CN100357436C - SiRNA for inhibiting Stat3 gene expression and preparation thereof - Google Patents

Abstract

The invention discloses a siRNA for inhibiting Stat3 gene expression and a preparation method thereof. The siRNA inhibiting the expression of the Stat3 gene, the siRNA expression plasmid comprising: pBS/U6 vector; the DNA fragment encoding the siRNA specifically inhibiting the expression of the Stat3 gene. The present invention uses DNA-mediated RNA interference technology to construct Stat3 gene-specific RNAi, which is used to observe the changes in signaling pathways when Stat3 is specifically inhibited in cells, thereby providing an effective method for studying the function of Stat3 in cell lines. It has broad application prospects in the field of treatment and research of tumors related to Stat3.

Description

siRNA inhibiting Stat3 gene expression and preparation method thereof

technical field

The present invention relates to an siRNA, in particular to an siRNA for inhibiting Stat3 (signal transducer and activator oftranscription) gene expression and a preparation method thereof.

Background technique

RNAi (RNA interference) technology is a new method of inhibiting gene expression developed in recent years. This method is to use the same gene promoter region (exon) or promoter region with unknown function in the way of reverse repetition. In this way, the RNA transcribed in the transgenic individual can form dsRNA, produce RNA interference, and silence the target gene. This phenomenon has been observed in plants, fungi, trypanosomes, planaria, hydra, drosophila, nematodes (canenorhabdit is elegans), zebrafish , mouse early embryos and other organisms have been found in different species.

The exogenous or endogenous dsRNA of RNAi binds to Dicer, an RNase III (RNAase III endonuclease) in the cell, and is then cut into 21-23 nt single-stranded tails with 3′ ends and The short dsRNA (double-stranded RNA) at the phosphorylated 5′ end is the initial inducer of RNAi, that is, siRNA (small interference RNA). siRNA and Dicer form RNA-induced silencing complex (RNAinduces silencing complex, RISC). siRNA acts as a guide sequence, which recognizes the mRNA transcribed from the target gene according to the principle of base complementarity, and guides RICS to bind to the mRNA. Then the siRNA and mRNA transpose in the complex, and the nuclease Dicer cuts the mRNA into 21-23nt fragments to specifically inhibit the expression of the target gene. The newly generated dsRNA fragments can form RISC again and continue to degrade mRNA, thereby producing a cascade amplification effect. Therefore, only a few siRNA molecules per cell are needed to elicit a strong RNAi effect. In addition, siRNA can also be amplified in large quantities under the action of RNA-dependent RNA polymerase (RdRp), and transported out of cells, so that RNAi can spread throughout the body and be passaged.

At present, RNAi technology has been widely used in the research fields of gene function, signal transduction pathway methods and tumor gene therapy.

RNAi recognition can be accurate to one nucleotide, and can produce accurate and effective blocking effects on oncogenes formed by wild-type point mutations, such as ras, p53, etc., while wild-type genes are not affected, and can be used to block certain mutations Gene expression, or malignant transformation caused by protein overexpression. Compared with traditional gene therapy methods such as gene replacement, antisense oligonucleotide therapy, and cytokine gene therapy, RNAi has the characteristics of specificity, high efficiency, and low toxicity in tumor gene therapy, so it can be used to treat some tumors. RNAi technology is used to inhibit the expression of tumor growth factors, which can inhibit the growth of tumors in animal models, and has been applied to rapidly identify new tumor targets. Since tumors are multi-gene and multi-factor diseases, the inhibition of a single oncogene is often difficult to achieve therapeutic effects. RNAi technology can inhibit multiple different genes at the same time, and the inhibitory effects do not interfere with each other.

The inhibitory effect of RNAi on tumor-related genes in human cervical cancer cell Hela, non-small cell lung H1299, head and neck squamous cell carcinoma C33, osteosarcoma U22OS, breast cancer MCF27 and other cell lines is also obvious.

The sequence specificity of siRNA is very strict, and a single base mismatch with the target mRNA will significantly weaken the effect of gene silencing.

Stat (signal transducer and activators of transcription) signal transducer and activator of transcription) is a class of potential signal transducer and activator of transcription existing in the cytoplasm. Its protein family has 7 members, Stat1, 2, 3, 4, 5a, 5b, and 6. Among them, Stat3 plays a very important role in cell proliferation, differentiation, survival and embryonic development. The overactivation of Stat3 signaling channel destroys the proliferation and survival activities of normal cells, and can induce the characteristics of some transformed cells; overexpressed Stat3 regulates the control of cell cycle and apoptosis, indicating that the activation of Stat3 may be related to carcinogenesis , Blocking the Stat3 signaling pathway is expected to prevent and treat human tumors. Therefore, using the above-mentioned RNAi technology to block the Stat3 signaling pathway can prevent and treat human tumors.

Contents of the invention

The purpose of the present invention is to provide a siRNA for inhibiting Stat3 gene expression. It uses DNA-mediated RNA interference technology to construct Stat3 gene-specific RNAi, which is used to observe the expression and signaling pathway changes when Stat3 is specifically inhibited in cells, and provides an effective tool for studying the function of Stat3 from cell lines , has broad application prospects in the field of research and treatment of tumors related to Stat3.

Another object of the present invention is to provide a method for preparing siRNA that inhibits Stat3 gene expression.

Above-mentioned purpose of the present invention can be realized by following measures:

Design double-stranded siRNA fragments to inhibit Stat3 gene expression. The designed sequence of Stat3 RNAi and its target mouse Stat3 gene (serial number AY299489) in Genbank database of NCBI website are as follows:

Article 1 (action site 157-174):

CACCAACGTGGCATGTGACTTTTTT＜3’5'>TCACATGCCACGTTGGTG

CACCAACGTGGCATGTGACTTTTTT<3'

Article 2 (Action site 1226-1243):

GGGTCAGGTGCTTGAACTCTTTTTT＜3’5'>AGTTCAAGCACCTGACCC

GGGTCAGGTGCTTGAACTCTTTTTTT<3'

Article 3 (Action site 1936-1953):

TGACATGTTGTTCAGCTGCTTTTTT＜3’5'>CAGCTGAACAACATGTCA

TGACATGTTGTTCAGCTGCTTTTTT<3'

Article 4 (Action site 2334-2351):

GTCACGTCTCTGCAGCTTCTTTTTT＜3’5'>AAGCTGCAGAGACGTGAC

GTCACGTCTCTGCAGCTTCTTTTTT<3'

The siRNA of the present invention can be chemically synthesized in vitro and expressed in vivo.

Wherein the U6 promoter is used to initiate the transcription of downstream DNA, the transcription start site is a G (guanine), and the transcription stopper is 5-6 consecutive T (thymine). The DNA in the coding region can be transcribed into mRNA with a specific sequence. The mRNA does not encode amino acids that can be translated, but automatically forms a hairpin RNA with a loop in the cell. mRNA degradation.

The vector containing the type III RNA polymerase eukaryotic promoter of the present invention is a pBS/U6 vector, which encodes a DNA fragment of siRNA that specifically inhibits Stat3 gene expression.

The molecular type of the siRNA expression plasmid of the present invention is circular DNA, with a size of 3303bp.

The inventor is committed to anti-tumor and genetic engineering research, and uses years of accumulated knowledge and experience to carry out RNAi anti-viral treatment research. A large number of studies and experiments have proved that using DNA-mediated RNA interference technology to construct Stat3 gene-specific RNAi can be used to observe the changes in expression and signaling pathways when Stat3 is specifically inhibited in cells, which is the purpose of the present invention. The cell line provides an effective tool for studying the function of Stat3, so the invention is listed as the third batch of scientific research fund projects in 2004 of the National Natural Science Foundation of China and the Chongqing Municipal Population and Family Planning Commission.

The present invention uses RNA interference (RNA interference, RNAi) to suppress the expression of the Stat3 gene. The degradation of the target mRNA is mediated by exogenously introducing dsRNA (double-stranded RNA-that is, double-stranded RNA) that is homologous to the endogenous gene, thereby leading to the silencing of specific genes in the organism. RNA interference shows extremely important application value in gene expression regulation, defect recognition and disease gene therapy by shutting down the expression of specific genes.

The invention aims at the effects of Stat3 on cell growth, survival and differentiation in the embryonic stage, and the RNA interference plasmid for mouse Stat3 can be used in mouse embryos, and designs a recombinant plasmid pBS/U6-Stat3-RNAi that inhibits Stat3 gene expression. Using siRNA as a guide sequence, it recognizes the mRNA transcribed from the target gene according to the principle of base complementarity, and guides RICS to bind to the mRNA. Then the siRNA and mRNA transpose in the complex, and the nuclease Dicer cuts the mRNA into 21-23nt fragments to specifically inhibit the expression of the target gene. The newly generated dsRNA fragments can form RISC again and continue to degrade mRNA, thereby producing a cascade amplification effect, which makes the RNA interference effect of Stat3 obvious, and can almost completely inhibit the exogenous Stat3.

The present invention uses the newly developed RNA interference technology to directly reduce the accumulation of Stat3 gene mRNA from the transcriptional level, and directly lead to the reduction of the accumulation of Stat3 protein, thereby inhibiting the Stat3 signaling pathway. The present invention uses Stat3 antisense oligonucleotides, Stat3 dominant negative protein Stat3β, antagonists of Stat3 upstream specific receptors and receptor neutralizing antibodies, or naturally occurring proteins such as SOCS and PIAS to inhibit Stat3 signal transduction The technology and method of pathway therapy for tumors are more convenient to operate and more effective.

Description of drawings

Fig. 1 is the electrophoresis diagram (positive result and negative control) of Stat3 gene interference plasmid digestion and identification;

Fig. 2 is that small interfering RNA suppresses the expression of Stat3 protein in 293T cells;

Fig. 3 is that RT-PCR detects that 4 kinds of Stat3 interference plasmids suppress the expression of Stat3 mRNA in 293T cells;

Figure 4 is the dosage effect of small interfering RNA inhibiting the expression of Stat3 protein in 293T cells in immunostaining experiments;

Fig. 5 is immunoblotting experiment detection Stat3 interfering plasmid inhibits the dose effect of Stat3 protein expression in 293T cells;

Fig. 6 is RT-PCR detection Stat3 interfering plasmid suppresses the dose effect of Stat3 mRNA expression in 293T cells;

Figure 7 is a simple schematic diagram of the siRNA stem-loop structure;

Fig. 8 is pBS/U6-Stat3-RNAi (2334) plasmid map;

Detailed ways

First synthesize the siRNA interference sequence, then process the RNAi vector, connect the processed vector to the processed dsRNA, then transform, select positive clones, extract the plasmid, and finally identify the Stat3 gene interference plasmid.

In the screening of the positive clones of the constructed interfering plasmids, we adopted the convenient and feasible double-enzyme-digestion negative identification method. For the identification of the effectiveness of this plasmid, we used Western blotting at the level of protein detection, semi-quantitative RT-PCR at the level of mRNA detection, and immunostaining methods with intuitive results under the microscope, and added a dose-effect comparison. Therefore, it can be clearly seen that the inhibitory effect of the constructed plasmid on the Stat3 gene.

The specific method is as follows:

1. Synthetic siRNA interference sequence:

The basic principles of synthesis are:

1. Starting from the AUG start codon of the mRNA, search for a sequence of 22 bp in length of the "AGN18TT" (wherein N is any base) structure;

A) Avoid selecting the target sequence at the start codon or nonsense region;

B) The GC content of the sequence is 30%-60%;

C) Since the 5' and 3' untranslated regions (UTRs) are rich in regulatory protein structural regions, these UTR binding proteins or translation initiation complexes may affect the binding of siRNA endonuclease complexes to mRNA and thus affect the effect of siRNA , so the design does not target the non-coding regions (UTRs) at the 5' and 3' ends;

D) Compare the selected sequences with corresponding genome databases such as human, mouse, rat, etc., and exclude sequences homologous to other coding sequences/ESTs, for example using BLAST;

F) select the appropriate target sequence to synthesize to find the most effective siRNA sequence;

G) Three or more Ts or As cannot appear consecutively on the sense strand and antisense strand sequence, otherwise it may lead to premature termination of siRNA transcription.

H) The terminal nucleotide of the interfering sequence is preferably C or T.

I) Try to avoid the interference sequence at the beginning and end of the target gene.

2. The DNA cloned into the siRNA expression vector includes two short inverted repeat sequences separated by a loop sequence in the middle to form a hairpin structure controlled by the polIII promoter. Then connect 5-6 T as the transcription terminator of RNA polymerase III.

3. Both ends of the two complementary oligonucleotides must have restriction enzyme sites (ApaI and EcoRI were selected in this experiment).

4. There is a C immediately below the enzyme cleavage site downstream of the promoter, so that there is a certain space between the inserted fragment and the promoter to ensure the occurrence of transcription. The first base of the siRNA target sequence must be G to ensure RNA polymerase transcription. If it does not start with a G, a G must be added immediately upstream of the justice chain.

5. The loop structure in the siRNA insert should be close to the center of the oligonucleotide. The size and nucleotide sequence of the circle can vary, but inclusion of a unique restriction site within it facilitates detection of clones with siRNA inserts. After comparing many loops with different lengths and sequences, it was found that the 5'TTCAAGAGA3' sequence was the most effective, so we adopted this sequence as the loop structure sequence.

After the interference sequence is determined, the linker connecting the positive sequence fragment and the reverse sequence fragment of the interference sequence will be obtained, and in order to stop the transcription of the interference sequence in time, polyT is added at the end of the interference sequence as a stop, and the pBS/U6 multiple cloning site is used As the endonucleases that can be used, ApaI and EcoRI are selected as the insertion sites, and appropriate bases matching the two endonucleases are respectively added at the end of the oligonucleotide sequence.

According to the above principles, four interference sequences were selected according to the full-length sequence of mouse Stat3 in GenBank.

The oligonucleotide sequences of four pairs of in vivo expression siRNA (short hairpin RNA) designed for Stat3 are:

157 position:

chain of justice

5'>TCACATGCCACGTTGGTG CACCAACGTGGCATGTGACTTTTTG<3';

antisense strand

CACCAACGTGGCATGTGA＜3’；5'>AATTCAAAAGTCACATGCCACGTTGGTG

CACCAACGTGGCATGTGA<3';

1226 position:

chain of justice

GGGTCAGGTGCTTGAACTCTTTTTTG＜3’；5'>AGTTCAAGCACCTGACCC

GGGTCAGGTGCTTGAACTCTTTTTTTG<3';

antisense strand

GGGTCAGGTGCTTGAACT＜3’；5'>AATTCAAAAAAGAGTTCAAGCACCTGACCC

GGGTCAGGTGCTTGAACT<3';

1936 site:

chain of justice

TGACATGTTGTTCAGCTGCTTTTTTG＜3’；5'>CAGCTGAACAACATGTCA

TGACATGTTGTTCAGCTGCTTTTTTG<3';

antisense strand

TGACATGTTGTTCAGCTG＜3’；5'>AATTCAAAAAAGCAGCTGAACAACATGTCA

TGACATGTTGTTCAGCTG<3';

2334 site:

chain of justice

GTCACGTCTCTGCAGCTTCTTTTTTG＜3’；5'>AAGCTGCAGAGACGTGAC

GTCACGTCTCTGCAGCTTCTTTTTTG<3';

antisense strand

GTCACGTCTCTGCAGCTT＜3’；5'>AATTCAAAAAAGAAGCTGCAGAGACGTGAC

GTCACGTCTCTGCAGCTT<3';

The sequence in the box is a loop structure sequence.

Interference sequence processing:

The 4 interference sequences were synthesized by Shanghai Sangong, all of which were 2OD (2 tubes, 1OD/tube, 1OD=33μg). Each synthesized oligonucleotide sense and antisense was made into a final concentration of 250umol/L solution, then take 100 μl each of the sense strand and the antisense strand and mix them evenly in a ratio of 1:1, put them in a beaker filled with water and boil for 10 minutes, then cool naturally at room temperature to anneal the oligonucleotide strands, and then The corresponding double-stranded DNA can be formed. That is, the insert fragment siRNA in the pBS/U6 vector was obtained.

During operation, siRNA fragments can be directly added to the enzyme-linked reaction system. This structure can produce siRNA in cells, and the siRNA expressed in vivo is shown in FIG. 7 .

2. Treatment of RNAi vector

Because the target gene driven by the U6 promoter must start with guanine, and there is exactly one guanine at the 3' end of the product after digestion with ApaI endonuclease, just cut off the 5' sticky end of the complementary strand, and then insert the target gene through blunt-end ligation The interfering sequence of the target gene can be used to construct the interfering plasmid of the target gene, so the pBS/U6 vector used is constructed by inserting the U6 promoter and an appropriate multiple cloning site based on pBlueScript. The pBS/U6 vector used is the vector described in Sui G et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 5515-5520.

The RNAi vectors are processed as follows:

1. Take 2 μg of pBS/U6 vector, digest with ApaI endonuclease overnight at room temperature, and cut Klenow enzyme after confirming that the enzyme digestion is complete. The enzyme digestion system is as follows:

20μl

2. After DNA recovery, take an appropriate amount of pBS/U6 after digestion with ApaI, and set up the following Klenow digestion system:

20μl

Digest at room temperature for 30 minutes to confirm that the digestion is complete. Place the reaction system in a water bath at 75 degrees Celsius to terminate the reaction, and then anneal the heat-shocked reaction system on ice.

3. Directly carry out solution system DNA recovery or gel recovery

NucleoTrap Gel Extraction Kit (purchased from BD Biosciences) was used for solution recovery, and DNA was recovered according to the experimental method provided by the manufacturer.

4. The recovered DNA fragments are directly digested with the endonuclease selected downstream. In this experiment, EcoRI enzyme is selected to ensure that the enzyme digestion is complete. The enzyme digestion system is:

37 degrees Celsius water bath, more than 2 hours

5. The digested product is recovered and purified by DNA electrophoresis

After digestion, take a small amount (such as 2 μl) to dilute and run DNA electrophoresis to judge whether the digestion is complete. If it is not complete, you can extend the digestion time or increase the amount of enzyme, if it is complete, you can go to the next step. Run all enzyme digestion systems on 1% DNA gel, 110V, 20-30 minutes. Strips were seen under UV light. Observe whether the band size is correct and whether the enzyme digestion is complete or not in a gel imager. Then use a knife to cut out the piece where the DNA is concentrated. Weigh a clean EP tube in advance, and then weigh the weight after adding the cut gel to obtain the weight of the DNA gel.

Then recover the DNA in the gel according to the usual experimental method.

So far the pBS/U6 vector has been processed.

3. Ligate the processed pBS/U6 vector with the processed dsDNA

The principle of connecting the treated pBS/U6 vector to dsDNA: the mass ratio of vector to dsDNA is 1:20 to 1:40.

Use the Takara Biotech DNA Ligation Kit Ver.2 kit (purchased from Takara) for rapid connection for more than 30 minutes to make the connection sufficient.

It can be left overnight at 16°C.

4. Transformation, selection of positive clones, and extraction of plasmids:

10 µl of the reaction solution left overnight for the ligation was used for all transformations by the usual method. After completion, place the coated plate in a 37°C incubator for 16-20 hours. The plate should be placed upright first, and the plate should be inverted after the bacterial solution is dry. After 16-20 hours, observe the plate, and there are colonies growing. Since the transformation of the ligation product is performed, the number of colonies is not as large as that produced by the transformation of the plasmid.

Pick a single clone, shake the bacteria, shake at 37 degrees Celsius, 9-12 hours, take out the LB tube, and extract the plasmid: use the plasmid small sample rapid extraction kit of Beijing Broadtech Biogene Technology Co., Ltd. to extract the plasmid according to the experimental method provided by the manufacturer , the plasmid map of its plasmid is shown in Figure 8. (Take the plasmid constructed from the fourth sequence as an example)

Five. Identification of Stat3 gene interference plasmid

In the screening of the positive clones of the constructed interfering plasmids, we used the negative identification method of double enzyme digestion. The endonucleases selected during interfering sequence insertion are ApaI and EcoRI. After the sequence is inserted, the endonuclease recognition sites between ApaI and EcoRI in the multiple cloning site will be replaced, which are XhoI, SalI, HindIII and EcoRV respectively. According to the pBS/U6 plasmid map, there are three endonuclease recognition sites in front of the U6 promoter, namely KpnI, XbaI and BamH I. Therefore, arbitrarily select an endonuclease in front of a U6 promoter and an endonuclease that can be replaced by an insert sequence to perform double digestion, and the interference vector will be single-digested to obtain a linear interference vector, and if the interference sequence is selected, it cannot be replaced. The endonuclease in front of the promoter and the endonuclease in front of the promoter should be able to cut out a fragment together with the U6 promoter and the interference sequence, and the size should be slightly larger than the U6 promoter. If there is no interfering sequence inserted, the result of enzyme digestion should show about 300bp of U6 promoter and about 3kb of pBlueScript backbone.

KpnI and EcoRV and KpnI and XhoI were used. The identification results are shown in Figure 1. After repeated screening, we got 4 positive interfering plasmids. In Figure 1, 1-marker; 2,3-pBS/U6 empty vector was digested with double enzymes (as a negative control); 4,5-plasmid pBS/U6-Stat3-RNAi was digested with double enzymes (positive result). The 2nd and 4th lanes are KpnI and EcoRV double enzyme digestion, and the 3rd and 5th lanes are KpnI and XhoI double enzyme digestion. The endonucleases selected during interfering sequence insertion are ApaI and EcoRI. After the sequence is inserted, the endonuclease recognition sites between ApaI and EcoRI in the multiple cloning site will be replaced, which are XhoI, SalI, HindIII and EcoRV respectively. According to the pBS/U6 plasmid map, there are three endonuclease recognition sites in front of the U6 promoter, namely KpnI, XbaI and BamHI. Therefore, arbitrarily select an endonuclease in front of a U6 promoter and an endonuclease that can be replaced by an insert sequence to perform double digestion, and the interference vector will be single-digested to obtain a linear interference vector, and if the interference sequence is selected, it cannot be replaced. The endonuclease in front of the promoter and the endonuclease in front of the promoter should be able to cut out a fragment together with the U6 promoter and the interference sequence, and the size should be slightly larger than the U6 promoter. If there is no interfering sequence inserted, the result of enzyme digestion should show about 300bp of U6 promoter and about 3kb of pBlueScript backbone. Therefore we use KpnI and EcoRV and KpnI and XhoI to identify the positive clones of the interference plasmid.

After successful ligation of pBS/U6 and insert, the EcoRV restriction site disappears, so KpnI and EcoRV double digestion cannot obtain 2 fragments, but pBS/U6 has an EcoRV site, so double digestion can obtain 2 fragments. The XhoI restriction site still exists, and pBS/U6 can be double digested with KpnI and XhoI to obtain a large fragment of about 3kb and a small fragment of U6 promoter of 350kb. After the insert is successfully connected, the EcoRV restriction site disappears, so KpnI and EcoRV double digestion cannot obtain fragments of 2 sizes, but pBS/U6 has an EcoRV site, so double digestion can obtain 2 fragments of size. Therefore, the insert was successfully cloned into the pBS/U6 vector to obtain pBS/U6-STAT3-siRNA.

Transform the constructed plasmid into the DH5α strain, and extract it with a plasmid mini-extraction kit (purchased from QIAGEN), without further purification. This plasmid can be directly used in experiments such as transfection.

6. Identification of the effectiveness of the pBS/U6-Stat3-RNAi plasmid:

The constructed plasmid was transfected into human embryonic kidney epithelial cells and mouse melanoma cells for experiments. Experimental part, including immunoblotting experiment, immunostaining experiment, RT-PCR experiment, etc.

1. pBS/U6-Stat3-RNAi specifically inhibits the expression of mouse Stat3 gene protein in cells

The mStat3-Flag expression plasmid and four pBS/U6-Stat3-RNAi plasmids were co-transfected into 293T cells, and the expression of Stat3 in 293T cells was analyzed by Western blotting to study the effect of the constructed interference plasmid on the expression of mouse Stat3. See Figure 2. In Figure 2, 1-Wild Type 293T; 2-pBS/U6; 3, 4, 5, and 6 are pBS/U6-Stat3-RNAi-I, II, III, and IV in sequence. After co-transfection with the interference plasmid pBS/U6-Stat3-RNAi, the expression of Stat3 protein in the cells decreased to varying degrees. Comparing lanes 3, 4, 5, and 6 in the upper row with 2, it shows that pBS/U6-Stat3-RNAi can Inhibit the expression of the Stat3 gene in the cells to varying degrees, thereby reducing the amount of protein synthesis; among them, the interference plasmid targeting the 2334-2351 site has the best effect, and almost reduces the expression of the mouse Stat3 to the lowest level (the comparison between lane6 and lane1 in the upper row ), because 293T cells contain endogenous human Stat3, and the pBS/U6-Stat3-RNAi interference plasmid is specific to the mouse Stat3 sequence and cannot interfere with the expression of human Stat3, so targeting the 2334-2351 site The interference plasmid almost suppressed the expression of mouse Stat3. At the same time, after co-transfection of GFP plasmids, it can be seen that pBS/U6-Stat3-RNAi only inhibits the expression of Stat3, and has no effect on the expression of EGFP protein (comparison of lanes 2, 3, 4, 5, and 6 in the lower row), which shows The Stat3 interference plasmid only specifically inhibits the expression of Stat3 protein but has no effect on the expression of other genes, indicating that DNA-mediated RNAi has a high degree of specificity. The expression of β-actin in the middle row shows that the amount of sample loaded in each lane is basically the same.

2. Using RT-PCR to semi-quantitatively detect the mRNA level of mouse Stat3 gene in 293T cells, in order to further clarify the reason for the decrease of mouse Stat3 protein expression in 293T cells after co-transfection of interference plasmids. 36 hours after the transfection of the plasmid, the 293T cells in each group were harvested, and the total RNA of the cells was extracted. Using this as a template, PCR amplification was carried out using specific primers for the mouse Stat3 gene. At the same time, it was used as a control for the sampling amount of the total RNA of the cells. The β-actin gene was amplified. See Figure 3. M-marker in Figure 3; Stat3 group 1-Wild Type 293T; 2-pBS/U6; 3, 4, 5, 6- pBS/U6-Stat3-RNAi-I, II, III, IV; β-actin Group - The same amount of sample is loaded on each channel. The expression of Stat3 gene mRNA in 293T cells of mice co-transfected with interference plasmids was significantly lower than that of the experimental group co-transfected with empty vector plasmids (lane3, 4, 5, 6 compared with 2), especially targeting 2334 The effect of the interference plasmid at the -2351 site is the best, which is consistent with the results of the western blotting experiment; in addition, although the human Stat3 gene exists in the wild-type 293T cell total RNA template used in the first pass, due to the designed The primers are specific to the mouse Stat3 gene, so no bands can be amplified. Similarly, the lower row of β-actin bands shows that the sampling amount of total RNA during RT-PCR amplification is basically the same.

3. The dosage effect of pBS/U6-Stat3-RNAi inhibiting the expression of Stat3 gene in cells

(1) Immunostaining can more intuitively observe the inhibitory effect of pBS/U6-Stat3-RNAi on Stat3 gene expression. Select the interference plasmid targeting the 2334-2351 site with the best effect to further test the inhibitory effect on mouse Stat3 gene in 293T cells. At the same time, different amounts of interfering plasmids were transfected to observe whether the inhibitory effect had a dosage effect. See Figure 4. The left column (a, d, g, j, m, p) in Figure 4 shows the expression of co-transfected GFP; the middle column (b, e, h, k, n, q) shows the immunostained Stat3 gene expression The result; the right column (c, f, i, l, o, r) is the result of the Merge of the left column and the middle column. Yellow light indicates the co-expression of GFP and Stat3. Compared with cells transfected with empty vector pBS/U6 and GFP-specific interference plasmid transfected with Stat3 interference plasmid, the expression of Stat3 protein was significantly reduced (compared with h in Figure 4 and b, e), and with the interference plasmid transfection As the amount of staining increased, the expression of Stat3 became lower and lower (from Figure 4-h to q), while the expression of GFP was almost equal to that of the control group (Figure 4-g, j, m, p compared with a). At the same time, the GFP interference plasmid can also efficiently and specifically inhibit the expression of the GFP gene (compared with Figure 4-d and a), but has no effect on Stat3 protein (compared with Figure 4-e and b). It shows that the interference plasmid targeting mouse Stat3 can specifically inhibit the expression of mouse Stat3, and presents an obvious dose effect. Immunostaining results visually showed that the constructed pBS/U6-Stat3-RNAi could efficiently and specifically inhibit the expression of Stat3 protein at the cellular level.

(2) The effect of different doses of interfering plasmids on the expression of Stat3 in mice was detected by immunoblotting. See Figure 5. In Figure 5, the dose effect of 1-GFPi; 2-pBS/U6; 3,4,5,6-pBS/U6-Stat3-RNAi-IV is consistent with the results of immunostaining, and the co-transfection of dry plasmid Compared with the control group, the expression of Stat3 decreased, and there was an obvious dose effect (3rd to 6th lane in the upper row of Figure 5), while pBS/U6-Stat3-RNAi had no effect on the expression of EGFP protein. Any inhibitory effect (lanes 3 to 6 compared to lane 2 in the lower row of Figure 5). The expression of GFP in the cells co-transfected with the GFp interference plasmid was inhibited (the ratio of the first lane and the second lane in the lower row of Figure 5), but the Stat3 protein was not affected in any way (the first lane and the second lane in the upper row of Figure 5 compared to). Similarly, the expression level of β-actin in the middle row shows that the amount of sample loaded in each lane is basically the same.

(3) Using the experimental technique of semi-quantitative RT-PCR to further detect the effect of different doses of interfering plasmids on the mRNA expression of mouse Stat3 in 293T cells. Mix equal amounts of Stat3 and βactin gene products amplified by RT-PCR from the total RNA template of the same experimental group and run agarose gel electrophoresis, see Figure 6. 1-pBS/U6 in Figure 6; 2-U6-GFPi; 3-U6-Stat3-RNAi-IV (0.5ug); 4-U6-Stat3-RNAi-IV (1.0ug); 5-U6-Stat3-RNAi - IV (2.0 ug); 6-U6-Stat3-RNAi-IV (3.0 ug). Consistent with the results of immunostaining and immunoblotting experiments, with the increase of the amount of co-transfection interference plasmid, the mRNA expression of Stat3 decreased compared with the control group, and showed a significant dose effect (the third in the upper row of Figure 6 Lane to Lane 6), the band of β-actin in the lower row shows that the sampling amount of total RNA during RT-PCR amplification is basically the same.

The pBS/U6-Stat3-RNAi plasmid prepared by the present invention can be used to inhibit the expression of tumor growth factors, inhibit the growth of tumors in animal models, and provide a theoretical basis for the rapid identification of new tumor targets, and can be used in the treatment of tumors Clinical trials and preparation of antitumor drugs.

sequence listing

<110> The Third Military Medical University of the Chinese People's Liberation Army

<120> siRNA inhibiting Stat3 gene expression and preparation method thereof

<130>

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caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagg 540

gggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttg 600

taaaacgacggccagtgagcgcgcgtaatacgactcactataggggcgaattgggtacccg 660

ctctagaactagtggatccgacgccgccatctctaggcccgcgccggccccctcgcacag 720

acttgtgggagaagctcggctactcccctgccccggttaatttgcatataatatttccta 780

gtaactatagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagc 840

tacattttacatgataggcttggatttctataagagatacaaatactaaattattatttt 900

aaaaaacagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagacta 960

taaatatcccttggagaaaagccttgtttgtcacatgccacgttggtgttcaagagacac 1020

caacgtggcatgtgacttttttgaattcctgcagcccgggggatccactagttctagagc 1080

ggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgct 1140

tggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccac 1200

acaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaac 1260

tcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagc 1320

tgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccg 1380

cttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctc 1440

actcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgt 1500

gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcc 1560

ataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaa 1620

acccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctc 1680

ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtgg 1740

cgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagc 1800

tgggctgtgtgcacgaacccccccgttcagcccgaccgctgcgccttatccggtaactatc 1860

gtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaaca 1920

ggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaact 1980

acggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcg 2040

gaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggttttt 2100

ttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatct 2160

tttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatga 2220

gattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaa 2280

tctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcac 2340

ctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtaga 2400

taactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacc 2460

cacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgca 2520

gaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagcta 2580

gagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcg 2640

tggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggc 2700

gagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcg 2760

ttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataatt 2820

ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagt 2880

cattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggata 2940

ataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggc 3000

gaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcac 3060

ccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaa 3120

ggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactct 3180

tcctttttcaatattattgaagcatttatcagggttatgtctcatgagcggatacatat 3240

ttgaatgtatttgaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgc 3300

cac 3303

<210>2

<211>3303

<212>DNA

<213> Artificial sequence

<400>2

ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctc 60

attttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccga 120

gatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactc 180

caacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcacc 240

ctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggag 300

cccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaa 360

agcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccac 420

cacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcg 480

caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagg 540

gggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttg 600

taaaacgacggccagtgagcgcgcgtaatacgactcactataggggcgaattgggtacccg 660

ctctagaactagtggatccgacgccgccatctctaggcccgcgccggccccctcgcacag 720

acttgtgggagaagctcggctactcccctgccccggttaatttgcatataatatttccta 780

gtaactatagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagc 840

tacattttacatgataggcttggatttctataagagatacaaatactaaattattatttt 900

aaaaaacagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagacta 960

taaatatcccttggagaaaagccttgtttgagttcaagcacctgacccttcaagagaggg 1020

tcaggtgcttgaactcttttttgaattcctgcagcccgggggatccactagttctagagc 1080

ggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgct 1140

tggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccac 1200

acaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaac 1260

tcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagc 1320

tgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccg 1380

cttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctc 1440

actcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgt 1500

gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcc 1560

ataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaa 1620

acccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctc 1680

ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtgg 1740

cgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagc 1800

tgggctgtgtgcacgaacccccccgttcagcccgaccgctgcgccttatccggtaactatc 1860

gtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaaca 1920

ggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaact 1980

acggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcg 2040

gaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggttttt 2100

ttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatct 2160

tttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatga 2220

gattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaa 2280

tctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcac 2340

ctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtaga 2400

taactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacc 2460

cacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgca 2520

gaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagcta 2580

gagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcg 2640

tggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggc 2700

gagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcg 2760

ttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataatt 2820

ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagt 2880

cattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggata 2940

ataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggc 3000

gaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcac 3060

ccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaa 3120

ggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactct 3180

tcctttttcaatattattgaagcatttatcagggttatgtctcatgagcggatacatat 3240

ttgaatgtatttgaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgc 3300

<210>3

<211>3303

<212>DNA

<213> Artificial sequence

<400>3

ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctc 60

attttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccga 120

gatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactc 180

caacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcacc 240

ctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggag 300

cccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaa 360

agcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccac 420

cacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcg 480

caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagg 540

gggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttg 600

taaaacgacggccagtgagcgcgcgtaatacgactcactataggggcgaattgggtacccg 660

ctctagaactagtggatccgacgccgccatctctaggcccgcgccggccccctcgcacag 720

acttgtgggagaagctcggctactcccctgccccggttaatttgcatataatatttccta 780

gtaactatagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagc 840

tacattttacatgataggcttggatttctataagagatacaaatactaaattattatttt 900

aaaaaacagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagacta 960

taaatatcccttggagaaaagccttgtttgcagctgaacaacatgtcattcaagagatga 1020

catgttgttcagctgcttttttgaattcctgcagcccgggggatccactagttctagagc 1080

ggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgct 1140

tggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccac 1200

acaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaac 1260

tcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagc 1320

tgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccg 1380

cttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctc 1440

actcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgt 1500

gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcc 1560

ataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaa 1620

acccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctc 1680

ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtgg 1740

cgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagc 1800

tgggctgtgtgcacgaacccccccgttcagcccgaccgctgcgccttatccggtaactatc 1860

gtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaaca 1920

ggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaact 1980

acggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcg 2040

gaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggttttt 2100

ttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatct 2160

tttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatga 2220

gattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaa 2280

tctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcac 2340

ctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtaga 2400

taactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacc 2460

cacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgca 2520

gaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagcta 2580

gagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcg 2640

tggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggc 2700

gagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcg 2760

ttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataatt 2820

ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagt 2880

cattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggata 2940

ataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggc 3000

gaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcac 3060

ccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaa 3120

ggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactct 3180

tcctttttcaatattattgaagcatttatcagggttatgtctcatgagcggatacatat 3240

ttgaatgtatttgaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgc 3300

cac 3303

<210>4

<211>3303

<212>DNA

<213> Artificial sequence

<400>4

ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctc 60

attttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccga 120

gatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactc 180

caacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcacc 240

ctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggag 300

cccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaa 360

agcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccac 420

cacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcg 480

caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagg 540

gggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttg 600

taaaacgacggccagtgagcgcgcgtaatacgactcactataggggcgaattgggtacccg 660

ctctagaactagtggatccgacgccgccatctctaggcccgcgccggccccctcgcacag 720

acttgtgggagaagctcggctactcccctgccccggttaatttgcatataatatttccta 780

gtaactatagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagc 840

tacattttacatgataggcttggatttctataagagatacaaatactaaattattatttt 900

aaaaaacagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagacta 960

taaatatcccttggagaaaagccttgtttgaagctgcagagacgtgacttcaagagagtc 1020

acgtctctgcagcttcttttttgaattcctgcagcccgggggatccactagttctagagc 1080

ggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgct 1140

tggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccac 1200

acaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaac 1260

tcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagc 1320

tgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccg 1380

cttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctc 1440

actcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgt 1500

gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcc 1560

ataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaa 1620

acccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctc 1680

ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtgg 1740

cgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagc 1800

tgggctgtgtgcacgaacccccccgttcagcccgaccgctgcgccttatccggtaactatc 1860

gtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaaca 1920

ggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaact 1980

acggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcg 2040

gaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggttttt 2100

ttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatct 2160

tttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatga 2220

gattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaa 2280

tctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcac 2340

ctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtaga 2400

taactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacc 2460

cacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgca 2520

gaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagcta 2580

gagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcg 2640

tggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggc 2700

gagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcg 2760

ttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataatt 2820

ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagt 2880

cattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggata 2940

ataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggc 3000

gaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcac 3060

ccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaa 3120

ggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactct 3180

tcctttttcaatattattgaagcatttatcagggttatgtctcatgagcggatacatat 3240

ttgaatgtatttgaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgc 3300

cac 3303

Claims (4)

1. a kind of siRNA that suppresses Stat3 gene expression is characterized in that its base sequence is as follows: Action site 2334-2351: chain of justice 5'>AAGCTGCAGAGACGTGAC

GTCACGTCTCTGCAGCTTCTTTTTTG<3'; antisense strand 5'>AATTCAAAAAAGAAGCTGCAGAGACGTGAC

GTCACGTCTCTGCAGCTT<3'. 2. A preparation method of siRNA inhibiting Stat3 gene expression as claimed in claim 1, characterized in that the siRNA is chemically synthesized in vitro and expressed in vivo. 3. the preparation method of siRNA according to claim 2 is characterized in that the plasmid that is used for expressing described siRNA in vivo comprises: Vector containing type III RNA polymerase eukaryotic promoter; A DNA fragment encoding an siRNA that specifically inhibits the expression of the Stat3 gene. 4. The preparation method of siRNA according to claim 3, characterized in that the carrier containing type III RNA polymerase eukaryotic promoter is pBS/U6.

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