CN106244688B - A kind of marker for assessing adenocarcinoma of colon risk - Google Patents
A kind of marker for assessing adenocarcinoma of colon risk Download PDFInfo
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Abstract
The present invention relates to a kind of markers for assessing adenocarcinoma of colon risk, and in particular to application of the mir-1538 and its maturation miRNA in preparation diagnosis and treatment adenocarcinoma of colon preparation.Invention obtains data to adenocarcinoma of colon tissue and the sequencing of two generation of cancer beside organism, in combination with database data with existing, after confluence analysis, obtains its miRNA expression data, filters out candidate mir-1538.RT-PCR demonstrates miR-1538 low expression in cancerous tissue, and cell experiment, which is overexpressed miR-1538 and interference miR-1538, can influence the in-vitro multiplication ability of colon cancer cell.The present invention provides the important target spot of new colon cancer gene diagnosis and treatment for clinic.
Description
Technical field
The present invention relates to tumor cells diagnostic fields, are specifically related to a kind of mark for assessing adenocarcinoma of colon risk
Object more particularly relates to the application of mir-1538 and its maturation miRNA in preparation diagnosis and treatment adenocarcinoma of colon preparation.
Background technique
Colon cancer, which is one, controlled by multiple genes, divides different phase, the extremely complex pathogenic course of formed for a long time.
If can early diagnose, and monitors the recurrence of tumour and effectively carry out new treatment method to the meanings of colorectal cancer patients very
Greatly.The tissue that oneself many microRNAs of discovery recent years are present in colon cancer tumours neutralizes in the blood of patient, and is tying
Very important effect is played in the mechanism of intestinal cancer.
MiRNA is a kind of endogenous non-coding small molecule, plays a part of to adjust feedback and buffering in normal cell physiology,
It is horizontal as accurate adjustment target gene protein, prevent it from exceeding the key factor of normal physiological scope.Each development of the miRNA in disease
Stage plays extremely important effect, and more and more evidences show that miRNA rises in the development of the carcinogenesis and tumour of tumour
Highly important effect, it is a variety of in various tumours by a large amount of tumor tissues and cell strain miRNA chip test experience
MiRNA is found to be up-regulation or downward.Researcher has found the unconventionality expression of miRNA in the experiment in vivo of transgenic mice
It would generally cause the generation of cancer.And some miRNA even play what difference was even completely contradicted in different tumour cells
Effect.So the relationship of miRNA and cancer is the problem of we must get clear during defeating this disease.
The present invention is based on high-flux sequence method, is carried out to the cancerous tissue and cancer beside organism of 6 adenocarcinoma of colon patients two generations
Sequencing, obtains the expression data of its miRNA, meanwhile, in conjunction with 337 adenocarcinoma of colon patient's miRNA data in TCGA database, into
Several progress RT-PCR verifyings and cell verifying, RT-PCR are picked out in the analysis of row Integrative Bioinformatics from candidate miRNA
MiR-1538 low expression in cancerous tissue is demonstrated, cell experiment, which is overexpressed miR-1538 and interference miR-1538, can influence colon
The in-vitro multiplication ability of cancer cell.The present invention provides the important target spot of new colon cancer gene diagnosis and treatment for clinic.
Summary of the invention
The purpose of the present invention is to provide the transcription of up-regulation mir-1538 and its maturation miRNA and/or promote mir-1538
And its application of the active reagent of maturation miRNA in preparation prevention and treatment colon cancer preparation.The sequence of mir-1538 is shown in sequence table
SEQ ID NO 1, miR-1538 sequence are shown in sequence table SEQ ID NO 2.
Further, colon cancer is adenocarcinoma of colon.
Further, prevention and treatment colon cancer preparation prevents and treats Colon Cancer Cells.
Preferably, using the acquired technology of microRNA function and/or gene specific miR Mimics skill based on RNA
Art raises the transcription of mir-1538 and its maturation miRNA and/or promotes the activity of mir-1538 and its maturation miRNA.It is preferred that people
Work synthesizes short hairpin RNA or raises mir-1538 and its maturation miRNA by regulation promoter.
The purpose of the present invention is to provide the preparations of detection mir-1538 and its maturation miRNA to prepare diagnosing colon examination
Application in agent.
Preferred 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 the transcription of mir-1538 and its maturation miRNA in adenocarcinoma of colon sample are detected based on probing procedure or are based on immune detection
Method detects the expression of the target gene of mir-1538 and its maturation miRNA regulation in 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 microballoon
The transcription of mir-1538 and its maturation miRNA in Flow cytometry adenocarcinoma of colon sample；Using ELISA and/or colloidal gold
Test strips detect the expression of the target gene of mir-1538 and its maturation miRNA regulation in adenocarcinoma of colon sample.
It preferably, include the primer of specific amplification mir-1538 and its maturation miRNA based on quantifying PCR method, into one
Preferably, the primer sequence of specific amplification miR-1538 is SEQ ID NO 3 to step；Include based on probing procedure and mir-
The probe of the nucleic acid array hybridizing of 1538 and its maturation miRNA.
The purpose of the present invention is to provide the target genes of mir-1538 and its maturation miRNA in preparation diagnosis and/or prevention and treatment
Application in adenocarcinoma of colon preparation.
The purpose of the present invention is to provide above-mentioned adenocarcinoma of colon diagnostic preparations to prepare answering in adenocarcinoma of colon diagnostic tool
With.
The purpose of the present invention is to provide a kind of prevention and treatment colon cancer reagents, and the reagent includes:
(a) it raises the transcription of mir-1538 and its maturation miRNA and/or promotes the work of mir-1538 and its maturation miRNA
The reagent of property；
(b) receptible carrier in pharmacy.
Preferably, using the acquired technology of microRNA function and/or gene specific miR Mimics skill based on RNA
Art raises the transcription of mir-1538 and its maturation miRNA and/or promotes the activity of mir-1538 and its maturation miRNA.It is preferred that people
Work synthesizes short hairpin RNA (short hairpin RNA, shRNA) or raises mir-1538 and its maturation by regulation promoter
The expression of miRNA.
Further, the colon cancer is adenocarcinoma of colon.
The purpose of the present invention is to provide a kind of adenocarcinoma of colon diagnostic reagent, adenocarcinoma of colon diagnostic reagent is able to detect colon
The transcription of mir-1538 and its maturation miRNA or immunologic detection method detect mir-1538 in adenocarcinoma of colon sample in gland cancer sample
And its expression of the target gene of maturation miRNA regulation.
Further, adenocarcinoma of colon diagnostic reagent is based on high-flux sequence method and/or is based on quantifying PCR method and/or base
The transcription of mir-1538 and its maturation miRNA in adenocarcinoma of colon sample are detected in probing procedure or are based on immunization method detection
The expression of the target gene of mir-1538 and its maturation miRNA regulation in adenocarcinoma of colon sample, it is preferred to use northern is miscellaneous
Friendship method, miRNA chip of expression spectrum, ribozyme protect analytical technology, RAKE method, in situ hybridization, bead-based flow-cytometry
Detect the transcription of mir-1538 and its maturation miRNA in adenocarcinoma of colon sample.
The purpose of the present invention is to provide the preparations of above-mentioned prevention and treatment adenocarcinoma of colon to prepare adenocarcinoma of colon therapeutic agent or examination
Application in agent.
Definition:
The method for detecting the expression of miRNA at this stage mainly includes based on high throughput sequencing technologies, based on nucleotide
The miRNA detection method of hybridization and based on PCR.MiRNA detection method based on probe hybridization technique is a kind of direct Detection Method,
It does not need to expand sample rna in advance, including northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analysis skill
The technologies such as art, RAKE method, in situ hybridization, bead-based flow-cytometry.
(1) Northern hybridizes
Also known as Northern blot is most classic detection eucaryote RNA size, estimates the experimental method of its abundance.Base
Present principles are as follows: fixing miRNA sample on carrier (such as silicon wafer, microballoon or film) first, then miscellaneous with the probe by label
It hands over, carries out signal detection after washing extra hybridization probe；It can also be first fixed complementary with target miRNA sequence on carrier
Then DNA probe hybridizes with the sample miRNA by label, then carries out signal detection.The method of signal label includes isotope
Label, fluorescent marker and nano gold mark etc..
(2) miRNA chip of expression spectrum
Principle is equally using the target molecule on label probe detection solid support.Pass through miRNA in design chips
Gene and internalcontrol sequence, can Accurate Analysis go out the expression of corresponding miRNA in sample.Genetic chip has the excellent of high throughput
Point can once detect whole expression of several hundred a genes in same sample.The liquid-phase chip that Luminex company develops
(Liquid chip) is also known as multifunctional suspending dot matrix (Multi analyte suspension array, MASA), is out new
Generation biochip technology.Liquid-phase chip system is made of many spherulas for main matrix, is fixed with not on every kind of spherula
With probe molecule, in order to distinguish different probes, each for label probe sphere matrix all have one it is unique
Color numbers constitute liquid-phase chip system as soon as these spherulas are suspended in a liquid-phase system.The system can be to same
Multiple and different molecules 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 carries out in aaerosol solution, detects speed pole
Fastly.
(3) ribozyme protection analytical technology (RPA)
The detection of miRNA can also protect analytical technology using ribozyme, and the probe marked and RNA sample to be measured are mixed
Close, hybridize after thermal denaturation, non-hybridized RNA and the single-chain nucleic acid enzymic digestion of extra probe, after heat inactivation nuclease purifying by
Probe, colour developing is separated by electrophoresis finally by denaturation PAGE in the RNA molecule of protection.This new method based on solution hybridization is simple
Quickly, high sensitivity, but be also only used for analyzing known miRNA.
(4) RAKE method
RAKE method (RNA primed array based Klenow emzyme) is the base in miRNA microarray
The Klenow segment of DNA polymerase i, the method for hybridizing miRNA with fixed DNA probe are utilized on plinth.RAKE can be sensitive
MiRNA is specifically detected, suitable for largely quickly screening all known miRNA.It can be in specific cell and tumour
Detect miRNA express spectra situation.Moreover, RAKE method can also be from the tissue of the paraffin embedding secured by formalin
It isolates miRNA and analyzes it, to analyze the door that miRNA opens hope from archive sample.
(5) in situ hybridization (in situ hybridization)
Hybridization in situ technique can intuitively understand miRNA expression way, be a kind of easier of observation miRNA spatial and temporal expression
Method, normal mark mode include digoxin, biotin, fluorescent marker 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) bead-based flow-cytometry (bead-based flow cytometry)
It is a kind of liquid-phase chip technology, FCM analysis is organically combined with chip technology, had concurrently by this method
The features such as flux is big, detection speed is fast, high sensitivity and specificity are good.
(7) Real-Time Fluorescent Quantitative PCR Technique (Real-time PCR, RT-PCR)
Fluorescence detection PCR instrument can be to the cumulative speed drafting dynamic changing curve of extension increasing sequence during entire PCR.Anti-
Answer the initial concentration of target sequence in mixed system bigger, it is desirable that the PCR cycle number for obtaining amplified production specific output is (general to use
Specific threshold recurring number Ct is expressed) it is fewer.Since miRNA length is only 22nt, it is such that traditional qRT-PCR is not suitable for amplification
Short segment.There are several real time quantitative PCR methods for miRNA, such as tailing method, neck ring method now.Neck ring method is a kind of
Ideal miRNA detects qRT-PCR method: special loop-stem structure primer is designed first, using miRNA to be measured as template reverse transcription
The first chain of cDNA is synthesized, which is stem Loop primer, and stem loop structure, which is opened, substantially increases the length of cDNA, then
Real-time quantitative PCR amplification is carried out by template design primer of the cDNA of synthesis.QRT-PCR has specificity height, sensitivity good, fast
A variety of advantages such as fast simple.
(8) PCR sequencing PCR
MiRNA known to major part is that discovery and identification is sequenced by cDNA clone.The method needs first to construct miRNA
CDNA library, then carry out PCR amplification, amplified production is then cloned on expression vector and is sequenced.Takada develops one kind and changes
Into amplification PCR cloning PCR (miRNA amplification profiling, mRAP), mRAP method first connects at the 3 ' ends of miRNA
Connector, then with the reverse transcription primer reverse transcription complementary with connector.Because specific reverse transcriptase has end deoxynucleotide
Transferase active, some nucleotide (mainly deoxycytidylic acid) can be connected to 3 ' ends of the cDNA chain that reverse transcription goes out.When 5 '
After the annealing of poly (C) cohesive end of end connector and cDNA chain, a pair of of general primer is added and can be realized, the PCR of cDNA is expanded
Increase.It, can be directly with the expression quantity of miRNA in clone and a small amount of tissue of sequencing technologies detection due to mRAP High sensitivity.Label
Sequence PCR cloning PCR is a kind of to have developed detection efficiency on the basis of serial analysis of gene expression (SAGE) technology higher
MiRAGE (miRNA SAGE) PCR cloning PCR, the method can detect multiple by generating big sub-series by single sequencing reaction
MiRNA, hence it is evident that improve detection efficiency.
High-flux sequence (High-throughput sequencing) is also known as next-generation sequencing technologies (next
Generation sequencing) it is the change for tradition being sequenced revolution, once to hundreds of thousands to millions of DNA
Molecule carries out sequencing, greatly improves sequencing efficiency.This kind of large scale sequencing technology greatly improves multiple species and loses
The solution reading rate of communication breath, for the sequence information for obtaining all miRNA, decryption miRNA map provides guarantee.It is high-throughput simultaneously
Sequencing to carry out the analysis of careful overall picture to the transcript profile and genome of species, so the depth that is otherwise known as
It is sequenced (deep sequencing).The representative of high-flux sequence platform is 454 sequenator (Roch of Roche Holding Ag (Roche)
GSFLX sequencer), the Solexa genome analysis instrument (Illumina Genome Analyzer) of Illumina company and
The SOLiD sequenator (ABI SOLiD sequencer) of ABI.
The acquired technology of microRNA function based on RNA be by precursor substance that exogenous supplement miRNAs is synthesized come
Increase the level of miRNAs.For example, can the artificial synthesized and consistent short hair clip sample RNA (short of endogenous miRNA sequence
Hairpin RNA, shRNA), promoter is done by polymerase II or III, is that carrier transfects cell with virus, by Dicer enzyme modification
It is loaded into RISC afterwards to play a role, is equivalent to the level for increasing pre-miRNA, function and effect are stable and lasting.
This technique avoids the nonspecific actions of miRNA and gene for gene specific miR Mimics technology.This people
The specific oligonucleotide chain of the combination complementary with 3 ' UTR of target gene of work synthesis, is adjusted after capable of playing transcription identical with miRNA
Section effect.
There are two types of the major ways of miRNA regulation: one is the not fully complementary pairing of 3 ' UTR with target gene mRNA, resistances
The translation of disconnected target gene, to adjust gene expression；Another kind be it is similar with siRNA, when miRNA and mRNA complete complementary match
When, Ago2 albumen directly results in its degradation by cutting mRNA, realizes gene silencing.In short, being presently believed to miRNA with which kind of side
Formula and target gene effect and miRNA are related with the pairing degree of target gene.When miRNA and target gene match incomplete,
MiRNA is just to inhibit the expression of target gene to play a role；When miRNA and target gene section sequence match complete, it is possible to
Cause target gene to be broken in complementary region and leads to gene silencing.
Include in the pharmacy of pharmaceutical composition of the invention the carrier permitted be the load usually utilized in preparation
Body, which includes lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbierite (sorbitol), sweet
Reveal alcohol (mannitol), starch, acacia gum, calcium phosphate, alginates (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
Oily (mineral oil) etc., but it is not limited to this.
Pharmaceutical composition of the invention can also include lubricant, wetting agent, sweetener, perfume (or spice) in addition to the above ingredients
Taste agent, emulsifier, suspending agent, preservative etc..The suitable carrier and preparation permitted in pharmacy is recorded in Lei Mingdengshi in detail
Pharmacy pandect.
Pharmaceutical composition of the invention can by it is oral or it is non-oral be administered, when as non-oral administration, can lead to
Cross intravenous injection, intranasal injection, locally injecting, intraventricular injection, spinal cavity injection is subcutaneously injected, intraperitoneal injection, percutaneously
The modes such as administration are administered.
The suitable dosage of pharmaceutical composition of the invention according to preparation ways, administration mode, patient year
The factor of age, weight, gender, morbid state, food, administration time, administration route, drainage rate and draw property etc and can be with
Carry out a variety of prescriptions, in general, skilled practitioner can be easily determined by and prescription to it is desired treatment or prevention effectively to
Pharmaceutical quantities.
Person of an ordinary skill in the technical field can be easy to implement pharmaceutical composition of the invention according to the present invention
Method, carried out using carrier receptible in pharmacy and/or excipient it is formulation, so as in the form of unit dose
It is prepared by preparation or interior be mounted in multicapacity container.At this point, dosage form be solution in oiliness or aqueous medium, suspension or
Emulsion form is either also possible to extract, powder agent, granule, tablet or capsule form, can also include dispersion
Agent or stabilizer.
Detailed description of the invention
Fig. 1 is cell growth curve figure after transfection miR-1538mimics
Fig. 2 is cell growth curve figure after transfection miR-1538inhibitor
Specific embodiment
Present invention will be further explained below with reference to specific examples, for explaining only the invention, and should not be understood as to this
The limitation of invention.It will be understood by those skilled in the art that: without departing from the principle and spirit of the present invention may be used
To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent
It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to item proposed by manufacturer
Part examinations.
The collection of 1 sample of embodiment and Total RNAs extraction
Hospital's in January, 2014 is collected to the adenocarcinoma of colon patient cancerous tissues of in August, 2015 and corresponding each 6 of cancer beside organism.Suffer from
Person is preoperative without radiation and chemotherapy, draws materials in art, is immediately placed in liquid nitrogen and saves, then continue at -80 DEG C of long-term preservations, to be used for
RNA is extracted.Sample is diagnosed as colon cancer through pathological diagnosis.
RNA extraction standard: RNA purity: OD260/280≤1.8,28S/18S≤1；RNA integrality: Zhi≤7.0 RIN.
RNA integrality detection method: Agilent 2100 (RNA 6000Nano kit), (Ago-Gel is dense for agarose gel electrophoresis
Degree: 1% agarose gel；Voltage: 5V/cm；Time: 20min).
The sequencing of embodiment 2 and Data Integration analysis
Sequencing: being sequenced miRNA with llumina Hiseq2500/Miseq second generation high throughput sequencing technologies,
By the processes such as removing connector, going low quality, depollute complete the processing of data, final data is obtained.
Confluence analysis: the nationwidechildrens.org_clinical_patient_coad text of TCGA database
The clinical information of 459 adenocarcinoma of colon patients is recorded in part altogether.This project exclude history_other_malignancy or
History_neoadjuvant_treatment is patient 62 of Yes, is Colon by histologic_diagnosis
337 patients of Adenocarcinoma are included in research, and (histologic_diagnosis is Colon Mucinous
Adenocarcinoma, [Not Available], [Discrepancy] 60 patients be not included in research).It is tied using 337
MiRNA data in enteraden cancer (COAD) patient's TCGA database, are analyzed.These data are all from adenocarcinoma of colon case group
The Solid Tissue Normal of Primary solid Tumor and control group.
After initial data and the progress background correction of confluence analysis data is sequenced to miRNA by transcript profile Data Analysis Software
It carries out t-test and obtains P value, then examined using Fisher and merge P value, screen differential expression miRNA.From candidate difference table
Late Stage Verification is carried out up to mir-1538 is selected in miRNA.
Embodiment 3Real-time PCR detects the expression of miR-1538 in adenocarcinoma of colon tissue
The acquisition of 1 sample:
36 adenocarcinoma of colon tumor patient cancerous tissues and cancer beside organism's (acquisition time in January, 2014 in August, -2015).
2 Total RNAs extractions:
The processing for removing Rnase of related experiment article:
1. 180 DEG C of high temperature dry 2 by soaking, 120 DEG C of high pressure 20min is rinsed with DEPC before the application of all glasswares
Hour or more.
2. will be needed before plastic ware (such as: EP pipe/pipette tips) use with 0.1%DEPC water enchroachment (invasion) bubble overnight, after drain liquid,
120 DEG C of high pressure 20min, oven is dried spare.
Leucocyte separation
(1) take 2m1 anticoagulation cirumferential blood (blood sampling time is no more than 3h)；
(2) isometric sterile PB S is added to be sufficiently mixed in peripheral blood, forms cell suspension；
(3) 4m1 lymphocyte separation medium is added in another centrifuge tube；
(4) draw 4m1 cell suspension be gently added to along tube wall lymphocyte separation medium surface (pay attention to not with lymphocyte
Separating liquid mixing).It is centrifuged 1500rpm 20min；
(5) boundary layer (tunica albuginea) is gently sucked out with suction pipe to enter in another centrifuge tube.Sterile cold PBS is washed 2 times, is washed for last 1 time
Washing can move into cell suspension in EP pipe, and supernatant is removed in centrifugation, for extracting RNA.
RNA is extracted
(1) sample is taken out in -80 DEG C of refrigerators, is shredded, and Trizol is added in EP pipe in the ratio of 1ml/50-100mg,
Homogenized is carried out, 5-l0min is stored at room temperature；
(2) 0.2m1 chloroform is added in every milliliter of Trizol, acutely shakes 15s, is stored at room temperature 2-3min, 1 2000 at 4 DEG C
Turn centrifugation 15min；
(3) the supernatant water 600ul that makes an appointment carefully is sucked out and moves into another centrifuge tube (being careful not to be extracted into albumin layer), equivalent is added
Isopropanol is mixed by inversion, and is stored at room temperature 10min；
(4) 4 DEG C of 1 2000g are centrifuged l 0min, abandon supernatant, bottom visible white substance；
(5) the rotation washing of the cold ethyl alcohol of lml 75% is added, cleans isopropanol；
(6) 4 DEG C of 12000g are centrifuged 5min, and dry in the air 5-l0min after removal ethyl alcohol, translucent, with the dissolution of 20u1DEPC water
RNA.3u1RNA sample is taken, the electrophoresis in 1.5% Ago-Gel；Lu1RNA sample in UV spectrophotometer measuring concentration,
It is considered as RNA sample qualification in 1.8-2.0 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；Aqua sterilisa is mended
It puts down to 20 μ l.
After 37 DEG C of heat preservation 60min keep reverse transcription reaction complete in 9700 type PCR instrument of ABI, 95 DEG C of 5min terminate reaction.Add
Enter 80 μ l Nuclease-free H2O is diluted to 100 μ l, and to be stored in -20 DEG C of refrigerators spare.
4 quantitative fluorescent PCRs
The preparation of the RT-PCR system of miRNA:
3 parallel tube reactions are arranged in the detection of expression of miRNAs 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 inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR: Δ
Δ Ct=Ct (miR-1538)-Ct (U6), fold=2-ΔΔCtIndicate that the multiple of experimental group and control group destination gene expression closes
System, compares expression of the miR-1538 in adenocarcinoma of colon tissue and cancer beside organism.As the result is shown: qRT-PCR amplification
Stablize, for target gene at about 4.5 times that the expression quantity of cancer beside organism is cancerous tissue, result above demonstrates high-flux sequence data
With the result of confluence analysis data analysis.
The culture and transient transfection of 4 human colon cancer cell strain of embodiment
One, material prepares:
Human colon cancer cell line HCT116 is purchased from ATCC cell bank.
LipofectamineTM2000Transfection Reagent(Invitrogen)。
RPMI 1640 and DMEM culture medium are purchased from GIBCO company, and newborn bovine serum and tire Niu Erqing are purchased from PAA company.
MiR-1538 sequence issues Synesis Company, asks its chemical synthesis miR-1538mimics, miR1538inhibitor
And its non specific control.
Two, experimental method
1, cell culture
Colon cancer cell line HCT116 uses 1640 culture medium of RPMI containing 10% newborn bovine serum, in 37 DEG C, 5%
CO2, secondary culture under conditions of saturated humidity, for testing when cell growth state is good.
2, miRNA is transiently transfected
Lipofectamine is pressed in operationTM2000 reagent specifications carry out.It is for 24 hours that growth conditions are good before transfection
For HCT116 cell inoculation into 6 orifice plates, cell count is about 4 × 105/ L, routine culture are to the same day, cell fusion degree is transfected
It is tested when 70-80%.100nM miR-1538mimics/miR1538inhibitor is added to 250 μ l opti-MEM
It is soft to mix in culture medium；It is another to dilute 5 μ l Lipofectamine with 250 μ l opti-MEM culture mediumsTM2000 liposomes,
It is soft to mix, it is incubated at room temperature 5min；Opti-MEM- liposome and Opti-MEM-miRNAs are mixed, 20min is incubated at room temperature, with shape
At transfection composite: and then said mixture is added in cell culture medium, it mixes gently, replaces culture completely after cultivating 6h
Base.Wherein, nonspecific mimics Negative Control (mimics NC) and inhibitor Negative
Control (inhibitor NC) sequence is as control.Cell total rna is extracted after culture 24-48h, reverse transcription is at cDNA, in real time
The change that miR-1538 is expressed after quantitative PCR detection transiently transfects.
3. experimental result:
It is transiently transfected using cationic-liposome method, respectively by miR-1538mimics or miR-
1538inhibitor and corresponding control sequence Negative Control (NC) transfect colon cancer cell line HCT116.Transfect 48h
Afterwards, cell total rna is extracted.Using U6 as internal reference, real-time quantitative PCR detects the expression of miR-1538.As the result is shown: with control group
It compares, after HCT116 transfects miR-1538mimics, the expression of miR-1538 increases about 5 times (P=0.001)；Transfect miR-
After 1538inhibitor, expression has dropped 81% (P=0.001).The above result shows that by transiently transfecting miR-
1538mimics and miR-1538inhibitor effectively can raise or lower the expression of miR-1538, after as a result can reliably carrying out
Continuous experiment.
Embodiment 5 transfects influence of the miR-1538 to Proliferation of Human Colon
1×103A cell inoculation cultivates 1,2,3,4,5d, the RPMI-1640 of serum-free is added in every hole in 96 orifice plates respectively
The 20 μ l of tetrazole indigo plant salt (MTT) of culture solution 200 μ l and 5mg/ml continue to cultivate 4h, inhale and abandon liquid, and DMSO150 μ is added in every hole
L is incubated at room temperature 10min, sets and gently shake 15min on shaking table, measures every hole in 490nm with Bio-Rad enzyme-linked immunosorbent assay instrument
Absorbance value (OD value), returned to zero with blank control wells, the size of ability of cell proliferation indicated with corresponding OD value.Every group sets
3 repeating holes, are averaged, and draw growth in vitro curve.
By the HCT116 cell inoculation after transient transfection into 96 orifice plates, using mtt assay detection miR-1538 overexpression pair
The influence of Colon Cancer Cells ability.As a result as shown in Figure 1, after transfection miR-1538mimics 5 days, with control group (transfection
Mimics NC) it compares, the speed of growth of HCT116 cell slows down about 36.9%, and difference is statistically significant, prompts miR-1538
It is overexpressed the in-vitro multiplication ability that can inhibit colon cancer cell.
By the HCT116 cell inoculation after transient transfection into 96 orifice plates, using mtt assay detection silencing miR-1538 to knot
The influence of colon-cancer cell proliferative capacity.As a result as shown in Fig. 2, colon cancer cell transiently transfects miR-1538inhibitor 5 days
Afterwards, the speed of growth of cell improves 43.7%, and difference is statistically significant, prompts silencing rniR-1538 expression that can enhance knot
The in-vitro multiplication ability of colon-cancer cell.
Although having referred to various preferred embodiments describes the present invention, it will be appreciated by those skilled in the art that can carry out each
Kind variation, and available equivalents substitute its component without departing from base region of the invention.Come in addition, many changes can be carried out
Specific condition or material is set to be suitable for the teachings of the present invention without departing from its base region.
Claims (7)
1. raising the transcription of miR-1538 and/or the active reagent of miR-1538 being promoted to prevent and treat adenocarcinoma of colon preparation in preparation
In application, which is characterized in that the miR-1538 sequence is shown in sequence table SEQ ID NO.2.
2. application according to claim 1, which is characterized in that prevention and treatment adenocarcinoma of colon preparation prevention and treatment colon adenocarcinoma cell increases
It grows.
3. application according to claim 1, which is characterized in that the acquired technology of microRNA function based on RNA and/or
Gene specific miR Mimics technology raises the transcription of miR-1538 and/or promotes the activity of miR-1538.
4. application according to claim 1, which is characterized in that artificial synthesized short hairpin RNA passes through in regulation promoter
Adjust the expression of miR-1538.
5. detecting application of the preparation of miR-1538 in preparation diagnosis adenocarcinoma of colon reagent, which is characterized in that the miR-
1538 sequences are shown in sequence table SEQ ID NO.2.
6. application according to claim 5, which is characterized in that diagnosis adenocarcinoma of colon reagent includes being based on high-flux sequence side
Method and/or the transcription that miR-1538 in adenocarcinoma of colon sample is detected based on quantifying PCR method and/or based on probing procedure.
7. application according to claim 6, which is characterized in that use northern hybridizing method, miRNA express spectra core
Piece, ribozyme protection analytical technology, RAKE method, in situ hybridization, bead-based flow-cytometry detect in adenocarcinoma of colon sample
The transcription of miR-1538.
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