CN118995721A - SiRNA for inhibiting interleukin-8 and application thereof - Google Patents
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Abstract
本发明涉及一种用于抑制白细胞介素‑8的siRNA及其应用，属于生物技术领域。本发明提供了一种用于抑制白细胞介素‑8的siRNA，所述siRNA的正义链包含核苷酸序列如SEQ ID NO.1~21任一项所示的核酸分子；所述siRNA的反义链包含核苷酸序列如SEQ ID NO.22~42任一项所示的核酸分子。实验证明，所述siRNA均对白细胞介素‑8具备高抑制活性。本发明还提供了用于抑制白细胞介素‑8的经修饰的siRNA，所述修饰包括甲氧基修饰、氟代修饰和/或硫代磷酸酯基连接。实验证明，经修饰的siRNA在0.1~10 nM的浓度下均对白细胞介素‑8具备高抑制活性。The present invention relates to a siRNA for inhibiting interleukin-8 and its application, belonging to the field of biotechnology. The present invention provides a siRNA for inhibiting interleukin-8, wherein the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NO.1~21; and the antisense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NO.22~42. Experiments have shown that the siRNAs all have high inhibitory activity on interleukin-8. The present invention also provides a modified siRNA for inhibiting interleukin-8, wherein the modification comprises methoxy modification, fluoro modification and/or thiophosphate group connection. Experiments have shown that the modified siRNAs all have high inhibitory activity on interleukin-8 at a concentration of 0.1~10 nM.
Description
Technical Field
The invention relates to siRNA for inhibiting interleukin-8 and application thereof, belonging to the technical field of biology.
Background
Interleukin-8 (interleukin-8, IL-8), also known as CXCL8, is an endogenous cytokine produced by a variety of cells, originally found in lipopolysaccharide-induced human peripheral blood mononuclear cell cultures, and is transcribed and encoded by the IL8 gene. The main biological function of interleukin-8 is to chemotaxis and activate neutrophils, causing them to migrate to the site of inflammation, involved in the regulation of immune and inflammatory responses. Studies have shown that abnormal levels of interleukin-8 expression are present in many autoimmune or infectious inflammatory patients, and studies have demonstrated that interleukin-8 inhibitors can achieve the effect of treating inflammation by inhibiting multiple pathways such as respiratory burst of leukocytes and free radical release.
In addition to being involved in the regulation of immune and inflammatory responses, interleukin-8 is also involved in the development and progression of tumors. Research shows that most tumors including liver cancer, breast cancer, nasopharyngeal cancer, colorectal cancer and gastric cancer can secrete interleukin-8 to promote the growth of the tumors, and the overexpression of the interleukin-8 is closely related to malignant biological behaviors such as proliferation, invasion, metastasis and the like of tumor cells. Meanwhile, research shows that interleukin-8 is an important inflammatory response factor and an immunosuppression factor which are indispensable in the tumor microenvironment, has the capability of recruiting immunosuppression cells to suppress anti-tumor immune responses, and is closely related to tumor resistance. Therefore, the interleukin-8 can be used as a target point of tumor immunotherapy, and can treat or assist in treating various cancers through various ways such as inhibiting tumor proliferation, invasion, metastasis, drug resistance and the like.
The small interfering RNA (SMALL INTERFERING RNA, SIRNA) is a double-stranded RNA with a length of 20-25 nucleotides, and is mainly used for mediating the specific gene silencing phenomenon participated by a specific enzyme through an RNA interference (RNAi) mechanism. In the RNAi pathway, siRNA interferes with gene expression by hybridizing to complementary mRNA molecules, which can trigger mRNA degradation, thereby inhibiting gene expression of a particular gene. The mode of regulating gene expression with specificity makes siRNA as one kind of targeted therapeutic medicine capable of regulating and controlling the expression of relevant disease gene specifically to treat disease. If an siRNA capable of effectively inhibiting the expression of interleukin-8 can be developed, the siRNA is a more effective and more targeted medicament for treating inflammation and cancer.
Disclosure of Invention
In order to solve the above problems, the present invention provides an siRNA for inhibiting interleukin-8, the siRNA comprising a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region; the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NO. 1-21; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NO. 22-42.
In one embodiment of the present invention, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.1 and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO. 22;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 23;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 24;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.4, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 25;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.5, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 26;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.6, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 27;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.7, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 28;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.8, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 29;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.9, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 30;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.10, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 31;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.11, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 32;
or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.12, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 33;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.13, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 34;
or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.14, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 35;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.15, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 36;
or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.16, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 37;
or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.17, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 38;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.18, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 39;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.19, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 40;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.20, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 41;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.21, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 42.
In one embodiment of the invention, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 22;
or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 23;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 24;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 25;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.5, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 26;
or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 27;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.7, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 28;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.8, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 29;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.9, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 30;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.10, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 31;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.11, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 32;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.12, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 33;
or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.13, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 34;
or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.14, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 35;
or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.15, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 36;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.16, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 37;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.17, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 38;
or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.18, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 39;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.19, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 40;
or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.20, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 41;
Or the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.21, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 42.
In one embodiment of the present invention, at least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide.
In one embodiment of the present invention, all of the nucleotides in the sense strand and/or antisense strand of the siRNA are modified nucleotides, and such modifications on the nucleotide groups do not result in a significant impairment or loss of the function of the siRNA of the present disclosure to inhibit interleukin-8 gene expression.
In one embodiment of the invention, the modification comprises methoxy modification, fluoro modification and/or phosphorothioate linkage.
In one embodiment of the present invention, the "phosphorothioate linkage" means that at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA are phosphate groups having a modifying group.
In one embodiment of the present invention, the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom.
In one embodiment of the invention, the fluoro-modified nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5 'end to the 3' end, the nucleotides at least at positions 7, 8, 9 of the sense strand are fluoro-modified nucleotides, and the nucleotides at least at positions 2, 6, 14, 16 of the antisense strand are fluoro-modified nucleotides;
The methoxy modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and at least nucleotides at positions 1,2,3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of the sense strand are methoxy modified nucleotides in the direction from the 5 'end to the 3' end, and at least nucleotides at positions 1,3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21 of the antisense strand are methoxy modified nucleotides;
The phosphorothioate-linked nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and at least between the 1 st and 2 nd, 2 nd and 3 rd nucleotides of the sense strand are linked by phosphorothioate groups according to the direction from the 5 'end to the 3' end, and at least between the 1 st and 2 nd, 2 nd and 3 rd, 19 th and 20 th, 20 th and 21 st nucleotides of the antisense strand are linked by phosphorothioate groups.
The invention also provides a recombinant plasmid which expresses the siRNA.
In one embodiment of the invention, the vector of the recombinant plasmid comprises at least one of a viral vector or a non-viral vector; the viral vector comprises at least one of a flavivirus vector, a retrovirus vector, a phage vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a hybrid virus vector, a baculovirus vector, a herpes simplex virus vector or a lentivirus vector; the non-viral vector includes a plasmid vector.
In one embodiment of the invention, the plasmid vector comprises a pUC plasmid, a pUC plasmid derivative plasmid, a pAAV plasmid derivative plasmid, a PGEM plasmid and/or a PGEM plasmid derivative plasmid.
In one embodiment of the present invention, the recombinant plasmid is prepared by the following method: designing shRNA according to siRNA; and (3) connecting the shRNA with a linearized vector to obtain a recombinant plasmid.
The invention also provides a host cell, wherein the genome of the host cell is integrated with the siRNA; or the host cell carries the recombinant plasmid.
In one embodiment of the invention, the host cell comprises a fungus, a bacterium, a plant cell and/or an animal cell.
The invention also provides application of the siRNA, the recombinant plasmid or the host cell in preparing a medicament for preventing and/or treating diseases, which is characterized in that the diseases are related to the expression of interleukin-8.
In one embodiment of the invention, the disease associated with the expression of interleukin-8 comprises cancer and/or inflammation.
In one embodiment of the invention, the cancer comprises liver cancer, breast cancer, nasopharyngeal cancer, colorectal cancer and/or gastric cancer.
The invention also provides an interleukin-8 inhibitor, the components of which comprise the siRNA, the recombinant plasmid and/or the host cell.
In one embodiment of the invention, the components of the inhibitor further comprise pharmaceutically acceptable excipients; the pharmaceutically acceptable auxiliary materials comprise a carrier, a diluent, a binder and/or a lubricant.
The present invention also provides a medicament for preventing and/or treating a disease associated with the expression of interleukin-8; the components of the drug comprise the siRNA, the recombinant plasmid and/or the host cell.
In one embodiment of the invention, the disease associated with the expression of interleukin-8 comprises cancer and/or inflammation.
In one embodiment of the invention, the cancer comprises liver cancer, breast cancer, nasopharyngeal cancer, colorectal cancer and/or gastric cancer.
In one embodiment of the invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable auxiliary materials comprise a carrier, a diluent, a binder and/or a lubricant.
The technical scheme of the invention has the following advantages:
the invention provides an siRNA for inhibiting interleukin-8, wherein the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NO. 1-21; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NO. 22-42. Experiments prove that the siRNA has high inhibition activity on interleukin-8, so that the siRNA has great application prospect in preparing medicaments for preventing and/or treating diseases (such as cancer, inflammation and the like) related to the expression of the interleukin-8.
Further, the nucleotides at positions 7, 8 and 9 of the sense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at positions 1,2, 3, 4,5,6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 are methoxy-modified nucleotides, and the nucleotides at positions 1 and 2, and the nucleotides at positions 2 and 3 are connected through phosphorothioate groups; the nucleotides at positions 2,6, 14 and 16 of the antisense strand of the siRNA are fluoro modified nucleotides, and the nucleotides at positions 1, 3, 4,5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20 and 21 are methoxy modified nucleotides, and the nucleotides at positions 1 and 2, the nucleotides at positions 2 and 3, the nucleotides at positions 19 and 20 and the nucleotides at positions 20 and 21 are connected through phosphorothioate groups. Experiments prove that the modified siRNA has high inhibition activity on interleukin-8 at the concentration of 0.1-10 nM, so that the modified siRNA has great application prospect in preparing medicaments for preventing and/or treating diseases (such as cancers, inflammations and the like) related to the expression of the interleukin-8.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
The capital letters C, G, U, A in the examples below represent ribonucleotides; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that phosphorothioate group modifications are between two nucleotides adjacent to the letter s.
Example 1: siRNA for inhibiting interleukin-8
This example provides an siRNA for inhibiting interleukin-8, whose nucleotide sequence was designed based on target mRNA, see table 1.
TABLE 1 siRNA inhibiting interleukin-8 and sequence thereof
Example 2: siRNA for inhibiting interleukin-8
This example provides an siRNA for inhibiting interleukin-8, wherein the siRNA is prepared by substituting nucleotides at positions 7, 8, and 9 of the sense strand of the siRNA with fluoro-modified nucleotides, substituting nucleotides at positions 1,2,3, 4,5,6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 with methoxy-modified nucleotides, connecting nucleotides at positions 1 and 2, and 3 via phosphorothioate groups, substituting nucleotides at positions 2, 6, 14, and 16 of the antisense strand of the siRNA with fluoro-modified nucleotides, substituting nucleotides at positions 1,3, 4,5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 with methoxy-modified nucleotides, and connecting nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 21 via phosphorothioate groups.
TABLE 2 siRNA inhibiting interleukin-8 and sequence thereof
Experimental example 1: inhibition activity assay for siRNA for inhibiting interleukin-8
The experimental example provides an inhibition activity detection experiment for inhibiting siRNA of interleukin-8, and the experimental process is as follows:
Hep3B cells (from the cell bank of the national academy of sciences of China) were inoculated into MEM medium (from Gibco, cat# 11095-080) containing 10% (v/v) fetal bovine serum (FBS, from Hyclone Co.) and 1% (v/v) Penicillin streptomycin cocktail (Penicillin-Streptomycin, from Gibco, cat# 15140122) and cultured in a 5% (v/v) CO 2, 37℃cell incubator for 48 h; after the end of the culture, the Hep3B cells obtained by the culture were digested with pancreatin (purchased from GIBCO under the trade designation 25200-072); after digestion, firstly using PBS buffer solution to rinse, and then using MEM culture medium to resuspend the digested Hep3B cells to obtain cell suspension with the cell concentration of 3X 10 5/mL;
diluting the different siRNAs with opti-MEM (available from Gibco under the trade name 31985-070) respectively to obtain siRNA dilutions containing the different siRNAs; mu.L opti-MEM was mixed with 0.25 mu L Lipofectamine RNAiMAX transfection reagent (available from Siemens, cat. No. 11668-019) to give a transfection reagent dilution; mixing 25 mu L of siRNA diluent containing different siRNAs with transfection reagent diluent respectively, and standing at room temperature (25 ℃) for 15min to obtain transfection solution containing different siRNAs;
After inoculating a cell suspension in an inoculum size of 50. Mu.L/well into a 96-well plate, setting a BLANK control group (BLANK), a transfection reagent control group (MOCK), a negative control group (NC), an IL8-H-1M1 test group, an IL8-H-2M1 test group, an IL8-H-3M1 test group, an IL8-H-4M1 test group, an IL8-H-5M1 test group, an IL8-H-6M1 test group, an IL8-H-7M1 test group, an IL8-H-8M1 test group, an IL8-H-9M1 test group, an IL8-H-10M1 test group, an IL8-H-11M1 test group, an IL8-H-12M1 test group, an IL8-H-13M1 test group, an IL8-H-14M1 test group, an IL8-H-15M1 test group, an IL8-H-16M1 test group, an IL8-H-17M1 test group, an IL8-H-9M1 test group, an IL8-H-1 test group, an IL 8-1M 1 test group, an IL8-H-9M1 test group, an IL8-H-10M1 test group, an IL8-H-1 test group and an IL8-H-1 test group in the 96M 1 test group in the 96-well, and a well-plate, respectively, in the 96-well, and setting each of the 96-well plates;
After the completion of the setting, 50. Mu.L of each of the transfection solutions containing different siRNAs was added to the wells of the experimental group (IL 8-H-1M 1-containing transfection solution was administered to the experimental group IL8-H-1M 1-containing transfection solution, IL8-H-2M 1-containing transfection solution was administered to the experimental group IL8-H-2M 1-containing transfection solution, and so forth, wherein the final concentration of the siRNA in the wells was 10 nM), no reagent was added to the wells of the BLANK control group (BLANK), 50. Mu.L of each of the transfection solution containing no siRNA was added to the wells of the transfection reagent control group (MOCK), 50. Mu.L of each of the transfection solutions containing NC (final concentration of NC in the wells was 10 nM), and 48H was cultured in a 5% (v/v) CO 2 and 37℃cell culture incubator to perform transfection; after transfection, discarding the liquid in the hole, collecting cells, and extracting total RNA in the cells by using a magnetic bead method cell total RNA extraction kit (Ji Ma gene-E31008-96) to obtain an RNA extract;
Preparing a genome DNA removal reaction system according to Table 3 to remove genome DNA in the RNA extract to obtain a treated RNA extract (the reaction procedure for removing genome DNA is 42 ℃ C., 2 min); preparing a cDNA synthesis reaction system according to Table 4, and performing reverse transcription on total RNA in the treated RNA extract to obtain cDNA solution (the reverse transcription condition is 50 ℃,15min;85 ℃,2 min); diluting cDNA solution with enzyme-free water for 5 times, taking diluted cDNA solution as a template, taking a gene (HGAPDH genes) encoding glyceraldehyde-3-phosphate dehydrogenase as an internal reference gene, configuring a RT-qPCR probe method reaction system (primers used in the reaction system are shown in a table 6) according to a table 5, and performing fluorescent quantitative PCR reaction on a Lightcycle 480II by using the configured RT-qPCR probe method reaction system (an amplification procedure of the fluorescent quantitative PCR reaction is that the fluorescent quantitative PCR reaction is pre-denatured for 10 min at 95 ℃, denatured for 30 s at 60 ℃ and annealed for 30 s at 72 ℃, and the modified and annealed for 30 s at 72 ℃ are repeated for 40 times, so that a product W containing amplified target genes IL8 and internal reference genes GAPDH is obtained, and sequentially carrying out incubation procedures at 95 ℃ for 15 s,60 ℃ for 1min and 15 s at 95 ℃ by using a real-time fluorescent quantitative PCR instrument to respectively collect the dissolution curves of the target genes IL8 and the internal reference genes GAPDH in the product W, thereby obtaining the Ct value of the target genes (IL 8) and the internal reference gene GAPDH of a test group; the relative quantitative calculation of the target gene (IL 8 mRNA) in each test group is carried out by adopting a comparative Ct (delta Ct) method, and the calculation results are shown in Table 7;
The relative quantitative calculation method comprises the following steps:
delta Ct (test group) =ct (test group target gene) -Ct (test group reference gene);
delta Ct (control) =ct (control target gene) -Ct (control reference gene);
ΔΔct (test group) =Δct (test group) - Δct (control group average);
ΔΔct (control) =Δct (control) - Δct (control average);
When calculation is carried out, the expression level of IL8 mRNA in a test group is normalized by taking a transfection reagent control group as a reference, and the expression level of IL8 mRNA in the transfection reagent control group is defined as 100%;
Relative expression level of test group IL8 mRNA = 2 -ΔΔCt( Test set ) x 100%;
For the same test group siRNA, the average value of the relative expression levels of the test group IL8 mRNA at each concentration is the arithmetic average value of the relative expression levels of 3 culture wells at that concentration;
The inhibition ratio of siRNA to IL8 mRNA expression amount was calculated as follows: inhibition = (relative expression level of IL8 mRNA of 1-test group) ×100%.
As is clear from Table 7, IL8-H-1M1 to IL8-H-21M1 each have an inhibitory activity against interleukin-8, and IL8-H-3M1, IL8-H-8M1, IL8-H-9M1, IL8-H-19M1 and the like have a higher inhibitory activity against interleukin-8 than the BLANK control (BLANK) and the transfection reagent control (MOCK).
TABLE 3 genome removal reaction System
TABLE 4 cDNA synthetic reaction System
TABLE 5 reaction System for RT-qPCR Probe method
TABLE 6 primer information
TABLE 7 inhibition of IL8 mRNA expression levels by different siRNAs (IL 8-H-1M1 to IL8-H-21M 1)
Experimental example 2: inhibition activity assay for siRNA for inhibiting interleukin-8 at different concentrations
The experimental example provides an inhibition activity detection experiment of siRNA for inhibiting interleukin-8 under different concentrations, and the experimental process is as follows:
Based on experimental example 1, IL8-H-3M1, IL8-H-8M1, IL8-H-9M1 and IL8-H-19M1 with higher inhibition activity are selected as study objects, and in the experimental process, the concentration of siRNA in the hole is respectively replaced by 0.1 nM, 1 nM and 10 nM from 10 nM by adjusting the concentration of siRNA after dilution by opti-MEM, and the experimental results are shown in Table 8.
As is clear from Table 8, IL8-H-3M1, IL8-H-8M1, IL8-H-9M1 and IL8-H-19M1 have high inhibitory activity against interleukin-8 at concentrations of 0.1 to 10nM, compared to the BLANK control (BLANK) and the transfection reagent control (MOCK).
TABLE 8 inhibition of IL8 mRNA expression levels by different siRNAs (IL 8-H-3M1/IL8-H-8M1/IL8-H-9M1/IL8-H-19M 1) at different concentrations (0.1 nM, 1 nM and 10 nM)
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. An siRNA for inhibiting interleukin-8, wherein the siRNA comprises a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region; the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NO. 1-21; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown in any one of SEQ ID NO. 22-42.
2. The siRNA of claim 1, wherein the sense strand of said siRNA comprises a nucleic acid molecule having a nucleotide sequence shown in SEQ ID No.1 and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence shown in SEQ ID No. 22;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 23;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 24;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.4, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 25;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.5, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 26;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.6, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 27;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.7, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 28;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.8, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 29;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.9, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 30;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.10, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 31;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.11, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 32;
or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.12, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 33;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.13, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 34;
or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.14, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 35;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.15, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 36;
or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.16, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 37;
or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.17, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 38;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.18, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 39;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.19, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 40;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.20, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 41;
Or the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.21, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 42.
3. The siRNA of claim 1 or 2, wherein at least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide.
4. The siRNA of claim 3, wherein said modification comprises methoxy modification, fluoro modification and/or phosphorothioate linkage.
5. The siRNA of claim 4, wherein the fluoro-modified nucleotides are located in the antisense and sense strands of the nucleotide sequence, and wherein the nucleotides at least at positions 7, 8, 9 of the sense strand are fluoro-modified nucleotides and the nucleotides at least at positions 2, 6, 14, 16 of the antisense strand are fluoro-modified nucleotides in the 5 'to 3' end direction;
The methoxy modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and at least nucleotides at positions 1,2,3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of the sense strand are methoxy modified nucleotides in the direction from the 5 'end to the 3' end, and at least nucleotides at positions 1,3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21 of the antisense strand are methoxy modified nucleotides;
The phosphorothioate-linked nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and at least between the 1 st and 2 nd, 2 nd and 3 rd nucleotides of the sense strand are linked by phosphorothioate groups according to the direction from the 5 'end to the 3' end, and at least between the 1 st and 2 nd, 2 nd and 3 rd, 19 th and 20 th, 20 th and 21 st nucleotides of the antisense strand are linked by phosphorothioate groups.
6. A recombinant plasmid expressing the siRNA of any one of claims 1 to 5.
7. A host cell having a genome into which the siRNA of any one of claims 1 to 5 has been integrated; or the host cell carries the recombinant plasmid of claim 6.
8. Use of the siRNA of any one of claims 1 to 5, the recombinant plasmid of claim 6 or the host cell of claim 7 for the preparation of a medicament for the prevention and/or treatment of a disease, wherein the disease is a disease associated with the expression of interleukin-8.
9. An interleukin-8 inhibitor, wherein the inhibitor comprises the siRNA of any one of claims 1 to 5, the recombinant plasmid of claim 6, and/or the host cell of claim 7.
10. A medicament for preventing and/or treating a disease, wherein the disease is a disease associated with the expression of interleukin-8; the composition of the drug comprises the siRNA according to any one of claims 1 to 5, the recombinant plasmid according to claim 6 and/or the host cell according to claim 7.
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| MEIYUE LOU: "Cancer‑Associated Fibroblast‑Derived IL‑8 Upregulates PD‑L1 Expression in Gastric Cancer Through the NF‑κB Pathway", ANN SURG ONCOL, vol. 31, no. 5, 31 May 2024 (2024-05-31), pages 2983 - 2995 * |
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