CN102533770A - Nucleic acid molecule and application thereof - Google Patents

Abstract

The invention provides a nucleic acid molecule. A basic group of the nucleic acid molecule consists of an SEQ ID NO.1, or the SEQ ID NO.1 and an SEQ ID NO.2, or an SEQ ID NO.3, or an SEQ ID NO.4, or an SEQ ID NO.9, or the basic group of the nucleic acid molecule consists of the SEQ ID NO.1, or the SEQ ID NO.1 and the SEQ ID NO.2, or the SEQ ID NO.3, or the SEQ ID NO.4, and the nucleic acid molecule contains modified nucleotide analogues, wherein the modified nucleotide analogues are glycosyl-modified or phosphate-backbone-modified or basic-group-modified or terminal-modified nucleotides. Anti-tumor pharmaceutical compositions prepared by using the nucleic acid molecule can be used for the diagnosis and prognosis of lung cancer.

Description

A kind of nucleic acid molecule and application thereof

Technical field

The invention belongs to biomedical sector, specifically relate to a kind of nucleic acid molecule and application thereof.

Background technology

MicroRNA (writing a Chinese character in simplified form miRNA) is one of the most popular little RNA of non-coding of research at present; It is that one type of length is about 22bp; Sophisticated by the dicer enzyme from the precursor shearing processing that specific secondary structure is arranged; Through RNA molecule complete with mRNA or that incomplete pairing comes regulatory gene to express, it belongs to a member of the little RNA of non-coding family, between species, guards.MiRNA participate in to regulate the growth of cell, breeding, and vital process such as die is transferred in differentiation.Surpass half the human miRNA gene and be considered to and related to cancer in genomic position, like fragile site, the narrow site district that amplification is relevant, perhaps broken site district.

The miRNA gene is transcribed into elementary precursor (primary miRNA by rna plymerase ii (Pol II) in nuclear; Write a Chinese character in simplified form pri-miRNA); Pri-miRNA contains the cap of one 7 methyl guanine (7-mG) and the tail (PolyA) of polyadenylic acid; These are characteristics of rna plymerase ii transcription; Pri-miRNA also comprises the stem-loop structure, and pri-miRNA is cut into the precursor miRNA (precursor miRNA writes a Chinese character in simplified form pre-miRNA) of about 70bp by III class RNA enzyme (RNase III) Drosha; Other flanking sequence is considered in nuclear, be degraded (Lee Y, et al.2004.MicroRNA genes are transcribed by RNA polymerase II.EMBO J 23:4051 4060).Pre-miRNA is delivered to outside the nuclear by the nuclear translocation factor acceptor export-5 (Exp5) that transport protein Ran relies on, and in tenuigenin, is processed as the sophisticated miRNA of about 22bp (He L and Hannon is RNAs with a big role in the gene regulation.Nature Gnetics 5:522-532 G.2004.MicroRNAs:Small) by nucleicacidase RNase III dicer.Sophisticated miRNAs and some albumen constitute complex body; The expression of miRISC regulatory gene (Mourelators et al., 2002.miRNPs:a novel class of ribonucleoproteins containing numerous microRNAs.Genes Dev.16 (6): 720-728.).In the complex body forming process, Dicer shears product (the double-stranded miRNA of about 22bp) and becomes strand, is also referred to as sophisticated miRNA, and another chain rapidly disappears.MiRNA can suppress the translation of mRNA; Perhaps through taking off adenylic acid(AMP); Raise one's hat etc. and to promote the mRNA degraded; RNA processing body (P-body is also referred to as GW body) possibly bring into play effect (BehM-Ansmant J, et al.2006.microRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay.Cold Spring Harb Symp Quant Biol 71:523-530) in the mRNA degradation process

Britten in 1969 and Dabidson (Britten RJ, Davidson EH.1969.Gene regulation for higher cells:a theory.Science 165 (891): 349-357.) year advance an idea: activate some protein expression genes from the viable rna s molecule and the regulating DNA sequence formation mixture of genome translation.Lee et al (Lee RC; Feinbaum RL; Ambros is C.elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell V.1993.The; 75 (5): 843-54.) find that from beautiful nematode (Caenorhabditis elegans) the small RNA molecular lin-4 of a non-proteins encoded regulates and control the growth of beautiful nematode through the proteic expression of negative regulation lin-14; Further find that there are two kinds of transcripts of 22nt and 61nt in lin4 and hold non-translational region (UTR) that the complementary pairing sequence is arranged with 3 ' of the proteic RNA of lin-14, and think the expression that lin4 and lin-14 come modulin lin-14 through the interactional form of RNA-RNA.Reinhart et al (Reinhart BJ et al.2000.The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.Nature.403 (6772): thus 901-906.) find that another non-coding small RNA molecular let-7 comes the expression of the lin-41 of modulin to regulate and control the growth of beautiful nematode through 3 ' UTR of regulation and control lin-41 protein rna molecule.Calendar year 2001 Bartel; Tuschl; Three of Ambros independently laboratory find a large amount of little RNA regulatory molecules of similar non-coding simultaneously; And it is referred to as microRNA (be called for short miRNA), do not re-use this title of stRNA (small temporal RNA) (Lau NC, Lim LP; Weinstein EG, Bartel DP.2001.An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.Science 294:858-862; Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl be of novel genes coding for small expressed RNAs.Science (New York, NY 294:853-858. T.2001.Identification; Lee RC; Ambros is extensive class of small RNAs in Caenorhabditis elegans.Science 294 (5543) V.2001.An: 862-864.) in the unified miRNA DB that is embodied in Britain Sanger center of miRNA (http://www.sanger.ac.ck/); The mankind's (Homo sapiens) that in this DB, register at present miRNA is 1921; Mouse (Mus musculus) be 1157; Rat (Rattus norvegicus) be 680 (Griffiths-Jones S; Saini HK, van Dongen S, Enright AJ.2008.miRBase:tools for microRNA genomics.Nucleic Acids Res 36:D154-158).

Research shows that microRNAs has participated in the regulation and control of cell signaling as important effector molecule.Transcription factor p53 and the relevant microRNAs of p65 regulation and control existing relevant report (Bhaumik, D., Scott, G.K.; Schokrpur, S., Patil, C.K.; Campisi, J., and Benz, C.C. (2008) .Expression of microRNA-146 suppresses NF-kappaB activity with reduction of metastatic potential in breast cancer cells.Oncogene 27; 5643-5647.Song, B., Wang, Y.; Kudo, K., Gavin, E.J.; Xi, Y.G., and Ju; J.F. (2008) .miR-192 Regulates Dihydrofolate Reductase and Cellular Proliferation through the p53-microRNA Circuit.Clinical Cancer Research 14,8080-8086.), yet mediation p53 and the mutual microRNAs that does of p65 do not appeared in the newspapers as yet.

NF-κ B transcription factor has important regulation in processes such as inflammation, immunoreation, cell proliferation apoptosis.NF-κ B family is one type of albumen that contains the RHD structural domain, and the member comprises NFkB1, NFkB2 (p52/p100), c-Rel, RelB and RELA (being p65), and wherein p65 has participated in the generation of transformation and tumour as most important member.Research shows; In inflammatory reaction, activate generation and development (Karin that NF-κ B helps tumour; M. (2006) .Nuclear factor-kappa B in cancer development and progression.Nature 441; 431-436), in lung cancer model, find the active mistake that needs NF-kB of lung cancer! Do not find Reference source.。In addition, in cancer chemotherapy and radiotherapy, usually can cause the activation of non-classical path NF-kB, thus a resistance mistake of cause cancer treatment! Do not find Reference source.。Target molecules (the Li that directly is considered to a cancer therapy and prevention; F.; And Sethi; G. (2010) .Targeting transcription factor NF-kappaB to overcome chemoresistance and radioresistance in cancer therapy.Biochim Biophys Acta 1805, a 167-180) mistake! Do not find Reference source., being set forth in the cancer therapy of p65 regulation mechanism has great importance.

Thymus nucleic acid (deoxyribonucleic acid; Be abbreviated as DNA) be a kind of long-chain deoxynucleoside acid polymer; 4 kinds of main composition Nucleotide comprise deoxyadenylic acid (dA); Deoxyguanytic acid (dG), deoxythymidine acid (dT), deoxycytidylic acid (dC); Yeast Nucleic Acid (Ribonucleicacid is abbreviated as RNA) is a kind of long-chain nucleotide polymer, and 4 kinds of main nucleic acid of forming comprise adenine nucleotide (A); Guanylic acid (G), uridylate (U), that cytidylic acid(CMP) (C) .DNA and the key distinction of RNA in based composition are that DNA uses is dA, dT, dG, dC; And RNA uses is A, U, C, G, dA wherein, dC; DG is respectively than A, C, and the G base is few hydroxyl-OH. on 2 in ribose

The ingredient Nucleotide of DNA or RNA is made up of three parts again: base, ribose, phosphoric acid.Ribose wherein generally is five-carbon sugar, and base combines to form nucleosides with ribose.2 carbon atoms of ribose be connected with hydroxyl (OH) be ribose, what do not have hydroxyl is exactly ribodesose.Phosphoric acid group through 5 in ribose between the Nucleotide and 3 hydroxyl condensation form phosphodiester bond with Nucleotide the connect together few nucleic acid that forms short chain or the DNA or the RNA of long-chain, and the two ends of chain are called 5 ' end, 3 ' ends respectively.

What application was more in molecular biology is used is the few nucleic acid of short chain, its base, and ribose, phosphoric acid can be modified respectively or group's replacement, in the stability that improves Nucleotide, does not influence this Nucleotide performance biological function.Naturally rna form that exists and common modifying method See Figure.

The modification of phosphoric acid mainly is sulfo-(P=O becomes P=S)

The modification of ribose comprises: 2 in ribose methylates (Me), methoxylation (MOE), and fluoro, halo, acetylize or longer carbochain are modified; Carry out optical dye, SUV, modifications such as PEG at few nucleic acid chains 3 ends of synthetic miRNA or 5 ends in addition.RNA structure and common modifying method, wherein B represents base, and two Nucleotide connect through phosphodiester bond.

Summary of the invention

The purpose of this invention is to provide a kind of nucleic acid molecule and the new application in medicine thereof.

The technical scheme that realizes above-mentioned purpose is following:

A kind of nucleic acid molecule, its based composition are SEQ ID NO.1 or SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.9.

Title sequence (5 '-3 ') SEQ ID

NO.

Hsa-miR-506-3p UAAGGCACCCUUCUGAGUAGA 1

Hsa-miR-506-3p simulates UAAGGCACCCUUCUGAGUAGA 1

Thing

AUUCCGUGGGAAGACUCAUCU 2

Hsa-miR-506-3p suppresses UCUACUCAGAAGGGUGCCUUA 3

Thing

Pre-hsa-miR-506 GCCACCACCAUCAGCCAUACUAUGUGUAGUGCCU 4

UAUUCAGGAAGGUGUUACUUAAUAGAUUAAUAUU

UGUAAGGCACCCUUCUGAGUAGAGUAAUGUGCAA

CAUGGACAACAUUUGUGGUGGC

shRNA?miR-506 GGATCCCGTAAGGCACCCTTCTGAGTAGATTGAT 9

ATCCGTCTACTCAGAAGGGTGCCTTATTTTTTCC

AAAAGCTT

A kind of nucleic acid molecule; Its based composition is SEQ ID NO.1 or SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4; And the nucleotide analog that contains modification, the nucleotide analog of this modification are glycosyl modified or backbone modification or base modification or end modified Nucleotide.

In certain embodiments; Said nucleotide analog is glycosyl modified ribonucleotide; 2 ' of ribonucleotide-hydroxyl is replaced into H, OR, R, halogen, SH, SR1, NH2, NHR, NR2 or CN, and wherein R is alkyl, thiazolinyl, conjugated radicle or the alkynyl of C1-C20, said halogen be F preferably; Said nucleotide analog is glycosyl modified ribonucleotide, and it is C that ribose is replaced into ribose analogue structure _3-30O _{2 -15}N _1-15S _1-8X _1-20, comprising but not only comprise dicyclo or many nucleolus sugar (LNA), acyclic ribose (UNA), peptide Nucleotide (PNA), wherein X is F, Cl, Br or I.

Or said modified nucleoside acid-like substance is the Nucleotide that contains the non-natural base of modification, and its base structure is C _2-30O _1-20N _1-20S _1-20X _1-20, comprise and not only comprise purine bases analogue, pyrimidine bases analogue, chemistry or photo-crosslinking group, wherein X is F, Cl, Br or I.

Or the structure of the Nucleotide of said end modified decorations is C _2-80O _1-20N _1-20S _1-20X _1-20, comprise and not only comprise SUV, vitamin H, cholic acid, folic acid, VITAMINs, fluorescence molecule, wherein X is F, Cl, Br or I.

Another object of the present invention provides the application of above-mentioned nucleic acid molecule.

The technical scheme that realizes above-mentioned purpose is following:

The application of said nucleic acid molecule in the medicine of preparation treatment tumour.

Said nucleic acid molecule is as the application of diagnosing tumor affinity tag.

In certain embodiments, said tumour is lung cancer, liver cancer, mammary cancer, cervical cancer, colorectal carcinoma.

Another object of the present invention provides a kind of medicinal compsns of treating tumour.

The technical scheme that realizes above-mentioned purpose is following:

A kind of medicinal compsns of treating tumour, its active one-tenth includes said nucleic acid molecule.

The miR-506 isolating nucleic acid is as the application of preparation antineoplaston medicine, and said tumour can be lung cancer, liver cancer, bladder cancer, cervical cancer, colorectal carcinoma, carcinoma of endometrium, white blood disease, ovarian cancer, the esophageal carcinoma, oral cancer, carcinoma of the pancreas, mammary cancer, cancer of the stomach, large celllymphoma, B cell lymphoma, chronic lymphatic parent cell property white blood disease, acute myeloid leukaemia, multiple myeloma, tumor of testis, star-like glucagonoma, melanoma, lymphoma mantle cell, meningioma, mesothelioma, neurofibroma, prostate cancer.

The present invention changes the miR-506mimics (SEQ ID NO.1 and SEQ ID NO.2) and the chemically modified synthetic negative control of design in the tumour cell over to; Compare with negative control; MiR-506 significantly suppresses tumor cell proliferation; Tumour cell is arrested in the G1 phase, and through Caspase signal path inducing apoptosis of tumour cell; Respectively through hairpin structure (shRNA miR-506; SEQ ID NO.9) form and miRNA precursor (pre-has-miR-506; SEQ ID NO.4) clone's method makes up the expression vector of miR-506; Find that the expression amount of expression vector in cell that these two kinds of methods make up is similar, and the expression amount of the miR-506 that has been significantly increased, and the miR-506 that obtains of the method through vector expression or the method through chemosynthesis all can significantly suppress the propagation of tumour cell; Suppress generation, the development of tumour in the body after the further confirmation of the experiment miR-506 administration pre-treatment in vivo; Through the adjustable a plurality of target genes of bioinformatics method analyses and prediction miR-506, further experiment confirm CDK-6, p65 are two target genes of miR-506 regulation and control; Expression through to the miR-506 in the different pathological tissue samples detects, and finds that the expression of miR-506 follows the generation of lung cancer that certain dependency is arranged, and prompting miR-506 can be used as the pulmonary cancer diagnosis affinity tag, is used for the judgement of pulmonary cancer diagnosis and prognosis.

Description of drawings

Fig. 1 is that miR-506 suppresses proliferation of lung cancer cells synoptic diagram as a result among the embodiment 1;

Fig. 2 is that miR-506 is arrested in G1 phase synoptic diagram as a result with cell among the embodiment 2;

Fig. 3 is that miR-506 promotes lung carcinoma cell apoptosis synoptic diagram as a result among the embodiment 3;

Fig. 4 is that miR-506 promotes apoptosis synoptic diagram as a result through the Caspase signal path among the embodiment 4;

Fig. 5 is that miR-506 suppresses potential target 3 ' the UTR reporter gene expression is synoptic diagram as a result among the embodiment 5;

Fig. 6 is miR-506 downward modulation p65 and CDK6mRNA and a proteic expression of results synoptic diagram among the embodiment 5;

Fig. 7 is that miR-506 and target gene thereof are arrested in G1 phase synoptic diagram as a result with cell among the embodiment 6;

Fig. 8 is that TNF-a stimulates the susceptibility synoptic diagram as a result can increase the miR-506 cell death inducing among the embodiment 7;

Fig. 9 is that miR-506 and target gene s iRNA thereof all can cause lung carcinoma cell apoptosis synoptic diagram as a result among the embodiment 8;

Figure 10 is miR-506 regulates and control through the NF-kB signal path among the embodiment 9 a gene synoptic diagram as a result;

Figure 11 is the synoptic diagram as a result that suppresses tumor growth among the embodiment 10 in the miR-506 body;

Figure 12 is that miR-506 suppresses cervical cancer, colorectal carcinoma, breast cancer cell propagation synoptic diagram as a result among the embodiment 11;

Figure 13 is that carrier format is crossed expression miR-506 inhibition tumor cell proliferation synoptic diagram as a result;

Figure 14 is the synoptic diagram as a result after the folding with RNAstructure 4.4 softwares of SEQ ID NO.4 and 9;

Figure 15 is that the miRNA of different modifying among the embodiment 13 all can suppress tumor cell proliferation synoptic diagram as a result;

Figure 16 be among the embodiment 14 miR-506 as molecular diagnosis affinity tag synoptic diagram as a result.

Embodiment

Come the present invention done further describing below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with describing.But these embodiment only are exemplary.Scope of the present invention is not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention the details of technical scheme of the present invention, but these modifications and replace and all fall into protection scope of the present invention.

Medicine by miR-506 isolating nucleic acid preparation of the present invention can be Nanoparticulate compositions, lipid composition, by biocompatiblity molecules and/or biodegradable molecular composition.

Medicine by miR-506 isolating nucleic acid preparation of the present invention can be in intestines or parenteral admin.Administration is that the oral administration parenteral admin is administration in administration in administration in administration in intravascular administration, encephalic administration, the pleura, the tumour, intraperitoneal administration, intramuscular administration, lymph administration, the gland, subcutaneous administration, topical, segmental bronchus administration, the tracheae, intranasal administration, inhalation or dropleting medicine-feeding in the intestines.

During the medicinal application by miR-506 isolating nucleic acid preparation of the present invention, to the 10mg/kg body weight, the miR-506 isolating nucleic acid obtains by chemical synthesis process is synthetic concentration in the 0.01mg/kg body weight; Perhaps expressing the back purifying through carrier construction, is 1X10 with every dosage then ⁵To 1X10 ¹⁴The DNA plasmid vector of individual virion or 100mg to 4000mg.

Embodiment one, miR-506 suppress tumor cell proliferation

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and methods such as the sequence process methylates, sulfo-, SUV are modified.Concrete sequence is following:

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol

Annotate: m represent methylideneization wherein, s represent sulfo-, and Chol represents the SUV modification.Following examples are all identical, do not repeat expression.

Human lung adenocarcinoma cell line A549, H1299, H1275 are from ATCC, and 95D is preserved by this laboratory.Routine is incubated in the F12 that contains 10% foetal calf serum, the DMEM nutrient solution, places the incubator of 5%CO2,37 ℃ of saturated humidities to cultivate and goes down to posterity.

The A549 that takes the logarithm vegetative period, 95D, H1299, H1275 cell are with every hole 4 * 10 ³Cell inoculation in 96 orifice plates, every porocyte suspension TV 100 μ L, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish Notarget contrast transfection group (i.e. NC among the figure), miR-506mimics group (i.e. miR-506 among the figure), establish 3 multiple holes for every group; Sucking-off substratum behind the 48h, every hole add 90 μ L fresh cultures, and simultaneously every hole adds the 10uL CCK-8 solution 10 μ L of new preparation, under 37 ℃, 5%CO2 saturated humidity, cultivate 1h; Put ELIASA 450nm place and measure absorbance (A value), get 3 hole MVs, relatively the difference of each transfection group and Notarget siRNA transfection group A value is calculated relative cell-proliferation activity, calculation formula=miRNA treatment group/Notarget control group.Experimental result shows that miR-506 significantly suppresses tumor cell proliferation (Fig. 1).

Embodiment two, miR-506 are arrested in the G1 phase with tumour cell

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and methods such as the sequence process methylates, sulfo-, SUV are modified.Concrete sequence is following:

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol.

The A549 that takes the logarithm vegetative period, 95D, H661, H1299 cell are with every hole 1 * 10 ⁵Cell inoculation in 6 orifice plates, every porocyte suspension TV 2mL, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish Notarget contrast transfection group, miR-506mimics organizes two groups; Transfection is carried out the cell cycle with the painted method of PI after 48 hours and is detected.Simply say to be exactly, clean once, dispel cell, add the 700uL absolute ethyl alcohol with 300uL PBS with PBS with 0.25% trysinization collecting cell, limit edged vibration mixing, 4 ℃ fixedly more than the 24h; Centrifugal collecting cell cleans cell 2 times with PBS-EDTA; 850uLPBS-EDTA dispels cell, adds 50ul 10mg/ml RNA enzyme solution, 37 ℃ of insulation 30min; Add 100uL PI, room temperature lucifuge dyeing 20min; Centrifugal collecting cell, 300uL PBS dispels cell, carries out streaming after 300 order nylon leaching films filter and detects; Detected result shows that miR-506 is arrested in the G1 phase (Fig. 2) with tumour cell.

Embodiment three, miR-506 inducing apoptosis of tumour cell

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and methods such as the sequence process methylates, sulfo-, SUV are modified.Concrete sequence is following:

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol

The A549 that takes the logarithm vegetative period, 95D, H661 cell are with every hole 1 * 10 ⁵Cell inoculation in 6 orifice plates, every porocyte suspension TV 2mL, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish Notarget contrast transfection group, miR-506mimics treatment group; Behind the transfection 36h, trysinization collecting cell, 1000rpm, 5min; PBS cleans once, 1000rpm, 5min; With 1X binding buffer (Apoptosis detection kit) suspension cell and adjust cell concn to 1X10 ⁶Individual/mL; Change the 100uL cell suspension to new EP pipe, add 5uL PE-Annexin V and 5uL 7-AAD; The vortex vibration makes the cell mixing, and the room temperature lucifuge leaves standstill 15min; Add 400uL 1X binding buffer and dispel and re-suspended cell, filter through gauze (300 order), fluidic cell is measured.Detected result shows miR-506 administration processing inducing apoptosis of tumour cell (Fig. 3).

Embodiment four, miR-506 induce relevant proteic degraded

The base sequence of the miR-506mimics group that present embodiment is used is that SEQ ID NO.1 and the concrete sequence of SEQ ID NO.2 are following:

The miR-506mimics sequence

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol

The 95D cell of taking the logarithm vegetative period is with every hole 1 * 10 ⁵Cell inoculation in 6 orifice plates, every porocyte suspension TV 2mL, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish Notarget contrast transfection group, miR-506mimics treatment group; Scrape cell with cell behind the 48h; 4 ℃, 3000rpm, 5min centrifugal collecting cell; 1mL precooling PBS cleans once, and 4 ℃, 3000rpm, 5min abandon upper strata liquid; Adding 50uL contains the cell pyrolysis liquid of PMSF, and 4 ℃ leave standstill 30min; 4 ℃, 12000rpm, 5min centrifuging and taking supernatant is the cellular proteins sample, gets the 40uL supernatant, adds 8uL (6X sample-loading buffer), and boiling water bath 10min uses perhaps-80 ℃ storage immediately; Institute's extracting albumen with SDS-PAGE carry out electrophoretic separation (150V, 60min); Gel piece after the separation with semidrying change film (2mA/cm2,90min); Change and seal respectively after film finishes, an anti-hybridization, two anti-hybridization; Develop with the X-ray sheet at last.Western blot result shows that miR-506 induces the proteic degraded of PARP, proves that miR-506 is through caspase signal path cell death inducing (Fig. 4).

The screening of embodiment five, target gene

The prediction of miR-506 target spot information biology

MiR-506 regulation and control target gene is with online software predictions such as picTar, TargetScan, miRnaDa; These softwares are all free; And the potential target gene of measurable a large amount of miR-506 through the analysis to the prediction target gene, screens some potential target spots and does further analysis.After test of many times, the present invention has screened 4 genes, comprises CCND2, CDK6, MAPK1, P65.

Target gene 3 ' the structure of UTR

Extract the 95D cell genomic dna, 3 ' UTR, and cloning between the XhoI and two restriction enzyme sites of NotI of the two reporter plasmids (Promega Company products) of psi-Check2, and the exactness of the product that method validation is cloned through order-checking of each gene of pcr amplification.

MiR-506 regulates target gene 3 ' UTR reporter gene carrier

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and the base sequence of used anta-miR-506 group is SEQ ID NO.3, and methods such as the sequence process methylates, sulfo-, SUV are modified.Concrete sequence is following:

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol

The 95D cell of taking the logarithm vegetative period is with every hole 4 * 10 ³Cell inoculation is in 96 orifice plates, and TV 100 μ L in every hole cultivate 24h in 37 ℃, 5%CO2 incubator.According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection; Target gene 3 ' UTR cloned plasmids and miR-506mimics and Notarget control co-transfection; The final transfection concentration of miR-506mimics and Notarget contrast is 50nM, and plasmid concentration is 100ng/mL, establishes 3 multiple holes with every group; Sucking-off substratum behind the 48h adds the 35uL/well fresh culture, and adds 35uL/well Luciferase substrate, and vibration 10min is with fluorescence luxmeter Dual-Glo program determination fluorescent value; Add 30uL Stop regent, vibration 10min measures fluorescent value with the fluorescence luxmeter.The result shows that miR-506 suppresses the expression of CCND2, CDK6, MAPK1, P65 reporter gene, explains the target gene (Fig. 5) that these four genes all possibly be miR-506.

The miR-506 post-transcriptional level is regulated CDK6, P65 expression of gene

The miR-506mimics sequence is the same,

The Anta-miR-506 sequence

umcmumamcmumcmamgmamamgmgmgmumgmcmcmumumam-Chol

Cell total rna extracts the Trizol reagent with Invitrogen company; Flow process is carried out to specifications; Reverse transcription is with the MMLV ThermoScript II of Promega company, and flow process is carried out to specifications, and the qPCR fluorescence quantitative detection kit adopts the Sybrgreen optical dye test kit of Tiangen company.The protein expression testing process is with embodiment four.Detected result shows that miR-506 has reduced CDK6 in the cell, P65mRNA and protein expression, and anta-miR-506 (i.e. A-506 group among the figure) has promoted CDK6, P65 protein expression (Fig. 6).Therefore miR-506 is in post-transcriptional level regulation and control CDK6 and the proteic expression of P65.

Embodiment six, siRNA are arrested in the G1 phase with tumour cell after disturbing CDK6, P65 gene

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and methods such as the sequence process methylates, sulfo-, SUV are modified.Concrete sequence is following:

MiR-506mimic s sequence

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol

The si-P65 sequence:

Positive-sense strand: CUCAAGAUCUGCCGAGUAA dTdT (SEQ ID NO.5)

Antisense strand: UUACUCGGCAGAUCUUGAG dTdT (SEQ ID NO.6)

si-CDK6

Positive-sense strand: GAUGUUGAUCAACUAGGAA dTdT (SEQ ID NO.7)

Antisense strand: UUCCUAGUUGAUCAACAUC dTdT (SEQ ID NO.8)

The 95D cell of taking the logarithm vegetative period is with every hole 1 * 10 ⁵Cell inoculation in 6 orifice plates, every porocyte suspension TV 2mL, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish Notarget contrast transfection group, miR-506mimics treatment group, siCDK6 treatment group, sip65 treatment group; Transfection after 48 hours collecting cell carry out the cell cycle and detect, detection method is referring to embodiment two.The result all is arrested in the G1 phase (Fig. 7) with tumour cell after showing miR-506 processing and target gene siRNA (sip65, siCDK6) interference thereof.

Cell death inducing behind embodiment seven, the s iRNA interference p65

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and methods such as the sequence process methylates, sulfo-, SUV are modified.

MiR-506mimics, anta-miR-506, the si-p65 sequence is with embodiment 5 and 6.

The A549 that takes the logarithm vegetative period, 95D, H661 cell are with every hole 1 * 10 ⁵Cell inoculation in 6 orifice plates, every porocyte suspension TV 2mL, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish Notarget contrast transfection group, miR-506mimics treatment group, anta-miR-506 treatment group (i.e. A-506 group among the figure), sip65 treatment group; Behind the transfection 36h, stimulate 2h with TNF-a, collecting cell carries out apoptosis to be measured, and the apoptosis detection method is referring to embodiment three.But detected result shows miR-506 and sip65 cell death inducing, and this promoter action can be stimulated collaborative promote (Fig. 8) by TNF-a.

Embodiment eight, miR-506 and sip65 induce relevant proteolytic degradation

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and methods such as the sequence process methylates, sulfo-, SUV are modified.

Use the base sequence of anta-miR-506 to be SEQ ID NO.3.

The miR-506mimics sequence

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol

The Anta-miR-506 sequence

UmCmUmAmCmUmCmAmGmAmAmGmGmGmUmGmCmCmUmUmAm-Chol

The 95D cell of taking the logarithm vegetative period is with every hole 1 * 10 ⁵Cell inoculation in 6 orifice plates, every porocyte suspension TV 2mL, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish Notarget contrast transfection group, miR-506 treatment group, anta-miR-506 treatment group, sip65 treatment group; Collecting cell carries out Western blot hybridization detection behind the 48h; Detection method is referring to embodiment four; The result shows that miR-506 induces the degraded of apoptosis-related proteins such as Caspae8, Caspase3, proves that miR-506 and sip65 are through caspase signal path cell death inducing (Fig. 9).

The gene that embodiment nine, miR-506 target p65 regulation and control NF-kB signal path are regulated and control

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and methods such as the sequence process methylates, sulfo-, SUV are modified.

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol

The 95D cell of taking the logarithm vegetative period is with every hole 1 * 10 ⁵Cell inoculation in 6 orifice plates, every porocyte suspension TV 2mL, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection; If No-target contrast transfection group, miR-506 treatment group; Collecting cell extracts total RNA behind the transfection 48h; QPCR detects Expression of Related Genes, and the result shows that miR-506 has reduced the signal path Expression of Related Genes that NF-kB regulated and control, like (Figure 10) such as Bcl2, Bclx, CDK2, COX-2, c-FLIPI, c-FLIPs, GM-CSF, c-IAP1, c-IAP2, TRAF2, VEGF, XIAP.

Suppress tumor growth in the embodiment ten miR-506 bodies

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and methods such as the sequence process methylates, sulfo-, SUV are modified

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol

The BALB/c nude mice is available from the Shanghai Experimental Animal Center, and safety certificate is numbered: the 0048536. A549-luc cell of taking the logarithm vegetative period is with 50% degrees of fusion kind diameter 15cm Tissue Culture Dish (Corning), four wares, incubated overnight; Carry out transfection according to LipofectamineTM 2000 reagent operational guidances; MiR-506mimics and Notarget contrast concentration are each transfection two ware of 50nM; Add fresh culture behind the 6h; Trysinization collecting cell behind the transfection 12h, and count after the two ware cytomixis with same treatment, with PBS adjustment cell concn to 1.5 * 10 ⁷Individual/mL; 200 μ L single cell suspensions are inoculated in the oxter under every nude mice, and cell divides 2 groups, every group of three nude mices; Adopt the smart promise true living imaging system of the U.S. (IVIS 200) to carry out the fluorescence detection of taking pictures; Every mouse abdominal injection 150 μ L 30mg/mL luciferase substrates; Carry out living imaging behind substrate injection 10～25min, carried out living imaging once in per four days, gross tumor volume of per two days mensuration; Observed for 6 weeks continuously, and the fluorescent value of each time point of quantitative analysis.Detected result shows that miR-506 administration pre-treatment has suppressed the ability (Figure 11) that tumour cell forms tumour in vivo.

Embodiment 11, miR-506 suppress tumor cell proliferations such as cervical cancer, colorectal carcinoma, mammary cancer

The base sequence of the miR-506mimics group that present embodiment is used is SEQ ID NO.1 and SEQ ID NO.2, and methods such as the sequence process methylates, sulfo-, SUV are modified.

The miR-506mimics sequence

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol

Human cervical carcinoma cell strain Hela, colon cancer cell HCT-116, breast cancer cell MCF-7 is from ATCC.Routine is incubated in the DMEM nutrient solution that contains 10% foetal calf serum, places the incubator cultivation of 5%CO2,37 ℃ of saturated humidities to go down to posterity.

The Hela cell of taking the logarithm vegetative period, colon cancer cell HCT-116, mammary cancer MCF-7 cell is with every hole 4 * 10 ³Cell inoculation in 96 orifice plates, every porocyte suspension TV 100 μ L, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish Notarget contrast transfection group, miR-506mimics treatment group, establish 3 multiple holes for every group; Sucking-off substratum behind the 48h, every hole add 90 μ L fresh cultures, and simultaneously every hole adds the 10uL CCK-8 solution 10 μ L of new preparation, under 37 ℃, 5%CO2 saturated humidity, cultivate 1h; Put ELIASA 450nm place and measure absorbance (A value), get 3 hole MVs, relatively the difference of each transfection group and Notarget siRNA transfection group A value is calculated relative cell-proliferation activity, calculation formula=miRNA treatment group/Notarget control group.Experimental result significantly suppresses cervical cancer, colorectal carcinoma after showing the miR-506 administration, tumor cell proliferations such as mammary cancer, so the miR-506 of chemical process preparation can be used for suppressing tumor cell proliferation (Figure 12).

Embodiment 12, vector expression miR-506 suppress tumor cell proliferation

The shRNA miR-506 of hairpin structure (SEQ ID NO.9) expressed sequence frame is synthetic by the design of Ribobio company, and pre-miR-506 (SEQ ID NO.4) sequence is synthetic through the method for chemosynthesis.MiR-506 obtains in the method for intracellular expression amount through qPCR, and the qPCR primer is provided by Ribobio company.

Ordinary method according to this area: the pre-miR-506 and the few nucleic acid strand of the shRNA miR-506 purifying after annealing of chemosynthesis are formed two strands; The agarose gel electrophoresis purifying is double-stranded and be connected into expression vector, and the result after sequence folds with RNAstructure 4.4 softwares sees Figure 14.

The 95D cell of taking the logarithm vegetative period is with every hole 4 * 10 ³Cell inoculation in 96 orifice plates, every porocyte suspension TV 100 μ L, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish bare contrast transfection group, shRNA miR-506 expression vector treatment group, and pre-miR-506 expression vector treatment group is established 3 multiple holes for every group, and the carrier working concentration is the 100ng/ hole; Sucking-off substratum behind the 48h, every hole add 90 μ L fresh cultures, and simultaneously every hole adds the 10uL CCK-8 solution 10 μ L of new preparation, under 37 ℃, 5%CO2 saturated humidity, cultivate 1h; Put ELIASA 450nm place and measure absorbance (A value), get 3 hole MVs, relatively the difference of each transfection group and bare transfection group A value is calculated relative cell-proliferation activity.The experimental result demonstration significantly suppresses tumor cell proliferation after crossing expression miR-506 with carrier, shows that the miR-506 with the carrier method preparation also can be used for suppressing tumor cell proliferation (Figure 13).

The miRNA of embodiment 13, different modifying all can suppress tumor cell proliferation

The used miR-506mimics sequence of present embodiment is following:

Positive-sense strand: UAAGGCACCCUUCUGAGUAGA

Antisense strand: AUUCCGUGGGAAGACUCAUCU is the basis, through modification, shown in the table specific as follows

Numbering	Sequence
			1	AmUmUmCCGUsGGGAAGACUsCAmUmCmU-Chol
2	AmUmUmCCGU FGGGAAGACU FCamUmCmU-Chol
		3	A MOEU MOEUCCGUsGGGAAGACUsCAU MOEC MOEU-PEG
4	AU LNAU LNACCGUsGGGAAGACUsCA LNAU LNACU-PEG

6	AU LNAU LNACCGU LNAGGGAAGACU LNACAU LNACU-Biotin
		7	AUUCCGUGGGAAGACUCAUCU

F represents fluorine, the MOE representation methoxy, and PEG represents polyoxyethylene glycol, LNA (locked nucleicacid) representative lock nucleic acid, Biotin is a vitamin H.

Human lung carcinoma cell line A549 is from ATCC.Routine is incubated in the RPMI-1640 that contains 10% foetal calf serum, places the incubator cultivation of 5%CO2,37 ℃ of saturated humidities to go down to posterity.

The Hela cell of taking the logarithm vegetative period is with every hole 4 * 10 ³Cell inoculation in 96 orifice plates, every porocyte suspension TV 100 μ L, overnight cultures in 37 ℃, 5%CO2 incubator; According to Lipofectamine ^TM2000 reagent operational guidances carry out transfection, establish Notarget contrast transfection group, miR-506mimics treatment group, establish 3 multiple holes for every group; Sucking-off substratum behind the 48h, every hole add 90 μ L fresh cultures, and simultaneously every hole adds the 10uL CCK-8 solution 10 μ L of new preparation, under 37 ℃, 5%CO2 saturated humidity, cultivate 1h; Put ELIASA 450nm place and measure absorbance (A value), get 3 hole MVs, relatively the difference of each transfection group and No-target siRNA transfection group A value is calculated relative cell-proliferation activity, calculation formula=miRNA treatment group/Notarget control group.Figure 15 shows that 1-7 numbers the 1-7 group in the corresponding present embodiment form respectively; All can suppress proliferation of lung cancer cells after the miR-506 administration that experimental result demonstration different chemical is modified; With compare without the 7th group that modifies; Different modifying is to a certain degree improved the miRNA action effect, has improved the stability (Figure 15) of miRNA simultaneously.

Embodiment 14, miR-506 are as the molecular diagnosis affinity tag

Get lung cancer and corresponding adjacent tissues sample 35 examples, respectively with after the liquid nitrogen grinding, with Trizol extracting total tissue RNA; Expression with miR-506 (SEQ ID NO.1) in the method detection by quantitative sample of qPCR; Detected result such as Figure 16, detected result shows miR-506 significantly low expression in the cancerous lung tissue of about 75% (26 routine sample), points out the significant dependency that has of miR-506 and lung cancer; Possibly carry out the diagnosis of lung cancer and the judgement of prognosis as the molecular marked compound of lung cancer.

The above embodiment has only expressed several kinds of embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with accompanying claims.

Claims (10)

1. a nucleic acid molecule is characterized in that, its based composition is SEQ ID NO.1 or SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.9.

2. nucleic acid molecule; It is characterized in that; Its based composition is SEQ ID NO.1 or SEQ ID NO.1 and SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4; And the nucleotide analog that contains modification, the nucleotide analog of this modification are glycosyl modified or phosphoric acid backbone modification or base modification or end modified Nucleotide.

3. nucleic acid molecule according to claim 2; It is characterized in that; Said nucleotide analog is glycosyl modified ribonucleotide; 2 ' of ribonucleotide-hydroxyl is replaced into H, OR, R, halogen, SH, SR1, NH2, NHR, NR2 or CN, and wherein R is alkyl, thiazolinyl, conjugated radicle or the alkynyl of C1-C20, and said halogen is F, Cl, Br or I.

4. nucleic acid molecule according to claim 2 is characterized in that, said nucleotide analog is glycosyl modified ribonucleotide, and it is C that ribose is replaced into ribose analogue structure _3-30O _2-15N _1-15S _1-8X _1-20, what wherein contain this structure comprises dicyclo or many nucleolus sugar, acyclic ribose, or peptide Nucleotide, and wherein X is F, Cl, Br or I.

5. nucleic acid molecule according to claim 2 is characterized in that, said modified nucleoside acid-like substance is the Nucleotide that contains the non-natural base of modification, and its base structure is C _2-30O _1-20N _1-20S _1-20X _1-20, what wherein contain this structure comprises purine bases analogue, pyrimidine bases analogue, chemistry or photo-crosslinking group, wherein X is F, Cl, Br or I.

6. nucleic acid molecule according to claim 2 is characterized in that, the structure of the Nucleotide of said end modified decorations is C _2-80O _1-20N _1-20S _1-20X _1-20, what wherein contain this structure comprises SUV, vitamin H, cholic acid, folic acid, VITAMINs or fluorescence molecule, wherein X is F, Cl, Br or I.

7. the application of each described nucleic acid molecule of claim 1-6 in the medicine of preparation treatment tumour.

8. application according to claim 7, said tumour are lung cancer, liver cancer, mammary cancer, cervical cancer, colorectal carcinoma.

9. each described nucleic acid molecule of claim 1-6 is as the application of diagnosing tumor affinity tag.

10. medicinal compsns of treating tumour, it is active to become to include each described nucleic acid molecule of claim 1-6.

Images