CN1281279C - Small interference ribonucleic acid molecule for epidermal growth facor gene and its use - Google Patents
Small interference ribonucleic acid molecule for epidermal growth facor gene and its use Download PDFInfo
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- CN1281279C CN1281279C CN 200410066528 CN200410066528A CN1281279C CN 1281279 C CN1281279 C CN 1281279C CN 200410066528 CN200410066528 CN 200410066528 CN 200410066528 A CN200410066528 A CN 200410066528A CN 1281279 C CN1281279 C CN 1281279C
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- nucleotide
- sirna
- acid molecule
- epidermal growth
- ribonucleic acid
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Abstract
The present invention provides a small disturbance ribonic acid molecule (SiRNA) used as the active component of anti-tumor medicaments, which can effectively kill tumor cells and aim at epidermal growth factor (Cripto) gene mRNA. The minus chain of the nucleotide sequence and a mutant of ribonucleotide thereof have no homology with the known human genes. The small disturbance RNA molecule (SiRNA) corresponding to the common sequence can effectively inhibit the expression of human colon cancer LS-174T cell Cripto genes through RISC in cells to help the treatment of patients with colon cancer aiming at high Cripto gene expression or cancers aiming at high Cripto gene expression, and can be used to prepare medicaments for treating human cancers related to the increase of Cripto expression and diseases related to cancers.
Description
Technical field
The present invention relates to the nucleic acid technical field, relate to small interference ribonucleic acid molecule (SiRNA) and the purposes in the medicine of the effective killing tumor cell of preparation thereof specifically at epidermal growth factor (Cripto) gene mRNA.
Background technology
Malignant tumor is a kind of principal disease of harm humans life and health, but this is not still had effective treatment means up to now.Scientists is attempting to develop the new drug that can effectively treat malignant tumor always for a long time.
According to World Health Organization (WHO) statistics, annual whole world New Development malignant tumor patient is about 1,000 ten thousand, dies from 600 of tumor～7,000,000.In China, annual neopathy is about 2,000,000, dead about 1,400,000/calculate by 1,300,000,000 populations).Along with the urbanization of aged tendency of population, smoking, life style and the further reinforcement of industrialization influence, this trend will continue.Tumor is the complex disease of multimachine system, still uses means such as early diagnosis, operation, radiotherapy, chemotherapy at present, and curative effect is limited, and brings bigger misery to patient.Colon cancer is one of fastest-rising tumor of present China's sickness rate.Transfer and chemotherapy resistance are the biggest problem of treatment of colon cancer, still lack effective treatment means and medicine at present, seek effective medicine and treatment means, are the task of top priority.Thoroughly capturing the challenge of malignant tumor such as colon cancer can only finish by general treatment measures such as above treatment means binding immunoassay treatment, gene therapies.
The generation development of most of solid tumors is relevant with amplification with oncogene active.Discover that recently the generation development of epidermal growth factor (Cripto) gene and malignant tumor is closely related.Cripto is a kind of autocrine type tumor growth factor, is one of EGF family member.This gene is found to express in people and mice embryonic tumor cell, and then finds all to be overexpression in human breast cancer, colon cancer, gastric cancer, cancer of pancreas, carcinoma of penis, bladder cancer and carcinoma of prostate etc., and normal tissue expression is very low or disappearance.Point out this gene in the formation development of malignant tumor such as colon cancer, playing the part of important role.Find in the experiment in vitro that this gene impels the migration of cell malignant proliferating cell, suppresses apoptosis.Prompting thus, this can be used as the novel targets of a malignant tumor therapy of tumor.
Summary of the invention
Technical problem to be solved by this invention provides a kind of small interference ribonucleic acid molecule (SiRNA) at epidermal growth factor (Cripto) gene mRNA of effective killing tumor cell of the active component as antitumor drug.This small interference ribonucleic acid molecule is a double stranded rna molecule, has arbitrary nucleotide sequence among the SEQ ID NO:1-12, and with the homology degree of nucleotide sequence at least 70% with following feature: the length of positive-sense strand and antisense strand is 21 nucleotide, positive-sense strand and antisense strand 3 ' end separately is two successive deoxythymidylic acids (TT), article two, chain remove 3 ' end TT beyond 19 nucleotide on base complementrity form two strands, 19 nucleotide sequences after the positive-sense strand nucleotide that continuous two bases are adenine (A) in the nucleotide sequence of 19 nucleotide sequences of 5 ' end beginning and SEQ ID NO:1 are in full accord, base in every chain is the nucleotide quantity of guanine (G) and nucleotide quantity sum that base is cytosine (C) ratio that accounts for 19 nucleotide quantity beyond the TT that removes 3 ' end for greater than 25% less than 75% (being the G/C ratio), and the mutant and the known person genoid of the minus strand of a nucleotide sequence provided by the invention and one nucleotides do not have homology.
" no homology " this coding mutation body that is meant nucleotide sequence provided by the invention and its any one position and known person genoid and gene expression fragment do not have 100% identical.
Above-mentioned siRNA molecule (SiRNA) is a synthetic, and positive-sense strand and antisense strand 3 ' end separately has two sweet acid of deoxidation breast (TT), and the base of other 19 nucleotide can be adenine (A), guanine (G), cytosine (C) or uracil (U); Article two, chain is removed base complementrity (A and U, C and G) on 19 nucleotide beyond the TT of 3 ' end and is formed double-strandedly, but two TT of their 3 ' ends exist with the form of strand.
When the nucleotide sequence of SEQ ID NO:1 occurs that successive base is the nucleotide of adenine (A) more than two, above-mentioned " 19 nucleotide sequences of positive-sense strand after the nucleotide that continuous two bases of the nucleotide sequence of 19 nucleotide sequences of 5 ' end beginning and SEQ ID NO:1 are adenine (A) are in full accord, " can be understood that " positive-sense strand from 19 nucleotide sequences of 5 ' end beginning and this more than two successive base be that latter two nucleotide 19 nucleotide sequences afterwards in the nucleotide of adenine (A) are in full accord.”
SiRNA molecule corresponding to this common sequences provided by the invention (SiRNA) can suppress human human colon's cancer LS-174T cell Cripto gene expression effectively by intracellular RISC (RNA-induced silencing complex), thereby helps to treat at the colon cancer of Cripto gene high expression or the malignant tumor patient of cripto gene high expression.
When nucleotide sequence provided by the invention also has following feature simultaneously, siRNA molecule provided by the present invention, aforesaid better effects if:
1), the positive-sense strand of nucleotide sequence is that base is a kind of in the nucleotide of guanine (G), cytosine (C) or uracil (U) from first nucleotide of 5 ' end beginning; That is to say when occurring in the sequence of SEQ ID NO:1 that successive base is the nucleotide of adenine (A) more than two, positive-sense strand from 19 nucleotide sequences of 5 ' end beginning and this more than two successive base be that 19 nucleotide sequences after latter two nucleotide in the nucleotide of adenine (A) are in full accord, ".
2), above-mentioned SiRNA sequence and other genes of the known mankind and expression fragment do not have 100% homology.
SEQ ID NO:1-12 is a nucleotide sequence provided by the invention:
The SiRNA sequence
| Numbering | Number | The positive-sense strand sequence | The antisense strand sequence | Mean molecule quantity | Firing area # |
| C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 | 1 2 3 4 5 6 7 8 9 10 11 12 | UUCGGCCUCGGUCUUCCCATT CAGAACCUGCUGCCUGAAUTT CUGUGAGCACGAUGUGCGCTT GAGAACUGUGGGUCUGUGCTT UGCUGGCACGGUCAGCUCCTT CUACCACCGUCUGCACGUATT UCGACAUUGACCUAUUUCCTT GGCUGCUGCUACAAUGUCCTT GUCAGCCAUAUCUCCAUUGTT CUGUGCAAGGCCUGGUGUUTT GUCCAUCUGCGUGUGUGCATT GAAUGAAGCCUCCUUAGAATT | UGGGAAGACCGAGGCCGAATT AUUCAGGCAGCAGGUUCUGTT GCGCACAUCGUGCUCACAGTT GCACAGACCCACAGUUCUCTT GGAGCUGACCGUGCCAGCATT UACGUGCAGACGGUGGUAGTT GGAAAUAGGUCAAUGUCGATT GGACAUUGUAGCAGCAGCCTT CAAUGGAGAUAUGGCUGACTT AACACCAGGCCUUGCACAGTT UGCACACACGCAGAUGGACTT UUCUAAGGAGGCUUCAUUCTT | 13347.2 13317.2 13347.2 13332.2 13362.2 13332.2 13287.2 13332.2 13302.2 13332.2 13332.2 13287.2 | 45-65 106-126 184-204 206-226 266-286 365-385 437-457 500-520 660-680 981-1001 1205-1225 1414-1434 |
*: as N is arranged in the infructescence, the mean molecule quantity 339.2 of then getting A, U, C, four kinds of nucleotidess of G calculates
#: firing area is meant that this nucleotides sequence is listed in the opposite position in the SEQ ID NO:1 sequence.
For the numerical value of above said homology degree, with more than 80% for better.The said homology degree of the present invention is meant any SiRNA, the positive-sense strand of its nucleotide sequence and the said nucleotide sequence of the present invention or antisense strand sequence, the identical rate of the nucleotide on the relevant position.
Effective inhibition provided by the present invention also comprises any part or whole formed siRNA molecules after chemical modification of the sequence of the small interference ribonucleic acid molecule at Cripto mRNA (SiRNA) at above mentioned effective killing tumor cell.
Described chemical modification comprises and mainly comprises following three classes: the first, the part of the phosphodiester bond that connects adjacent two nucleotide is modified or with other any chemical bond replacement.The part modification of typical phosphodiester bond is that the oxygen on the two keys of phosphoric acid is replaced as sulfur (sulfuration) or other element, or the oxygen on the phosphoric acid singly-bound is become nitrogen (nitrogenize) or other element and chemical group; Whole replacements to phosphodiester bond itself, also comprise part or all of change to itself or its part and two ribose linking to each other with it, typical illustration is that phosphodiester bond and two adjacent ribose are become peptide bond, make nucleic acid become peptide nucleic acid(PNA) (peptidenucleic acid, PNA); The second, to examine sweet in the five-membered ring of ribose change or its side chain on chemical group modify.The case history that changes ribose ring is as becoming five yuan ribose ring on the Morpholino ring of six rings; Modification on the ribose side chain is meant that mainly 2 ' in ribose is gone up OH becomes other element (as halogens), or with the H (for example alkyl) among the alternative OH of other chemical group.Three, the base ring on the nucleotide is done the whole change or the modification of side chain.
The generation development of most of solid tumors is relevant with amplification with oncogene active.The generation development of epidermal growth factor (Cripto) gene overexpression and many malignant tumor is closely related.Seal this expression of gene effectively, will help to treat with Cripto and cross the malignant tumor that expression has substantial connection.Therefore, siRNA cripto SiRNA provided by the present invention can be applicable to prepare treatment and Cripto and expresses in the medicine that increases the disease that relevant human malignancies and malignant tumor be correlated with.
Description of drawings
Fig. 1 is the expression of Cripto mRNA behind 12 kinds of SiRNA processing cancerous cell.
Fig. 2 is the concentration effect (48 hour) of C1 to LS-174T cell cripto mRNA horizontal force.
Fig. 3 is the concentration effect (48 hour) of C12 to LS-174T cell cripto mRNA horizontal force.
Fig. 4 is C1, and C12 is to the time effect (100nM) of LS-174T cell cripto mRNA horizontal force.
The specific embodiment
The present invention is further described in conjunction with the embodiments.
Embodiment 1 SiRNA's is synthetic
Synthetic can the trust of SiRNA openly externally carried out synthetic professional commercial company, such as U.S. Dharmacon company, specifically can pass through
Www.dharmacon.comKnow that all SiRNA molecules all through 2 upward deprotection, desalination, purification and double-stranded processing of annealing formation, are dissolved in the distilled water of pyrocarbonic acid diethyl ester processing then.
The preparation method of siRNA molecule provided by the invention (SiRNA) also can adopt conventional solid state chemistry synthetic method.
SiRNA molecule (SiRNA) with SEQ ID NO:1 is an example: whole chemosynthesis can roughly be divided into four process (1) oligomerization ribonucleic acid synthetic; (2) deprotection; (3) purifies and separates; (4) the desalination aseptic sterilization of annealing.
Concrete preparation manipulation is as follows:
(1), oligomerization ribonucleic acid is synthetic: the synthetic of SiRNA at automated DNA/RNA synthesizer (for example: carry out Applied Biosystems EXPEDITE 8909) is, (positive-sense strand 5 '-UUCGGCCUCGGUCUUCCCATT, the order of antisense strand 5 '-UGGGAAGACCGAGGCCGAATT) couples together the nucleotide of correspondence one by one according to the nucleotide sequence of c1 number siRNA molecule.Because SiRNA is made up of one section 19 poly-oligomerization ribonucleic acid and one 2 poly-deoxythymidylic acid.Therefore starting material is that 5 '-O-of connecting of solid phase (CPG) is to dimethoxytrityl-thymidine (1-2umol).Concrete each circulation is synthetic can be divided into for four steps and to finish.The first step be with thymidine that solid phase is connected on protecting group eluting under the effect of 3% trichloroacetic acid of 5 '; Second step was coupled to 5 '-O-on the last thymidine of sloughing protection to dimethoxytrityl-thymidine phosphoramidite under the effect of active catalyst S-ethyl tetrazolium, formed two thymidine tris phosphites.Coupling time and coupling circulation time number average provides program to finish by instrument producer; The 3rd step was that two thymidine tris phosphites with coupling are oxidized to two thymidine phosphotriesters under the effect of 0.05M iodine water; The 4th step was acetylation, and a small amount of unreacted active group on the solid phase (for example: hydroxyl and amido) is formed ester or amide under the effect of acetic anhydride.Thereby reach sealing process, in order to reduce whole production of by-products.Repeat this circulation until finishing the synthetic of whole nucleotide sequences.
(2) deprotection: will synthesize good solid phase SiRNA and put into a bottle that can seal, and with the methylamine water solution (10M, 50% ethanol) that adds 1 milliliter, rest on room temperature.After two hours, take out solution, and solid phase CPG is used ethanol once more; The mixed liquor drip washing of water and acetonitrile, and leacheate and the solution that takes out previously merged a place, its solvent is drained.In bottle, continue to add the tetrahydrofuran solution (1M) of 1 milliliter of tetrabutyl ammonium fluoride.Solution was left standstill 12 hours in room temperature.Slough the protecting group (comprising base, the silanization protecting group that nucleoside phosphorylase and nucleoside are 2 ') on all oligomerization ribonucleic acid.Pass through ethanol precipitation again, produce the crude product of SiRNA.
(3) purifies and separates: the crude product of SiRNA is dissolved in 2 milliliters the aqueous solution of ammonium acetate, passes through the separation of anti-phase C18 high pressure liquid chromatography then.The method of utilization gradient elution, the principal product (ammonium acetate of leacheate A:0.1M of collection SiRNA; The ammonium acetate of the 0.1M of leacheate b:20% and 80% acetonitrile).The solvent of the principal product of SiRNA is removed, and added 5 milliliter of 80% acetic acid aqueous solution, left standstill 15 minutes in room temperature.Then this solution is carried out the separation (DEAE-5PW, anion-exchange column) of anion exchange, can obtain purity at the SiRNA more than 90% (gradient elution, the Tris-HCl of leacheate A:0.025M, 0.025M NaCl pH=8,5% acetonitrile; The Tris-HCl of leacheate b:0.025M, 2.0M NaCl, pH=8,5% acetonitrile).
(4) the desalination aseptic sterilization of annealing: the SiRNA of purification removes salt through dialysis, and the solution of SiRNA carries out filter-sterilized and drying crystalline.Oligomerization ribonucleic acid with positive-sense strand and antisense strand carries out the SiRNA that annealing in process forms stable bifilar interlinkage then.Its method is the oligomerization ribonucleic acid mixed dissolution (10mM Tris, pH=7.5-8.0,50mM NaCl) in the buffer solution of 1-2 milliliter with positive-sense strand and antisense strand.This solution is heated to 95 ℃, slowly this solution cooling is caused room temperature (this process should be no less than hour) then.At last this solution is left in 4 ℃ of refrigerators and preserve, so that can use at any time.
Purity and evaluation through the SiRNA behind the purification have two kinds of ways relatively more commonly used.One is identified the purity of SiRNA with the capillary gel electrophoresis method, this method can be referring to Paulus A, Ohms JI.Analys isof oligonucleotides by capillary gelelectrophoresis.J Chromatogr1990,507:113-123.
It two is accurately to measure its molecular weight with the MALDI-TOF mass spectrum, thus the chemical constitution of definite SiRNA composition.
Said all SiRNA of the present invention can adopt method for preparing according to its sequence, and authentication method is the same.
The transfection of embodiment 2 cell culture and SiRNA
1, cell culture human colon cancer LS-174T cell has the Cripto gene high expression.This cells in vitro adopts the RPMI1640 culture medium (containing each 100U/ml of penicillin and streptomycin) of 10% hyclone to cultivate, and changes liquid every day.
2, the transfection of SiRNA: SiRNA changes tumor cell over to and carries out by means of the Oligofectamine of Invitrogen (www.invitrogen.com) company, and concrete steps are carried out to specifications.Be summarized as follows 1.0 * 10
5Tumor cell inoculation on 24 well culture plates, spend the night, changed over to SiRNA in second day, after continuing then to cultivate different time, collecting cell detects the level of cripto mRNA in the cell.
3, the detection of LS-174T cell cripto mRNA expression: the detection of tumor cell Cripto mRNA expression is carried out with the technology of PCR in real time.Specifically can be divided into following three steps: the first, the extraction of RNA in the cell, this method can carry out with reference to existing standard method (seeing chapter 4, Currentprotocols in Molecular Biology, John ﹠amp; Wiley, 2003).In second step, cDNA's is synthetic, but synthetic method also the methodology of reference standard carry out (seeing chapter 5, Current protocolsin Molecular Biology, John ﹠amp; Wiley, 2003).The 3rd step, the carrying out of PCR in real time, PCR in real time is to carry out on ABI7700 Sequence Detection instrument, concrete grammar carries out with reference to the standardization program of ABI company.The Cripto primer: the upstream, 5 '-CAATTCGGCCTCGGTCTTC-3 '; The downstream, 5 '-TTCAGGCAGCAGGTTCTGTTT-3 '.FAM probe primer 5 '-CTCCTTACTGTGCTGTATCCCCATGG-3 '.The sequence of PGR probe is 5 '-CCGCTTCTCGTCGATGTAGGTCACG-3 ' in real time, corresponding to 1710 to 1734 in the cripto cDNA sequence.The circular response condition: 50 ℃, 2min, 1 circulation; 95 ℃, 10min; 95 ℃, 15sec, 60 ℃, 1min.Totally 40 circulations.
1, the selection of SiRNA: the present invention has selected at 12 of the SiRNA molecules of cripto mRNA coding region, i.e. 12 listed SiRNA molecules of SEQ ID NO:1-12 totally.Attack the site between the 45-1414 of initiation codon, the G/C in each SiRNA molecule in the nucleotide sequence of 19 complementary one-tenth two strandss is between 42%-68%.
2,12 effects that SiRNA expresses LS-174T cell cripto mRNA:
A, concentration are the effects of 12 SiRNA of 100nM to SW480 cell cripto mRNA:
These 120 kinds of SiRNA with 100nM acted on the LS-174T cell after 48 hours, and observing them all has certain inhibitory action to the time spent 12 kinds of SiRNA that do of cripto mRNA level to cripto RNA.The most obvious with C1 number (SEQ ID NO:1) and C12 number (SEQ ID NO:12) to cripto mRNA inhibitory action, reach 86%, 84% respectively.The result is referring to Fig. 1,12 kinds of SiRNA of diagram 100nM act on the influence of cripto mRNA level in the pair cell after 48 hours of colon cancer LS-174T cell, matched group (control) is that phalangeal cell deals with transfection reagent under condition of equivalent, vertical coordinate represents that the mRNA level of SiRNA processed group cripto gene expression accounts for the relative percentage of matched group, the meansigma methods of three repeated experiments of each data representation among the figure.
B, C1 SiRNA are to the inhibiting concentration effect of LS-174T cell cripto mRNA:
In order to observe the concentration effect that No. 1 SiRNA suppresses Cripto mRNA, we have selected concentration from 1.56nM to 200nM.Found that C1 SiRNA has the significant concentration effect to Cripto mRNA inhibitory action.The result is referring to Fig. 2, and vertical coordinate represents that the mRNA level of SiRNA processed group cripto gene expression accounts for the relative percentage of matched group among the figure, the meansigma methods of three repeated experiments of each data representation among the figure.
C, C12 SiRNA are to the inhibiting concentration effect of LS-174T cell cripto mRNA:
In order to observe the concentration effect that C12 SiRNA suppresses Cripto mRNA, we have selected concentration from 1.56nM to 200nM.Found that C12 SiRNA has the significant concentration effect to Cripto mRNA inhibitory action.The result is referring to Fig. 3, and vertical coordinate represents that the mRNA level of SiRNA processed group cripto gene expression accounts for the relative percentage of matched group among the figure, the meansigma methods of three repeated experiments of each data representation among the figure.
D, C1, C12 SiRNA are to the inhibiting time effect of LS-174T cell cripto mRNA:
In order to observe the time effect that the 1st and No. 12 (100nM) SiRNA suppresses Cripto mRNA, we have selected 24,48,72 hours three time points.Found that C1 and C12 promptly have inhibitory action at 24 hours cripto genes, inhibitory action obviously strengthened (with comparison in 24 hours) in 48 hours, 72 hours obvious more (with comparison in 24 hours).But 72 hours with 48 hours inhibitory action relatively, difference is little, the result is referring to Fig. 4, and vertical coordinate represents that the mRNA level of SiRNA processed group cripto gene expression accounts for the relative percentage of matched group among the figure, the meansigma methods of three repeated experiments of each data representation among the figure.
The modification of embodiment 4 SiRNA
The kind of the modification of SiRNA can be divided three major types, and each apoplexy due to endogenous wind has many variations, so their synthetic methods separately are the same not to the utmost.Here enumerate two kinds of the most common SiRNA method of modifying.And following method does not limit the relevant protection domain that SiRNA is modified of the present invention.
1, sulfuration
Sulfuration is meant that an oxygen atom on the nucleoside phosphorylase diester linkage is transformed into sulphur atom forms nucleoside D2EHDTPA diester.Do not change because other of whole SiRNA formed structure, so its is synthetic the same substantially with the SiRNA building-up process of embodiment 1.Only need the oxidation reaction in the building-up process is become vulcanization reaction, in this reaction, add suitable sulfuration reagent, 3H-1 for example, 2-benzodithiol-3-one1,1-dioxide (claiming Beaucage reagent again).
2, the modification of 2 ' hydroxyl of nucleoside
The modification of 2 ' hydroxyl of nucleoside is meant 2 ' the hydroxyl that comes five Yuans sugar of substituted nucleosides to encircle with various saturated alkoxyls or unsaturated alcoxyl.Wherein modal is the hydroxyl that comes 2 ' of substituted nucleosides with methoxyl group.The synthesis step basically identical of the SiRNA of the synthetic and embodiment 1 of 2 ' methoxylation of nucleoside of SiRNA only need replace 2 '-tertiary butyl dimethyl Si yl nucleosides phosphoramidite with 2 '-methoxyl group nucleoside phosphoramidites and get final product in coupling reaction.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the embodiment of front is interpreted as only illustrating, but not limits the scope of the invention by any way.
Claims (7)
1, the small interference ribonucleic acid molecule at epidermal growth factor mRNA of effective killing tumor cell, it is a double stranded rna molecule, has arbitrary nucleotide sequence among the SEQ ID NO:1-12, homology degree with the nucleotide sequence at least 70% with following feature: the length of positive-sense strand and antisense strand is 21 nucleotide, positive-sense strand and antisense strand 3 ' end separately is two successive deoxythymidylic acids (TT), article two, chain remove 3 ' end TT beyond 19 nucleotide on base complementrity form two strands, 19 nucleotide sequences after the positive-sense strand nucleotide that continuous two bases are adenine (A) in the sequence of 19 nucleotide sequences of 5 ' end beginning and SEQ ID NO:1 are in full accord, and the ratio that the nucleotide quantity sum that the base in every chain is the nucleotide quantity of guanine (G) with base is cytosine (C) accounts for 19 nucleotide quantity beyond the TT that removes 3 ' end is less than 75% greater than 25%.
2, the small interference ribonucleic acid molecule at epidermal growth factor mRNA of the described effective killing tumor cell of claim 1, described homology degree is greater than 80%.
3, the small interference ribonucleic acid molecule at epidermal growth factor mRNA of the arbitrary described effective killing tumor cell in the claim 1 to 2, it is any part of described small interference ribonucleic acid molecule or all formed after chemical modification.
4, the small interference ribonucleic acid molecule at epidermal growth factor mRNA of the effective killing tumor cell described in claim 3, described chemical modification comprise to the part modification of the phosphodiester bond that connects nucleotide or with other any chemical bond and replacing.
5, the small interference ribonucleic acid molecule at epidermal growth factor mRNA of the effective killing tumor cell described in claim 3, described chemical modification comprises that the OH on to ribose on these molecular side chains 2 ' does on the modification of any chemistry and change and the base any position and does chemical modification or change.
6, the described effective killing tumor cell of claim 1 at the small interference ribonucleic acid molecule of epidermal growth factor mRNA preparation treatment tumor or with the medicine of tumor relevant disease in application.
7, the application of small interference ribonucleic acid molecule in the medicine of the preparation treatment disease relevant at epidermal growth factor mRNA of effective killing tumor cell as claimed in claim 4 with epidermal growth factor expression increase.
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