adenocarcinoma of colon diagnostic reagent can detect colon
In adenocarcinoma sample, mir-3620's and ripe miRNA thereof transcribes or mir-3620 in immunologic detection method detection adenocarcinoma of colon sample
And the expression of the target gene of maturation miRNA regulation and control.
Further, adenocarcinoma of colon diagnostic reagent is based on high-flux sequence method and/or based on quantifying PCR method and/or base
In probing procedure detection adenocarcinoma of colon sample, mir-3620's and ripe miRNA thereof transcribing or detecting based on immunization method
The expression of the target gene of mir-3620 and ripe miRNA regulation and control thereof in adenocarcinoma of colon sample, it is preferred to use northern is miscellaneous
Friendship method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, flow cytometry based on microsphere
In detection adenocarcinoma of colon sample, mir-3620's and ripe miRNA thereof transcribes.
The preparation that it is an object of the invention to provide above-mentioned preventing and treating adenocarcinoma of colon is preparing adenocarcinoma of colon medicine or examination
Application in agent.
Definition:
The method of the expression of present stage detection miRNA mainly includes based on high throughput sequencing technologies, based on nucleotide
Hybridization and the miRNA detection method of PCR-based.MiRNA detection method based on probe hybridization technique is a kind of direct Detection Method,
Need not sample rna is carried out pre-amplification, analyze skill including northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection
The technology such as art, RAKE method, in situ hybridization, flow cytometry based on microsphere.
(1) Northern hybridization
It is the most classical detection eukaryote RNA size also known as RNA engram technology, estimates the experimental technique of its abundance.Base
Present principles is as follows: first at the upper fixing miRNA sample of carrier (such as silicon chip, microsphere or film etc.) more miscellaneous with the probe through labelling
Hand over, after washing unnecessary hybridization probe, carry out signal detection；Can also be first fixing and the complementation of target miRNA sequence on carrier
DNA probe, then hybridizes with the sample miRNA through labelling, then carries out signal detection.The method of signal labelling includes isotope
Labelling, fluorescent labeling and nano gold mark etc..
(2) miRNA chip of expression spectrum
Principle is to use the target molecule on label probe detection solid support equally.By miRNA in design chips
Gene and internal reference sequence, Accurate Analysis can go out the expression of corresponding miRNA in sample.Gene chip has high-throughout excellent
Point, once can detect whole expression of hundreds of gene in same sample.The liquid-phase chip that Luminex company develops
(Liquid chip) also known as multifunctional suspending dot matrix (Multi analyte suspension array, MASA), is new
Generation biochip technology.Liquid-phase chip system is that main matrix is constituted by many spherulas, and every kind of spherula is fixed with not
With probe molecule, in order to distinguish different probes, each is all unique with one for the sphere matrix of label probe
These spherulas are suspended in a liquid-phase system, just constitute liquid-phase chip system by color numbers.This system can be to same
Multiple different moleculars in one trace sample carry out quick qualitative and quantitative analysis simultaneously, and this detection technique is referred to as
FMAP (Flexible multianalyte profiling) technology.Molecule hybridization is carried out in aaerosol solution, detects speed pole
Hurry up.
(3) ribozyme protection analytical technology (RPA)
The detection of miRNA can also use ribozyme to protect analytical technology, and the probe that labelling is good and RNA sample to be measured mix
Closing, hybridize after thermal denaturation, non-hybridized RNA and unnecessary probe single-chain nucleic acid enzymic digestion, after heat inactivation nuclease, purification is subject to
The RNA molecule of protection, is separated by electrophoresis probe, colour developing finally by degeneration PAGE.This new method based on solution hybridization is simple
Quickly, highly sensitive, but be also only used for analyzing known miRNA.
(4) RAKE method
RAKE method (RNA primed array based Klenow emzyme) is the base at miRNA microarray
The Klenow fragment of DNA polymerase i is utilized, the method making miRNA and the hybridization of fixing DNA probe on plinth.RAKE can be sensitive
Detect miRNA specifically, it is adaptable to a large amount of quickly all miRNA that oneself knows of screening.Can be in specific cell and tumor
Detection miRNA express spectra situation.Moreover, RAKE method can also be from the paraffin-embedded tissue secured by formalin
Isolate miRNA and it is analyzed, opening the door of hope for analyzing miRNA from archive specimen.
(5) in situ hybridization (in situ hybridization)
Hybridization in situ technique can intuitively understand miRNA expression way, is a kind of easier of observation miRNA spatial and temporal expression
Method, normal mark mode includes digoxin, biotin, fluorescent labeling etc..In situ hybridization (Locked on the basis of locked nucleic acid
Nucleic Acid (LNA) based in situ hybridization (LNA-ISH)) it is the more probe side of current application
Formula.
(6) flow cytometry based on microsphere (bead-based flow cytometry)
Being a kind of liquid-phase chip technology, FCM analysis is organically combined by the method with chip technology, has concurrently
The features such as flux is big, detection speed is fast, highly sensitive and specificity is good.
(7) Real-Time Fluorescent Quantitative PCR Technique (Real-time PCR, RT-PCR)
The cumulative speed of extension increasing sequence during whole PCR can be drawn dynamic changing curve by fluoroscopic examination PCR instrument.Instead
Answer the initial concentration of target sequence in mixed system the biggest, it is desirable to the PCR cycle number obtaining amplified production specific output (is typically used
Specific threshold period Ct is expressed) the fewest.Amplification is not suitable for so owing to miRNA length is only 22nt, traditional qRT-PCR
Short fragment.There is several real time quantitative PCR method for miRNA now, such as tailing method, neck ring method etc..Neck ring method is a kind of
Preferably miRNA detects qRT-PCR method: first design special loop-stem structure primer, with miRNA to be measured for template reverse transcription
Synthesis cDNA the first chain, this cDNA one end is stem Loop primer, and stem circulus is opened and substantially increases the length of cDNA, subsequently
Real-time quantitative PCR amplification is carried out for template design primer with the cDNA of synthesis.QRT-PCR has that specificity is high, sensitivity is good, fast
The multiple advantages such as speed is simple.
(8) sequencing
The known miRNA of major part is found by cDNA clone order-checking and identifies.This method needs first to build miRNA
CDNA library, then carry out PCR amplification, amplified production is cloned on expression vector order-checking subsequently.Takada develops one and changes
The amplification cloning (miRNA amplification profiling, mRAP) entered, mRAP method first connects at the 3 ' of miRNA ends
Joint, then with the reverse transcription primer reverse transcription complementary with joint.Because specific reverse transcription has end deoxynucleotide
Transferase active, some nucleotide (mainly dCMP 1beta-Deoxyribofuranosylcytosine-5'-phosphate) can be connected to 3 ' ends of the cDNA chain that reverse transcription goes out.When 5 '
After poly (C) the sticky end annealing of end connector and cDNA chain, add a pair general primer and can realize the expansion of the PCR to cDNA
Increase.Due to mRAP High sensitivity, can be directly with the expression of miRNA in clone and a small amount of tissue of sequencing technologies detection.Label
Sequence cloning is a kind of to have developed detection efficiency on the basis of serial analysis of gene expression (SAGE) technology higher
MiRAGE (miRNA SAGE) cloning, this method, by generating big sub-series, can detect multiple by single sequencing reaction
MiRNA, hence it is evident that improve detection efficiency.
High-flux sequence (High-throughput sequencing) is also known as sequencing technologies (next of future generation
Generation sequencing) it is the change to tradition order-checking revolution, once to hundreds of thousands to millions of DNA
Molecule carries out sequencing, greatly improves order-checking efficiency.This kind of large scale sequencing technology greatly improves multiple species and loses
Delivering a letter the solution reading rate of breath, for obtaining the sequence information of all miRNA, deciphering miRNA collection of illustrative plates provides guarantee.High flux simultaneously
The analysis that order-checking makes the transcript profile to species and genome carry out careful overall picture is possibly realized, so the degree of depth that is otherwise known as
Order-checking (deep sequencing).The representative of high-flux sequence platform is 454 sequenator (Roch of Roche Holding Ag (Roche)
GSFLX sequencer), the Solexa gene element analyzer (Illumina Genome Analyzer) of Illumina company and
The SOLiD sequenator (ABI SOLiD sequencer) of ABI.
MicroRNA gain-of-function technology based on RNA is i.e. come by the precursor substance of exogenous supplementary miRNAs synthesis
Raise the level of miRNAs.For example, it is possible to the bob folder sample RNA (short that synthetic is consistent with endogenous miRNA sequence
Hairpin RNA, shRNA), polymerase II or III do promoter, with virus as vector-transfected cell, by Dicer enzyme modification
Rear loading RISC plays a role, and is equivalent to raise the level of pre-miRNA, and action effect is stably lasting.
Gene specific miR Mimics technology this technique avoids the nonspecific action of miRNA and gene.This people
The specific oligonucleotide chain being combined with target gene 3 ' UTR complementation of work synthesis, it is possible to play tune after identical with miRNA transcribing
Joint effect.
The major way of miRNA regulation and control has two kinds: a kind of is 3 ' the not fully complementary pairings of UTR with target gene mRNA, resistance
The translation of disconnected target gene, thus regulator gene is expressed；Another kind is similar with siRNA, when miRNA Yu mRNA complete complementary matches
Time, Ago2 albumen directly results in its degraded by cutting mRNA, it is achieved gene silencing.In a word, miRNA it is presently believed to which kind of side
Formula is relevant with the pairing degree of genes of interest with genes of interest effect and miRNA.When miRNA is incomplete with genes of interest pairing,
MiRNA just plays a role with the expression of suppression genes of interest；When miRNA is complete with the pairing of genes of interest section sequence, it is possible to
Genes of interest is caused to cause gene silencing in complementary region fracture.
On the pharmaceutics of the pharmaceutical composition being included in the present invention, the carrier of license is the load generally utilized when preparation
Body, this carrier comprises lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbitol (sorbitol), sweet
Dew alcohol (mannitol), starch, acacia gum, calcium phosphate, alginate (alginate), gel (gelatin), calcium silicates,
Microcrystalline Cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, first
Base cellulose (methyl cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxy benzene
Propyl formate (propyl hydroxybenzoate), Talcum, magnesium stearate (stearic acid magnesium) and mineral
Oil (mineral oil) etc., but it is not limited to this.
The pharmaceutical composition of the present invention can also comprise lubricant, wetting agent, sweeting agent, perfume (or spice) in addition to mentioned component
Taste agent, emulsifying agent, suspending agent, preservative etc..On pharmaceutics, the carrier being suitable for and the preparation of license are recorded in Lei Mingdengshi in detail
Pharmacy pandect.
The pharmaceutical composition of the present invention, can be able to be led to during as non-oral administration by oral or parenteral be administered
Cross intravenous injection, intranasal injection, local injection, intracerebral ventricle injection, spinal cavity injection, subcutaneous injection, lumbar injection, percutaneous
The modes such as administration are administered.
The dosage being suitable for of the pharmaceutical composition of the present invention is according to preparation ways, administering mode, the year of patient
The factor of age, body weight, sex, morbid state, food, administration time, route of administration, drainage rate and being quick on the draw property etc and permissible
Carry out multiple prescription, generally, skilled practitioner can be easily determined by and prescription to desired treatment or prevention effectively to
Pharmaceutical quantities.
The pharmaceutical composition of the present invention can easily be implemented according to general technical staff of the technical field of the invention
Method, utilize receptible carrier and/or excipient on pharmaceutics formulation to carry out such that it is able to unit dose form
Preparation or in be contained in multicapacity container in prepare.Now, solution during dosage form is oiliness or aqueous medium, suspension or
Emulsion form, or can also be extractum, powder agent, granule, tablet or capsule form, it is also possible to include dispersion
Agent or stabilizer.
Accompanying drawing explanation
Fig. 1 is cell growth curve figure after transfection miR-3620-3p mimics
Fig. 2 is cell growth curve figure after transfection miR-3620-3p inhibitor
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further, be only used for explain the present invention, and it is not intended that to this
The restriction of invention.It will be understood by those skilled in the art that: can in the case of without departing from the principle of the present invention and objective
These embodiments to carry out multiple change, revises, replace and modification, the scope of the present invention is limited by claim and equivalent thereof
Fixed.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer
Part examinations.
The collection of embodiment 1 sample and Total RNAs extraction
Collect each 6 examples of hospital's adenocarcinoma of colon patient cancerous tissue in August, 2015 in January, 2014 to and corresponding cancer beside organism.Suffer from
Person is preoperative to draw materials in art without radiation and chemotherapy, is immediately placed in liquid nitrogen preservation, then continues at-80 DEG C long-term preservations, for
RNA extracts.Specimen is diagnosed as colon cancer through pathological diagnosis.
RNA extracts standard: RNA purity: OD260/280 1.8,28S/18S 1；RNA integrity: RIN value 7.0.
RNA integrality detection method: Agilent 2100 (RNA 6000Nano kit), (agarose gel is dense for agarose gel electrophoresis
Degree: 1% agarose gel；Voltage: 5V/cm；Time: 20min).
Embodiment 2 checks order and Data Integration analysis
Order-checking: use llumina Hiseq2500/Miseq second filial generation high throughput sequencing technologies that miRNA is checked order,
By removing joint, going low quality, the process such as depollute to complete the process of data, obtain final data.
Confluence analysis: the nationwidechildrens.org_clinical_patient_coad literary composition of TCGA data base
Part records the clinical information of 459 example adenocarcinoma of colon patients altogether.This project get rid of history_other_malignancy or
History_neoadjuvant_treatment is patient 62 example of Yes, is Colon by histologic_diagnosis
The 337 example patients of Adenocarcinoma include research in, and (histologic_diagnosis is Colon Mucinous
Adenocarcinoma, [Not Available], the 60 example patients of [Discrepancy] do not include research in).Use 337 example knots
MiRNA data in enteraden cancer (COAD) patient TCGA data base, are analyzed.These data are all from adenocarcinoma of colon case group
The Solid Tissue Normal of Primary solid Tumor and matched group.
After miRNA order-checking initial data and confluence analysis data being carried out background correction by transcript profile data analysis software
Carry out t-test and obtain P value, then utilize Fisher inspection to merge P value, screen differential expression miRNA.From the difference table of candidate
Reach and miRNA selects mir-3620 carry out Late Stage Verification.
The expression of miR-3620-3p in embodiment 3 Real-time PCR detection adenocarcinoma of colon tissue
1 sample collecting:
36 example adenocarcinoma of colon tumor patient cancerous tissues and cancer beside organism's (acquisition time in August ,-2015 in January, 2014).
2 Total RNAs extraction:
The process removing Rnase of related experiment article:
1. all rinsing with DEPC before being applied by all glass drying ovens and invade bubble, 120 DEG C of high pressure 20min, 180 DEG C of high temperature dry 2
More than hour.
2. will plastic ware (such as: EP pipe/rifle head) use before need with 0.1%DEPC water enchroachment (invasion) steep overnight, after drain liquid,
120 DEG C of high pressure 20min, baking box is dried standby.
Leukocyte separates
(1) 2m1 anticoagulation cirumferential blood (blood sampling time is less than 3h) is taken；
(2) add equal-volume aseptic PB S to be sufficiently mixed in peripheral blood, form cell suspension；
(3) 4m1 lymphocyte separation medium is added in another centrifuge tube；
(4) draw 4m1 cell suspension join gently along tube wall lymphocyte separation medium surface (note not with lymphocyte
Separate liquid mixing).Centrifugal 1500rpm 20min；
(5) enter in another centrifuge tube with suction pipe sucking-off boundary layer (tunica albuginea) gently.Aseptic cold PBS washes 2 times, washes for last 1 time
Wash and cell suspension can be moved in EP pipe, be centrifuged and remove supernatant, be used for extracting RNA.
RNA extracts
(1) from-80 DEG C of refrigerators, sample is taken out, chopping, in the ratio addition Trizol of 1ml/50-100mg in EP pipe,
Carrying out homogenized, room temperature stands 5-l0min；
(2) every milliliter of Trizol adds 0.2m1 chloroform, acutely shakes 15s, and room temperature stands 2-3min, at 4 DEG C 1 2000
Leave heart 15min；
(3) the careful sucking-off supernatant water 600ul that makes an appointment moves into another centrifuge tube (being careful not to be extracted into albumin layer), adds equivalent
Isopropanol, reverse mixing, room temperature stands 10min；
(4) 4 DEG C of 1 2000g are centrifuged l 0min, abandon supernatant, bottom visible white material；
(5) add the cold ethanol of lml 75% and rotate washing, clean isopropanol；
(6) 4 DEG C of 12000g are centrifuged 5min, and dry in the air after removing ethanol 5-l0min, translucent, with 20u1DEPC water dissolution
RNA.Take 3u1RNA sample, electrophoresis in 1.5% agarose gel；Lu1RNA sample in UV spectrophotometer measuring concentration,
It is considered as RNA sample at 1.8-2.0 qualified with A260/280.
3 reverse transcriptions
The preparation of RT system: 1 μ g total serum IgE is as template ribonucleic acid；5×miScript HiSpec Buffer 4μl；10×
Nucleics Mix 2μl；miScript Reverse Transcriptase Mix 2μl；Aquesterilisa filling-in is to 20 μ l.ABI
After in 9700 type PCR instrument, 37 DEG C of insulation 60min make reverse transcription reaction completely, 95 DEG C of 5min terminate reaction.Add 80 μ l
Nuclease-free H2O is diluted to 100 μ l, and to be stored in-20 DEG C of refrigerators standby.
4 quantitative fluorescent PCRs
The preparation of the RT-PCR system of miRNA:
The detection of expression of miRNAs arranges 3 parallel pipe reactions every time, using snRNA U6 as internal reference.
Amplification program: 95 DEG C of 10min；40 circulations (95 DEG C of 10s, 60 DEG C of 30s).
5 statistical analysis
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: Δ
Δ Ct=Ct (miR-3620-3p)-Ct (U6), fold=2-ΔΔCtRepresent the multiple of experimental group and matched group destination gene expression
Relation, compares miR-3620-3p expression in adenocarcinoma of colon tissue and cancer beside organism.Result shows: qRT-PCR expands
Result is stable, and genes of interest is at expression is cancerous tissue about 4 times of cancer beside organism, and result above demonstrates high-flux sequence number
According to the result with confluence analysis data analysis.
The cultivation of embodiment 4 human colon cancer cell strain and transient transfection
One, material prepares:
CCL188 HCT116 is purchased from ATCC cell bank.
LipofectamineTM2000Transfection Reagent(Invitrogen)。
RPMI 1640 and DMEM culture medium is purchased from GIBCO company, new-born calf serum and tire Niu Erqing purchased from PAA company.
MiR-3620-3p sequence issues Synesis Company, please its chemosynthesis miR-3620-3p mimics, miR3620-3p
Inhibitor and non specific control thereof.
Two, experimental technique
1, cell is cultivated
Colon cancer cell line HCT116 uses containing RPMI 1640 culture medium of 10% new-born calf serum, 37 DEG C, 5%
CO2, Secondary Culture under conditions of saturated humidity, be used for when cell growth state is good testing.
2, miRNA transient transfection
Lipofectamine is pressed in operationTM2000 reagent description are carried out.Before transfection, 24h is good by growth conditions
HCT116 cell is inoculated in 6 orifice plates, and cell counting is about 4 × 105/ L, cellar culture is to transfecting the same day, and cell degrees of fusion is
Test during 70-80%.100nM miR-3620-3p mimics/miR3620-3p inhibitor is joined 250 μ l
In opti-MEM culture medium, softly mix；Separately dilute 5 μ l Lipofectamine by 250 μ l opti-MEM culture mediumTM 2000
Liposome, softly mixes, incubated at room 5min；Mixing Opti-MEM-liposome and Opti-MEM-miRNAs, incubated at room
20min, to form transfection composite: be then added in cell culture medium by said mixture, mix gently, changes after cultivating 6h
Complete medium.Wherein, nonspecific mimics Negative Control (mimics NC) and inhibitor
Negative Control (inhibitor NC) sequence is as comparison.Extracting cell total rna after cultivating 24-48h, reverse transcription becomes
CDNA, the change that after real-time quantitative PCR detection transient transfection, miR-3620-3p expresses.
3. experimental result:
Cationic-liposome method is used to carry out transient transfection, respectively by miR-3620-3p mimics or miR-3620-
3pinhibitor and corresponding control sequence Negative Control (NC) transfects colon cancer cell line HCT116.Transfection 48h
After, extract cell total rna.With U6 as internal reference, the expression of real-time quantitative PCR detection miR-3620-3p.Result shows: with right
Comparing according to group, after HCT116 transfection miR-3620-3p mimics, the expression of miR-3620-3p increases about 4.5 times of (P=
0.001)；After transfection miR-3620-3p inhibitor, express and have dropped nearly 76% (P=0.001).Result above shows, logical
Cross transient transfection miR-3620-3p mimics and miR-3620-3p inhibitor can effectively raise or lower miR-3620-
The expression of 3p, reliable results can carry out subsequent experimental.
Embodiment 5 transfects the miR-3620-3p impact on Proliferation of Human Colon
1×103Individual cell is inoculated in 96 orifice plates, cultivates 1,2,3,4,5d respectively, and every hole adds the RPMI-1640 of serum-free
Tetrazole indigo plant salt (MTT) the 20 μ l of culture fluid 200 μ l and 5mg/ml, continues to cultivate 4h, inhales and abandon liquid, and every hole adds DMSO150 μ
L, incubated at room 10min, put and on shaking table, be shaken gently for 15min, measure every hole with Bio-Rad enzyme-linked immunosorbent assay instrument at 490nm
Absorbance (OD value), with blank control wells return to zero, represent the size of ability of cell proliferation with corresponding OD value.Often group sets
3 repeating holes, average, and draw growth in vitro curve.
HCT116 cell after transient transfection is inoculated in 96 orifice plates, uses mtt assay detection miR-3620-3p process LAN
Impact on Colon Cancer Cells ability.Result as it is shown in figure 1, transfection miR-3620-3p mimics after 5 days, and compares
Group (transfection mimics NC) is compared, and the speed of growth of HCT116 cell slows down about 35.7%, and difference is statistically significant, prompting
MiR-3620-3p process LAN can suppress the in-vitro multiplication ability of colon cancer cell.
HCT116 cell after transient transfection is inoculated in 96 orifice plates, uses reticent miR-3620-3p pair of mtt assay detection
The impact of Colon Cancer Cells ability.Result is as in figure 2 it is shown, colon cancer cell transient transfection miR-3620-3p
After inhibitor 5 days, the speed of growth of cell improves 34.5%, and difference is statistically significant, the reticent rniR-of prompting
3620-3p expresses the in-vitro multiplication ability that can strengthen colon cancer cell.
Although describing the present invention with reference to various preferred embodiments, it will be appreciated by those skilled in the art that can carry out each
Plant change, and available equivalents substitutes its assembly elemental range without departing from the present invention.Come additionally, many changes can be carried out
Particular case or material is made to be suitable for the teachings of the present invention without departing from its elemental range.
Claims (10)
1. the preparation of detection mir-3620 and ripe miRNA thereof application in preparation diagnosing colon cancer reagent.
Application the most according to claim 1, it is characterised in that colon cancer is adenocarcinoma of colon.
Application the most according to claim 1, it is characterised in that the sequence of mir-3620 is shown in sequence table SEQ ID NO 1,
Mir-3620 maturation miRNA is miR-3620-5p and/or miR-3620-3p, and miR-3620-5p sequence is shown in sequence table SEQ ID
NO 2；MiR-3620-3p sequence is shown in sequence table SEQ ID NO 3.
Application the most according to claim 1, it is characterised in that diagnosing colon cancer reagent includes based on high-flux sequence method
And/or based on quantifying PCR method and/or based on mir-3620 and maturation thereof in probing procedure detection colon cancer sample
MiRNA transcribes.
Application the most according to claim 3, it is characterised in that use northern hybridizing method, miRNA express spectra core
Mir-in sheet, ribozyme protection analytical technology, RAKE method, in situ hybridization, Flow cytometry colon cancer sample based on microsphere
3620 and ripe miRNA transcribe.
6. raise the examination of the activity transcribing and/or promoting mir-3620 and ripe miRNA thereof of mir-3620 and ripe miRNA thereof
Agent prevents and treats the application in colon cancer preparation in preparation.
Application the most according to claim 6, it is characterised in that colon cancer is adenocarcinoma of colon.
Application the most according to claim 6, it is characterised in that prevent and treat colon cancer preparation preventing and treating Colon Cancer Cells.
Application the most according to claim 6, it is characterised in that microRNA gain-of-function technology based on RNA and/or
Gene specific miR Mimics technology raise mir-3620 and ripe miRNA thereof transcribe and/or promote mir-3620 and
The activity of ripe miRNA.
Application the most according to claim 6, it is characterised in that synthetic short hairpin RNA or by regulation and control promoter on
Adjust mir-3620 and the expression of ripe miRNA thereof.
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| CN103667459A (en) * | 2013-11-26 | 2014-03-26 | 上海中医药大学附属岳阳中西医结合医院 | Application of microRNA to hypertension diagnose and antihypertension medication preparation |
| WO2015194627A1 (en) * | 2014-06-18 | 2015-12-23 | 東レ株式会社 | Esophageal cancer detection kit or device, and detection method |
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