CN105505929A - Small interfering RNA aiming at HPV16E6 gene and application of small interfering RNA - Google Patents

Abstract

The invention discloses a small interfering RNA aiming at an HPV16E6 gene and an application of the small interfering RNA. According to the small interfering RNA aiming at the HPV16E6 gene, a target sequence of the small interfering RNA molecule is separated from the HPV16E6 gene, and is the sequence shown in SEQ ID NO:2. The small interfering nucleic acid molecule aiming at the HPV16E6 gene expression provided by the invention can efficiently and specifically inhibit the expression of the HPV16E6 gene, has relatively low toxic and side effects, and can be used for preparing the medicine for inhibiting the expression of the HPV16E6 gene.

Description

For siRNA and the application thereof of HPV16E6 gene

Technical field

The present invention relates to double stranded RNA (dsRNA), and relate to its purposes of relative disease be used for caused by Prevention and Curation human papilloma virus 16 hypotype (HPV16) infection by RNA interference (RNAi), the cancers such as wherein said disease such as cervical cancer, carcinoma vulvae, the esophageal carcinoma, mammary cancer, lung cancer, and the disease such as precancerous lesion and Genital warts caused by HPV16.

Background technology

1998, AndrewZ.Fire and CraigC.Mello found the mechanism of action of RNA interference in body jointly, and jointly obtains Nobel Prize in Physiology or Medicine in 2006.Thus open a fan gate for the research and development of the medicine of new generation (RNA interference class medicine) of serious diseases such as opposing virus, cancer etc.This RNA disturbs that class medicine has mode of action novelty, mechanism of action is clear and definite, targeting is strong and the advantage such as side effect is little.

Human papillomavirus's (HumanPapillomavirus is called for short HPV), belongs to papovaviridae Papillomavirus, a kind of small-sized nonencapsulated virion, spherical in shape, icosahedral symmetry, diameter is 45-55nm about, and the core of virus is double-stranded DNA; 72 shell particulates that viral capsid is made up of two kinds of structural protein form.Viral DNA comprises about 8000 base pairs, comprising 8 early stage open reading frames (E1-E8), 2 single open reading frame in late periods (L1 and L2) and 1 non-coding Chang Kong district (LCR).In early days in open reading frame, E6 and E7 gene pairs Growth of Cells stimulates the most important, and the E7 albumen of independent E7 coding just can cause cervical epithelial cells immortalization.And late period reading frame L1 and L2 gene to encode respectively the main of HPV and secondary capsid protein, be assembled into the capsid of HPV.

HPV is one of most popular sexually transmitted disease source in the world, is also the important human tumor virus of a class simultaneously.Identify the hypotype more than 100 kinds of HPV viruses at present.Have common antigen between HPV is various, be present in L1 albumen conservative region, it is special that these antigens have genus.Position, epithelium place according to it infects: be divided into skin epidermis type HPV and mucous epithelium type HPV.

Determine the earliest and most important HPV related neoplasms is cervical cancer, its sickness rate is only second to mammary cancer, is the killer of serious threat women's health.About have 490 in annual world wide according to statistics, the new cases of 000 example left and right, wherein about 130,000 occurs in China, about has 270 every year, and 000 people dies from this disease, and about 50,000 is Chinese.Subsequently, more find carcinoma vulvae, the esophageal carcinoma, the mammary cancer even generation of the malignant tumour such as lung cancer, development all infects relevant to HPV.In more than the 100 kind of HPV hypotype found so far, high carcinogenic HPV hypotype (i.e. high-risk HPV) has kind more than 10, comprises HPV16,18,31,58 etc.Think at present, it is necessary but non-sufficient to cervical cancer that HPV infects.Show according to the study, 99.7% cervical cancer patient exists HPV to be infected.

The women's initial symptoms having infected HPV is not obvious.By the existence of HPV can be determined based on to the Pap of abnormal cervical cells Histological assessment test and based on the PCR of nucleic acid and hybridization technique and determines HPV hypotype.Abnormal cervical cells is classified as low SIL (LSIL) and HSIL (HSIL).Wherein LSIL comprises the cell of Cervical intraepitheliaI neoplasia one-level (CINI); HSIL comprises the cell of Cervical intraepitheliaI neoplasia secondary (CINII) and three grades (CINIII).

The medicine and the therapy that are used for the treatment of HPV infection at present mainly contain: 1, resin of podophyllium (podophyllin); 2, podophyllotoxin podophyllotoxin (podofilox); 3, trichoroacetic acid(TCA) (TCA)/dichloro acetic acid (BCA); 4, psychrotherapy; 5, excision; 6, Interferon, rabbit (IFN); 7, Imiquimod hniquimod [1-(2-methylpropyl)-1H-imidazo (4,5-quinolin-4-amine)]; 8,5 FU 5 fluorouracil/suprarenin; 9, Acyclic nucleotide analogues Cidofovir (HPMPC:[5]-1-[3-hydroxy-2-phosphonylmethoxy-propyl]-cytosine) etc.But these the not only specificitys that are antiviral and other antitumor class medicine applied clinically are not strong, and side effect is large.The medicine of a kind of special, efficient suppression HPV is not had to reduce the generation and development that even suppress its associated cancer at present in the world.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, a kind of siRNA for HPV16E6 gene is provided.

Second object of the present invention is to provide the composition of the siRNA comprised for HPV16E6 gene.

3rd object of the present invention is to provide the application of the siRNA for HPV16E6 gene.

Technical scheme of the present invention is summarized as follows:

For the siRNA of HPV16E6 gene, the target sequence of this siRNA molecule is separated from HPV16E6 gene, and wherein said target sequence is the sequence shown in SEQIDNO:2.

Described siRNA instructs the shearing of the HPV16E6mRNA sequence existed in cell, thus the suppression realized HPV16E6 genetic expression, this siRNA molecule comprises the sequence with the complementary shown in SEQIDNO:2, positive-sense strand is the sequence shown in SEQIDNO:13, and antisense strand is the sequence shown in SEQIDNO:14.

In described siRNA, at least one Nucleotide is the Nucleotide of chemically modified, and wherein said chemically modified is at least one in following modification:

(1) modification of the phosphodiester bond of Nucleotide is connected;

(2) modification of the 2'-OH of ribose;

(3) modification of base.

A kind of composition comprising the above-mentioned siRNA for HPV16E6 gene and pharmaceutically acceptable carrier composition.

Described pharmaceutically acceptable carrier is liposome or high molecular polymer.

SiRNA for HPV16E6 gene is preparing the application of cancer therapy drug.

For solving the problem, the invention provides the HPV16E6 genetic expression being caused the optimum and neoplasm of cell by small RNA molecules in inhibiting, carrying out the solution that prevention and therapy HPV16 infects relative disease.

In a first aspect of the present invention, provide small RNA molecule for the preparation of the application in the preparation suppressing HPV16E6 gene at cells, described small RNA molecule instructs the shearing of the HPV16E6mRNA sequence existed in described cell, thus the suppression described in realizing.In the present invention, described small RNA molecule comprises sense nucleic acid fragment and antisense nucleic acid fragment, described sense nucleic acid fragment and described antisense nucleic acid fragment contain complementary region, and complementary region can form double-strandednucleic acid structure, and this double-strandednucleic acid can suppress the expression of HPV16E6 gene in cell.The sense nucleic acid fragment of small RNA and antisense nucleic acid fragment may reside in two different nucleic acid chains, also may reside in same nucleic acid chains.When sense nucleic acid fragment and antisense nucleic acid fragment lay respectively on two chains, at least one chain of small RNA has the 3 ' overhang that length is 0 to 6 Nucleotide, and under preferable case, two chains all have the 3 ' overhang that length is 2-3 Nucleotide.When sense nucleic acid fragment and antisense nucleic acid fragment are present in same nucleic acid chains, under preferable case, this small RNA is hair clip type single stranded nucleic acid molecule, and wherein the complementary region of sense nucleic acid fragment and antisense nucleic acid fragment forms double-strandednucleic acid structure.In above-mentioned small RNA molecule, the length of sense nucleic acid fragment and antisense nucleic acid fragment is 19 to 29 Nucleotide, can be 19,20,21,23,25,27 and 29, is preferably 19,21,25,27 Nucleotide.Above-mentioned small RNA molecule can be imported directly in described cell, also can be to produce in cell after the nucleotide sequence of this small RNA molecule of coding is imported described cell; Described cell is preferably mammalian cell, is more preferably human cell.Above-mentioned cell can be in vitro, as clone, also may reside in mammalian body, as in human body.This human body is the patient suffering from HPV infection symptoms, and described small RNA molecule is applied enough amounts to realize the treatment to described symptom, and described HPV infection symptoms is cervical cancer or pointed condyloma.

Second aspect present invention provides a kind of HPV16E6 gene small molecule interference nucleic acid target sequence of separation, wherein said target sequence is the upper arbitrary continuation 19-30 nucleotide sequence of mRNA (cDNA) of HPV16E6, preferably, described target sequence is any sequence in SEQIDNO:1-10 in table 1.

In a third aspect of the present invention, provide a kind of small RNA molecule, it instructs the shearing of the HPV16E6mRNA sequence existed in cell, thus the suppression realized HPV16E6 expression, under preferable case, this small RNA molecule comprises the sequence with any complementary in SEQIDNO:1-10 in table 1.Nucleotide all in above-mentioned small RNA molecule can be the natural Nucleotide without chemically modified, and a Nucleotide also can be had at least to be the Nucleotide of chemically modified, and chemically modified mode is selected from least one in following modification:

(1) modification of the phosphodiester bond of Nucleotide is connected in the nucleotide sequence to described small RNA molecule;

(2) to the modification of 2 '-OH of the ribose in the nucleotide sequence of described small RNA molecule;

(3) to the modification of the base in the nucleotide sequence of described small RNA molecule.

The expression amount of HPV16E6mRNA effectively can be reduced after the cells contacting of small RNA of the present invention and expression HPV16E6mRNA.

The present invention finally additionally provides a kind of composition comprising small RNA molecule described in above-mentioned any one and pharmaceutically acceptable carrier, and under preferable case, pharmaceutically acceptable carrier is liposome or high molecular polymer.

Beneficial effect of the present invention

Small RNA molecule for HPV16E6 genetic expression provided by the invention, can suppress the expression of HPV16E6 gene efficiently, specifically, have lower toxic side effect simultaneously, can be used for preparing the medicine suppressing HPV16E6 genetic expression.

Embodiment

The present invention can cause the HPV16E6 genetic expression of the optimum and neoplasm of cell by RNA conflicting mode silence, carrys out prevention and therapy HPV16 and infects this problem of relative disease.In the present invention, HPV16E6 gene is sometimes also referred to as HPV target gene.

Small RNA molecule provided by the invention comprises and has length lower than 30 Nucleotide and be substantially complementary to the nucleic acid fragment (antisense fragments) in the region of at least part of HPV said target mrna transcript.The use of these small RNA molecules can cause HPV target gene mRNA to be sheared by the process being called as RNA interference (RNAi).Small RNA molecule provided by the invention can be natural not modified nucleic acid molecule, also can carry out chemically modified to wherein at least one Nucleotide.According to the present invention, described chemically modified is at least one in following modification:

(1) modification of the phosphodiester bond of Nucleotide is connected in the nucleotide sequence to described siRNA;

(2) to the modification of 2 '-OH of ribose in the nucleotide sequence of described siRNA;

(3) to the modification of base in the nucleotide sequence of described siRNA.

Described chemically modified is conventionally known to one of skill in the art, and the modification of described phosphodiester bond refers to modifies the oxygen in phosphodiester bond, comprises thiophosphoric acid and modifies, as shown in Equation 1; With boranated phosphate modified, as shown in Equation 2.Two kinds of modifications can stablize siRNA structure, keep high specific and the high-affinity of base pairing.

Described ribose modifies the modification referred to 2 '-OH in Nucleotide pentose, that is, introduce some substituting group in the hydroxy position of ribose, and such as, 2 '-fluoro is modified, as shown in Equation 3; 2 '-oxygen methyl is modified, as shown in Equation 4; 2 '-oxygen ethylenemethoxy is modified, as shown in Equation 5; 2,4 '-dinitrophenol(DNP) is modified, as shown in Equation 6; Lock nucleic acid (LNA), as shown in Equation 7; 2 '-amido modified, as shown in Equation 8; 2 '-deoxidation is modified, as shown in Equation 9.

Described base modification refers to be modified the base of Nucleotide, and such as, 5 '-bromouracil is modified, as shown in Equation 10; 5 '-iodouracil is modified, as shown in Equation 11; N-methyl uracil is modified, as shown in Equation 12; 2,6-diaminopurine is modified, as shown in Equation 13.

These modifications can increase the bioavailability of described small RNA molecule, improve the affinity with target sequence, strengthen the ability of resisting nuclease hydrolysis in cell.

In addition, in order to promote that small RNA molecule enters cell, can on the above basis modified, introduce the lipophilic groups such as cholesterol at the end of the positive-sense strand of small RNA molecule and be beneficial to cytolemma by being made up of lipid bilayer and intracellular mRNA has an effect.

By the test based on cell and animal level, contriver confirms that these small RNA molecules can mediate rna i effectively, causes the remarkable suppression of HPV16E6 genetic expression, and suppresses the growth of implanting knurl body.Therefore, comprise small RNA molecule of the present invention and can treat by target HPV target gene the pathologic process being infected mediation by HPV.

The preparation method of small RNA molecule provided by the invention comprises sequences Design and sequent synthesis.

The synthesis of described small RNA molecular sequences can adopt the method for chemosynthesis, or entrusts the biotech company's synthesis specializing in nucleic acid synthesis, synthesizes as entrusted Ai Bosi bio tech ltd, Shanghai.

In general, the method for described chemosynthesis comprises following Four processes: the synthesis of (1) Oligoribonucleotides; (2) deprotection; (3) purifies and separates; (4) desalination.

Such as, the concrete steps with the siRNA chemosynthesis of nucleotide sequence shown in HPV16E6si01 are as follows:

(1) synthesis of Oligoribonucleotides

At automated DNA/RNA synthesizer (such as, AppliedBiosystemsEXPEDITE8909) RNA of upper setting synthesis 1 mmole, the coupling time simultaneously setting each circulation is 10-15 minute, initiator is that 5 '-O-of solid diffusivity is to dimethoxy-thymidine upholder, first circulates in a connection base on solid support, then in n-th time (19 >=n >=2) circulation, the base that (n-1)th circulation connects connects a base, repeats this circulation until complete the synthesis of whole nucleotide sequence.

(2) deprotection

The solid support being connected with siRNA is joined in test tube, and in this test tube, add the ethanol/ethamine (volume ratio is 1:3) of 1 milliliter, then seal, be placed in 55-70 DEG C of incubator, hatch 2-30 hour, fech connection has the solid support of siRNA and with distilled water drip washing 2 times (each 1 milliliter), collects elutriant, and at room temperature dry 30 minutes.Then, add the tetrahydrofuran solution (1M) of 1 milliliter of tetrabutyl ammonium fluoride, room temperature places 4-12 hour, then adds 2 milliliters of ethanol, and namely collecting precipitation obtains the crude product of siRNA.

(3) purifies and separates

The crude product of the siRNA obtained being dissolved in 2 ml concns is in the ammonium acetate solution of 1 mole/milliliter, is then separated by C18 high pressure liquid chromatography, obtains the siRNA product of purifying.

(4) desalination

Be siRNA product 2-4 time (each 2 milliliters) of the aqueous ethanolic solution washing purifying of 75 % by weight by concentration, to remove salt, and dry under room temperature.Then by oligonucleotides mixed dissolution (10mMTris in the damping fluid of 1-2 milliliter of positive-sense strand and antisense strand, pH=7.5-8.0,50mMNaCl), this solution is heated to 95 DEG C, then slowly this solution is cooled to room temperature, and maintain room temperature 16-22 hour, obtain the solution containing siRNA.

This research finds, after above-mentioned siRNA transfectional cell, and can the effectively expression of HPV16E6RNA and albumen in T suppression cell and animal heteroplastic transplantation knurl.

Below in conjunction with specific embodiment and accompanying drawing, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.

Embodiment 1: design HPV16E6 small RNA sequence

HPV16E6mRNA sequence obtains from the biomedical website ncbi database (http://www.ncbi.nlm.nih.gov/) that U.S. National Institutes is built, at the siRNA sequence of siDESIGNCenter and DSIR Photographing On-line specificity for this gene.By the principle choosing small RNA molecular sequences of online software definition be: mainly based on the linear model being combined with small RNA molecular sequences and target sequence feature.The high stability of small RNA molecule 5 ' end may block small RNA molecule positive-sense strand and be loaded into RNA induction silencing complex (RISC); The low stability of small RNA Molecules Antisense chain 5 ' end may promote that small RNA Molecules Antisense chain is loaded into RISC; And the low stability of small RNA molecule region intermediate can promote that RISC-small RNA molecular complex discharges.

According to the principle of design of above-mentioned small RNA molecular sequences, design 10 Smal linterference ribonucleic acid sequences for HPV16E6 (siRNA) (the seeing the following form 2) of the dsRNAHPV16E6si01 ~ HPV16E6si10 comprising positive-sense strand and antisense strand, described small RNA molecule comprises 2 deoxythymidylic acids (T) at 3 ' end of positive-sense strand and antisense strand.In described dsRNA, HPV16E6si01 ~ HPV16E6si10 total length is the structure with two Nucleotide 3 ' protruding terminuses of 21bp.What in positive-sense strand, overstriking represented be through Nucleotide that 2 '-methoxyl group modifies.

Table 1.HPV16E6 gene target sequence

Sequence names	Target sequence (5 '-3 ')	Corresponding sequence number
			HPV16E6 01	CCGTTGTGTGATTTGTTAA	SEQ ID NO:1
HPV16E6 02	GCAAACAACTATACATGAT	SEQ ID NO:2
			HPV16E6 03	CAATGTTTCAGGACCCACA	SEQ ID NO:3
HPV16E6 04	CGATGTATGTCTTGTTGCA	SEQ ID NO:4
			HPV16E6 05	GACCCAGAAAGTTACCACA	SEQ ID NO:5
HPV16E6 06	GTACTGCAAGCAACAGTTA	SEQ ID NO:6
			HPV16E6 07	GTGTGTACTGCAAGCAACA	SEQ ID NO:7
HPV16E6 08	AGCAAAGACATCTGGACAA	SEQ ID NO:8
			HPV16E6 09	GGATTTATGCATAGTATAT	SEQ ID NO:9
HPV16E6 10	AGATCATCAAGAACACGTA	SEQ ID NO:10

Table 2HPV16E6 Smal linterference ribonucleic acid sequence

Embodiment 2: the efficiency that dsRNA sequence described in Validation in vitro suppresses HPVE6 to express

(1) cell cultures

Human cervical cancer cell lines (SiHa, ATCC) application contains the Pen .-Strep (PS of 1%, Gibco) antibiotic mixture and 10% calf serum (FBS, Gibco) MEM substratum (Gibco), 37 DEG C, cultivate in the cell culture incubator of 5% carbonic acid gas and 95% atmospheric moisture.

(2) cell transfecting

(1) day before transfection, SiHa cell is inoculated in the medium, and the degree of converging to cell during transfection is 30 ~ 50%.

(2) transfection sample is prepared:

Be siRNA (see table 1) and the 0.5 μ l lipofectamine Lipofectamine of 20 μMs respectively by 1 μ l concentration ^tM2000 (Invitrogen) are diluted in 0.05mlOpti-MEM (Invitogen), mix gently, hatch 5 minutes.Mix gently, hatch 5 minutes.

With meaningless contrast siRNA, (its positive-sense strand comprises: 5-UUCUCCGAACGUGUCACGU-3; Antisense strand comprises: 5-ACgUgACACgUUCggAgAA-3) and lipofectamine Lipofectamine ^tM2000 (Invitrogen) carry out transfection, as Normal group (NC group).

Get siRNA group and the NC group of dilution, mix gently, at room temperature hatch 20 minutes, form siRNA-Lipofectamine2000 (Invitrogen) mixture and meaningless siRNA-Lipofectamine2000 (Invitrogen) mixture.Incubation time is no more than 30 minutes, and longer incubation time may reduce activity.

SiRNA-LipofectamineTM2000 mixture being joined each comprises in the hole of cell and substratum, and by the culture plate mixing that rocks back and forth lightly, at 37 DEG C, CO2 incubator hatches 24 hours.Do not need remove mixture or change substratum.But, within 4 ~ 6 hours, change the activity that substratum also can not lose transfection after transfection.

Table 3 gives preferred amount and volume by using reagent during various tissue culture mode transfectional cell.

Amount and the volume of reagent is used during table 3 transfectional cell

(3) Total RNAs extraction, reverse transcription and real-time quantitative PCR gene expression detection

After transfection 24 hours, collecting cell.The cultured cells RNA extraction agent (RNAisoPlus, TAKARA, Cat.No.9108) of TAKARA company carries out the extracting of cell RNA.Sucking-off cell culture fluid, cleans once with 1 × PBS.Add the RNAisoPlus of 1-2ml in the culturing cell of every 10cm2 growth, weak vibrations, guarantees to make lysate be uniformly distributed in cell surface.Interior celliferous lysate is transferred in centrifuge tube, with liquid-transfering gun repeatedly pressure-vaccum until without obvious sediment in lysate.Room temperature (15-30 DEG C) leaves standstill 5 minutes, then isolation of RNA from nucleoprotein.In homogenate lysate, add chloroform (1/5 volume of RNAisoPlus), cover tightly centrifuge tube lid, be mixed to emulsifying soln and be creamy white.Room temperature leaves standstill 5 minutes, and 12,000g4 DEG C centrifugal 15 minutes.From whizzer, take out centrifuge tube, now homogenate is divided into three layers, that is: colourless supernatant liquor (containing RNA), middle white egg white (major part is DNA) and be with coloured lower floor organic phase.Aspirate supernatant is transferred in another new centrifuge tube.Isopyknic Virahol is added in supernatant, after the centrifuge tube that turns upside down fully mixes, left at room temperature 10 minutes.12,000g4 DEG C centrifugal 10 minutes.Supernatant discarded, adds 75% ethanol with RNAisoPlus equivalent, and turn upside down washing centrifuge tube tube wall gently, and 7,500 × g4 DEG C of careful supernatant discarded after centrifugal 5 minutes, open centrifuge tube lid, drying at room temperature precipitation several minutes.After precipitation drying, add appropriate RNase-free water dissolution precipitation.

PrimeScriptTMRTMasterMix (PerfectRealTime) (Cat.No.RR036A) reverse transcription of the RNA TAKARA company of 100ng extracting is become cDNA.The RNA DEPC water of extraction is blown afloat (8 μ l) totally, add 2 μ l5 × PrimeScriptRTMasterMix (containing PrimeScriptRTase, RNaseInhibitor, Random6mers, OligodTPrimer, dNTPMixture, reaction Buffer), put into 37 DEG C of water-baths 15 minutes, 85 DEG C of water-baths 5 seconds, then ice bath fast.

With this cDNA for template carries out the expression level that real-time quantitative polymerase chain reaction (RealtimePCR) carrys out detection by quantitative HPV16E6mRNA.First adopt SDS2.1 software relative standard curve method to calculate mrna expression level, from corresponding curve, determine Ct value (Ct), Ct value reflects the relative expression levels of each target gene.Then, divide aim parameter by β globin gene (glyceraldehyde-3-phosphate dehydrogenase is abbreviated as GAPDH) contrast and obtain normality target value, thus determine the relative expression levels of mRNA.

The primer sequence used in experiment:

HPV16E6 upstream primer: 5-CACAGGAGCGACCCAGAAAG-3

HPV16E6 downstream primer: 5-GCATAAATCCCGAAAAGCAA-3

GAPDH upstream primer: 5-GGCATGGACTGTGGTCATGAG-3

GAPDH downstream primer: 5-TGCACCACCAACTGCTTAGC-3

Calculate the efficiency of institute siRNA inhibition of gene expression by following formulae discovery:

In formula, " process cell " refers to the cell of siRNA in difference transfection table 1, and " compared with control cells " refers to the cell of transfection meaningless contrast siRNA, and " Relative Expression values " is the ratio of HPVE6mRNA and GAPDHmRNA in phalangeal cell.

The efficiency that the suppression HPVE6mRNA that table 4 shows transfection described siRNA after 24 hours expresses.

The suppression efficiency that table 4siRNA expresses HPVE6mRNA

Double-strand title	Suppression efficiency	Standard deviation
			HPV16E6si01	62.7％	10.4％
HPV16E6si02	87.3％	4.0％
			HPV16E6si03	83.2％	1.4％
HPV16E6si04	65.7％	7.7％
			HPV16E6si05	84.6％	6.0％
HPV16E6si06	87.6％	1.6％
			HPV16E6si07	99.8％	0.2％
HPV16E6si08	80.4％	7.3％
			HPV16E6si09	85.1％	4.3％
HPV16E6si10	79.3％	13.9％

Embodiment 3: dsRNA sequence described in Validation in vitro suppresses the propagation of Human cervical cancer cell lines SiHa

(1) cell cultures

The same

(2) cell transfecting

The same

(3) tetrazolium salts (MTT) measures cell inhibitory rate

Every hole adds the MTT solution (5mg/ml, i.e. 0.5%MTT) of 1/10 culture volume, continues cultivation 4 hours.Stop cultivating, suck nutrient solution in hole, every hole adds 150 μ lDMSO, to put on shaking table low-speed oscillation 10 minutes, crystallisate is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD570nm place.Be calculated as follows cell inhibitory rate: cell inhibitory rate=(1-treatment group absorbance/control group absorbance) × 100%.Experimental result such as following table 5 shows,

Table 5siRNA is to cervical cancer tumer line (SiHa) growth inhibition ratio

Double-strand title	Suppression efficiency	Standard deviation
			HPV16E6si01	24.8％	4.6％
HPV16E6si02	39.1％	3.7％
			HPV16E6si03	33.8％	4.7％
HPV16E6si04	23.5％	5.7％
			HPV16E6si05	18.6％	4.1％
HPV16E6si06	14.7％	10.2％
			HPV16E6si07	11.3％	4.6％
HPV16E6si08	4.7％	7.3％
			HPV16E6si09	6.4％	6.1％
HPV16E6si10	7.0％	14.1％

Embodiment 4: in Mice Body, the growth that described siRNA sequence can suppress to implant cervical cancer cell is injected in experimental verification

Cervical cancer cell (SiHa) is expelled to 6 weeks large BALB/c Female nude mice dorsal sc lotus knurls, each injected in mice 2x10 ⁶individual cell.Within after lotus knurl the 14th day, in knurl, use microneedle injection HPV16E6si02 and meaningless dsRNA, every injection in three days once, per injection 5 μ lsiRNA solution (10 μ g/ μ l).Within after lotus knurl the 23rd day, analyze tumor weight.Experimental result such as following table 6 shows, and HPV16E6si02dsRNA can suppress the growth of implanting knurl body.

Table 6HPV16E6si02 suppresses the growth of implanting knurl body

Double-strand title	Tumor weight	Standard deviation
			HPV16E6si02	0.129g	0.019g
Meaningless dsRNA	0.266g	0.031g

Claims (6)

1., for the siRNA of HPV16E6 gene, it is characterized in that the target sequence of this siRNA molecule is separated from HPV16E6 gene, wherein said target sequence is the sequence shown in SEQIDNO:2.

2. the siRNA for HPV16E6 gene according to claim 1, it is characterized in that described siRNA instructs the shearing of the HPV16E6mRNA sequence existed in cell, thus the suppression realized HPV16E6 genetic expression, this siRNA molecule comprises the sequence with the complementary shown in SEQIDNO:2, positive-sense strand is the sequence shown in SEQIDNO:13, and antisense strand is the sequence shown in SEQIDNO:14.

3. the siRNA for HPV16E6 gene according to claim 2, is characterized in that in described siRNA, at least one Nucleotide is the Nucleotide of chemically modified, and wherein said chemically modified is at least one in following modification:

(1) modification of the phosphodiester bond of Nucleotide is connected;

(2) modification of the 2'-OH of ribose;

(3) modification of base.

4. one kind comprises the composition of the siRNA for HPV16E6 gene described in Claims 2 or 3 and pharmaceutically acceptable carrier composition.

5. composition according to claim 4, is characterized in that described pharmaceutically acceptable carrier is liposome or high molecular polymer.

6. the siRNA for HPV16E6 gene of one of claim 1-3 is preparing the application of cancer therapy drug.