CN101804208A - Application of miR-451 in preparing medicine for treating non-small cell lung cancer - Google Patents

Abstract

The invention belongs to the field of genetic engineering, in particular to the application of miR-451 in preparing a medicine for treating non-small cell lung cancer. Since the recombinant of Pre-miR-451 has influence on propagation, apoptosis, migration and drug resistance of non-small cell lung cancer cells, it is proved that the miR-451 prohibits the propagation, migration and drug resistance of the non-small cell lung cancer cells and promotes the apoptosis of the non-small cell lung cancer cells; and living body level also indicates that the miR-451 has excellent anti-cancer effect.

Description

MiR-451 is in the application of preparation treatment non-small cell lung cancer drug

Technical field

The invention belongs to the genetic engineering field, particularly miR-451 is in the application of preparation treatment non-small cell lung cancer drug.

Background technology

RNA is one of most important material base in the organism, and it constitutes the framework of life with DNA and protein.But for a long time, RNA once had been regarded as merely " transition " between DNA and the protein, and it changes into protein with hereditary information then from the order that DNA obtains oneself there.Yet, a series of studies show that, these microRNAs are in fact being handled many cell functions, and it can react on DNA by the combination of complementary series, thereby closes or the expression of regulator gene.The RNA family of the non-encoding histone of one class of recent findings, microRNA (miRNA, i.e. Microrna) can be by coming regulate gene expression with the protein translation process that specific mRNA combined or regulated specific mRNA.Confirmed miRNA has kind more than 1,000 in the human genome at present, may participate in the expression of human 30% protein coding gene.

MicroRNA abbreviates miRNA as, and normal length is 19～25 nucleotide, extensively is present in various animals and plants even the unicellular eukaryote, is the micromolecule single stranded RNA of class high conservative on evolving.MiRNA is positioned at the gene intron zone, and the genetic transcription of coding miRNA becomes pri-microRNA (pri-miRNA) in nucleus.Pri-miRNA is cut into about 70 length of nucleotides under the effect of a kind of Drosha RNase, have the miRNA precursor (pre-miRNA) of loop-stem structure.Pre-miRNA is transported to the kytoplasm in examining under the effect of caryoplasm/Cytoplasm transport protein Exportin 5 that Ran-GTP relies on.Under the effect of Dicer enzyme, pre-miRNA is sheared into the double-stranded miRNA:miRNA* of 21～25 length of nucleotides.Double-stranded subsequently miRNA:miRNA* untwists, and sophisticated miRNA enters into endochylema RISC (the inductive reticent complex of RNA) performance gene silencing function.3 ' end noncoding region (3 ' UTR) of the messenger RNA of miRNA and downstream specific gene (mRNA) produces the base complementrity pairing, causes the degraded of this mRNA or suppresses its translation, causes the protein expression failure.We are called the gene in these downstreams the target gene of miRNA, they have important biological function usually in cell, miRNA exercises important function by regulating target gene expression in cell, with embryo's growth, the propagation of growth promoter, cell, differentiation are closely related.Scientist confirms that the nucleotides sequence of miRNA is listed in high conservative between different plant species, and mutation rate is extremely low, and its expression has gene cluster collection phenomenon, space-time and tissue specificity.MiRNA decides the process and the multiformity of a series of important vital movements such as cell differentiation, fetal development in post-transcriptional level regulation protein expression of gene spectrum.It is found that miRNA is not only the important regulating and controlling factor of cell proliferation, differentiation and apoptosis, the miRNA more than 50% is positioned at tumor-related gene group zone, comprises LOH district, chromosome amplification district and fragile site etc.Through identifying, the express spectra of miRNA in multiple hematological system tumor and solid tumor changes, and this provides fundamental basis for we disclose the tumor mechanism from angles of RNA regulation and control, and provides new gene target spot and molecular marker for diagnosing tumor and treatment.

In human genomic sequence, miR-451 is a kind of MicroRNA, is positioned at 17q11.2, in rat and the Brachydanio rerio genome, lays respectively at No. 11 and No. 5 chromosomes.The sequence of miR-451 be 5 '-aaaccguuaccauuacugaguu-3 ' (GeneBank:GeneID:574411).MiR-451 is high conservative on evolving, and has homology between different plant species.The data of announcing according to The World Health Organization (WHO) shows that the M ﹠ M of pulmonary carcinoma all is the trend of obvious rising in countries in the world, especially the flourishing country of industry.In developed country, pulmonary carcinoma is one of modal malignant tumor, first of row male common cancer, the 2nd, 3 of row women common cancer.20 the end of the century pulmonary carcinoma accounted for the first place of malignant tumor death.Nonsmall-cell lung cancer (NSCLC) accounts for 80% of all pulmonary carcinoma, can be divided into scale cancer, adenocarcinoma, maxicell pulmonary carcinoma etc.Many patients with lung cancer the time have shifted in diagnosis, often lose surgical engine meeting, and also not good enough at the chemotherapy regimen effect of pulmonary carcinoma, the patient is easy to generate drug resistance, and five year survival rate is lower than 5%, need badly searching new, effectively treat the method for pulmonary carcinoma.Along with going deep into of genetic engineering research, scientist shows keen interest to utilization genetic engineering development tumor medicine.The inventor finds that miR-451 has good application prospect aspect the treatment nonsmall-cell lung cancer.

Summary of the invention

Technical purpose

The purpose of this invention is to provide the application of miR-451 in preparation treatment non-small cell lung cancer drug.

The objective of the invention is to realize through the following steps:

1, amplification Pre-miR-451:Pre-miR-451, the gene order of the miR-451 precursor pre-miR-451 that promptly encodes, its RT and PCR primer are synthetic by invitrogen company, and primer sequence is seen RT-PCR in the specific embodiment.The RT-PCR Pre-miR-451 that increases, purified product show through agarose gel electrophoresis, about 72bp place amplify one with the big or small corresponding to band of expection.

2, connect: pcDNA ^TM6.2-GW/EmGFP-miR carrier is (available from Shanghai Ji Ma company, carrier is seen Fig. 1, and wherein EmGFP is the green fluorescent protein reporter gene, in order to detect transfection efficiency), behind BamHI and Xho I enzyme action, obtain linear carrier, the PCR product is connected to constitutes pcDNA on the carrier ^TM6.2-GW-Pre-miR-451 recombinant.10ul linked system: 2ulPCR product, 1ul linear carrier, 1ul 10*T ₄Ligase buffer, 1ulT ₄DNA ligase, 5ul ddH ₂O.

3, transform:

A, prepare fresh competent escherichia coli cell (JM109), mixing competent cell.

B, get the 20ul competent cell in new centrifuge tube, add the 1ul linked system, leave standstill the 30min ice bath.

C, competent cell take out in the rapid 4 ℃ of frozen water in back and place 2min in 42 ℃ of heating 42 seconds.

D, add the blank LB culture fluid of 400ul, 37 ℃ of joltings, constant temperature culture 30min, resuspended back is applied on the agar plate with the sterilization glass rod, treat the plate drying after, the inversion plate is hatched 12～16h for 37 ℃.

4, amplification: according to the characteristic of carrying the blasticidin S resistance on this plasmid vector, select positive colony 37 ℃ of constant temperature jolting 12～16h in the 5mlLB culture fluid, pcDNA in a large number increases ^TM6.2-GW-Pre-miR-451 recombinant.Recombinant extracts, purification does not have the little extraction reagent kit of endogenous toxin plasmid (PD1214) description with reference to BIOMIGA.

5, carrier pcDNA ^TM6.2-GW-Pre-miR-451 obtain two fragments through BamHI and Xho I enzyme action, be respectively the Pre-miR-451 fragment of 72bp and the linear pcDNA of 5.7kb ^TM6.2-GW carrier, the result conforms to expected results, pcDNA ^TM6.2-GW-Pre-miR-451 sequence is in full accord among sequencing result and the GenBank.

6, the recombinant transfection tumor cell that order-checking is correct uses liposome method, and the transfection step is with reference to the LipofectamineTM2000 description.The cell of transfection success has egfp expression, the transfection efficiency flow cytometry analysis, and different cell line transfection efficiencies all can reach 60%.

7, after Pre-miR-451 goes into tumor cell with plasmid transfection, the CMV promoter is quick in tumor cell, efficient, continuous expression Pre-miR-451, effect forms the pre-miR-451 (about 72nt) with loop-stem structure through Drosha (RNaseIII), form sophisticated miR-451 (22nt) through the Dicer effect again, performance gene silencing function.

One, the basic demonstration test of the anti-nonsmall-cell lung cancer of miR-451

(1): the express spectra of miR-451 in non-small cell lung cancerous tissue and the other normal structure of cancer:

1. sample is prepared: collect 60 routine nonsmall-cell lung cancer (NSCLC) excision specimen, all belong to IIIb and IV phase through Clinical and Pathological Analysis, specimen is provided by Pathology Deparment of The Second Affiliated Hospital of Nanjing Medical University.

2. chip preparation and analyzing: the cancerous lung tissue specimen is prepared in the requirement according to the LC Sciences company of the U.S., transfers to LC Sciences company and carries out the chip preparation, and Sanger miRBase V10.0 version is finished chip analysis.

3. chip results: experimental data is from LC Sciences company, and the result is shown in Fig. 2 and form 1.

4. interpretation of result: miR-451 signal in normal structure (Sample A) is 26,894.73, signal is 157.30 in tumor tissues (Sample B), log2 (Sample B/Sample A) is-7.41, expression miR-451 high expressed in normal structure, significantly low expression the in tumor tissues, the two difference reaches 150 times, and prompting miR-451 may be as an important antitumor action of antioncogene performance in pulmonary carcinoma.

Table 1 significant difference tabulation (significant difference of p value＜0.01 is expressed transcripton, and wherein hsa is for detecting the probe of people's specimen)

??No.	??Probe_ID	??Sample?A??Signal	??Sample?B??Signal	??log2(Sample?B??/Sample?A)
					??1	??hsa-miR-451	??26,894.73	??157.30	??-7.41
??2	??hsa-miR-196a	??33.02	??3,362.17	??6.67
					??3	??hsa-miR-335*	??81.08	??4,720.06	??5.90
??4	??hsa-miR-205	??94.21	??5,112.51	??5.79
					??5	??hsa-miR-335	??162.56	??8,041.86	??5.59
??6	??hsa-miR-483-5p	??49.00	??1,331.34	??4.70
					??7	??hsa-miR-30a	??2,588.28	??115.34	??-4.54
??8	??hsa-miR-134	??5.37	??107.00	??4.35
					??9	??hsa-miR-126	??27,320.62	??1,279.92	??-4.35
??10	??hsa-miR-182	??68.22	??1,297.40	??4.10
					??11	??hsa-miR-21	??889.51	??12,086.79	??3.90
??12	??hsa-miR-30d	??4,000.27	??319.67	??-3.64
					??13	??hsa-miR-574-5p	??48.52	??546.44	??3.54

(2): RT-PCR analyzes the express spectra of miR-451 in non-small cell lung cancerous tissue and the other normal structure of cancer

1. method: Trizol reagent extracts total tissue RNA, and the RT-PCR utilization Ambion SuperTaq of company polymerase system and mirVana detect box, and people U6 is as confidential reference items.

2.RT-PCR result: as shown in Figure 3.

3. interpretation of result: miR-451 increases in the normal tissue expression amount, expresses in the tumor tissues to descend, and the result is consistent with gene chip.

Two, miR-451 is to non-small cell lung cancer cell propagation, apoptosis, migration, drug-fast influence

(1): cell proliferation experiment

1.MTT method is surveyed cell proliferation: select four strain NSCLC cell lines, wherein lung adenocarcinoma cell is A549, H3255, Lung Squamous Carcinoma Cells is H157, lung large cell carcinoma cell line H1299, four strain cell line transfection Pre-miR-451 are experimental group, and (negative control NC) is made as matched group to the empty carrier sequence.With these cell strain kinds on 96 orifice plates, 2500 cells/well.Transfection is MTT staining monitoring cell proliferation situation after 3 days.

2. the result measures: as shown in Figure 4.

3. interpretation of result: mtt assay shows, compare with matched group, transfection the A549 cell proliferating number of Pre-miR-451 obviously reduce.H157, H1299 also observes this phenomenon in three cell lines of H3255, and prompting miR-451 can suppress the growth of tumor cell.

Experiment two: apoptosis experiment

1.Caspase3/7 active method is surveyed apoptosis: at NSCLC cell line A549, H157, H1299, difference transfection Pre-miR-451 and empty carrier sequence among the H3255, the latter is made as matched group.Behind the transfection 72h these cell strain branches are put in the culture medium of 0 μ M DDP (cisplatin, chemotherapeutic) and 2 μ M DDP and cultivate, utilization ApoONE Homogeneous Caspase3/7Assay measures caspase3/7 activity in the cell.

2.caspase3/7 determination of activity: as shown in Figure 5.

3. interpretation of result: compare with matched group, behind the transfection Pre-miR-451, all occur the active rising of caspase3/7 in four cell lines, prompting miR-451 can promote the apoptosis of tumor cell.Although single active increasing of caspase3/7 that causes with Pre-miR-451 do not have list unobvious with the DDP effect, but after uniting use DDP and Pre-miR-451, caspase3/7 is active significantly to rise, being higher than list can increase the sensitivity of tumor cell to chemotherapeutic with Pre-miR-451 and DDP prompting miR-451, can be used as the intervention target spot of oncotherapy.

Experiment three: cell invasion experiment

1.Transwell the migration experiment: A549 cell line is transfection Pre-miR-451 and empty carrier sequence respectively, and the latter is made as contrast, is inoculated into Transwell and goes up in the chamber, selects inoculation back 24h, 48h, 72h time point observe A549 cell invasion situation.

2. cell counting: measure under the Transwell number of tumor cell in the chamber with direct counting method.Experimental group is starkly lower than matched group.Count results as shown in Figure 6.

3. interpretation of result: compare with matched group, the invasive ability of A549 cell descends behind the transfection Pre-miR-451, and miR-451 can suppress invasion by tumor cells, shifts.

Experiment four: the live body level is observed the antitumaous effect of miR-451

(1), subcutaneous tumors experiment

1. life: we choose the female nude mice of four 5-6 sizes in age in week, with transfection the A549 cell of Pre-miR-451 and empty carrier (NC) be inoculated into respectively in the subcutaneous tissue of same mice both sides ribbed back portion, every side joint kind cell number is 1*10 ⁵Or 5*10 ⁴Individual, the growing state of observation tumor cell.

2. tumor growth assessment of scenario: observe once every day, continued at least five weeks.Measure the length L and the width W of tumor kitchen range, judge that according to the volume that formula (L*W^2) * 0.5 calculates the tumor kitchen range miR-451 is in the influence of live body level to growth of tumour cell.

3. measurement result: to observe finish till, 4 mice transfections the side of Pre-miR-451 do not see the formation of obvious tumor kitchen range, and have 3 when 22-27 days of observing in 4 mices, the tangible formation of inoculation NC one side to the tumor kitchen range, observe when finishing, these tumor stoves are long-pending to be 35-145cm ²Size.Gross tumor volume as shown in Figure 7.

4. interpretation of result: from the experiment made on the living result as can be seen, miR-451 can suppress the lung carcinoma cell growth in the mice body, consistent with the result who obtains in the cell line level before us, has confirmed that miR-451 has cancer suppressing action.

(2) lung cancer in situ experiment

1, the material of the foundation of lung cancer model: 4 Lox-stop-lox K-Ras G12D (LSL K-Ras G12D) transgenic mice is by human cancer association mouse model storehouse (Mouse Models of Human CancerConsortium, MMHCC) present, HEK293 (American Type Culture Collection, cat#CRL.1573) incasing cells of adenovirus is presented by Biochemistry and Molecular Biology teaching and research room of Third Military Medical University, ADCre, the AdPre-miR-451 recombinant adenovirus is provided by Microbix company.Have the beta galactosidase operon on the Ad adenovirus vector, in order to detect transfection efficiency.Utilize the Cre recombinase in the transgenic mice body, to induce K-Ras gene expression, promote the principle of lung tumors generation to set up the model of adenocarcinoma of lung.Reference literature: (1) The let-7microRNA reduces tumor growth in mouse modelsof lung cancer.Cell Cycle 7:6, the foundation of 759-764.2008 (2) mouse lung adenocarcinoma animal model.Mountain Western Medicine S University's journal.1007-6611(2009)05-0408-03。

2, the intervention of modeling procedure and miR-451: with pentobarbital sodium 45mg/kg intraperitoneal injection of anesthesia mice in 3 age in week, (Ad adenovirus: CaPi: titre is that the Ad adenovirus 50ul of 2.5 * 10 ' pfu joins among the MEM of 69ul with Ad adenovirus: CaPi again, the calcium chloride that adds the 2.5mol/L of 6ul again) coprecipitate splashes into the nasal cavity of mice, splash into about 125ul altogether, divide and drip the people 2 times, after 5 days, repeat twice totally.Experimental group: AdCre+AdPre-miR-451, n=2, matched group: AdCre+AdNC, n=2.

3, the result measures: 7 week of nasal cavity instillation back execution mices, and get the mouse lung tissue and weigh, paraffin embedding, section, HE dyes.Experimental group (Cre/Pre-miR-451) is compared gross tumor volume than matched group (Cre/NC) and significantly dwindled lung tumor: full lung tissue ratio as shown in Figure 8.

4, interpretation of result: this experiment utilization transgenic mice LSL K-Ras G12D sets up the adenocarcinoma of lung model, proves that once more miR-451 can suppress the generation of adenocarcinoma of lung, suppresses the growth of tumor cell.

The expression vector of described miR-451, these expression vectors include but not limited to plasmid, antibacterial, host cell.

The pharmaceutical composition that the adjuvant that contains the described miR-451 that treats effective dose or its expression vector and pharmaceutically allow is formed.

Described pharmaceutical composition, its preparation adopts any one dosage form that pharmaceutically allows, and includes but not limited to tablet, pill, capsule, injection, oral liquid or liposome.

The application in the preparation tumor-targeting drug of above-mentioned miR-451 sequence or their expression vector.

Statistical analysis among the present invention adopts SPSS 11.0 softwares (Release 11.0, SPSS Inc.) to carry out date processing, and data representation is with means standard deviation.Carry out variance analysis and t check, the result is that difference has the significance meaning with P＜0.05.

Beneficial effect

1, the method for preparing miRNAs at present comparatively commonly used has external preparation miRNAs and also transcribes in vivo to cell for the template transfection with DNA to obtain miRNAs.The present invention has adopted back a kind of method, and this law is easy, and existing lot of documents report can successfully prepare miRNAs, does not need straightforward manipulation RNA, has overcome synthetic miRNA and has degraded easily, the shortcoming of cost costliness.But and owing to contain bacterial strain long preservation and a large amount of amplification of expression vector, for from now on research provides great convenience.

2, because miR-451 generally exists with precursor forms (pre-miR-451) in vivo, and Pri-miR-451 is cut into about 72 length of nucleotides under the effect of a kind of Drosha RNase, have the miRNA precursor (pre-miRNA) of loop-stem structure.Pre-miR-451 is transported to the kytoplasm in examining under the effect of caryoplasm/Cytoplasm transport protein Exportin 5 that Ran-GTP relies on.Under the effect of Dicer enzyme, pre-miR-451 is sheared into the double-stranded miRNA:miRNA* of 22 length of nucleotides.Double-stranded subsequently miRNA:miRNA* untwists, and sophisticated miR-451 enters into endochylema RISC (the inductive reticent complex of RNA) performance gene silencing function.Because Pre-miR-451 can successfully be processed into ripe body miR-451 in vivo, the present invention also adopts Pre-miR-451 to replace miR-451.Obtain a large amount of Pre-miR-451 by recombinant transfection Escherichia coli JM109 cell in the test, ready for carrying out corresponding pharmacological testing.For fear of carrier this experiment is influenced simultaneously, adopt empty carrier to do controlled trial, the result shows that empty carrier is to not influence of the present invention.

3, by miR-451 to non-small cell lung cancer cell propagation, apoptosis, migration, drug-fast influence, say that miR-451 all has inhibitory action to non-small cell lung cancer cell propagation, migration, drug resistance, and promote the apoptosis of non-small cell lung cancer cell, the live body level shows that also miR-451 has antitumaous effect simultaneously.

Description of drawings

Fig. 1 is for expressing the plasmid vector of miR-451.

(the Sample A on the left side is a normal structure by the cancer to Fig. 2, and signal value is Cy3 for gene chip is analyzed the situation of miRNA express spectra in cancerous lung tissue and the other normal structure of cancer; Intermediary Sample B is a cancerous lung tissue, and signal value is Cy5, the right be by the cancer/the signal ratio Cy3/Cy5 of cancer; In Cy3/Cy5 signal ratio figure, when the Cy3 signal was higher than the Cy5 signal, color was shown in green; When the Cy3 signal was suitable with the Cy5 signal, color was shown as yellow; When the Cy5 signal was higher than the Cy3 signal, color was shown in red).

Fig. 3 RT-PCR surveys cancerous lung tissue (T) and the middle miR-451 level of the other normal structure of cancer (N), and GAPDH is made as confidential reference items.

Fig. 4 surveys miR-451 to lung cancer cell line A549, H1299, H3255, the effect of H157 cell proliferation for mtt assay.

Fig. 5 be the miR-451 precursor to lung cancer cell line A549, H1299, H3255, the detection of H157 apoptosis

Fig. 6 surveys the effect of miR-451 precursor to lung cancer cell line A549 cell migration for the Transwell method

Fig. 7 surveys the effect of miR-451 to the A549 cell proliferation for experiment made on the living, and vertical coordinate is the subcutaneous tumors volume, and abscissa is the mice code name

Fig. 8 observes the effect of miR-451 to the adenocarcinoma of lung growth on the LSL K-Ras G12D transgene mouse model, wherein vertical coordinate is lung tumor (Tumor): the ratio of full lung tissue (Total Lung Area); Abscissa is the transgenic mice code name, and #1, #2 are experimental group (Cre/Pre-miR-451), and #3, #4 are matched group (Cre/NC).

The specific embodiment

The invention will be further elaborated by the following examples, but do not limit the present invention.

General explanation:

Among the embodiment end indicate actual conditions experimental technique, basically all according to Sambrook, " molecular cloning experiment guide (the 3rd edition) " (Molecular Cloning:A Laboratory Manual, 3 that people such as J write ^RdEd. yellow training hall waits translates the .2002.8 of Science Press) described in condition and method or carry out according to condition and method that material provider is advised, other not have technology of detailed description is standard methods of knowing corresponding to those skilled in the art.

Material of the present invention: the microorganism of mentioning among the application, cell line, plasmid or other expression vectors and culture medium all have supply of commodities or can be public's gained with other approach; they are only given an example; to the present invention is not unique, can replace with other instrument and biomaterial that is fit to respectively.

Embodiment 1

1, amplification Pre-miR-451:Pre-miR-451, its RT and PCR primer are synthetic by invitrogen company, and primer sequence is seen following RT-PCR.The RT-PCR Pre-miR-451 that increases, purified product show through agarose gel electrophoresis, about 72bp place amplify one with the big or small corresponding to band of expection.

RT-PCR

Design miR-451 and U6 (as confidential reference items) primer respectively, synthetic by Invitrogen company.

1) primer sequence and reaction condition

The RT primer of MiR-451:

5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAACTCAG-3’

The RT primer of GAPDH is Oligo (dT) ₁₅

Table 1.PCR primer sequence and reaction condition

2) cDNA's is synthetic

5X?RT?Buffer????????????????????4μl

dNTP????????????????????????????2μl

Inhibitor???????????????????????1μl

Primer??????????????????????????1μl

RNA 1 μ l (quality is less than 1 μ g)

RT?ace?????????????????????????1μl

H ₂O(RNase?Free)????????????????10μl

????????????????????????????????????????

3) RT-PCR reaction system (50 μ l system)

With cDNA is template, the PCR reaction system:

10×Ex?Taq?Buffer(Mg2+Free)????5μl

MgCl2(25mM)????????????????????5μl

dNTP(2.5mM)????????????????????5μl

Forward primer (20pM/ μ l) 2 μ l

Downstream primer (20pM/ μ l) 2 μ l

Template???????????????????????2μl

Ex?Taq(5U/μl)?????????????????0.5μl

ddH ₂O??????????????????????????28.5μl

??????????????????????????????????????????

Cumulative volume 50 μ l

Adopt following reaction condition:

94 ℃ of pre-degeneration of 2min, after connect 35 circulations (25 circulations of 18S RNA), the circulation condition is each time: 94 ℃ of 30s degeneration, 54 ℃ of 45s annealing, 72 ℃ were extended in 2 minutes.

4) evaluation of PCR product

The preparation of 50 * TAE stock solution: Tris 60.5g, glacial acetic acid 14.3ml, 0.5mol/L EDTA 25ml adds ddH2O to 250ml.

The preparation of 1.0% agarose gel: agarose 1.0g, 1 * TAE solution 100ml is heated to agarose and melts, and adds ethidium bromide to 0.5 μ g/ml when being cooled to 50 ℃ of left and right sides, and bed board solidifies the back to be used.

Agarose gel electrophoresis: inject 1 * TAE solution in the electrophoresis tank to being higher than the gel face, get 10 μ l PCR products, 10 * DNA the sample-loading buffer that adds 1 μ l, sample on the mixing, with DL2000DNA Marker as reference, image is observed and gathered to 80V electrophoresis, Jetta gel imaging system, the experiment triplicate.

2, connect: pcDNA ^TM6.2-GW/EmGFP-miR carrier is available from Shanghai Ji Ma company, (carrier is seen Fig. 1, and wherein EmGFP is the green fluorescent protein reporter gene, in order to detect transfection efficiency), behind BamHI and XhoI enzyme action, obtain linear carrier, the PCR product is connected to constitutes pcDNA on the carrier ^TM6.2-GW-Pre-miR-451 recombinant.10ul linked system: 2ulPCR product, 1ul linear carrier, 1ul 10*T4ligase buffer, 1ulT4DNA ligase, 5ul ddH ₂O.

3, transform: 1. prepare fresh escherichia coli competence (JM109) cell, mixing competent cell 2. is got the 20ul competent cell in new centrifuge tube, adds the 1ul linked system, leaves standstill the 30min ice bath.3. in 42 ℃ of heating 42 seconds, take out in the rapid 4 ℃ of frozen water in back and place 2min.4. add the blank LB culture fluid of 400ul, 37 ℃ of joltings, constant temperature culture 30min, resuspended back is applied on the agar plate with the sterilization glass rod, treat the plate drying after, the inversion plate is hatched 12～16h for 37 ℃.

4, amplification: according to the characteristic of carrying the blasticidin S resistance on this plasmid vector, select positive colony 37 ℃ of constant temperature jolting 12～16h in the 5mlLB culture fluid, pcDNATM6.2-GW-Pre-miR-451 recombinant in a large number increases.Recombinant extracts, purification does not have the little extraction reagent kit of endogenous toxin plasmid (PD1214) description with reference to BIOMIGA.

5, carrier for expression of eukaryon pcDNATM6.2-GW-Pre-miR-451 obtains two fragments through BamHI and Xho I enzyme action, is respectively the Pre-miR-451 fragment of 72bp and the linear pcDNA of 5.7kb ^TM6.2-GW carrier, the result conforms to expected results, pcDNA ^TM6.2-GW-Pre-miR-451 sequence is in full accord among sequencing result and the GenBank.

6, the recombinant transfection tumor cell that order-checking is correct uses liposome method, and the transfection step is with reference to the LipofectamineTM2000 description.The cell of transfection success has egfp expression, the transfection efficiency flow cytometry analysis, and different cell line transfection efficiencies all can reach 60%.

Embodiment 2

Mtt assay is surveyed cell proliferation:

1) inoculating cell: with 0.25% trypsinization single-layer culturing cell, be made into the individual cells suspension with the RPMIt604 culture fluid that contains 10% hyclone, with every hole 100-1000 cell inoculation in 96 well culture plates, every pore volume 200ul.Be divided into four experimental grouies: empty carrier group, Pre-miR-451 group, DDP group, Pre-miR-451+DDP group.

2) cultured cell; Culture plate is moved in the COz incubator, at 37 ℃, 5%CO2: and under the damp condition, cultivated 3-5 days.

3) colour generation: after cultivating 2-5 days, every hole adds MTT solution (5mg/m1) 20ul, and 37 ℃, continue to hatch 4h, stop cultivating, the careful suction abandoned culture supernatant in the hole.Add 150 μ l DMSO (dimethyl sulfoxide) vibration 10min, crystal is fully dissolved.

4) colorimetric: 490nm wavelength place measures optical density value (OD), 3 every group multiple holes, triplicate experiment.Infect matched group in contrast with empty carrier, calculate survival rate.Survival rate=experimental group OD value/matched group OD value * 100%.

Select four strain non-small cell lung cancer cells (NSCLC system by as above method, wherein lung adenocarcinoma cell is A549, H3255, Lung Squamous Carcinoma Cells is H157, lung large cell carcinoma cell line H1299, four strain cell line transfection miR-451 precursor sequence (Pre-miR-451), empty carrier sequence (negative control) is made as matched group.With these cell strain kinds on 96 orifice plates, 2500 cells/well.Transfection is MTT staining monitoring cell proliferation situation after 3 days.Mtt assay shows: compare with matched group, transfection the A549 cell proliferating number of Pre-miR-451 obviously reduce.H157, H1299 also observes this phenomenon in three cell lines of H3255, and prompting miR-451 can suppress the growth of tumor cell.

Embodiment 3

Apoptosis experiment (detected by Western blot mensuration)

1. extraction total protein of cell

2.SDS-PAGE electrophoresis

1) cleans, installs glass plate, potsherd.

2) preparation perfusion separation gel, 37 ℃ leave standstill 25min.

3) the preparation perfusion concentrates glue, and room temperature leaves standstill 20min.

4) add protein sample and molecular weight marker, each, the albumen applied sample amount was 40 μ g in period.

5) constant voltage electrophoresis (concentrating glue 80V, separation gel 160V).

3. half dry type changes film

1) after electrophoresis finishes, wear glove, cut glue (corner cut labelling) according to the destination protein molecular weight ranges with reference to molecular weight marker, cut is with 2 of one of big or small nitrocellulose filter (corner cut labelling) and filter paper.

2) soak glue, film and filter paper 10min in the commentaries on classics film buffer.

3) changeing tiling from bottom to up on the film instrument: filter paper, film, gel, filter paper is caught up with except that bubble, especially notes prohibiting between film and the gel and stays bubble.Dry unnecessary liquid, 0.8mA * membrane area (cm2) electric current changeed film 1.5 hours.

4. immunoblotting

1) change film and finish, whether check marker has gone on the film (or Ponceaux dyeing) is changeed the film effect to judge.After TBST cleaned film, 4 ℃ of sealings of 5% defatted milk powder (TBST preparation) were spent the night.

2) add anti-Caspase-3/7 (activated form) monoclonal antibody (1: 500 and 1: 500), room temperature is shaken 2h.

3) the TBST rinsing is 3 times, each 10min.

4) add two anti-(1: 200), room temperature is shaken 1h.

5) the TBST rinsing is 3 times, each 10min.

5.ECL detect

1) AB liquid mixes, treat that film does not drip after, tile on it.

2) behind the reaction 5min, according to fluorescence intensity darkroom tabletting 5s～30min.

3) development 15～30s, washing, photographic fixing 1～3min.

4) picture scanning and quantitative analysis.

Press as above method, at NSCLC cell line A549, H157, H1299, difference transfection Pre-miR-451 and empty carrier sequence among the H3255, the latter is made as matched group.Behind the transfection 72h these cell strain branches are put in the culture medium of 0 μ M DDP (cisplatin, chemotherapeutic) and 2 μ M DDP and cultivate, utilization ApoONEHomogeneous Caspase3/7Assay measures caspase3/7 activity in the cell.Caspase3/7 determination of activity: as shown in Figure 5.Compare with matched group, behind the transfection Pre-miR-451, all occur the active rising of caspase3/7 in four cell lines, prompting miR-451 can promote the apoptosis of tumor cell.Although single active increasing of caspase3/7 that causes with Pre-miR-451 do not have list unobvious with the DDP effect, but after uniting use DDP and Pre-miR-451, caspase3/7 is active significantly to rise, prompting miR-451 can increase the sensitivity of tumor cell to chemotherapeutic, can be used as the intervention target spot of oncotherapy.

Embodiment 4

Transwell (cell invasion experiment)

1.Transwell cell is prepared

1. wrap by basement membrane: by the last chamber face of Transwell cell bottom film, 4 ℃ air-dry with 1: 8 diluent bag of 50mg/L Matrigel.

2. aquation basement membrane: residual liquid in the sucking-off culture plate, every hole adds the serum-free medium that 50ul contains 10g/LBSA, 37 ℃, 30min.

2. preparation cell suspension

1. can allow cell remove serum starvation 12-24h earlier before preparing cell suspension, further remove influence of serum.2. peptic cell stops the centrifugal culture fluid that discards in digestion back, washes 1-2 time with PBS, and is resuspended with the serum-free medium that contains BSA.Adjust cell density to 1-10 * 105.

3. inoculating cell

1. obtained cell suspension 100-200 μ l adds the Transwell cell,

2. the chamber generally adds the culture medium that 500 μ l contain FBS or chemotactic factor under 24 orifice plates,

3. cultured cell: the conventional 12-48h (mainly deciding) that cultivates according to the cancerous cell invasive ability.

4. the result adds up:

" adherent " cell counting

Here so-called " adherent " is after phalangeal cell passes film, can not drop to down in the chamber attached to the following chamber side of film.By giving cell dyeing, can be at counting cells under the mirror

1. wipe matrigel and last indoor cell with cotton swab, violet staining is arranged.

2. cell counting: we use is that Leica DC 300F is just putting microscope and observes and take pictures, and the end just can be known the cell of seeing that the chamber side is adhered to about the cell counterdie up conversely the Transwell cell.

" non-adherent " cell counting is owing to the reason of some cell self or the relation of some film, and cell can not be attached on the film after passing film sometimes, but falls into down the chamber.

By as above method is with A549 cell line difference transfection Pre-miR-451 or empty carrier sequence, the latter is made as contrast, is inoculated into Transwell and goes up in the chamber, selects inoculation back 24h, 48h, 72h time point observation A549 cell invasion situation.Cell counting: with the number of tumor cell in the chamber under the direct counting method mensuration Transwell.Experimental group is starkly lower than matched group.Count results as shown in Figure 6.Compare with matched group, the invasive ability of A549 cell descends behind the transfection Pre-miR-451, and miR-451 can suppress invasion by tumor cells, shifts.

Embodiment 5

The live body level is observed the antitumaous effect of miR-451

The subcutaneous tumors experiment

1, life: we choose the female nude mice of four 5-6 sizes in age in week, with transfection the A549 cell of miR-451 and empty carrier (NC) be inoculated into respectively in the subcutaneous tissue of same mice both sides ribbed back portion, every side joint kind cell number is 1*10 ⁵Or 5*10 ⁴Individual, the growing state of observation tumor cell.

2, tumor growth assessment of scenario: observe once every day, continued at least five weeks.Measure the length L and the width W of tumor kitchen range, judge that according to the volume that formula (L*W^2) * 0.5 calculates the tumor kitchen range miR-451 is in the influence of live body level to growth of tumour cell.

3, measurement result: till the observation end, transfection one side of miR-451 precursor do not see the formation of obvious tumor kitchen range, and have 3 when 22-27 days of observing in 4 mices, the tangible formation of inoculation NC one side to the tumor kitchen range, observe when finishing, these tumor stoves are long-pending to be 35-145cm ²Size.Gross tumor volume as shown in Figure 7.

4, interpretation of result: from the experiment made on the living result as can be seen, miR-451 can suppress the lung carcinoma cell growth in the mice body, consistent with the result who obtains in the cell line level before us, has confirmed that miR-451 has cancer suppressing action.

The experiment of lung cancer in situ

1, the material of the foundation of lung cancer model: 4 Lox-stop-lox K-Ras G12D (LSL K-Ras G12D) transgenic mice by Mus by human cancer association mouse model storehouse (Mouse Models of Human CancerConsortium, MMHCC) present, HEK293 (American Type Culture Collection, cat#CRL.1573) incasing cells of adenovirus is presented by Biochemistry and Molecular Biology teaching and research room of Third Military Medical University, ADCre, the AdPre-miR-451 recombinant adenovirus is provided by Microbix company.Have the beta galactosidase operon on the Ad adenovirus vector, in order to detect transfection efficiency.Utilize the Cre recombinase in the transgenic mice body, to induce K-Ras gene expression, promote the principle of lung tumors generation to set up the model of adenocarcinoma of lung.Pertinent literature: (1) The let-7microRNA reduces tumor growth in mouse models of lung cancerCell Cycle 7:6, the foundation of 759-764.2008 (2) mouse lung adenocarcinoma animal model.Mountain Western Medicine S University's journal.1007-6611(2009)05-0408-03。

2, the intervention of modeling procedure and miR-451: with pentobarbital sodium 45mg/kg intraperitoneal injection of anesthesia mice in 3 age in week, (Ad adenovirus: CaPi: titre is that the Ad adenovirus 50ul of 2.5 * 10 ' pfu joins among the MEM of 69ul with Ad adenovirus: CaPi again, the calcium chloride that adds the 2.5mol/L of 6ul again) coprecipitate splashes into the nasal cavity of mice, splash into about 125ul altogether, divide and drip the people 2 times, after 5 days, repeat twice totally.Experimental group: AdCre+AdPre-miR-451, n=2, matched group: AdCre+AdNC, n=2.

3, the result measures: 7 week of nasal cavity instillation back execution mices, and get the mouse lung tissue and weigh, paraffin embedding, section, HE dyes.Experimental group (Cre/Pre-miR-451) is compared gross tumor volume than matched group (Cre/NC) and significantly dwindled lung tumor: full lung tissue ratio as shown in Figure 8.

4, interpretation of result: this experiment utilization transgenic mice LSL K-Ras G12D sets up the adenocarcinoma of lung model, proves that once more miR-451 can suppress the generation of adenocarcinoma of lung, suppresses the growth of tumor cell.

Claims (1)

1.miR-451 application at preparation treatment non-small cell lung cancer drug.

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