CN103224983A - Application of miR-96 gene in non-small cell lung cancer - Google Patents

Abstract

The invention relates to an application of an miR-96 gene in a non-small cell lung cancer. The complementation of a target gene CREB1, miR-96 gene of miR-96 in an H1299 cell line with 3'-UTR of mRNA of the target gene inhibits the translation of the target gene mRNA or directly degrades the mRNA of the target gene. A case that the interaction of the miR-96 of the H1299 cell line with CREB1 influences the vital activities comprising propagation, apoptosis and the like of H1299 cells is determined in the invention. A case that the CREB1 is the target gene of the miR-96 is preliminarily verified through software prediction, vector construction and the analysis by utilizing a luciferase reporter gene in an HEK293T cell, and the case is further verified through utilizing an miR-96 over-expression and qRT-PCR technology in the H1299 cells. The case that the CREB1 is the target gene of the miR-96 is reported in the invention for the first time, and the report provides certain application values for the clinic utilization of the miRNA to diagnose and treat the non-small cell lung cancer and the provision of drug targets.

Description

The application of miR-96 gene in the nonsmall-cell lung cancer

Technical field

The present invention relates to the application of miRNA gene in a kind of nonsmall-cell lung cancer.

Background technology

(microRNA, miRNA) function controlling is the important front edge of current life science to microRNA.MiRNA is that a class length is the non-coding RNA molecule of 21-22 nt, and it is by the activity of PTGS regulation and control target gene.To the target gene of miRNA biological function and regulation and control thereof studies show that miRNA plays an important role in the tumour generating process, miRNA probably becomes the new way of cancer therapy.The non-coding sequence of miRNA and target gene mRNA (3 '-UTR or 5 '-UTR) exists the relation of one-to-many.MiRNA is normally transcribed by the RNA polymerase II in nucleus, the primary transcription product is pri-miRNA, pri-miRNA is processed into hair fastener type precursor miRNA (pre-miRNA) by nuclease RNase III Drosha and its cofactor Pasha, pre-miRNA is outputed to tenuigenin by translocator Exportin 5 from nucleus, sheared by the Dicer enzyme complex afterwards and become short miRNA two strands, the miRNA two strands is dissociated under the help of helicase, sophisticated miRNA and RNA inductive silencing complex (RISC) combination.MiRNA can combine with the complementary sequence among target gene mRNA 3 '-UTR by base pairing with the complex body of RISC, immediately according to the complementary height of miRNA and its target-gene sequence, or the translation of arrestin matter, or cause the mRNA degraded, thus the negative regulation target gene expression.

Lung cancer is the main reason that causes global cancer mortality, and annual number because of lung cancer death surpasses 1,000,000, and annual newly-increased case 1,200,000.Adenocarcinoma of lung (lung adenocarcinoma) is modal cancer, wherein about 80% lung cancer is nonsmall-cell lung cancer (non small cell lung cancer, NSCLC), its 5 annual survival rate only is 15%, and major cause is to lack effective early diagnosis and therapy means at lung cancer.MiRNA plays a significant role in lung cancer generation, development and transfer by the translation of regulation and control target gene mRNA.MiRNA may become the new lung cancer early diagnosis marker relevant with the cancer process, helps the 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 purpose of the present invention is to provide the application of a kind of miR-96 gene in suppressing the nonsmall-cell lung cancer cancer cell multiplication.

Two of purpose of the present invention is to provide the application of a kind of miR-96 gene in promoting the nonsmall-cell lung cancer cancer cell-apoptosis.

Three of purpose of the present invention is to provide a kind of miR-96 gene to reduce the application of CREB1 expression level in H1299 clone.

The present invention at first utilizes the Solexa sequencing technologies, utilize the expression amount of miR-96 in the multiple nonsmall-cell lung cancer of the qRT-PCR technology for detection system to change, and therefrom select the most tangible nonsmall-cell lung cancer of a kind of miR-96 down-regulated expression system, in this clone, cross expression miR-96 to detect its function.

The used transfection reagent of cell transfecting is Lipo2000(Invitrogen).In order to reduce the hole differences that factors such as cell density, reagent dosage and transfection cause, guarantee the reliability and the repeatability of experiment, each transfection sample is provided with 3 multiple holes in this experiment.During 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 Trizol reagent of giving birth to worker biotech firm is adopted in the extraction of cell total rna.Concrete extraction step is as follows:

Directly in culture plate, add Total RNA Extractor lysing cell, per 10 cm ²Area adds 1 ml Total RNA Extractor, blows and beats mixing with pipettor;

Sample after the cracking or homogenate room temperature are placed 5-10 min, make nucleoprotein separate fully with nucleic acid;

Add 0.2 ml chloroform, thermal agitation 15 sec, room temperature is placed 3 min.4 ℃ of centrifugal 10 min of 12,000 rpm;

Draw water and be transferred in the clean centrifuge tube, add the equal-volume Virahol, mixing, room temperature is placed 20 min;

4 ℃ of centrifugal 10 min of 12,000 rpm abandon supernatant;

Add 1 ml, 75% washing with alcohol precipitation, 4 ℃ of centrifugal 3 min of 12,000 rpm abandon supernatant, drying at room temperature 5-10 min;

Add 30-50 l RNase-free ddH ₂O fully dissolves RNA, places-70 ℃ to preserve or be used for follow-up test resulting RNA solution.

Reverse transcription PCR adopts the Takara One Step PrimeScript miRNA cDNA Synthesis Kit of company.Because miRNA is different with mRNA, miRNA does not have Poly(A) structure, but this transcript reagent box can carry out Poly(A to miRNA in the sample and its small non-coding RNA simultaneously) add end reaction, utilize Universal Adaptor Primer to carry out reverse transcription reaction then and obtain cDNA through the RNA of tailing and mRNA, and the binding site of introducing Uni-miR qPCR Primer, utilize this position that any cDNA in the sample is carried out the quantitative PCR reaction.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.The Taq archaeal dna polymerase is contained, dNTP mix, SYBR Green dye in this reagent the inside.U6 is used as internal control gene.This tests used primer: miR-96 primers F orward:5 '-TTTGGCACTAGCACATTTTTGCT-3 ', Reverse: be Uni-miR qPCR primer; The U6 primer is Forward:5 '-CTCGCTTCGGCAGCACA-3 ', Reverse:5 '-AACGCTTCACGAATTTGCGT-3 '.

Second step, utilize CCK8 technology and low cytometric analysis, detected the propagation of expressing H1299 cell behind the miR-96 and the variation of apoptosis situation respectively.

Nonsmall-cell lung cancer H1299 cell is cultured in 1640 substratum that contain 10% foetal calf serum.CO ₂Contain 5% CO in the incubator ₂And moistening, temperature is 37 ℃.In order to reduce the hole differences that factors such as cell density, reagent dosage and transfection cause, guarantee the reliability and the repeatability of experiment, each transfection sample is provided with 3 multiple holes in this experiment.During 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 transfection step is as follows:

The day before yesterday of transfection non-small cell lung cancer cell, the cell of inoculation proper amt is to culture plate, and every hole adds and do not contain antibiotic substratum, and the cell density when making transfection can reach 50%.During transfection, cell density is one of key factor that influences transfection efficiency, and the cell overgrowth can weaken cell viability, reduces the transfection efficiency of cell; Prepare the mimic-lipo2000 mixed solution: a. dilution miRNA mimic: do not contain blood serum medium Opti-MEM dilution miRNA mimics with 50 l, making the final concentration in the adding cell is 50 nmol/L, mixing gently, incubated at room 5 min; B. dilute lipo2000: the Opti-MEM that does not contain serum with 60 l dilutes 1 l lipo2000, gently mixing and incubated at room 5 min; C. with a and b mixing gently, incubated at room 20 min.Attention: the long-time placement of lipo2000 that dilution is good may cause the active reduction of transfection reagent, should mix with dilution mimics well within 25 min as far as possible.When mix reagent, can not acutely blow and beat or shake, light finger bomb tube wall gets final product, and Overexertion may destroy the structure of liposome and the formation of miRNA-mimics-lipo2000 mixture;

The adding of miRNA-mimics-lipo2000 mixed solution is contained in the culture hole of cell and nutrient solution, gently mixing;

Culture plate placed 37 ℃ CO ₂48 h in the incubator.After cultivating 6 h, the substratum that contains the mimics-lipo2000 mixed solution in the hole is removed, changed fresh culture.

The CCK8(Cell Counting Kit-8 of DOJINDO company is adopted in this experiment) test kit.This test kit has utilized water-soluble four file salt-WST-8(2-(2-methoxyl group-4-nitre phenyl)-3-(4-nitre phenyl)-5-(2, the 4-disulfobenzene)-the single sodium salt of 2H-four files).It can be reduced into water miscible first a ceremonial jade-ladle, used in libation dyestuff under the situation that electron carrier 1-Methoxy PMS exists.It can directly be read in 96 orifice plates under 450 nm absorbancys, need not extra process, and the amount of the CCK8 that is determined by 450 nm place absorbancys directly is directly proportional with the quantity of viable cell in the culture.

Fluorescence dye propidium iodide (PI) is a kind of can the painted nuclei dyeing toner of DNA being usually used in apoptosis and detecting.PI is a kind of analogue of ethidium bromide, discharges red fluorescence after embedding double-stranded DNA.PI can not pass membrane, but can pass damaged cytolemma and to nuclear staining.AnnexinV is the apoptotic reagent of a kind of detection, and in normal cell, phosphatidylserine only is distributed in the double-deck inboard of cell membrane lipid, and cell generation apoptosis is early stage, and membrane phospholipid acyl Serine by rollover in the adipose membrane laterally.AnnexinV has high affinity as a kind of phospholipids incorporate albumen with phosphatidylserine, and it combines with the after birth of the early stage cell of apoptosis by the phosphatidylserine that the cell outside exposes.AnnexinV and PI coupling are used, and the cell that can apoptosis is early stage and the cell in late period and dead cell make a distinction.

In the 3rd step,, and 3 of its mRNA '-UTR is connected to the pGL-3 carrier by the target gene of Targetscan software prediction miR-96.MiR-96 and recombinant plasmid corotation are gone into the HEK-293T cell, fluorescence intensity behind 48 h.

HEKC HEK-293T(is available from biochemical cell institute of Chinese Academy of Sciences cell bank) be cultured in the DMEM substratum that contains 10% foetal calf serum.CO ₂Contain 5% CO in the incubator ₂And moistening, temperature is 37 ℃.By searching ncbi database, find the mRNA sequence of CREB1 gene, and then find 3 of this mRNA '-UTR sequence.Search the TargetScan database, find on the 1691-1697 site of 3 of this mRNA '-UTR sequence, to contain the binding site of miR-96 kind subsequence.The design primer carries out PCR, in this site is included in.CREB1-3 '-UTR primers F orward:5 '-GCTCTAGAGCCACAACCTGAAAGACAAA-3 ', Reverse:5 '-CGGAATTCGTCCCACATTCAAATCCTCTC-3 '.This primer has been introduced XbaI and EcoTwo restriction enzyme sites of R I.The annealing temperature of PCR is 55 ℃, and product length is 619 bp.

The 4th step crossed expression miR-96 in the H1299 cell, detect CREB1 mRNA level and change.

Non-small cell lung cancer cell strain H1299 is cultured in the DMEM substratum that contains 10% foetal calf serum.CO ₂Contain 5% CO in the incubator ₂And moistening, temperature is 37 ℃.The used transfection reagent of cell transfecting is Lipo2000(Invitrogen).In order to reduce the hole differences that factors such as cell density, reagent dosage and transfection cause, guarantee the reliability and the repeatability of experiment, each transfection sample is provided with 3 multiple holes in this experiment.During 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 RNA afterwards, utilize the Primescript RT Master Mix perfect Real Time test kit of Takara company to carry out reverse transcription, carry out qRT-PCR again and detect.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.The Taq archaeal dna polymerase is contained, dNTP mix, SYBR Green dye in this reagent the inside.18S is used as internal control gene.This tests used primer: CREB1 primers F orward:5 '-CCACTGTAACGGTGCCAACT-3 ', Reverse:5 '-GCTGCATTGGTCATGGTTAATGT-3 '; 18S primers F orward:5 '-CAGCCACCCGAGATTGAGCA-3 ', Reverse:5 '-TAGTAGCGACGGGCGGTGTG-3 '.

In the H1299 clone miR-96 gene by with 3 of the mRNA of its target gene CREB1 '-UTR complementation, suppress the translation or the said target mrna of directly degrading of target gene mRNA.In conjunction with biochemistry and molecular biology experiment, determined that miR-96 and CREB1 have interaction in H1299 clone, this interaction has influenced vital movements such as the propagation, apoptosis of H1299 cell.This experiment is by software prediction, carrier construction, simultaneously to 3 of target gene '-UTR utilizes test kit to suddenly change, in the HEK293T cell, utilize the luciferase reporter gene analysis verification then CREB1 be the target gene of miR-96, and in the H1299 cell, utilize the qRT-PCR technology, further verified this discovery.Disclosed for CREB1 in H1299 clone is the reported first of the target gene of miR-96, this invention is clinically for utilizing miRNA to diagnose and treat nonsmall-cell lung cancer, and provides the drug target aspect that certain application value is provided.

Description of drawings

Relative expression's content of miR-96 in Fig. 1 non-small cell lung cancer cell system;

Fig. 2 crosses and expresses the propagation that suppresses the H1299 cell behind the miR-96;

Fig. 3 crosses and expresses the apoptosis that promotes the H1299 cell behind the miR-96, wherein the situation that influences of pair cell apoptosis behind the A negative control NC that has been transfection; The situation that influences of pair cell apoptosis behind B the has been transfection miR-96 mimics;

Fig. 4 is the conservative binding site of miR-96 between different plant species;

Fig. 5 is the binding site situation of miR-96 and target gene CREB1 wild-type and mutant;

Fig. 6 is miR-96 and target gene CREB1 wild-type and the two luciferase report analysis histograms of mutant.

Embodiment

Further set forth the present invention below in conjunction with specific examples.These examples only are used to set forth the present invention, limit the scope of the invention and be not used in.The experimental technique of unreceipted concrete experiment condition in the following example, usually according to normal condition, the condition described in the molecular cloning (Molecular Cloning:A Laboratory Manual, 3rd ed.), or the condition of advising according to manufacturer.

Embodiment one: according to the Solexa sequencing result, determine miR-96 low expression in non-small cell lung cancer cell system

The used k-ras mutant mouse of the present invention lung cancer model (L703T2) and normal lung cancer model (L1805) are provided by the biochemical cell Ji Hongbin of institute professor seminar of the Chinese Academy of Sciences.

With the lung tissue of normal mouse and the lung tissue sampling of the mouse that suffers from nonsmall-cell lung cancer,, add chloroform with TRIZOL method cracking tissue, treat albumen and nucleic acid layering, draw supernatant and add isopropanol precipitating after centrifugal, precipitate with washing with alcohol behind the recentrifuge, dry, obtain total RNA.Utilizing the cDNA library of two kinds of tissues of reverse transcription test kit structure of TaKaRa company, is that primer carries out reverse transcription with oligo dT, obtains the cDNA of subsequent experimental by reaction of degeneration and 2 steps of reverse transcription.

Sample is utilized Solexa method order-checking (the Solexa order-checking is finished by the big genome company of China), discover that further (referring to Fig. 1) significantly reduced in the expression of miR-96 in non-small cell lung cancer cell system.

Embodiment two: miR-96 is to the influence of H1299 cell proliferation

The H1299 cell is layered in 96 orifice plates uniformly, and the substratum of use is 1640.Mimics with miR-96 behind 24 h passes through the lipo2000 transfection reagent, change in the H1299 cell by after the hatching altogether of serum-free medium, change cell culture fluid behind transfection 6 h, put into fresh nutrient solution, add CCK8 reagent then immediately, in incubator, cultivate 2.5 h, suck out in enzyme plate again and survey, measure once every 24 h, detecting wavelength is 450 nm.

Check finds that transfection group is compared with control group, and its absorbancy has significant decline, and the rate of propagation of the cell of the miR-96 that is transfection also significantly is lower than control group, illustrates that miR-96 has the effect (referring to Fig. 2) of inhibition H1299 cell proliferation.

Embodiment three: miR-96 is to the apoptotic influence of H1299

The H1299 cell is layered in 6 orifice plates equably, changes in the H1299 cell after behind 24 h mimics of miR-96 being hatched altogether by lipo2000 and serum-free medium 1640, behind transfection 6 h cell culture fluid is changed to fresh medium.Behind transfection 48 h, with the cell of collecting annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Sigma-Aldrich, USA) staining cell.Detect the apoptosis situation of H1299 cell with Flow Cytometry.The result shows, transfection the H1299 apoptosis rate of miR-96 obviously greater than control group (referring to Fig. 3).

Embodiment four: two luciferase reporter gene analyses

With 3 of CREB1 mRNA '-UTR(619 bp) be connected on the pGL-3 plasmid, contain the promotor of SV40 on this plasmid, 3 in the connection '-UTR comprised the binding site of miR-96 and CREB1.With the lipo2000 transfection reagent mimics of miR-96 is gone in the HEK-293T cell with the plasmid corotation that builds, and change pRL plasmid signal as a setting simultaneously over to, behind transfection 6 h, nutrient solution is changed to fresh medium.Behind transfection 48 h, detect the luciferase expression of pGL and pRL with two luciferase reporter gene assay kits of Promega company.The final concentration of the plasmid of transfection is 400 nmol/L, and the final concentration of mimics is 20 nmol/L.Found that, transfection in the HEK-293T cell of miR-96 the expression of two luciferases significantly be lower than control group.Utilize the sudden change test kit to do the point mutation of CREB1 3 '-UTR afterwards, the mimics of miR-96 is gone in the HEK-293T cell with the point mutation plasmid corotation that builds with the lip2000 transfection reagent.Behind transfection 48 h, with two luciferase reporter gene assay kits detections of promega company.Found that, transfection in the HEK-293T cell of miR-96 the expression of two luciferases do not have what variation with control group, these illustrate that all CREB1 is the target gene (referring to Fig. 4) of miR-96.

Embodiment five: qRT-PCR checking miR-96 is to the restraining effect of the endogenous CREB1 gene of H1299 cell

The H1299 cell is layered in 6 orifice plates equably, change in the H1299 cell after behind 24 h miR-96 mimics, NC, miR-96 inhibitor, anti-NC being hatched altogether by lipo2000 and serum-free medium 1640, behind transfection 6 h cell culture fluid is changed to fresh medium.Behind transfection 48 h, collecting cell extracts RNA, utilizes the test kit reverse transcription of Takara company to be cDNA, carries out qRT-PCR afterwards and detects.The result shows that miR-96 is inhibited (referring to Fig. 5 and Fig. 6 to CREB1.

Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to 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 (3)

1. the application of miR-96 gene in suppressing the nonsmall-cell lung cancer cancer cell multiplication.

2. A kind ofThe application of miR-96 gene in promoting the nonsmall-cell lung cancer cancer cell-apoptosis.

3. A kind ofThe miR-96 gene is reduced the application of CREB1 expression level in H1299 clone.

Images