CN104069506A - Application of miR-486-5p in non-small cell lung carcinoma cell line - Google Patents
Application of miR-486-5p in non-small cell lung carcinoma cell line Download PDFInfo
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Abstract
The invention relates to application of miR-486-5p in a non-small cell lung carcinoma cell line. The miR-486-5p performs specific low-expression in a non-small cell lung carcinoma cell and a series of lung carcinoma tissue clinical specimen; in an H1299 cell, the over-expressed miR-486a-5p can inhibit cell proliferation of H1299 and inhibit G1/S conversion to block the cell in the G1 phase. Experiments based on bioinformatics analysis discovers that the miR-486a-5p is complementary with 3'-UTR () of a target gene mRNA (messenger ribonucleic acid), so that translation of the mRNA of the target gene can be inhibited or the mRNA of the target gene can be directly degraded; furthermore, western blotting and other experiments prove that the CDK4 gene is the target gene of the miR-486a-5p. The experiment first proves that the CDK4 gene is the garget gene of miR-486a-5p, and the miRNA is utilized to diagnose and treat non-small cell lung carcinoma in clinical application, and a certain application value is created for providing the aspect of drug targets.
Description
Technical field
The present invention relates to the application of miR-486-5p in Lines.
Background technology
MicroRNA (microRNA, miRNA) function controlling is the important front edge of current life sciences.MicroRNA (microRNA, miRNA) is the non-coding microRNA of a class approximately 22 nt, is extensively present in more high eukaryote, mostly has higher conservative.MiRNA acts on special mRNA, makes its degraded more or suppresses its translation, expresses at post-transcriptional level negative regulator gene, and what some miRNA can activation target gene transcribes.5 of ripe miRNA ' end 2nd ~ 7 bit bases are called as " Seed Sequences ", are the keys of Effective Regulation mRNA, and the binding site of miRNA can be not only 3 of target gene '-UTR, can be also 5 of target gene '-UTR or CDS region.In mammal, approximately 30% protein coding gene is subject to the regulation and control of miRNA.
Pulmonary carcinoma is the main reason that causes global cancer mortality, annual because the number of lung cancer death exceedes 1,000,000, and increases every year case 1,200,000 newly.Adenocarcinoma of lung (lung adenocarcinoma) is modal cancer, wherein approximately 80% pulmonary carcinoma is nonsmall-cell lung cancer (non-small cell lung cancer, NSCLC), its 5 annual survival rate is only 15%, and main cause is to lack the effectively early diagnosis and therapy means for pulmonary carcinoma.MiRNA, by the translation of regulation and control target gene mRNA, occurs, develops and shift playing a significant role in pulmonary carcinoma.MiRNA may become the new pulmonary carcinoma early diagnosis label relevant with cancer process, contributes to Accurate Diagnosis and the personalized treatment of disease.The realization of everything imagination must be based upon on the basis of miRNA target gene functional study work.
Summary of the invention
One of object of the present invention is to provide a kind of miR-486-5p to suppress the application in H1299 cell proliferation medicine in nonsmall-cell lung cancer in preparation.
Two of object of the present invention is to provide a kind of miR-486-5p to suppress H1299 cell G1/S in nonsmall-cell lung cancer in preparation and transforms the application in medicine.
Three of object of the present invention is to provide a kind of miR-486-5p in the application of preparing in the endogenous CDK4 gene expression dose of targeting downward H1299 cell medicine.
Four of object of the present invention is to provide the application of a kind of miR-486-5p in preparation is treated or prevention nonsmall-cell lung cancer is used compositions or test kit.
First the present invention utilizes Solexa sequencing technologies, utilize the expression of miR-486-5p in the multiple nonsmall-cell lung cancer of qRT-PCR technology for detection system to change, and therefrom select the most obvious nonsmall-cell lung cancer of a kind of miR-486-5p down-regulated expression system, in this cell line, cross expression miR-486-5p to detect its function.
Cell transfecting transfection reagent used is Lipo2000(Invitrogen).In order to reduce difference between the hole that the factors such as cell density, reagent dosage and transfection cause, ensure the reproducibility and reliability of experiment, in this experiment, each transfection sample is provided with 3 and answers holes.When inoculating cell, the cell quantity of every hole inoculation is consistent as far as possible, and cell is evenly distributed on the surface in each hole.
The extraction of cell total rna adopts the Trizol reagent of Sheng Gong biotech firm.Concrete extraction step is as follows:
directly in culture plate, add Total RNA Extractor cell lysis, every 10 cm
2area adds 1 ml Total RNA Extractor, blows and beats and mixes with pipettor;
sample after cracking or homogenate room temperature are placed to 5-10 min, nucleoprotein is separated completely with nucleic acid;
add 0.2 ml chloroform, thermal agitation 15 sec, room temperature is placed 3 min.Centrifugal 10 min of 12,000 4 DEG C of rpm;
draw water and be transferred in clean centrifuge tube, add equal-volume isopropyl alcohol, mix, room temperature is placed 20 min;
centrifugal 10 min of 12,000 4 DEG C of rpm, abandon supernatant;
add 1 ml 75% washing with alcohol precipitation, centrifugal 3 min of 12,000 4 DEG C of rpm, abandon supernatant, drying at room temperature 5-10 min;
add 30-50 μ l RNase-free ddH
2o, fully dissolves RNA, by obtained RNA solution be placed in-70 DEG C preserve or for follow-up test.
Reverse transcriptional PCR adopts the One Step PrimeScript miRNA cDNA Synthesis Kit of Takara company.Because miRNA is different from mRNA, miRNA does not have Poly(A) structure, but this transcript reagent box can carry out Poly(A to the miRNA in sample and its small non-coding RNA simultaneously) add end reaction, then utilize Universal Adaptor Primer to obtain cDNA by carrying out reverse transcription reaction through the RNA of tailing and mRNA, and introduce the binding site of Uni-miR qPCR Primer, utilize this position to carry out quantitative PCR reaction to any cDNA in sample.The instrument that is used for detecting is the iQ5 system of Bio-Rad company, and reagent is the SYBR Green Mix of TaKaRa company.Taq archaeal dna polymerase is contained, dNTP mix, SYBR Green dye in this reagent the inside.U6 is used as reference gene.This tests primer used: miR-486-5p primers F orward:5 '-TCCTGTACTGAGCTGCCCCGAG-3 ', Reverse: be Uni-miR qPCR primer; U6 primer is Forward:5 '-CTCGCTTCGGCAGCACA-3 ', Reverse:5 '-AACGCTTCACGAATTTGCGT-3 '.
Second step, utilizes CCK8 technology and low cytometric analysis, detects respectively and expresses the propagation of H1299 cell and the variation of cycle situation after miR-486-5p.
Nonsmall-cell lung cancer H1299 cell is cultured in 1640 culture medium that contain 10% hyclone.CO
2in incubator, contain 5% CO-
2and moistening, temperature is 37 DEG C.In order to reduce difference between the hole that the factors such as cell density, reagent dosage and transfection cause, ensure the reproducibility and reliability of experiment, in this experiment, each transfection sample is provided with 3 and answers holes.When inoculating cell, the cell quantity of every hole inoculation is consistent as far as possible, and cell is evenly distributed on the surface in each hole.
Transfection step is as follows:
the previous day of transfection non-small cell lung cancer cell, the cell of inoculation right quantity is to culture plate, and every hole adds not containing antibiotic culture medium, and the cell density while making transfection can reach 50%.When transfection, cell density is one of key factor affecting transfection efficiency, and Growth of Cells excessively can weaken cell viability, reduces the transfection efficiency of cell;
prepare mimic-lipo2000 mixed liquor: a. dilution miRNA mimic: not containing blood serum medium Opti-MEM dilution miRNA mimics, making to add the final concentration in cell is 50 nmol/L, mixes gently incubated at room 5 min with 50 μ l; B. dilute lipo2000: the Opti-MEM that does not contain serum with 50 μ l dilutes 1 μ l lipo2000, mixes gently and incubated at room 5 min; C. a and b are mixed gently to incubated at room 20 min.Attention: the long-time placement of lipo2000 of having diluted may cause the reduction of transfection reagent activity should mix with the mimics having diluted as far as possible within 25 min.In the time of mix reagent, can not acutely blow and beat or shake, finger flicks tube wall, and Overexertion may destroy the structure of liposome and the formation of miRNA-mimics-lipo2000 mixture;
miRNA-mimics-lipo2000 mixed liquor is added in the culture hole that contains cell and culture fluid, mix gently;
culture plate is placed in to the CO of 37 DEG C
248 h in incubator.Cultivate after 6 h, the culture medium that contains mimics-lipo2000 mixed liquor in hole is removed, change fresh culture.
This experiment adopts the CCK8(Cell Counting Kit-8 of DOJINDO company) test kit.This test kit has utilized water solublity four to file salt-WST-8(2-(2-methoxyl group-4-nitre phenyl)-3-(4-nitre phenyl)-5-(2,4-disulfobenzene)-2H-tetra-files single sodium salt).It can be reduced into water miscible first a ceremonial jade-ladle, used in libation dyestuff in the situation that electron carrier 1-Methoxy PMS exists.It can directly read under 450 nm absorbances in 96 orifice plates, without extra process, is directly directly proportional to the quantity of living cells in culture by the amount of the definite CCK8 of 450 nm place absorbances.
Fluorescent dye propidium iodide (PI) is a kind of nuclei dyeing toner that can dye to DNA, is usually used in cell cycle and detects.PI is a kind of analog of ethidium bromide, after embedding double-stranded DNA, discharges red fluorescence.PI can not pass living cells film, but can be through damaged cell membrane and to nuclear staining.Due to the DNA content difference of cell cycle phase when each, common Normocellular G1/G0 phase has the DNA content (2N) of diploid cell, and G2/M phase has four times.By Flow cytometry to the fluorescence intensity of the PI of being combined with DNA directly reflected the number of DNA content in cell.
The 3rd step, by the target gene of Targetscan software prediction miR-486-5p, and is connected to pGL-3 carrier by 3 of its mRNA '-UTR.MiR-486-5p and recombiant plasmid corotation are entered to HEK-293T cell, fluorescence intensity after 48 h.
HEKC HEK-293T is cultured in the DMEM culture medium that contains 10% hyclone.CO
2in incubator, contain 5% CO-
2and moistening, temperature is 37 DEG C.HEKC HEK-293T is purchased from biochemical cell institute of Chinese Academy of Sciences cell bank.
By searching ncbi database, find the mRNA sequence of CDK4 gene, and then find 3 of this mRNA '-UTR sequence.Search TargetScan data base, find the binding site that contains miR-486-5p Seed Sequences on the 3520-3526 site of 3 of this mRNA '-UTR sequence.Design primer, carries out PCR, and this site is included.CDK4-3 '-UTR primer is Forward:5 '-GCTCTAGAGCCATTTCCCTTCTGGACACTG-3 ', Reverse:5 '-GGAATTCATCTCGGCTCACCGCAACCT-3 '.This primer has been introduced
xbai and
ecotwo restriction enzyme sites of R I.
The 4th step is crossed expression miR-486-5p in H1299 cell, detects CDK4 mRNA level and changes.
Non-small cell lung cancer cell strain H1299 is cultured in the DMEM culture medium that contains 10% hyclone.CO
2in incubator, contain 5% CO-
2and moistening, temperature is 37 DEG C.Cell transfecting transfection reagent used is Lipo2000(Invitrogen).In order to reduce difference between the hole that the factors such as cell density, reagent dosage and transfection cause, ensure the reproducibility and reliability of experiment, in this experiment, each transfection sample is provided with 3 and answers holes.When inoculating cell, the cell quantity of every hole inoculation is consistent as far as possible, and cell is evenly distributed on the surface in each hole.Extract afterwards RNA, utilize the Primescript RT Master Mix perfect Real Time test kit of Takara company to carry out reverse transcription, then carry out qRT-PCR detection.The instrument that is used for detecting is the iQ5 system of Bio-Rad company, and reagent is the SYBR Green Mix of TaKaRa company.Taq archaeal dna polymerase is contained, dNTP mix, SYBR Green dye in this reagent the inside.18S rRNA is used as reference gene.This tests primer used: CDK4 primers F orward:5 '-AATGTTGTACGGCTGATGGA-3 ', Reverse:5 '-AGAAACTGACGCATTAGATCCT-3 '; 18S primers F orward:5 '-CAGCCACCCGAGATTGAGCA-3 ', Reverse:5 '-TAGTAGCGACGGGCGGTGTG-3 '.
Quantitatively albumen adopts Lowry method.Detect with the multi-resistance of CDK4.This antibody is that Cell Signal company produces.This experiment adopt 12% SDS-PAGE glue carry out after protein electrophoresis, by protein delivery to nitrocellulose filter.The preparation of transferring film buffer and film cleanout fluid is all with reference to molecular cloning.The antibody of CDK4 albumen dilutes according to 1:800, and the antibody of Gapdh albumen dilutes according to 1:1000.What adopt is two anti-with HRP labelling, dilutes according to 1:10000.With the chemiluminescence agent colour developing of Milipore.
In H1299 cell line miR-486-5p gene by with the 3 '-UTR complementation of the mRNA of its target gene CDK4, suppress translation or the said target mrna of directly degrading of target gene mRNA.In conjunction with biochemistry and molecular biology experiment, determine that miR-486-5p and CDK4 have interaction in H1299 cell line, this interaction has affected the vital movement such as propagation, cycle of H1299 cell.This experiment is by software prediction, carrier construction, 3 '-the UTR to target gene utilizes test kit to suddenly change simultaneously, then in HEK293T cell, utilize luciferase reporter gene analysis verification CDK4 be the target gene of miR-486-5p, and in H1299 cell, utilize qRT-PCR, western blot technology, further verify this discovery.Disclosed for CDK4 in H1299 cell line is the reported first of the target gene of miR-486-5p, this invention is clinically for utilizing miRNA to diagnose and treating nonsmall-cell lung cancer, and provides drug target aspect that certain using value is provided.
Brief description of the drawings
Fig. 1 is the relative expression's content of miR-486-5p in Lines;
Fig. 2 is the expression of miR-486-5p in 32 pairs of cancerous lung tissue clinical samples;
Fig. 3 was the propagation that suppresses H1299 cell after expression miR-486-5p;
Fig. 4 was that expression miR-486-5p is suppressed at the G1 phase H1299 cell;
Fig. 5 is that CDK4 is the target gene of miR-486-5p, figure A:miR-486-5p and CDK4 wild type and saltant type 3 '-UTR binding sequence schematic diagram.Figure B: two fluorescence report analyses.MiR-486-5p family lowers the reporter gene expression that contains wild type 3 '-UTR, and the reporter gene expression that contains saltant type 3 '-UTR is not affected.Figure C: cross the protein level of expressing CDK4 in miR-486-5p downward H1299.The mimics of transfection miR-486-5p, by the protein content of western blotting detection CDK4.
Detailed description of the invention
Further set forth the present invention below in conjunction with instantiation.These examples only, for setting forth the present invention, limit the scope of the invention and be not used in.The experimental technique of unreceipted specific experiment condition in following example, conventionally according to normal condition, the condition described in molecular cloning (Molecular Cloning:A Laboratory Manual, 3rd ed.), or the condition of advising according to manufacturer.
embodiment mono-: according to Solexa sequencing result, determine miR-486-5p low expression in Lines
The present invention's k-ras mutant mouse used lung cancer model (L703T2) and normal lung cancer model (L1805) are provided by the biochemical cell Ji Hongbin of institute of Chinese Academy of Sciences teach problem group.
The lung tissue of normal mouse and the lung tissue of the mice that suffers from nonsmall-cell lung cancer are sampled, with TRIZOL method cracking tissue, add chloroform, treat albumen and nucleic acid layering, draw supernatant and add isopropanol precipitating after centrifugal, after recentrifuge, precipitate with washing with alcohol, dry, obtain total RNA.Utilize the reverse transcription test kit of TaKaRa company to build the cDNA library of two kinds of tissues, carry out reverse transcription taking oligo dT as primer, obtain the cDNA of subsequent experimental by reaction of degeneration and reverse transcription 2 steps.
Sample is utilized to Solexa method order-checking (Solexa order-checking is completed by Hua Da genome company), and further research finds that (referring to Fig. 1 and Fig. 2) significantly lowered in the expression of miR-486-5p in Lines.
the impact of embodiment bis-: miR-486-5p on H1299 cell proliferation
H1299 cell is layered in 96 orifice plates equably, and the culture medium of use is 1640.After 24 h, the mimics of miR-486-5p is passed through to lipo2000 transfection reagent, proceed in H1299 cell by after the hatching altogether of serum-free medium, after transfection 6 h, change cell culture fluid, put into fresh culture fluid, then add immediately CCK8 reagent, in incubator, cultivate 2.5 h, then solution is transferred to ELISA Plate, detect the absorbance of 450 nm, measure once every 24 h.
Inspection finds, transfection group is compared with matched group, and its absorbance has significant decline, and the growth rate of the cell of the miR-486-5p that is also transfection, significantly lower than matched group, illustrates that miR-486-5p has the effect (referring to Fig. 3) of inhibition H1299 cell proliferation.
the impact of embodiment tri-: miR-486-5p on H1299 cell cycle
H1299 cell is layered in 6 orifice plates equably, and each sample repeats for three times.The mimics of transfection miR-146a-5p after 24 h.Collecting cell after 48 h.Washing twice, 70% ethanol with PBS fixes 4 DEG C and spends the night.PBS washes once, and the RNase of 50 mg/ml digests 1 h, room temperature.50 mg/ml PI, 30 min that dye,, detect with flow cytometer by 4 DEG C.(referring to Fig. 4).
embodiment tetra-: Relative luciferase activity assay
By 3 of CDK4mRNA '-UTR(533 bp) be connected on pGL-3 plasmid, on this plasmid, contain the promoter of SV40, the binding site that 3 in connection '-UTR has comprised miR-486-5p and CDK4.The mimics of miR-486-5p is entered in HEK-293T cell with the plasmid corotation that builds with lipo2000 transfection reagent, and proceed to pRL plasmid signal as a setting simultaneously, after transfection 6 h, culture fluid is changed to fresh medium.After transfection 48 h, detect the luciferase expression of pGL and pRL with the Relative luciferase activity assay test kit of Promega company.The final concentration of the plasmid of transfection is 400nmol/L, and the final concentration of mimics is 20 nmol/L.Found that, transfection in the HEK-293T cell of miR-486-5p the expression of Dual-Luciferase significantly lower than matched group.Utilize afterwards sudden change test kit to do the point mutation of CDK43 '-UTR, the mimics of miR-486-5p and the point mutation plasmid corotation that builds are entered in HEK-293T cell with lip2000 transfection reagent.After transfection 48 h, with the Relative luciferase activity assay test kit detection of Promega company, found that, transfection in the HEK-293T cell of miR-486-5p the expression of Dual-Luciferase and matched group do not have anything to change, these all illustrate that CDK4 is the target gene (referring to Fig. 5) of miR-486-5p.
the inhibitory action of embodiment five: qRT-PCR checking miR-486-5p to the endogenous CDK4 gene of H1299 cell
H1299 cell is layered in 6 orifice plates equably, after miR-486-5p mimics, NC, miR-486-5p inhibitor, anti-NC being hatched altogether by lipo2000 and serum-free medium 1640 after 24 h, proceed in H1299 cell, after transfection 6 h, cell culture fluid is changed to fresh medium.After transfection 48 h, collecting cell, extracts RNA, and utilizing the test kit reverse transcription of Takara company is cDNA, carries out afterwards qRT-PCR detection.
H1299 cell is layered in 6 orifice plates equably, and each sample repeats for three times.The mimics of transfection miR-486-5p after 24h.After 48h, collect, cell lysis, and CDK4 protein content in quantitative each sample.QRT-PCR and western blot result show, miR-486-5p is to CDK4 inhibited (referring to Fig. 5).
Although the invention describes concrete example, having is a bit significantly to those skilled in the art, can the present invention be made various changes and be changed under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these variations within the scope of the present invention.
Claims (4)
1. a miR-486-5p suppresses the application in H1299 cell proliferation medicine in nonsmall-cell lung cancer in preparation.
2. a miR-486-5p suppresses the application in H1299 cell G1/S conversion medicine in nonsmall-cell lung cancer in preparation.
3. a miR-486-5p is in the application of preparing in the endogenous CDK4 gene expression dose of targeting downward H1299 cell medicine.
4. the miR-486-5p application in compositions or test kit that preparation treatment or prevention nonsmall-cell lung cancer are used.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104726454A (en) * | 2015-02-03 | 2015-06-24 | 湖州市中心医院 | Action of hsa-miR-1280 in non-small cell lung cancer and application of hsa-miR-1280 |
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| CN101804208A (en) * | 2010-02-26 | 2010-08-18 | 南京医科大学 | Application of miR-451 in preparing medicine for treating non-small cell lung cancer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104726454A (en) * | 2015-02-03 | 2015-06-24 | 湖州市中心医院 | Action of hsa-miR-1280 in non-small cell lung cancer and application of hsa-miR-1280 |
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