CN1663957A - Micromolecule interference RNA aiming at middle period factor gene and use thereof - Google Patents

Abstract

The invention provides small interfering ribonucleic acid molecules for midkine gene mRNA, including any part of the sequence of small interfering ribonucleic acid molecules for midkine gene mRNA, or small interfering RNA molecules formed after chemical modification. The siRNA molecule of the present invention is chemically synthesized, which is beneficial to commercial production. The raw materials and synthesis equipment are simple, and it is four kinds of ribonucleotides. The synthesis and purification technology is mature, and there is no industrial hazard in the production process, and the product can be Dry storage is conducive to transportation and long-term storage. The small interfering ribonucleic acid molecules provided by the invention can effectively eliminate the MK gene mRNA molecules of tumor cells, find new molecules for treating tumors, and can be used as active ingredients in the preparation of drugs that effectively kill tumor cells.

Description

Small interfering ribonucleic acid molecule targeting midkine gene and use thereof

technical field

The present invention relates to the field of nucleic acid technology, in particular to effectively killing tumor cells, the characteristics of small interfering ribonucleic acid molecules (SiRNA) directed at midkine (midkine, MK) gene mRNA and its application in the preparation of drugs for effectively killing tumor cells use.

technical background

Tumor is a major disease that endangers human life and health, but so far there is still no effective treatment for it. Scientists have long been trying to develop new drugs that can effectively treat tumors. It has been known that the occurrence and development of tumors is related to many factors, and the effects of these factors lead to abnormal expression of tumor-related genes, and the inhibition of tumor positive-related gene expression can play a certain role in tumor intervention. For the negative regulation of gene expression, stem cell gene knockout and sense-antisense RNA interference technologies commonly used at present have not been ideal solutions due to their long cycle, high cost, or ineffective effects. The recently discovered double-stranded RNA interference (RNAi) phenomenon has a more efficient effect on the expression of mRNA of the target gene, and the established siRNA technology can inhibit the expression of the gene from the abundance of the mRNA expression of the target gene, and has become a A new, high-impact scientific achievement.

Human MK gene is a newly discovered tissue growth positively related gene, and its expression product has the characteristics of promoting nerve cell growth and inhibiting apoptosis, which determines the close relationship between the gene and tumor. Existing studies have shown that in adults, except for the detectable expression in the kidney, the rest are highly expressed in tumor tissues, including almost all human tumor tissues. What has been known so far is that the gene is closely associated with the occurrence, development, and prognosis of liver cancer. In cancer tissues, paracancerous tissues, and normal tissues, the expression of MK gene presents high, medium, and low levels. , with the recovery of liver cancer patients, the expression level of this gene tends to be normal. Based on the characteristics and expression of MK gene, inhibiting its expression has important practical significance in tumor therapy.

Contents of the invention

The object of the present invention is to provide a kind of small interfering ribonucleic acid molecule (siRNA) that suppresses targeting midkine (midkine MK) gene mRNA, the double strand formed by pairing of forward and reverse two RNA strands, is artificially synthesized, has SEQ ID NO : Nucleotide sequence of 1-6, with less than 70% homology with other genes, the length of the sense strand and the negative sense strand are both 21 nucleotides, and the respective 3' ends of the sense strand and the negative sense strand are Two consecutive unpaired deoxythymidylic acid (TT), the bases on the 19 nucleotides of the two strands except TT at the 3' end are complementary to form a double strand, and the bases of these 19 nucleotides can be adeno Purine (A), Guanine (G), Cytosine (C), or Uracil (U), the number of nucleotides in each chain where the base is Guanine (G) is the same as the number of nucleotides where the base is Cytosine (C) The sum of the number of nucleotides, accounting for the proportion of 19 nucleotides except TT at the 3' end, is between 25% and 75% (i.e. G/C ratio), SEQ ID NO: 1-6 nucleotides The sequence is:

SEQ ID NO 1: CUUCUUCACCUUAUCUUUCTT

SEQ ID NO 2: UCCAAACUCCUUCUUCCAGTT

SEQ ID NO 3: CCAGUUCUCAAACUUGUACTT

SEQ ID NO 4: GGUCUCCUGGCACUGAGCATT

SEQ ID NO 5: AGGCUUGGCGUCUAGUCCUTT

SEQ ID NO 6: GCUCUGGGACUCACAUUGCTT

Table 1 is the nucleotide sequence of the present invention: SiRNA No. serial number Sense strand sequence (5'->3') Antisense strand sequence (5'->3') molecular weight attack site MDK1 1 GAAAGAUAAGGUGAAGAAGTT CUUCUUCACCUUAUCUUUCTT 13429.2 95-104 MDK2 2 CUGGAAGAAGGAGUUUGGATT UCCAAACUCCUUCUUCCAGTT 13459.4 248-267 MDK3 3 GUACAAGUUUGAGAACUGGTT CCAGUUCUCAAACUUGUACTT 13444.3 278-297 MDK4 4 UGCUCAGUGCCAGGAGACCTT GGUCUCCUGGCACUGAGCATT 13504.7 362-381 MDK5 5 AGGACUAGACGCCAAGCCUTT AGGCUUGGCGUCUAGUCCUTT 13489.6 448-467 MDK6 6 GCAAUGUGAGUCCCAGAGCTT GCUCUGGGACUCACAUUGCTT 13489.6 674-693

The attack site refers to the sequence position where the nucleotide sequence recognizes and binds in the MK gene mRNA sequence.

The present invention also includes small interfering RNA molecules formed after any part or all of the siRNA sequence directed at the MK gene mRNA is chemically modified.

The chemical modification mainly includes the following three categories: first, partial modification of the phosphodiester bond connecting two adjacent nucleotides or replacement with any other chemical bond. The typical partial modification of the phosphodiester bond is to Replace the oxygen on the phosphoric acid double bond with sulfur (sulfurization) or other elements, or change the oxygen on the phosphoric acid single bond into nitrogen (nitridation) or other elements and chemical groups, all substitutions of the phosphodiester bond itself, also Including partial or complete changes to itself or part of it, as well as the two ribose sugars connected to it, a typical example is to change a phosphodiester bond and two adjacent ribose sugars into a peptide bond, so that a nucleic acid becomes a peptide Nucleic acid (peptide nucleic acid, PNA); Second, the five-membered ring of ribose in nucleosides is changed or the chemical groups on the side chain are modified. A typical example of changing the ribose ring is such as changing the five-membered ribose ring into a six-ring Morpholino ring; the modification on the ribose side chain mainly refers to changing the OH on the ribose 2' position to other elements (such as halogen elements), or using Other chemical groups replace the H in OH (such as alkyl); third, the base ring on the nucleotide is changed as a whole or the side chain is modified.

The small interfering ribonucleic acid molecule directed at the midkine (MK) gene mRNA provided by the invention can be used as an active ingredient in the preparation of drugs for effectively killing tumor cells.

The technical scheme that realizes the object of the present invention:

1. Design the siRNA targeting the expression mRNA of the target gene MK,

2. Use software to screen SiRNAs that meet certain conditions,

3. Purify the synthetic product and test the content,

4. SiRNA transfected SMMC-7721 cells,

5. RT-PCR analysis of MK gene mRNA abundance in cells,

6. Evaluate the effect of siRNA.

The present invention has the following characteristics:

(1) The present invention is a double-stranded RNA molecule and its nucleotide sequence has the following characteristics: siRNA is artificially synthesized, has less than 70% homology with other genes, and the length of both positive and negative strands is 21 nuclei Nucleotide, the 3' end of the sense strand and the negative sense strand are two consecutive unpaired deoxythymidylic acid (TT), and the bases on the 19 nucleotides except TT at the 3' end of the two strands are complementary to form The base of these 19 nucleotides can be adenine (A), guanine (G), cytosine (C) or uracil (U), and the base in each strand is guanine (G ) and the sum of the nucleotides whose base is cytosine (C), the proportion of the 19 nucleotides except TT at the 3' end is between 25% and 75% (i.e. G /C ratio).

(2) SiRNA molecule of the present invention is by chemical synthesis, is conducive to commercialized production, and raw material and synthesis instrument are simple, is four kinds of ribonucleotides: adenine (A), guanine (G), cytosine (C) or uracil (U) nucleotides. Synthesis and purification technology is mature.

(3) The essence of this product is the nucleic acid component, which is an integral part of the human body. Due to its high efficiency and low dosage, there are few side effects, and there will be no industrial hazards during the production process. Moreover, the product can be stored dry, which is conducive to transportation and long-term storage.

(4) The siRNA directed at the MK gene mRNA molecule provided by the present invention can effectively eliminate the MK gene mRNA molecule of tumor cells, and finds a new type of molecule for treating tumors. Its maximum inhibitory concentration is several to tens of nanomolar (nM), which is 10-100 times lower than the effective concentration of antisense nucleic acid.

Description of drawings

Figure 1 shows the mRNA levels of MK gene expression in the cells after 6 kinds of siRNAs acted on liver cancer cell lines for 48 hours.

Figure 2 shows the mRNA levels of MK gene expression in cells after the three siRNAs acted at different concentrations for 48 hours.

Figure 3 shows the mRNA levels of MK gene expression in the cells after the three siRNAs acted on liver cancer cell lines for different periods of time.

Detailed ways

The present invention is described further in conjunction with embodiment.

Example 1 Synthesis of siRNA

The synthesis of siRNA can be entrusted to commercial companies that openly carry out synthesis business, such as Dharmacon in the United States. The details can be learned through www.dharmacon.com . All siRNA molecules are deprotected, desalted, purified, and annealed at the 2-position to form double strands treated and then dissolved in DEPC-treated distilled water.

The siRNA preparation method provided by the present invention can also adopt the existing solid-phase chemical synthesis method. The method is available in: Wincott F, DiRenzo A, Shaffer C, Grimm S, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S and Usman N. Synthesis, degradation, analysis and purification of RNA and ribozymes. Nucleic Acids Res . 1995, 23:2677-84.

The siRNA of the former table number MDK2 is an example: the whole chemical synthesis can be roughly divided into four processes (1) synthesis of oligoribonucleic acid; (2) deprotection; (3) purification and separation; (4) desalination annealing aseptic disinfection.

Concrete preparation operation is as follows:

(1) Synthesis of oligoribonucleic acid: the synthesis of siRNA is carried out on an automatic DNA/RNA synthesizer (for example: Applied Biosystems EXPEDITE 8909), according to the nucleic acid sequence of the No. 2 small interfering RNA molecule (sense strand 5'-GAAAGAUAAGGUGAAGAAGTT , antisense strand 5'-CUUCUUCACCUUAUCUUUCTT) order to connect the corresponding nucleotides one by one. Since siRNA is composed of a 19-mer oligoribonucleic acid and a 2-mer deoxythymidylic acid. Therefore, the starting material is 5'-O-p-dimethoxytrityl-thymidine (1-2umol) linked to the solid phase (CPG). Specifically, each cycle synthesis can be divided into four steps to complete. The first step is to elute the 5' protecting group on the thymidine connected to the solid phase under the action of 3% trichloroacetic acid; the second step is to elute the 5' -O-P-dimethoxytrityl-thymidine phosphoramidite is coupled to the last thymidine that has been deprotected to form a dimhymidine phosphite triester. The coupling time and the number of coupling cycles are according to The instrument instructions provide procedures to complete; the third step is to oxidize the coupled dimymidine phosphite triester under the action of 0.05M iodine water into dimymidine phosphotriester; the fourth step is acetylation, the solid phase A small amount of unreacted active groups (for example: hydroxyl and amine groups) form esters or amides under the action of acetic anhydride, so as to achieve blocking and reduce the overall by-product generation. This cycle is repeated until the synthesis of the entire nucleic acid sequence is complete.

(2) Deprotection: put the synthesized solid-phase siRNA into a sealable vial, and add 1 ml of methylamine aqueous solution (10M, 50% ethanol), and let it stand at room temperature. After two hours, take out the solution, The solid-phase CPG was rinsed again with ethanol; a mixture of water and acetonitrile, and the rinse solution was combined with the solution taken out earlier, and the solvent was drained. Continue to add 1 milliliter of tetrabutylammonium fluoride tetrahydrofuran solution (1M) in the vial, and the solution is allowed to stand at room temperature for 12 hours to remove all protective groups (including bases, nucleoside phosphates and silylated protecting group at the 2' position of the nucleoside). After ethanol precipitation, the crude product of siRNA was produced.

(3) Purification and separation: the crude product of siRNA is dissolved in the aqueous solution of 2 milliliters of ammonium acetate, then through the separation of reversed-phase C18 high pressure liquid chromatography, the method for using gradient elution to collect the main product of siRNA (eluent) A: 0.1M ammonium acetate; eluent b: 20% 0.1M ammonium acetate and 80% acetonitrile), the solvent of the main product of SiRNA was removed, and 5 milliliters of 80% acetic acid aqueous solution was added, and allowed to stand at room temperature After 15 minutes, this solution was subjected to anion-exchange separation (DEAE-5PW, anion-exchange column) to obtain SiRNA with a purity of more than 90% (gradient elution, eluent A: 0.025M Tris-HCl, 0.025 M NaCl pH=8, 5% acetonitrile; eluent b: 0.025M Tris-HCl, 2.0M NaCl, pH=8, 5% acetonitrile).

(4) Desalting and annealing aseptic disinfection: the purified siRNA is dialyzed to remove salt, the solution of siRNA is filtered and sterilized and dried and crystallized, and then the oligoribonucleic acid of the sense strand and the antisense strand is annealed to form a stable double strand Cross-linked siRNA. The method is to mix and dissolve the oligoribonucleic acid of the sense strand and the antisense strand in 1-2 ml of buffer solution (10mM Tris, pH=7.5-8.0, 50mM NaCl). Heat the solution to 95°C, then slowly cool the solution to room temperature (this process should not be less than one hour), and finally store the solution in a refrigerator at 4°C so that it can be used at any time.

There are two commonly used methods for the purity and identification of purified siRNA. One uses capillary gel electrophoresis to identify the purity of siRNA, which can be found in Paulus A, Ohms JI. Analysis of oligonucleotides by capillary gelelectrophoresis. J Chromatogr 1990, 507: 113-123. The second is to use MALDI-TOF mass spectrometry to accurately measure its molecular weight, so as to determine the chemical structure of siRNA.

All the siRNAs mentioned in the present invention can be prepared according to the above method according to their sequences, and the identification methods can be capillary gel electrophoresis and MALDI-TOF mass spectrometry.

Example 2 Cell culture and transfection of siRNA

(1) Cell culture: SMMC-7721 is a liver cancer cell line isolated from liver cancer tissue [Dong Rongchun et al., Establishment of SMMC-7721 human liver cancer cell line and preliminary observation of its biological characteristics, Journal of Second Military Medical University, 1980.6;(1):5-9]. The cells were cultured in vitro with 1640 medium containing 10% fetal bovine serum.

(2) SiRNA transfection: SiRNA was transferred into tumor cells by means of Oligofectamine from Invitrogen (www.invitrogen.com), and the specific steps were performed according to the instructions. Briefly described as follows, 5×10 ⁴ tumor cells were seeded on 24-well culture plates overnight, transferred into siRNA the next day, and then continued to culture for a certain period of time, then real-time quantitative PCR was used to detect the level of MK gene mRNA in the cells.

(3) Detection of the expression level of MK gene mRNA in SMMC-7721 cells: the detection of the expression level of MK gene mRNA in tumor cells was carried out by real-time quantitative PCR technology. Specifically, it can be divided into the following three steps: first, the extraction of RNA in cells, which can be carried out by referring to the existing standard methodology [see Chapter 4, Current protocols in Molecular Biology, John & Wiley, 2003]; second The first step is the synthesis of cDNA, and the synthesis method can also be carried out according to the standard methodology [see Chapter 5, Current protocols in Molecular Biology, John & Wiley, 2003]; the third step is the real-time PCR, which is based on the ABI7700 sequence It is carried out on the detector, and the specific method is carried out with reference to the standard procedure of ABI Company. The sequence of the 5' primer used to detect the MK gene in this patent is 5'-GCGCGCTACAATGCTCAGT-3', corresponding to positions 372 to 391 in the cDNA sequence of the MK gene, and the sequence of the 3' primer is 5'-CCCTTCCCTTTCTTGGCTTT- 3', corresponding to positions 467 to 447 in the cDNA sequence of the MK gene, and the sequence of the real-time PCR probe is 5'-CCAGGAGACCATCCGCGTCACC-3', corresponding to positions 392 to 414 in the cDNA sequence of the MK gene.

Example 3 Inhibitory effect of siRNA on tumor cell MK gene mRNA

(1) Selection of SiRNA: the present invention has selected 6 SiRNA molecules directed at the mRNA coding region of MK gene, i.e. 6 SiRNA molecules listed in SEQ ID NO: 1-6, and the attack site is from 95- Between 693 and 19 complementary double-stranded nucleotide sequences in each SiRNA molecule, the G/C is between 32% and 74%.

(2) Effects of 6 SiRNAs on MK gene mRNA expression in SMMC-7721 cells:

A. The effect of 6 SiRNAs with a concentration of 75nM on the MK gene mRNA of SMMC-7721 cells: after 48 hours of acting on SMMC-7721 cells with these 6 SiRNAs of 75nM, when observing their effects on the MK gene mRNA level, it was found that the third No. 4, No. 4 and No. 6 SiRNA molecules have the most obvious inhibitory effects on MK, reaching 97%, 98% and 98% respectively. In addition, No. 1, No. 4, and No. 6 have an inhibitory effect on MK gene mRNA greater than 50%. See Figure 1. Figure 1 shows the MK gene expression in the cells after 48 hours of action of 6 kinds of 75nM siRNA on the liver cancer cell line SMMC-7721. mRNA level, the control group refers to the cells treated with transfection reagent only under the same conditions. The vertical axis represents the relative percentage of the mRNA level of MK gene expression in the SiRNA treatment group to the control group. Each data in the graph represents the mean of three replicate experiments.

B, the effect of 3 siRNAs of different concentrations on the MK gene mRNA of SMMC-7721 cells:

For the No. 3, No. 4, and No. 6 SiRNA molecules that highly inhibited the mRNA level of the MK gene, 5 gradients were diluted from 10 nM concentration to 0.625 nM. It was found that all groups had an inhibitory effect on the mRNA level of the MK gene. No. 3 No. 4 and No. 4 reached 76% and 81% respectively at 5nM, and No. 6 reached 73% at 10nM. See Fig. 2. Fig. 2 shows the effects of three siRNAs on liver cancer cell line SMMC-7721 at different concentrations. Hours later, the mRNA level of MK gene expression in the cells, and the control group refers to the cells treated with the transfection reagent under the same conditions. The vertical axis represents the relative percentage of the mRNA level of MK gene expression in the SiRNA treatment group to the control group. Each data in the graph represents the mean of three replicate experiments.

The effect of three SiRNAs of C, 10nM concentration on the MK gene mRNA of SMMC-7721 cells at different times:

Take 10nM of No. 3, No. 4, and No. 6 SiRNA, and after acting on SMMC-7721 cells for 24, 48, and 72 hours, observe the level of MK gene mRNA in SMMC-7721 cells. SiRNA can inhibit MK gene mRNA from 24 hours Effect, until 72 hours, No. 3 and No. 6 have the strongest inhibition of 70% and 74% after 48 hours of action, and No. 4 has the strongest effect of 79% after 72 hours, see Figure 3, Figure 3 is three 10nM siRNA pairs The mRNA levels of MK gene expression in the cells after the liver cancer cell line SMMC-7721 has been treated for different times. The control group refers to the cells treated with the transfection reagent under the same conditions. The vertical axis represents the relative percentage of the mRNA level of MK gene expression in the SiRNA treatment group to the control group. Each data in the graph represents the mean of three replicate experiments.

Example 4 Modification of siRNA

The methods for modifying siRNA in the present invention can be divided into three categories, and there are many changes in each category, so their respective synthesis methods are not the same. Here are two of the most common siRNA modification methods. Moreover, the following methods do not limit the protection scope of the modification of siRNA described in the present invention.

(1) Vulcanization

Sulfurization refers to converting an oxygen atom on the nucleoside phosphodiester bond into a sulfur atom to form a nucleoside phosphorothioate. Since the other composition structures of the entire SiRNA remain unchanged, its synthesis is the same as the SiRNA synthesis process of Example 1 Basically the same. It is only necessary to change the oxidation reaction in the synthesis process into a sulfuration reaction, and add an appropriate sulfurization reagent, such as 3H-1,2-benzodithiol-3-one1,1-dioxide (also known as Beaucage reagent), to this reaction.

(2) Modification of nucleoside 2' hydroxyl

The modification of the 2' hydroxyl group of the nucleoside refers to the replacement of the 2' hydroxyl group on the five-membered sugar ring of the nucleoside with various saturated alkoxy or unsaturated alkoxy groups. The most common one is to replace the nucleoside with a methoxy group The hydroxyl group at the 2' position. The synthesis of the methoxylation of the nucleoside 2' position of SiRNA is basically the same as the synthesis steps of SiRNA in Example 1, only need to replace 2'-tert-butyl with 2'-methoxynucleoside phosphoramidite in the coupling reaction Dimethylsiloxynucleoside phosphoramidites are sufficient.

The present invention is described in conjunction with the best embodiment, but after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the appended claims of the application within the bounds of the book.

Claims (8)

1. A small interfering ribonucleic acid molecule directed at the midkine gene mRNA, which is a double strand formed by the pairing of forward and reverse RNA strands, is artificially synthesized, has the nucleotide sequence of SEQ ID NO 1-6, and is compatible with other genes Less than 70% homology, the length of the sense strand and the negative sense strand are both 21 nucleotides, the 3' ends of the sense strand and the negative sense strand are two consecutive unpaired deoxythymidylates, two The bases on the 19 nucleotides other than TT at the 3' end of the chain are complementary to form a double strand. The bases of these 19 nucleotides can be adenine, guanine, cytosine or uracil. The sum of the number of nucleotides whose base is guanine and the number of nucleotides whose base is cytosine accounts for between 25% and 75% of the 19 nucleotides except TT at the 3' end, SEQ The nucleotide sequence of ID NO 1-6 is: SEQ ID NO 1: CUUCUUCACCUUAUCUUUCTT SEQ ID NO 2: UCCAAACUCCUUCUUCCAGTT SEQ ID NO 3: CCAGUUCUCAAACUUGUACTT SEQ ID NO 4: GGUCUCCUGGCACUGAGCATT SEQ ID NO 5: AGGCUUGGCGUCUAGUCCUTT SEQ ID NO 6: GCUCUGGGACUCACAUUGCTT 2. The small interfering ribonucleic acid molecule according to claim 1, characterized in that it includes any part or all of the sequence of the midkine gene mRNA being chemically modified to form a small interfering RNA molecule. 3. The small interfering ribonucleic acid molecule according to claim 2 is characterized in that: said chemical modification refers to partial modification of the phosphodiester bond connecting two adjacent nucleotides or replacement with any other chemical bond, Or change the oxygen on the phosphoric acid single bond to nitrogen, nitriding or other elements and chemical groups 4. The total substitution of the phosphodiester bond itself according to claim 3, also includes partial or total changes to itself or its part, and the two ribose sugars linked to it. 5. The small interfering ribonucleic acid molecule according to claim 2, characterized in that: said chemical modification refers to modifying the five-membered ring of ribose in the nucleoside or modifying the chemical group on its side chain. 6. The modification on the ribose side chain according to claim 5 mainly refers to changing the OH on the ribose 2' position into other elements, or replacing the H in the OH with other chemical groups. 7. The small interfering ribonucleic acid molecule according to claim 2, characterized in that: the chemical modification refers to the overall change of the base ring on the nucleotide or the modification of the side chain. 8. The small interfering ribonucleic acid molecule according to any one of claims 1-7, characterized in that it is used in the preparation of drugs for effectively killing tumor cells.

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