CN103146702A

CN103146702A - Application of MiR-181a-5p in non-small cell lung cancer

Info

Publication number: CN103146702A
Application number: CN2013100570392A
Authority: CN
Inventors: 马中良; 金由辛; 赵波涛; 邱翔; 田珂
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2013-02-22
Filing date: 2013-02-22
Publication date: 2013-06-12

Abstract

The invention discloses miR-181a-5p in A549 cell lines. The miR-181a-5p has the advantages of inhibiting cell proliferation and migration, promoting cell apoptosis and inhibiting size and quantity of cell colonies. The miR-181a-5p inhibits the translation of a target gene or directly degrades the mRNA of the target gene through complementation with 3'-UTR of mRNA of a target gene. The luciferase reporter gene experiment proves that Kras is the target gene of the miR-181a-5p; the down-regulation effect of the miR-181a-5p is further proved through qRT-PCR and western-blot; the report that the Kras in the disclosed A549 cell lines is the target gene of the miR-181a-5p is provided for the first time; and the miR-181a-5p provides a certain theoretical basis for drugs for target treatment of non-small cell lung cancer in clinic.

Description

The application of MiR-181a-5p in nonsmall-cell lung cancer

Technical field

The invention belongs to biological technical field.The present invention relates to a kind of miRNA, target gene and application thereof.

The present invention relates to the target gene of target gene, Relative luciferase activity assay and the western-blot technical identification miRNA of solexa order-checking and software prediction miRNA and cross and express impact, the low cytometric analysis that the variation, the soft agar assay that utilize CCK-8 kit detection cell propagation after miR-181a-5p detect cell cluster formation and detect apoptotic impact.

Background technology

MicroRNA(is called for short miRNA) be one of member of the little RNA of non-coding family, length is 18 ~ 25 bases, can be incorporated into the 3'-end non-coding region (3'-UTR) of target gene mRNA by the base complementrity principle, thereby suppress the translation of target gene mRNA or induce its degraded to change the protein expression level of target gene, and the network regulation system of mixing by time multiplexed cell, the physiological activities such as the growth of organism, differentiation, propagation, apoptosis, immunity are carried out fine adjustment.

Lung cancer is the No.1 killer of cancer mortality, and 5 years survival rates are less than 15 %, more than wherein nonsmall-cell lung cancer (non small cell lung cancer, NSCLC) accounts for 80 % of all lung cancer cases.The variation that is accompanied by the miRNA abundance of lung cancer, the expression of miRNA has Space-time speciality.The level of miRNA is variant in different tissues, different developmental phases.The abnormal expression of miRNA can make cell phenotype that corresponding the variation occurs, and then causes the formation with tumour brought out of disease.

Summary of the invention

One of purpose of the present invention is to provide a kind of miRNA, and it has the function that suppresses nonsmall-cell lung cancer propagation.This miRNA picks out by the solexa sequencing analysis, is miR-181a-5p, and its mature sequence is (5 '-aacauucaacgcugucggugagu-3 ').Determine through experimental analyses such as CCK-8 and cell colonies.

Two of purpose of the present invention is to provide the application of miR-181a-5p in promoting cancer cell-apoptosis.

It is Kras that three of purpose of the present invention is to provide the target gene of miR-181a-5p in A549 clone.A549 is available from biochemical cell institute of Chinese Academy of Sciences cell bank.

For achieving the above object, the present invention adopts following technique means:

The first step by the solexa sequencing result, is utilized the qRT-PCR technology to seek out the Lines of miR-181a-5p down-regulated expression, and is crossed expression miR-181a-5p in this clone, to detect its function.

Second step utilizes low cytometric analysis and CCK-8 technology, detects respectively apoptosis, the propagation situation of expressing A549 cell after miR-181a-5p.

In the 3rd step, studied cell colony situation after expression miR-181a-5p with soft agar assay.

The 4th step was connected to the pGL-3 carrier with 3 of the mRNA of Kras '-UTR, had on it and miR-181a-5p Seed Sequences complementary base mutually.With miR-181a-5p and recombinant plasmid cotransfection HEK-293T cell, detect fluorescent signal after 48 h.

In the 5th step, western-blot detects at protein level Kras by the downward situation.

Description of drawings

The relative expression quantity of miR-181a-5p in Fig. 1 Lines;

Fig. 2 crosses and expresses the propagation that suppresses the A549 cell after miR-181a-5p;

Fig. 3 crosses the cell colony form of expressing microscopically after transfection NC, miR-181a-5p after miR-181a-5p and Anti-miR-181a-5p;

Fig. 4 was the cell colony form of expressing under miR-181a-5p transfection rear plate;

Fig. 5 was the counting diagram of expressing colony number of cell after miR-181a-5p;

Fig. 6 crosses and expresses the apoptosis that promotes the A549 cell after miR-181a-5p, and wherein A is transfection NC; B is transfection miR-181a-5p;

Fig. 7 is for passing through two fluorescence report gene test miR-181a-5p to the inhibition of Kras;

Fig. 8 is the expressing quantity of Kras after transfection miR-181a-5p in western experiment;

Fig. 9 is the relative expressing quantity of the Kras that arrives by software detection.

Embodiment

Further set forth the present invention below in conjunction with specific examples.Ordinary method is with reference to " molecular cloning " (Molecular Cloning:A Laboratory Manual, 3rd ed.) and test kit correlation step in concrete experimental procedure.

Embodiment one: according to the solexa sequencing result, determine miR-181a-5p low expression in Lines

First the lung tissue of normal mouse is taken a sample with the lung tissue of suffering from the mouse of nonsmall-cell lung cancer, extract total RNA with the TRIZOL method.Utilize the reverse transcription test kit of TaKaRa company to build the cDNA library of two kinds of tissues, carry out reverse transcription take oligo dT as primer, obtain the cDNA of subsequent experimental by reaction of degeneration and 2 steps of reverse transcription.

Sample utilizes Solexa method order-checking (the Solexa order-checking is completed by the large genome company of China), the data that analysis obtains, found that with mouse normal lung model (L1805) and compare, the relative expression quantity of miR-181a-5p has been lowered 65.6 % in the lung cancer model (L703T2) of mouse, (L822T1) lower 85.6 %, (L903T1) lowered 69.7 %.MiR-181a-5p all has downward at the expression amount of mice lung cancer model, and further research finds that (Fig. 1) significantly lowered in the expression of miR-34a in Lines.

Embodiment two: cell proliferation has restraining effect to miR-181a-5p to A549

The A549 cell is layered in 96 orifice plates uniformly, and the mimics with miR-181a-5p after 24 h changes in the A549 cell, changes cell culture fluid after transfection 4-6 h.First remove the nutrient solution that contains serum before transfection, replace with not containing serum and antibiotic nutrient solution, to improve transfection efficiency.After transfection 48 h, begin to detect with the CCK-8 method vigor of cell, detect once every 24 h, detecting wavelength is 450 nm.The result demonstration, the rate of propagation of the A549 cell of transfection miR-181a-5p illustrates that lower than control group cell proliferation has restraining effect (Fig. 2) to miR-181a-5p to A549.

Embodiment three: miR-181a-5p can suppress the formation of A549 cell colony

First configure 1.2% low melting-point agarose solution, the low melting-point agarose of 1.2 g is added in 100 ml ultrapure waters, 4 ℃ of preservations are put in sterilization.Then configure 2 * DMEM/E12 nutrient solution, add 25 ml FBS and 2 % microbiotic in every 100 ml serum-free DMEM/E12 nutrient solutions, prepare again single cell suspension, then prepare bottom-layer agar (final concentration 0.6%), add isopyknic 2 * DMEM/E12 nutrient solution in the agarose of 1.2 %, then spread by six orifice plates, 2 ml/ holes, be placed in cell culture incubator 30 min, standby.Preparation at last contains the top-agar (final concentration is 0.3%) of cell, add two volumes 2 * DMEM/E12 nutrient solution in the agarose solution of 1.2 %, and add the single cell suspension for preparing, the adjustment cell concn is 5000/ml, be positioned over incubator and cultivate 14-20 day, observe weekly.Result shows, crosses the A549 cell colony of expressing miR-181a-5p and diminishes, reduced number.Illustrate that miR-181a-5p can suppress the formation of A549 cell colony (Fig. 3, Fig. 4 and Fig. 5).

Embodiment four: miR-181a-5p promotes the apoptosis of A549 cell

The A549 cell is layered in 6 orifice plates uniformly, and the mimics of transfection miR-181a-5p after 24 h is after 48 h, with the cell of collecting BD FITC Annexin V Apoptosis Detection Kit I apoptosis detection kit staining cell.Detect the apoptosis situation of A549 cell with Flow Cytometry.Result proves, transfection the A549 apoptosis rate of miR-181a-5p greater than control group (Fig. 6).

Embodiment five: the analysis of luciferase reporter gene

3 of Kras mRNA '-UTR is connected on the pGL-3 plasmid, the promotor that contains SV40 on this plasmid, because find to have two binding sites of miR-181a-5p on 3 of Kras '-UTR, main binding site in order to study that binding site, clone's 3 '-UTR is three kinds, the first only comprises miR-181a-5p at first binding site of Kras 3 '-UTR, be 280 bp, the second only comprise its 3 '-second binding site of UTR, size is 113 bp, and the third not only comprises first binding site and also comprises second binding site, size 395 bp.With the lip2000 transfection reagent, the mimics of miR-181a-5p is entered in the HEK-293T cell with the plasmid corotation that builds, after 48 h, with the Relative luciferase activity assay test kit detection of promega company.The final concentration of the plasmid of transfection is 200 nmol/L, and the final concentration of mimics is 80 nmol/L.Found that, transfection in the HEK-293T cell of miR-181a-5p the expression of Dual-Luciferase lower than control group, and the decline degree that comprises two binding sites is the most obvious, illustrate that two sites all can be combined with miR-181a-5p, be miR-181a-5p at Kras 3 '-target site (Fig. 7, Fig. 8 and Fig. 9) of UTR.

Embodiment six: the restraining effect of western-blot checking miR-181a-5p to the endogenous Kras gene of A549 cell

Non-small cell lung cancer cell strain A549 is cultured in the F-12K substratum that contains 10% calf serum.CO ₂Contain 5 % CO in incubator ₂And moistening, temperature is 37 ℃.

The monolayer adherence cell is sucked nutrient solution, and PBS cleans twice.Add 10 l PMSF by every 1 ml lysate, shake up and be placed on ice.Every bottle of cell adds 200 l to contain the lysate of PMSF, in 30 min of cracking on ice.After cracking is complete, scrape a side of culturing bottle with clean scraper, centrifugal 15 min of 12000 rpm under 4 ℃.After this experiment adopts 12% SDS-PAGE glue to carry out protein electrophoresis, with protein delivery to nitrocellulose filter.The preparation of transferring film damping fluid and film scavenging solution is all with reference to molecular cloning.The antibody of Kras albumen dilutes according to 1:1000, and the antibody of GAPDH albumen dilutes according to 1:1000.The two anti-weaker concns that adopt are 1:10000.

First the A549 cell is layered in 6 orifice plates equably, after 24 h with the mimics of miR-181a-5p by changing in the A549 cell with hatching altogether of Lip2000 and serum-free medium, change cell culture fluid after transfection 6 h.After transfection 48 h, collect, lysing cell, and the quantitative protein content in each sample adopts the Bradford method.Come the protein content of Kras genes encoding in test sample with the antibody of Kras.This antibody is produced by Abcam company.Result show transfection in the A549 cell of miR-181a-5p the protein content of Kras genes encoding lower than control group.Explanation Kras in the A549 cell is the target gene ((Fig. 7, Fig. 8 and Fig. 9)) of miR-181a-5p.

Although the invention describes concrete example, having is a bit significantly to those skilled in the art, namely can make various changes and change the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.

Claims

1. the application of miR-181a-5p gene in the propagation of anticancer and clone form in a nonsmall-cell lung cancer.

2. the application of miR-181a-5p gene in the apoptosis that promotes cancer cells in a nonsmall-cell lung cancer.

3. the application that in a nonsmall-cell lung cancer, the miR-181a-5p gene is lowered the Kras gene expression dose.