CN117384904A - Application of nucleotide capable of simultaneously inhibiting 3 RNAs in preparation of antitumor drugs - Google Patents
Application of nucleotide capable of simultaneously inhibiting 3 RNAs in preparation of antitumor drugs Download PDFInfo
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- CN117384904A CN117384904A CN202311373732.0A CN202311373732A CN117384904A CN 117384904 A CN117384904 A CN 117384904A CN 202311373732 A CN202311373732 A CN 202311373732A CN 117384904 A CN117384904 A CN 117384904A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/36—Arsenic; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/31—Combination therapy
Abstract
The invention discloses a nucleotide for inhibiting the expression of FNDC3bmRNA and circRNA and application thereof, which is characterized in that: the nucleotide achieves the effect of inhibiting lung cancer under the condition of not affecting normal cells by inhibiting the expression of 3 RNAs such as FNDC3bmRNA, circRNA and the like, and the sequence of the nucleotide is as follows: GCUGCACAACAGGUAUACAADTdT and its complementary sequence UUGUAUACUUUGUGCAGCdTdT. The nucleotide can effectively inhibit the expression of FNDC3bmRNA and circRNARNA in the lung cancer representative cell A549 cells, and can kill the A549 cells under the condition of not influencing the normal cell 16HBE, and the anti-tumor effect can be increased by combining with an anti-tumor drug.
Description
Technical Field
The invention belongs to the field of molecular tumor treatment and medicines, and particularly relates to a short-segment RNA. The siRNA can be effectively targeted and combined with the mRNA of FNDC3B and 2 circRNAs thereof, so that the expression of 3 RNAs is inhibited, and the siRNA plays an anti-tumor role with other anti-tumor medicines.
Background
In recent years, the treatment means of lung cancer mainly comprise operation treatment, immunotherapy, targeted treatment and chemotherapy, and the effects are obvious in a short period of time, but the toxic and side effects are obvious, other injuries of organisms are easy to cause, and a new treatment method is urgently needed to be searched.
siRNA is a double-stranded non-coding RNA, also called short interfering RNA or silencing RNA, consisting of 19-23 base pairs, with phosphorylated 5 'and hydroxylated 3' ends. The interference mechanism of siRNA is to make gene silence through the targeted complementation of double-stranded RNA and mRNA, so as to achieve the aim of inhibiting gene expression. In 1998, the phenomenon of RNA interference was found, in 2001 RNA interference was beginning to be applied to the treatment of diseases, in 2004 the first siRNA based drug entered into a first clinical trial, in 2010 the siRNA drug against tumor entered into a first clinical trial, and in 2023 the drug small interfering RNA cholesterol lowering drug for the treatment of adult primary hypercholesterolemia or mixed dyslipidemia was marketed in china. Research for over 20 years proves that RNA interference is of great significance for treatment of clinical diseases. At present, siRNA drugs are used for treating lung cancer, and specific siRNA is mainly delivered through a viral vector, a polymer nano-vector and a lipid nano-vector and combined with a specific target, so that the inhibition of lung cancer cells, the promotion of lung cancer cell apoptosis and the inhibition transfer are realized.
FNDC3B, also called FASD104, has a proline rich region, nine fibronectin type III domains and a transmembrane region. Initially, FNDC3B was identified as a regulator of adipocyte differentiation and osteoblast differentiation, and FNDC 3B-deficient mice died at birth due to lung abnormalities. New studies have shown that the fibronectin type III domain of the FNDC3B protein is involved in cell adhesion and growth signaling and is up-regulated in expression in a variety of human tumors. Furthermore, FNDC3B has also been shown to be an oncogene that plays an important role in regulating tumor cell motility and invasion. Single inhibition of FNDC3B resulted in death of normal cells as well, leading to large side effects that were not clinically useful. Often normal epithelial and cancerous cells are not delivered accurately during delivery, and therefore reducing side effects is critical to the applicability of the RNA.
Disclosure of Invention
The invention aims to provide a nucleotide for inhibiting the expression of FNDC3bmRNA and circRNA and application thereof.
The object of the invention is achieved in that the sequence comprising the synthetic nucleotide is represented by GCUGCACAACAGGUAUACAADTdT (SEQ ID NO. 1) and its complement UUGUAUACUGUUGGUAGGCdTdT (SEQ ID NO. 2). The nucleotide can be applied to preparing anti-lung cancer drugs, and can also be combined with other anti-tumor therapeutic drugs, for example, the nucleotide can be used in alkaline solution sodium arsenite of arsenic trioxide.
The invention (advantage): the nucleotide has good FNDC3bmRNA and circRNA inhibiting effect, acts on specific target sites, has no effect on normal cells by inhibiting the expression of 3 RNAs, has a strong treatment effect on lung cancer cells, achieves the effect of reducing side effects of treating cancers, and can be used with various antitumor drugs.
Drawings
FIG. 1 is a western assay of SiRNA for FNDC3b protein inhibition in A549 cells.
Detailed Description
The invention is further described below without limiting the invention in any way, and any alterations or substitutions based on the teachings of the invention are within the scope of the invention.
Example 1
The RNA sequences in this example are: the siRNA group GCUGCACAACAGAUACAADTdT (SEQ ID NO. 1) and its complementary sequence UGUAUACUGUUGUGCAGCdTdT (SEQ ID NO. 2), the control sequence GUAUGUCGACGCGCUCAUA (SEQ ID NO. 3) and its complementary sequence all are 5-to 3-end product number A06001, shanghai Ji Ma pharmaceutical technology Co., ltd. In the synthesis process, dTdT is added to all the siRNA sequences at the tail end, so that SEQ ID NO.1 and SEQ ID NO.2 have dTdT, UUGUAUACUGUUGUGCAGC as useful sequences, and other products such as UU and the like have the same effect.
The experimental steps are as follows:
1. and (5) carrying out cell subculture. Culturing A549 cells and 16HBE cells in T25 on an ultra-clean workbench, discarding partial cell liquid according to experimental requirements and cell growth period, supplementing a proper amount of culture liquid, and culturing in a constant-temperature CO2 incubator. Collecting cells when the cell abundance reaches 80% -90%, inoculating 3000 cells/well into 96-well plate, inoculating 2.5X105 cells/well into six-well plate, culturing in 5% CO2 incubator at 37deg.C.
2. And (5) transfection. After 22 hours from the inoculation of the cells, the cells were transfected, the transfection solution was prepared on an ultra-clean bench, 4. Mu.L of RFect small nucleic acid transfection reagent was added per 100. Mu.L of double culture solution, and 2. Mu.L of siRNA fragment solution was added per 100. Mu.L of double culture solution, and after 5 minutes of standing, the two were mixed and mixed uniformly, and the mixture was kept stand for 20 minutes. The transfection of a 96-opening plate is to add the mixed transfection reagent after standing for 20 minutes into 800 mu L of double-antibody-free culture solution, suck the liquid in the 96-opening plate, add 50 mu L of the liquid into each hole, the transfection of a six-opening plate is to suck the old culture solution, add 800 mu L of double-antibody-free culture solution into each hole, add 200 mu L of mixed transfection reagent into each hole, and put into constant temperature CO 2 The silencing effect was detected after the incubator continued to incubate for 72 hours.
3. And (5) detecting the cell viability. Cell viability was measured 72 hours after transfection, 20 μl of the prepared CCK-8 solution was added per 100 μl, incubated at 37 ℃ for 15 minutes, absorbance was measured at 450 nm, and once every 10 minutes until the absorbance of the control reached 1.
4. Total RNA extraction and real-time fluorescent quantitative PCR. And (3) adding 1 mL of Trizol reagent into each hole 72 hours after transfection to extract total RNA, using a Roche company to produce a PCR instrument to run a reverse transcription program, carrying out reverse transcription on the extracted total RNA into cDNA, preparing the transcribed cDNA into a Real-Time fluorescent quantitative PCR reaction system, and carrying out Q-PCR on the prepared system in a LightCycle 96 Real-Time system to detect the relative expression of target gene mRNA.
5. Total protein extraction and Western blot. Cells were lysed using IP lysate from bi yun tian company, and the lysed cell solution was centrifuged using a high-speed ultra-low temperature centrifuge manufactured by Thermo Scientific company to obtain protein supernatant, the same amount of protein was electrophoresed by SDS-PAGE, transferred onto PVDF membrane, incubated with primary antibody specific for FNDC3B (proteontech company) at 4 ℃ overnight, and then incubated with appropriate HRP conjugated rabbit secondary antibody (hangzhou wakame biotechnology ltd) at room temperature for 2 hours. Images were obtained by Omni-ECL ™ chemiluminescent detection kit (Shanghai elegance Biotechnology Co., ltd.) and using Western blot imaging system (Shanghai Jiapeng technologies Co.).
The sequence of the detection primer is as follows: hsa_circ_0067990 primer sequence: TTCAGGCAAGAGCAGTTGTG (SEQ ID NO. 4), TTGCTGTACTGGTCTTCTCG (SEQ ID NO. 5), hsa_circ_0006156 primer sequence TCAGATAGGGAGGGAGATGA (SEQ ID NO. 6), GTACTATCTTCAATCACCTTGC (SEQ ID NO. 7), mRNA detection sequence: ACTGAAAGACCGCCAGATCG (SEQ ID NO. 8) and TCTTGCTCGTCGCTCTGTTT (SEQ ID NO. 9).
Results: FNDC3BmRNA relative expression level (x+ -s) in A549 cells was 0.99+ -0.06 in control group, siRNA group was 0.33+ -0.09, as a result, hsa_circ_0006156 relative expression level (x ̅ + -s) in A549 cells was 1.01+ -0.07 in control group, siRNA group was 0.39+ -0.02 in control group, hsa_circ_0067990 in A549 cells was 1.03+ -0.04 in control group, siRNA group was 0.32+ -0.03 in control group, above fluorescence was quantified for 3 sub-wells, A549 cell survival rate (x+ -s,%) was 100.01+ -3.22 in control group, siRNA group was 69.75+ -6.40, CCK8 was 6 sub-wells, above data were statistically different.
Results: the control group of the relative expression level (x ̅ + -s) of FNDC3BmRNA in the 16HBE cells is 1.01+ -0.07, the siRNA group is 0.40+ -0.03, the result is that the control group of the relative expression level (x ̅ + -s) of hsa_circ_0006156 in the 16HBE cells is 1.04+ -0.05, the siRNA group is 0.29+ -0.01, the control group of hsa_circ_0067990 in the 6HBE cells is 1.03+ -0.05, the siRNA group is 0.37+ -0.02, the above fluorescence quantifies more than 3 secondary wells with statistical differences, the control group of the survival rate (x ̅ + -s,%) of the 16HBE cells is 100+ -2.30, the siRNA group is 99.88+ -2.50, and the CCK8 is 6 secondary wells with no statistical differences.
Example 2
The procedure was as in example 1, using only a549 cells grouped as control, arsenic-plus-group, siRNA interference-plus-arsenic group, with concentration of sodium arsenite-plus-arsenic 10 μm, and time of addition 6 hours after interference, CCK8 (x ̅ ±s,%) data for four groups were detected: the control group 101.00, + -3.21, arsenic group 92.22+ -2.23, siRNA interference group 71.55 + -3.21, siRNA interference and arsenic group 50.33+ -1.23 have remarkable synergistic effect.
Claims (4)
1. A nucleotide for inhibiting the expression of 3 RNAs related to FNDC3B is characterized in that the sequence is GCUGGACAACAGGUACAADTdT (SEQ ID NO. 1) and the complementary sequence is UUGUAUACUGUUGUGCAGCdTdT (SEQ ID NO. 2).
2. The use of a nucleotide for inhibiting the expression of 3 RNAs associated with FNDC3B according to claim 1, characterized by the use in the preparation of a medicament for the treatment of lung cancer.
3. Use of a pharmaceutical composition for the preparation of a medicament for treating lung cancer, comprising the nucleotide for inhibiting expression of 3 RNAs associated with FNDC3B of claim 1 and sodium arsenite.
4. A pharmaceutical composition according to claim 3, characterized in that the concentration of sodium arsenite in the composition is 10uM.
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| CN202311373732.0A CN117384904A (en) | 2023-10-23 | 2023-10-23 | Application of nucleotide capable of simultaneously inhibiting 3 RNAs in preparation of antitumor drugs |
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