CN100395335C - siRNA duplexes inhibiting bcl-2 gene expression - Google Patents

Abstract

The present invention discloses four sets of siRNA double-stranded sequences, which can inhibit the expression of bcl-2 anti-apoptosis gene, effectively inhibit the expression and translation of Bcl-2 protein, thereby reducing the synthesis of Bcl2 protein, in order to restore the normal apoptosis of cells It is a good anti-leukemia and tumor cell drug resistance small molecule double-stranded nucleic acid drug.

Description

siRNA duplexes inhibiting bcl-2 gene expression

The patent application of the present invention is a divisional application with the application number "2004100511978", the application date of the original application is "August 24, 2004", the application number is "2004100511978", and the invention name is "siRNA for inhibiting bcl-2 gene expression Double chain and its application".

technical field

The present invention relates to siRNA double strand sequence, especially siRNA double strand for inhibiting bcl-2 gene expression.

Background technique

RNAi is double-stranded RNA-mediated post-transcriptional gene silencing (Post-transcriptional gene silencing, PTGS), in which case the promoter is active, the target gene can be transcribed, but mRNA cannot be accumulated normally. Various in vivo and in vitro experiments have confirmed that short double-stranded RNA of 21-23 nt, namely siRNA (short interference RNA, siRNA), can cause gene closure in mammalian cells and tissues, and its structure is stable without the need for antisense nucleotides. Such extensive chemical modification can increase its half-life, and at a concentration several orders of magnitude lower than antisense nucleotides, the target gene can be reduced to a very low level or even completely "knocked out", thereby producing a deletion mutant phenotype. The intervention effect of siRNA depends critically on the selection of its target gene sequence. Any nt error will lead to the loss of RNAi effect. This high sequence specificity makes it very useful for selective gene silencing of point mutations, sequence insertions and deletions. important pharmacological effects. It has been proven that siRNA technology can be used alone or in combination with other existing treatments for diseases caused by mutations, such as viral infection, SBMA, and can also be used for the treatment of tumors.

Bcl-2 is a proto-oncogene that can inhibit cell apoptosis and is closely related to the occurrence of tumors and the formation of drug resistance. Gene therapy can increase the sensitivity of drug-resistant cells to drugs, and has strong specificity and non-toxicity. It opens up a new way for the reversal of tumor MDR and optimizes chemotherapy, so it has broad application prospects.

Contents of the invention

The purpose of the present invention is to provide the siRNA duplex sequence for inhibiting bcl-2 gene expression, and further provide the application of the siRNA duplex in pharmacy.

In order to achieve the above-mentioned purpose, the sequences of the four pairs of siRNA targeting the bcl-2 gene of the present invention are respectively:

siRNA2: antisense strand 5'-AAG CCG GCG ACG ACT TCT CCC CCT GTC TC-3',

  Justice Chain 5’-AAG GGA GAA GTC GTC GCC GGC CCT GTC TC-3’;

SIRNA3: antisense strand 5'-AAC ATC GCC CTG TGG ATG ACT CCT GTC TC-3,

  Justice Chain 5’-AAA GTC ATC CAC AGG GCG ATG CCT GTC TC-3’;

SIRNA5: antisense strand 5'-AAA GCG TTC ACT CCC AAC CTG CCT GTC TC-3',

Justice Chain 5'-AAC AGG TTG GGA GTG AAC GCT CCT GTC TC-3';

SIRNA6: antisense strand 5'-AAG AAT GCA AAG CAC ATC CAA CCT GTC TC-3',

Strand of justice 5'-AAT TGG ATG TGC TTT GCA TTC CCT GTC TC-3'.

The above pairs of siRNA sequences can be used to prepare medicines for treating tumors and leukemia.

According to the design tool software provided by Ambion Company on the Internet, the present invention designs and synthesizes 6 pairs of siRNAs targeting the bcl-2 gene, transfers the synthesized siRNAs into the U251 cell line through liposomes, and uses untransfected cells and bcl-2 The antisense drug G3139 was used as a control. The inhibition of siRNA on cell growth was detected by MTT method and the change of cell cycle by flow cytometry. The inhibitory effect of siRNA on bcl-2 expression was detected by RT-PCR and immunohistochemistry. The results showed that: MTT showed cell growth and survival rate at each time point, and there were significant differences among siRNA2, 3, 5, 6, siRNA1, 4 groups, control group and liposome group (P<0.05). There were also differences between the siRNA1 and 4 groups, the control group and the liposome group (P<0.05). There were differences between the siRNA1-6 group and the antisense group at 24 and 48 hours (P<0.05), but there was no difference at 72 hours (P>0.05). RT-PCR and immunohistochemical methods showed that the expression of bcl-2 gene in siRNA2, 3, 5, and 6 groups was significantly lower than that between control group\liposome group\antisense group and siRNA1 and 4 (P<0.05). The results of flow cytometry showed that the cells in the siRNA1-6 group and the antisense group were arrested in S phase. Conclusion: siRNA2, 3, 5, and 6 synthesized by in vitro transcription can inhibit the expression of bcl-2 gene in U251 cells, and the efficiency can reach more than 50%.

RNAi is double-stranded RNA-mediated post-transcriptional gene silencing. RNAi has strong cell penetration ability and can be transmitted and maintained over long distances between different cells. 21-23 nt siRNA can cause gene closure in mammalian cells and tissues. The intervention effect of siRNA depends on the selection of its target gene sequence. Any nt error will lead to the loss of RNAi effect. This height Sequence specificity makes it pharmacologically important for selective gene silencing of point mutations, sequence insertions, and deletions. The stable structure of siRNA does not require extensive chemical modification like antisense nucleotides to increase its half-life, and can reduce the target gene to an extremely low level or even completely "knockout" at a concentration several orders of magnitude lower than antisense nucleotides. remove". siRNA is mainly transfected by liposome, electroporation, microinjection and plasmid transfer.

Translocation and high expression of bcl-2 gene are closely related to tumorigenesis. It has been found that it is closely related to the occurrence and poor prognosis of various malignant tumors of the blood and lymphatic system, prostate cancer, colon cancer, ovarian cancer and neuroblastoma. The high expression of anti-apoptosis genes such as bcl-2 is closely related to the homeostasis mechanism of tumor escape from the body. bcl-2 can inhibit the apoptosis induced by apoptotic factors under various physiological conditions, thus providing conditions for the continued survival of metastatic tumor cells. Another study found that tumor cells with high expression of Bcl-2 increased resistance to chemotherapy drugs. Therefore, reducing or eliminating the overexpression of Bcl-2 gene in tumor cells can relieve the inhibitory effect of Bcl-2 on tumor cell apoptosis, promote tumor cell death and enhance the sensitivity of tumor cells to chemotherapy drugs. The present invention designs and synthesizes 6 siRNAs for the bcl-2 gene, transfers them into glioma U-251 through liposomes, and compares them with G3139 to evaluate their bcl-2 inhibiting effect. The results of MTT and RT-PCR showed that siRNA2, 3, 5, and 6 had obvious inhibitory effects, and the expression of bcl-2 was reduced by more than 50% compared with the blank control group. The results of flow cytometry showed that the cells were arrested in S phase. The experimental results indicated that the inhibitory effect of antisense nucleotides on bcl-2 mostly occurred after 48 hours, while siRNA had a significant inhibitory effect 12 hours after administration, but the MTT results suggested that the inhibitory efficiency decreased with time. The present invention uses a brand-new means of gene therapy in recent years --- siRNA, to inhibit the expression of bcl-2, an anti-apoptotic gene, and can effectively inhibit the expression and translation of Bcl-2 protein, thereby reducing the synthesis of Bcl2 protein, in order to restore cells The ability to regulate normal apoptosis can finally achieve the purpose of delaying tumor growth, and then apply it in the pharmaceutical production of tumor drugs.

Description of drawings

Figures 1 to 9 are pictures of bcl-2 protein expression patterns of U251 cells in each group observed under a microscope when the expression of bcl-2 protein was detected by immunohistochemical method.

Fig. 10 is an electrophoresis diagram of bcl-2 mRNA expression levels of transfected cells in each group detected by RT-PCR.

Figures 11 to 19 are graphs of cell cycle changes observed by flow cytometry after transfection.

Figure 20 is a comparison chart of the percentages of cells in S phase in each group.

Detailed ways

The present invention will be further described in detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

Example 1: Synthesis of siRNA.

Regarding siRNA sequence design, Ambion provides design tool software on the Internet (see www.ambion.com/techlib/misc/siRNA finder.html) and synthesizes the following six pairs of siRNA based on the kit (Ambion, USA):

Table 1. Six pairs of siRNA sequences designed and synthesized according to Ambion's design software

Example 2: MTT detection of sensitivity of cells to each pair of siRNA.

Take U251 cells in the logarithmic growth phase, prepare 1.5×105/mL initial concentration of cell suspension and add it to a 96-well culture plate (manufactured by Corning, USA), add 100 μL to each well, set up blank control group, liposome (USA) Invitrogen) control group, antisense group (15 μmol/L), and siRNA1-6 group (1 μmol/L). There were 5 wells in each group, and after 24 hours of inoculation, the supernatant was discarded, and siRNA1-6 or G3139 was added, and placed in a 37°C, saturated humidity, 5% CO2 incubator without serum for 6 hours, and then supplemented with no double antibody containing 100 μL of 1640 with 10% fetal bovine serum was added to 20 μL of MTT at 24, 48, and 72 hours respectively. After 4 hours, the supernatant was centrifuged and 150 μL of DMSO was added, and the absorbance was read with a microplate reader. Cell survival rate=light absorption value of experimental group/light absorption value of control group×100%.

The results showed that there were significant differences between siRNA2, 3, 5, 6 and the control group and liposome group at each time point (P<0.001); There is a difference (P<0.05) and a difference (P<0.05) between the control group and the liposome group. There was no difference between the siRNA1-6 group and the antisense group at 72 hours (P=0.073, 0.133, 0.239, 0.054, 0.225, 0.137), but there were differences at 24 and 48 hours (P<0.05). See Table 2 and Table 3.

※Indicates that the OD value of each group is the result after deducting the basic background of blank culture

★Indicates that there is a difference from the control group (P<0.05) *Indicates that there is a significant difference from the pair (P<0.001) in the control group

Table 2. OD value of cell proliferation activity detected by MTT method at each time point after transfection of siRNA ^※

★Indicates that there is a difference with the control group (P<0.05) *Indicates that there is a significant difference with the control group (P<0.01)

Table 3. Cell viability detected by MTT method at each time point after transfection of siRNA

Example 3: Detection of the expression of bcl-2 protein (manufactured by Wuhan Boster Company) by immunohistochemical method.

Cells in each group were inoculated in a 6-well plate at an initial concentration of 1.5×105/mL, and a 1.5×1.5 cm coverslip was built in. After transfection according to the above culture method, culture was continued for 48 hours, and the supernatant was discarded. Fix the coverslip with 3:1 methanolic glacial acetic acid solution for 15 seconds, rinse with PBS; permeabilize with 1% Tris-Triton 100 at 4°C for 5 minutes, rinse with PBS; block with peroxidase for 10 seconds, rinse with PBS; block with non-immune animal serum for 10 seconds Seconds, discard and add bcl-2 primary antibody dropwise, incubate at 4°C for 12 hours, rinse with PBS; add secondary antibody, biotin-labeled antibody in turn, and rinse with PBS, develop color with DAB, and counterstain nuclei with hematoxylin. Each batch of experiments had a blank control with PBS instead of the primary antibody.

Figures 1 to 9 are the expression patterns of bcl-2 protein in U251 cells of each group observed under a microscope by detecting the expression of bcl-2 protein by immunohistochemical method. Table 4 shows the differences in protein expression in each group.

★ means there is a difference from the control group (P<0.05); * means there is a significant difference from the control group (P<0.01)

Table 4 Protein expression of immunohistochemical bcl-2 in each group

Example 4: Detection of expression level of bcl-2 mRNA in transfected cells by RT-PCR (Shanghai Shenergy Gaming Company).

Cells in each group were inoculated in 6-well plates at an initial concentration of 1.5×10 5 /L, cultured for 12 hours after transfection according to the culture method described in Example 2, and collected by trypsinization and centrifugation. The bcl-2 primer sequence is as follows: 5' end primer: CGACGACTTCTCCCGCCGCTACCGC, 3' end primer: CCGCATGCTGGGGCCGTACAGTTCC amplification product is 318bp. RNA extraction: successively use RNA extraction solution (Life Technology Company), chloroform, isopropanol, and 75% ethanol to separate and wash the precipitate, and the specific steps are carried out according to the instructions. Finally, the RNA was precipitated. Use an appropriate amount of DEPC-treated sterile deionized water to dissolve in a water bath at 56°C. Take a small amount of RNA to measure the OD value (OD260/OD280 ratio > 1.8) and use 1% agarose gel (containing 0.5 μg/ml EB ) Electrophoresis to observe two clear bands of 28S and 18SrRNA to ensure the purity and integrity of the extracted RNA. (2) cDNA synthesis and PCR amplification were carried out in strict accordance with the instructions of the reverse transcription kit. Using β-actin as an internal reference, denature at 94°C for 30 seconds; anneal at 60°C for 1 minute; extend at 72°C for 1 minute. After 30 cycles, extend at 72°C for 5 minutes. The electrophoresis product was observed and photographed on 2% agarose gel electrophoresis, observed and analyzed on a Gel-Doc1000 ultraviolet gel image analyzer, and the ratio of Bcl-2 to β-actin amplification band was calculated, and photographed. See Figure 10. Take 5 μl of RT-PCR product and electrophoresis on 2% agarose gel, the results show that there are clear bands at 318bp in the blank, liposome control group and antisense group, and the bands in each group of siRNA are weakened. Groups 5 and 6 are obvious. The bands of the control β-actin in each group were consistent. It shows that the expression of bcl-2 in cells is inhibited after siRNA transfection.

Example 5, Flow Cytometry Observation of Cell Cycle Changes After Transfection

The cells of each group were inoculated in 25cm ² culture flasks at an initial concentration of 1.5×105/L, and continued to culture for 48 hours after transfection according to the culture method described in Example 2. They were collected by trypsinization and centrifuged and suspended in PBS. Fix with ethanol for 30 min, stain with staining solution containing RNase and propidium iodide for 30 min, measure the change of DNA content by flow cytometry (EPICS-ELITE-ESP), and process the results with MULTICYCLE software. The results showed that the growth of cells in siRNA2, 3, 5, and 6 groups was arrested in S phase, as shown in Figure 11-Figure 19 and Figure 20.

Example 6. Application of siRNA in the preparation of drugs for treating tumors.

The siRNA2, siRNA3, siRNA5 and siRNA6 obtained in the present invention are used for preparing medicine for treating tumor or leukemia. It is used for human body administration, and can be used alone or in combination with other drugs.

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Claims (1)

1. A kind of siRNA duplex that suppresses bcl-2 gene expression, its sequence is: Antisense strand 5'-AAC ATC GCC CTG TGG ATG ACT CCT GTC TC-3, Sense strand 5'-AAA GTC ATC CAC AGG GCG ATG CCT GTC TC-3'.

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