CN114134146B - Long-chain non-coding RNA and application thereof - Google Patents

Long-chain non-coding RNA and application thereof Download PDF

Info

Publication number
CN114134146B
CN114134146B CN202111358287.1A CN202111358287A CN114134146B CN 114134146 B CN114134146 B CN 114134146B CN 202111358287 A CN202111358287 A CN 202111358287A CN 114134146 B CN114134146 B CN 114134146B
Authority
CN
China
Prior art keywords
mapkapk5
lncrna
mir
expression
apoptosis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111358287.1A
Other languages
Chinese (zh)
Other versions
CN114134146A (en
Inventor
刘健
文建庭
万磊
王馨
王杰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
First Affiliated Hospital of AHUTCM
Original Assignee
First Affiliated Hospital of AHUTCM
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by First Affiliated Hospital of AHUTCM filed Critical First Affiliated Hospital of AHUTCM
Priority to CN202111358287.1A priority Critical patent/CN114134146B/en
Publication of CN114134146A publication Critical patent/CN114134146A/en
Application granted granted Critical
Publication of CN114134146B publication Critical patent/CN114134146B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention provides a long-chain non-coding RNA and application thereof, belonging to the field of rheumatism molecular biology. The cDNA sequence of lncRNA MAPKAPK5-AS1 is shown in SEQ ID No: 1 is shown. The long-chain non-coding RNA and the miR-146a-3p as molecular intervention targets can be applied to regulation and control of cell inflammation reaction and apoptosis deficiency, and the miR-146a-3p sequence is shown as SEQ ID No: 2, respectively. The invention utilizes the total-transcription high-throughput sequencing technology to analyze rheumatoid arthritis patients to obtain lncRNA MAPKAPK5-AS1 which is closely related to the cell inflammation reaction and apoptosis deficiency, and can play a role of miRNA molecules in a sponge-like manner and competitively combine miR-146a-3p, thereby antagonizing the functions of miRNA, inhibiting the expression of SIRT1 gene, promoting the cell inflammation reaction and inhibiting the cell apoptosis.

Description

Long-chain non-coding RNA and application thereof
Technical Field
The invention belongs to the field of rheumatism molecular biology, and relates to a long-chain non-coding RNA and application thereof.
Background
Long non-coding RNA (lncNA) is a regulatory non-coding RNA with a length of more than 200nt, which was considered to be a "noise" or "dark material" and has no ability to code for protein. In recent years, many studies show that lncRNA can play a sponge-like role at epigenetic, transcriptional and post-transcriptional levels, competitively bind to microRNA (microRNA, miRNA), influence the expression of target genes, regulate downstream pathways, and participate in the progress of various diseases.
Fibroblast-like synoviocytes (FLS) are inflammatory cells, which are the main components of synoviocytes, secrete a large amount of inflammatory cytokines, and long-term inflammatory stimulation can cause joint swelling and pain, deformity, loss of function and the like. The stimulation of inflammatory signals can lead FLS to abnormally proliferate and lack apoptosis, thereby generating a plurality of inflammatory cytokines, and the generated inflammatory cytokines continuously activate FLS, and the two factors mutually promote to form a vicious circle. Immune inflammatory response and apoptosis deficiency are associated with the pathogenesis of a variety of rheumatic diseases, such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gouty arthritis, and the like. Therefore, the research finds that lncRNA related to cell inflammation and apoptosis has great clinical application value.
Disclosure of Invention
The invention utilizes the total transcription high-throughput sequencing technology to analyze the lncRNAs of the RA patient, obtains an lncRNA MAPKAPK5-AS1 related to the insufficient cell inflammatory reaction and apoptosis, and has the potential of becoming a new target for inhibiting the synovial inflammatory reaction of the rheumatism patient and promoting the synovial cell apoptosis.
The invention adopts the following specific technical scheme: a long non-coding RNA, is lncRNA MAPKAPAK 5-AS1, and the cDNA sequence is shown in SEQ ID No: 1 is shown.
The invention also aims to provide application of the long-chain non-coding RNA and miR-146a-3p as molecular intervention targets in regulation and control of cell inflammatory reaction and apoptosis deficiency, wherein the miR-146a-3p sequence is shown in SEQ ID No: 2, respectively.
The long-chain non-coding RNA is taken as miRNA molecule sponge to be specifically combined with miR-146a-3p, so that the function of miRNA is inhibited; after the function of the miR-146a-3p is inhibited, the expression of the SIRT1 gene can be promoted, so that the cell inflammatory response and the apoptosis deficiency can be regulated.
The invention also aims to provide application of the long non-coding RNA and miR-146a-3p as molecular intervention targets in preparation of medicines for treating rheumatism related to cell inflammation reaction and apoptosis deficiency.
The rheumatism associated with the cellular inflammatory reaction and the apoptosis deficiency includes rheumatoid arthritis, ankylosing spondylitis, osteoarthritis or gouty arthritis.
The invention relates to a specific research application, in particular to an application of long-chain non-coding RNA as a molecular intervention target in drugs for inhibiting inflammatory reaction of fibroblast-like synoviocytes and promoting apoptosis of the fibroblast-like synoviocytes.
The invention utilizes a full-transcription high-throughput sequencing technology to analyze RA patients to obtain 30 lncRNAs with differential expression, wherein the lncRNA MAPKAPK5-AS1 is expressed and reduced in the RA patients. RT-qPCR demonstrated that the expression change of lncRNA MAPKAPK5-AS1 is consistent with the high-throughput sequencing result, and the expression trend of fibroblast-like synoviocytes (FLS) is consistent with that of PBMCs. RACE reaction obtains the full-length sequence of lncRNA MAPKAPK5-AS1, which is located at 12q24.12-q 24.13. Subcellular localization analysis lncRNA MAPKAPK5-AS1 is mainly expressed in the cytoplasm of FLS.
The research of the invention shows that the lncRNA MAPKAPK5-AS1 is overexpressed in vitro, the inflammatory reaction of the FLS is obviously inhibited, the apoptosis of the cell is promoted, the lncRNA MAPKAPK5-AS1 is interfered in vitro, the inflammatory reaction of the FLS is obviously promoted, and the apoptosis of the cell is inhibited. Sequencing result analysis and ceRNA prediction combined with Luciferase detection suggest that lncRNA MAPKAPK5-AS1 is combined with miR-146a-3p to regulate the expression of a target gene SIRT1 related to inflammation reaction and apoptosis deficiency.
The research of the invention suggests that lncRNA MAPKAPK5-AS1 has the potential to be a new target for inhibiting inflammatory response and promoting apoptosis by influencing inflammation and apoptosis of FLS through regulating gene expression related to cell inflammatory response and apoptosis deficiency.
Drawings
FIG. 1 shows a schematic representation of the inventionExpression trends and distributions of ncRNA MAPKAPK5-AS1 (MK 5-AS1 represents lncRNA MAPKAPK5-AS1 in the figure, the same below). FIG. 1A shows that RT-qPCR detected the expression change of IncRNA MAPKAPK5-AS1 (referred to AS beta-actin reference) in 30 RA patients and 30 normal human PBMCs, and the expression of IncRNA MAPKAPK5-AS1 was reduced in the RA patients PBMCs compared with the normal human (see: beta-actin reference) (see: FIG.)***p<0.001), consistent with the sequencing results. FIG. 1B shows RT-qPCR detection of lncRNA MAPKAPK5-AS1 expression in RA-FLS (using. beta. -action reference), and after TNF-alpha stimulation, lncRNA MAPKAPK5-AS1 expression was reduced (see (B)) ***p<0.001), consistent with expression in PBMCs. FIG. 1C shows the results of FISH experiments, that lncRNA MAPKAPK5-AS1 is located in the cytoplasm of RA-FLS.
FIG. 2 shows the effect of interference and overexpression of lncRNA MAPKAPK5-AS1 on the inflammation and apoptosis of RA-FLS according to the present invention. FIG. 2A shows that interference and overexpression of lncRNA MAPKAPK5-AS1 in ELISA assay were detected to affect the inflammation of RA-FLS, which are inflammatory cytokines IL-8 and IL-10, respectively. FIG. 2B shows Western Blotting experiment to detect the effect of interference and overexpression of lncRNA MAPKAPK5-AS1 on the apoptotic proteins of RA-FLS, including the apoptotic proteins Bax and Bcl-2. FIG. 2C is Annexin V-TITC/PI staining, flow cytometry is used for detecting the influence of interference IncRNA MAPKAPK5-AS1 on RA-FLS apoptosis, and FIG. 2D is a histogram which is a statistical graph of the apoptosis rate of RA-FLS cells in different experimental groups of flow detection. (in comparison with the control group,***p<0.001, compared to si-NC group,###p<0.001, compared with OV-NC group,▲▲▲p<0.001, ns means no statistical significance).
FIG. 3 shows that lncRNA MAPKAPK5-AS1 affects the expression of gene SIRT1 by regulating miR-146a-3 p. FIG. 3A is RT-qPCR to detect the effect of interference and over-expression of lncRNA MAPKAPK5-AS1 on miR-146a-3p expression in RA-FLS. FIG. 3B shows RT-qPCR detection of interference and over-expression of lncRNA MAPKAPK5-AS1 in RA-FLS affecting the expression of gene SIRT 1. FIG. 3C shows the binding of IncRNA MAPKAPK5-AS14 and miR-146a-3p detected by Luciferase reporter assays. FIG. 3D is a graph of Luciferase reporter assays for detecting the binding of miR-146a-3p to the 3' UTR of SIRT 1. FIG. 3E shows the expression of SRIT1 after RT-qPCR detection of miR-146a-3p transfection in RA-FLS. (in comparison to the control group, ***p<0.001, compared to si-NC group,###p<0.001, compared with OV-NC group,▲▲▲p<0.001, ns means no statistical significance).
FIG. 4 shows that lncRNA MAPKAPK5-AS1 regulates RA-FLS inflammation and apoptosis through miR-146a-3 p. FIG. 4A is an ELISA experiment for detecting the effect of miR-146a-3p overexpression on MAPKAPPK 5-AS1 overexpression on the regulation of RA-FLS inflammation, namely inflammatory cytokines IL-8 and IL-10 respectively. FIG. 4B is a Western Blotting experiment to detect the effect of miR-146a-3p overexpression on MAPKAPPK 5-AS1 overexpression, i.e., the regulation of RA-FLS apoptotic proteins, i.e., apoptotic proteins Bax and Bcl-2, respectively. FIG. 4C shows Annexin V-TITC/PI staining, flow cytometry is used for detecting the influence of overexpression miR-146a-3p on the regulation of RA-FLS apoptosis through overexpression of lncRNA MAPKAPK5-AS1, and a histogram in FIG. 4D is a statistical chart for the apoptosis rate of RA-FLS cells in different experimental groups through flow detection. (in comparison with the control group,***p<0.001, compared with OV-NC group,###p<0.001, compared to the imic-NC group,▲▲▲p<0.001, ns means no statistical significance).
FIG. 5 shows that lncRNA MAPKAPK5-AS1 regulates RA-FLS inflammation and apoptosis through SIRT 1. FIG. 5A is an ELISA experiment to examine the effect of interfering SIRT1 on the regulation of RA-FLS inflammation by overexpressing lncRNA MAPKAPK5-AS1, which are inflammatory cytokines IL-8 and IL-10, respectively. FIG. 5B is a Western Blotting experiment to examine the effect of interfering SIRT1 on the regulation of RA-FLS apoptotic proteins, respectively apoptotic proteins Bax and Bcl-2, by overexpressing lncRNA MAPKAPK5-AS 1. FIG. 5C is Annexin V-TITC/PI staining, flow cytometry is used for detecting the influence of interfering SIRT1 on the regulation of RA-FLS apoptosis by overexpressing lncRNA MAPKAPK5-AS1, and FIG. 5D is a histogram which is a statistical graph of the apoptosis rate of RA-FLS cells in different experimental groups of flow detection. (in comparison to the control group, ***p<0.001, compared with OV-NC group,###p<0.001, compared to si-NC group,▲▲▲p<0.001, ns means no statistical significance).
Detailed Description
Example 1
Investigating the expression and distribution characteristics of lncRNA MAPKAPK5-AS1
1. RNA extraction and RT-qPCR in PBMCs of RA patients
Collecting peripheral blood of RA patients by using an EDTA anticoagulant tube, extracting PBMCs, extracting RNA in the PBMCs according to TRIzol Reagent (Invitrogen) instructions, performing reverse transcription, performing RT-qPCR by using PrimeScript RT-PCR Kit (Takara), performing the operation according to the Kit instructions (taking beta-actin as an internal reference), and performing the reaction program of a PCR instrument: stage 1: 95 ℃ for 1min, Stage2 (Cycle: 40): 95 ℃ for 20s, 60 ℃ for 1 min. The primer sequence of lncRNA MAPKAPK5-AS1 is shown in SEQ ID 1. RT-qPCR results are shown in FIG. 1A, and the expression of lncRNA MAPKAPK5-AS1 in PBMCs of RA patients is reduced compared with that of normal people.
Figure BDA0003355434030000041
2. Isolation, identification, preparation and culture of RA-FLS
Taking tissues of knee joints of patients with RA operation, soaking in 75% alcohol for about 2min, placing in PBS containing P/S, cutting tissue blocks into squares with side length of about 0.1cm, repeatedly cleaning, discarding supernatant, placing in a culture dish containing complete culture medium, incubating at 37 deg.C for 30-60 min, and placing upside down in 5% CO2Incubating for 2h in incubator, 2ml fibroblast complete culture medium, infiltrating tissue block, placing in 5% CO 2In the cell culture box, the liquid is changed every 3 days, when the cells growing around the tissue block are fused into slices, the tissue block can be removed, and the cells are digested by trypsin. After cell climbing, sucking out the culture medium, adding 4% PFA, fixing at 4 ℃ for 30min, taking 50uL of broken membrane sealing liquid, dripping on a waterproof membrane, covering one surface with cells on a glass slide for 2h, after breaking membrane, taking 50uL of primary antibody on the waterproof membrane, incubating secondary antibody (PBS: 1:500) for 2h at room temperature in a dark place, washing with PBS for 3 × 5 min/time, staining with DAPI (DAPI: PBS: 1:1000) for 5min, washing with PBS for 3 × 5 min/time, dripping 1 drop of fluorocount-G on each glass slide, covering one surface with cells, and identifying the cells as P1 generation cells. Cells were seeded into 6-well plates at approximately 1X 10 cells per well5After the cells attached to the wall, 1mL of complete medium was added, 20. mu.L of SV40 overexpressed lentivirus was added, after the cells grew to the bottom of the plate, they were passed to a T25 flask, and fresh medium containing puromycin (1ug/mL, 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL, 7ug/mL) at various concentrations was added to a 24-well plate with the cells already plated, the smallest purinThe using concentration of the puromycin is the lowest screening concentration for killing all cells within 1-4 days from the puromycin screening, and the result is that the using concentration of the puromycin is 1ug/mL, the action time is 2d, the RA-FLS immortalized cell line is prepared, and after the cells are fused, the purity is over 95 percent and the purities are used for subsequent experiments. RA-FLS at 37 ℃ 5% CO 2The cell culture flasks of (1) were cultured in high-glucose DMEM medium supplemented with 15% (V/V) heat-inactivated Fetal Bovine Serum (FBS) and penicillin streptomycin solution.
3. RNA extraction and RT-qPCR in RA-FLS
RNA in RA-FLS was extracted according to TRIzol Reagent (Invitrogen) instructions, reverse transcription was performed, RT-qPCR was performed using PrimeScript RT-PCR Kit (Takara), the procedure was performed according to the Kit instructions (with. beta. -actin as an internal reference), PCR instrument reaction program: stage 1: 95 ℃ for 1min, Stage2 (Cycle: 40): 95 ℃ for 20s, 60 ℃ for 1 min. The RT-qPCR result is shown in FIG. 1B, after TNF-alpha stimulation, lncRNA MAPKAPK5-AS1 expression in RA-FLS was reduced, which is consistent with the expression trend in PBMCs of RA patients.
4. FISH experiment for detecting subcellular localization of lncRNA
Fixing the cell slide: fixing the cell slide in 4% paraformaldehyde (DEPC) for 20min, and washing on a decolorizing shaker for 5min for 3 times in PBS (pH 7.4); digestion: the gene pen draws circles, protease K (20ug/ml) is dripped to digest for 5min according to different index characteristics of different tissues, and PBS is washed for 3 times and multiplied by 5min after pure water washing; pre-hybridization: dropwise adding a prehybridization solution into a constant temperature box at 37 ℃ for 1 h; and (3) hybridization: pouring out the pre-hybridization solution, dripping the hybridization solution (containing 500nM concentration of the probe MAPKAPK5-AS 1) and hybridizing at 42 ℃ overnight; washing after hybridization: the hybridization solution was washed off, 2 XSSC, 10min at 37 ℃, 2 XSSC, 2X 5min at 37 ℃ and 10min at 0.5 XSSC 37 ℃. If the number of non-specific hybrids is large, formamide washing can be increased; and (3) secondary standard incubation: and (3) dropwise adding hybridization solution containing two standard probes, wherein the dilution ratio is 1: 400. incubating at 42 deg.C for 3h, washing at 37 deg.C for 10min, washing at 37 deg.C for 2 × 5min, washing at 37 deg.C for 10min, and washing at 0.5 × SSC for 37 deg.C for 10 min; dropwise adding a confining liquid: adding dropwise normal rabbit serum containing closed serum at room temperature for 30 min; the mouse anti-digoxin labeled peroxidase (anti-DIG-HRP) was added dropwise: pouring out the blocking solution, dropwise adding anti-DIG-HRP, incubating at 37 ℃ for 50min, and washing with PBS for 3 times and 5 min; adding CY3-TSA dropwise: adding CY3-TSA reagent dropwise, keeping out of the sun, reacting at room temperature for 5min, and washing with PBS for 3 times and 5 min; DAPI counterstaining nuclei: dripping DAPI dye solution into the slices, incubating for 8min in a dark place, and dripping an anti-fluorescence quenching sealing agent into the slices after washing; and (5) microscopic examination and photographing: the section is observed under a Nikon positive fluorescence microscope and an image is collected (CY3 red light excitation wavelength 510-560, emission wavelength 590nm and red light); interpretation of cell slide digoxin fluorescence in situ hybridization experiment results: DAPI-stained nuclei were blue under UV excitation and positively expressed as red light labeled with the corresponding fluorescein (CY 3). The results are shown in FIG. 1C, and indicate that lncRNA MAPKAPK5-AS1 is mainly expressed in cytoplasm
Example 2
Investigation of the Effect of interference and overexpression of lncRNA MAPKAPK5-AS1 on inflammation and apoptosis of RA-FLS
1. RA-FLS transfection
Small molecule RNA transfection: in this experiment, Lipofectamine TMRNAiMAX was used to transfer siRNA (final concentration: 100nM), miR-146a-3p imic (purchased from Ribo Biotechnology Co., Ltd., Guangzhou, final concentration: 20nM) into RA-FLS, which was changed to complete medium the next day, and the subsequent experiments were performed as needed. The sequences of siRNAs si-1, si-2 and si-3 to LncRNA MAPKAPK5-AS1 are shown below, and the sequences of siRNAs si-1, si-2 and si-3 to SIRT1 are shown below:
Figure BDA0003355434030000061
Figure BDA0003355434030000062
plasmid DNA transfection: inoculating cells AS required, when the density of RA-FLS reaches above 85%, using Lipofectamine TM2000 to mix pcDNA3.1-NC, pcDNA3.1-MAPKAPK5-AS1 according to lip 3000: plasmid 2 μ l: uniformly dripping 1 mu g of the culture medium into the cell culture solution, slightly mixing the culture medium and the cell culture solution uniformly, changing the culture medium into a new complete culture medium after 4-6 hours, changing the culture medium into the new complete culture medium again the next day, and carrying out subsequent experiments according to the needs.
2. ELISA experiments
Taking cell supernatant, centrifuging at 4000rpm for 20min, removing cell particles and polymers, and storing the supernatant at below-20 ℃ to avoid repeated freeze thawing; removing the required panels from the aluminum foil bags: setting a standard product hole, a 0-value hole, a blank hole and a sample hole, wherein the standard product hole is respectively added with 50 mu L of standard products with different concentrations, the 0-value hole is added with 50 mu L of the diluent, the blank hole is not added, and the sample hole is added with 50 mu L of a sample to be detected; adding 100 mu L of detection antibody marked by horseradish peroxidase (HRP) into the standard wells, the 0-value wells and the sample wells except for blank wells; covering the reaction plate with a sealing plate film, and incubating for 60min in a 37 deg.C water bath or thermostat; uncovering the unsealing plate film, discarding liquid, patting dry on absorbent paper, filling each hole with cleaning solution, standing for 20S, throwing off the cleaning solution, patting dry on the absorbent paper, and repeating the steps for 5 times; fully mixing the substrates A and B according to the volume of 1:1, adding 100 mu L of substrate mixed solution into all the holes, covering the reaction plate with a sealing plate film, and incubating for 15min in a water bath kettle or a thermostat at 37 ℃; the stop solution was added to 50. mu.L of each well, and the absorbance (OD value) of each well was read on a microplate reader. The siRNA (si-2) and siRNA Negative Control (NC) with the highest interference efficiency of lncRNA MAPKAPK5-AS1 and overexpression plasmids pcDNA3.1-MAPKAPK5-AS1 and pcDNA3.1-NC are used for transfecting RA-FLS, and after transfection is carried out for 48h, operation is carried out according to a kit method, and the ELISA experiment result is shown in FIG. 2A. The results show that: compared with a control group, the model group has higher IL-8 expression and lower IL-10 expression, compared with a si-NC group, the model group has higher IL-8 and lower IL-10 expression after interfering lncRNA MAPKAPPK 5-AS1, and compared with a pcDNA3.1-NC group, the model group has lower IL-8 and higher IL-10 expression after over-expressing lncRNA MAPKPK 5-AS 1.
3. Western blot experiment
After collecting cells, adding RIPA cell lysate (containing 1mM PMSF) into a 6-well plate at 150uL per well for lysis, centrifuging at 12,000rpm for 10min, and collecting supernatant containing total cell proteins; preparing SDS-PAGE gel; adding a 5X SDS-PAGE protein loading buffer solution into a cell protein sample according to the ratio of 1:4, and heating in a boiling water bath for 10 minutes to fully denature proteins; after the sample is cooled to room temperature, directly loading the protein sample into SDS-PAGE gel loading holes, and loading 30ug of sample into each hole; cutting filter paper and a PVDF film (which are soaked in methanol for 3 minutes in advance) which have the same size as the adhesive tape in advance, soaking the filter paper and the PVDF film in a film-transferring buffer solution for 5 minutes, sequentially placing an anode plate, 3 layers of filter paper, the PVDF film, gel, 3 layers of filter paper and a cathode plate by a film-transferring device from top to bottom, accurately aligning the filter paper, the gel and the PVDF film, and transferring the film at a constant current of 300 mA; after the membrane conversion is finished, immediately placing the protein membrane into a prepared Western washing solution, rinsing for 5 minutes to wash the membrane conversion solution on the membrane, adding a Western confining liquid (5% skimmed milk powder), slowly shaking on a shaking table, and sealing for 2 hours at room temperature; diluting with beta-actin (1: 1000), Bax (1: 1000) and Bcl-2 (1: 1000) primary antibody, and incubating overnight at 4 deg.C under slow shaking; horseradish peroxidase (HRP) -labeled secondary antibodies were diluted with secondary antibody diluent according to beta-actin (1: 1000), Bax (1: 3000) and Bcl-2 (1: 3000); mixing ECL A solution and ECL B solution in a centrifuge tube at a ratio of 1:1 in a dark room, putting the PVDF membrane with the protein surface facing upwards in the center of an exposure plate of an automatic exposure instrument, adding the mixed ECL solution for full reaction, and analyzing the film strip by using Image J software. The siRNA-NC with the highest interference efficiency of lncRNA MAPKAPK5-AS1 and the overexpression plasmids pcDNA3.1-MAPKAPK5-AS1 and pcDNA3.1-NC were used for transfection of RA-FLS, and after transfection for 48h, the operation was carried out according to a kit method, and the ELISA experiment results are shown in FIG. 2A. The results show that: compared with a control group, the model group has increased Bcl-2 expression and reduced Bax expression, compared with a si-NC group, the model group has increased Bcl-2 expression and reduced Bax expression after interfering with lncRNA MAPKAPK5-AS1, and compared with a pcDNA3.1-NC group, the model group has reduced Bcl-2 expression and increased Bax expression after over-expressing lncRNA MAPKPK 5-AS 1.
4. Annexin V-FITC apoptosis assay
Digesting RA-FLS by pancreatin, and transferring to 5ml centrifuge; centrifuging for 5 minutes at 1000g, discarding supernatant, collecting cells, resuspending the cells with a proper amount of PBS and counting; taking 5-10 ten thousand of resuspended cells, centrifuging for 5 minutes at 1000g, abandoning the supernatant, and adding 195 mul Annexin V-FITC binding solution to gently resuspend the cells; add 5. mu.l Annexin V-FITC and mix gently. Adding 10 μ l PI (propidium iodide) staining solution, and mixing gently; incubating at room temperature (20-25 deg.C) in dark for 10-20 min, placing in ice bath, and incubating in dark with aluminum foil for 2-3 times to improve staining effect; then, the fluorescent probe is detected by a flow cytometer, Annexin V-FITC is green fluorescence, and Propidium Iodide (PI) is red fluorescence. After transfection of siRNA-NC and over-expression plasmids pcDNA3.1-MAPKAPK5-AS1 and pcDNA3.1-NC with siRNA-FLS with highest interference efficiency of lncRNA MAPKAPK5-AS1 for 48h, flow cytometry is performed to detect apoptosis of RA-FLS, and flow results are shown in FIG. 2C, wherein compared with a control group, apoptosis rate of model group is reduced, compared with a si-NC group, apoptosis rate of lncRNA MAPKPK 5-AS1 is interfered, and compared with a pcDNA3.1-NC group, apoptosis rate of over-expression lncRNA is increased (FIG. 2D).
Example 3
Investigation of the influence of lncRNA MAPKAPK5-AS1 on the expression of the gene SIRT1 by regulating miR-146a-3p
1. Target Gene screening
For lncRNAs obtained by high-throughput sequencing, 341 genes which are more than 2 times lower are screened, and 16 lncRNAs related to inflammation and apoptosis are obtained by GO and KEGG analysis. According to the correlation between the full-length sequence of lncRNA MAPKAP 5-AS1 and the gene co-expression network, 15 lncRNA MAPKAPK5-AS1 which can be regulated in a cerRNA mode are selected by a method for predicting the cerRNA by using bioinformatics established by Tay Y and the like (Tay Y, Kats L, Salmena L, et al. coding-independent regulation of the tumor-competent PTEN b + complex mRNAs [ J ] Cell,2011,147(2):344 + 357.), and are combined with three public databases of miase RBS, targetScan and RNAhybrid, the two miRNAs are intersected by 5 miRNAs and are further confirmed by RT-qPCR and dual luciferase report to be miR-146a-3 p; reference is next made to Yang Y et al (Yang Y, Li L. depletion microRNA-146a-3p proteins lipid polysaccharides-induced amino acids in nucleic acid up-regulating SIRT1 and mediating NF-. kappa.B pathway [ J ]. J Drug Target,2021,29(4):420-429.) to predict that the miRNA downstream Target gene is SIRT1 and verified by SIRT-qPCR and dual-luciferase report.
2. RT-qPCR detection of interference and expression of miR-146a-3p and SIRT1 after overexpression of lncRNA MAPKAPK5-AS1
RA-FLS was transfected with siRNA (si-2) specific to lncRNA MAPKAPK5-AS1 and pcDNA3.1-MAPKAPK5-AS1, cells were harvested 48h after transfection, RNA was extracted, and RT-qPCR was used to detect expression of miR-146a-3p and SIRT1, and the primer sequence of gene SIRT1 is shown. The results are shown in FIGS. 3A and 3B, and show that after lncRNA MAPKAPK5-AS1 is interfered, miR-146a-3p expression is increased, and SIRT1 expression is reduced, and after lncRNA MAPKAPK5-AS1 is over-expressed, miR-146a-3p expression is reduced, and SIRT1 expression is increased. The lncRNA MAPKAPK5-AS1 can regulate the expression of miR-146a-3p and SIRT 1.
Figure BDA0003355434030000091
3. Dual luciferase reporter assay
Detection was carried out using the Dual-Luciferase Reporter Assay System (Promega) kit. After 293T cells are transfected, the cells are continuously cultured for 24h, the cell state is observed, and the cells are washed once by precooled PBS; adding 100 μ l of 1 XPLB, and shaking the plate shaker vigorously at room temperature for 15 min; each hole was blown and punched 5 times, and the lysate was transferred to an EP tube; centrifuging at 4 ℃, 13000rpm, 5 min; adding 20 mul LABII into a 96-well plate, adding 20 mul cell lysate, blowing and beating each well for 5 times, uniformly operating, and placing the well on an enzyme labeling instrument to detect the fluorescence intensity of Fireflyfiuferase (Firefly) by using software Gene 5; then 20 mul of Stop & Glo is added, and the fluorescence intensity of Renilla luciferase (Renilla) is detected by a microplate reader; the two fluorescences were compared (relative luciferase). The experiment is repeated for more than 3 times by 3 times of repeated wells each time, and the analysis result is counted. The full length of lncRNA MAPKAPK5-AS1 was constructed on a pmirGLO vector, pmirGLO-lncRNA MAPKAPK5-AS1-full, miR-146a-3p mix and mix NC were co-transfected in 293T cells, and after 24h of cell culture, the fluorescence values of Firefly and Renilla were detected and Relative luciferase (Firefly/Renilla) was calculated using the dual luciferase reporter system according to the above method, and the results are shown in FIG. 3C. The results show that: compared with the mimic NC, the experimental group transfected with the miR-146a-3p mimic has the advantage that the standardized luciferase activity is remarkably reduced, which indicates that the lncRNA MAPKAPK5-AS1 can be combined with the miR-146a-3p to a certain extent. Then, the binding condition of the miR-146a-3p and the 3' -UTR of the corresponding target gene SIRT1 is detected through luciferase experiment, and the result is shown in figure 3D. The result shows that compared with the mimic NC, the luciferase activity is remarkably reduced after the miR-146a-3p is transfected, and the miR-146a-3p can be combined with SIRT 1.
4. RT-qPCR detection of influence of miRNAs on expression of target gene SIRT1
In RA-FLS, miR-146a-3p mimic, mimic NC, miR-146a-3p inhibitor and inhibitor NC are transfected. 48h after miRNA transfection, cell harvesting, RNA extraction, reverse transcription, and RT-qPCR detection of gene SIRT1 expression by the same method. The RT-qPCR detection result is shown in figure 3E, and the result shows that the expression of SIRT1 is obviously reduced after miR-146a-3p imic is transfected, and the expression of SIRT1 is obviously improved after miR-146a-3p inhibitor is transfected. This shows that miR-146a-3p can inhibit the expression of SIRT1 by binding with 3' -UTR of SIRT1, and lncRNA MAPKAPK5-AS1 can competitively bind to miR-146a-3p, thereby antagonizing the inhibition of miR-146a-3p on the expression of SIRT 1.
Example 4
Investigation of lncRNA MAPKAPK5-AS1 Regulation of RA-FLS inflammation and apoptosis by miR-146a-3p
Transfecting overexpression lncRNA MAPKAPK5-AS1 in RA-FLS, changing back to a conventional culture medium after 12h, continuing to culture, transfecting miR-146a-3p imic and Negative Control (NC) in the RA-FLS overexpressing lncRNA MAPKAPK5-AS1, and carrying out ELISA, Western blot and Annexin V-FITC apoptosis experiments after 48h according to the method. The ELISA experiment result is shown in figure 4A, after lncRNA MAPKAPK5-AS1 is over-expressed, the expression of IL-8 is reduced, the expression of IL-10 is increased, and after miR-146a-3p is over-expressed, the influence of lncRNA MAPKAPK5-AS on RA-FLS inflammatory response can be antagonized; western blot and apoptosis experiment results are shown in FIG. 4B and FIG. 4C, after lncRNA MAPKAPK5-AS1 is over-expressed, Bcl-2 expression is reduced, Bax expression is increased and apoptosis rate is increased, and after miR-146a-3p is over-expressed, the influence of lncRNA MAPKPK 5-AS on RA-FLS apoptosis can be antagonized, so that lncRNA MAPKPK 5-AS1 regulates RA-FLS inflammatory response and apoptosis through miR-146a-3p (FIG. 4D).
Example 5
Investigation of lncRNA MAPKAPK5-AS1 Regulation of RA-FLS inflammation and apoptosis by SIRT1
Transfecting the overexpression lncRNA MAPKAPK5-AS1 in RA-FLS, changing back to a conventional culture medium after 12h, continuing to culture, transfecting si-SIRT1 and Negative Control (NC) in the RA-FLS of the overexpression of lncRNA MAPKAPK5-AS1, and carrying out ELISA, Western blot and Annexin V-FITC apoptosis experiments after 48h according to the method. The ELISA experiment result is shown in figure 5A, after lncRNA MAPKAPK5-AS1 is over-expressed, the IL-8 expression is reduced, the IL-10 expression is increased, and after SIRT1 is interfered, the influence of lncRNA MAPKAPK5-AS1 on RA-FLS inflammatory response can be antagonized; western blot and apoptosis experimental results are shown in FIG. 5B and FIG. 5C, after the IncRNA MAPKAPK5-AS1 is over-expressed, Bcl-2 expression is reduced, Bax expression is increased and apoptosis rate is increased, and after SIRT1 is interfered, the influence of IncRNA MAPKPK 5-AS1 on RA-FLS apoptosis can be antagonized, which indicates that the IncRNA MAPKK 5-AS1 regulates RA-FLS inflammatory response and apoptosis through SIRT1 (FIG. 5D).
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Sequence listing
<110> first subsidiary Hospital of Anhui TCM university (traditional Chinese medicine of Anhui province)
<120> long-chain non-coding RNA and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 841
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agcctttatc tccactctgc ggagattcac gcctggaaaa cgctcttctg agaggatctg 60
tggaggtcaa cccaggagag agaagacagg actgaagcac tgaaagggtc ctgccgttaa 120
gggcgcagga ttgtatagaa tatataatag cagtagcagc tctgtttacg gagcattaac 180
cttacgtgga gttatttcct gcatttcctc ctttcgtctt tacaaggtag ccgtttggcg 240
tcgtgaggta tggatgttcc catccctaat ttgcaaaata ggtaactgag gctcaggaga 300
gctagactag tttgtgcaca gccgagaagt ggtcaagcta gaatgggacc ccaggtttgt 360
gctccctact ctccaccgat aacctatcaa agggctttgc aagagctttt gactaatcgt 420
ctgtcaatgg ttcttcattc acgtattaat tgcacatcag tatgtgccaa atctactaga 480
cattggagaa acagtaggaa caacacatgg taccggcaca tggatctttc aggaaaacga 540
agtaggtaat aaacaggaaa aagcccgagt ctgatgctaa gaagaacata aacaggatgc 600
tgagcggaaa gtgaccagaa gaggtgtgca gtttccaggc ctggcccata cagacctcca 660
acaggtgctc ccctgtgctg ttactccttc tgccaactgg aagcagatgg tgaccaggct 720
ctggagaagg caaggcctga agatgggaga ttcctaagtg gaggagaact gtgccttact 780
gacctaaata tccactcagt attgttatgt gagaataaat aaacttgtgt tgaccgttta 840
c 841
<210> 2
<211> 99
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccgatgtgta tcctcagctt tgagaactga attccatggg ttgtgtcagt gtcagacctc 60
tgaaattcag ttcttcagct gggatatctc tgtcatcgt 99

Claims (1)

1. A polypeptide with a sequence shown as SEQ ID No: 1 as miRNA molecule sponge, the long-chain non-coding RNA has a specific binding sequence shown as SEQ ID No: 2, and is characterized in that the function of the miR-146a-3p can promote the expression of SIRT1 gene after being inhibited, so as to regulate the cell inflammation reaction and apoptosis deficiency.
CN202111358287.1A 2021-11-15 2021-11-15 Long-chain non-coding RNA and application thereof Active CN114134146B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111358287.1A CN114134146B (en) 2021-11-15 2021-11-15 Long-chain non-coding RNA and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111358287.1A CN114134146B (en) 2021-11-15 2021-11-15 Long-chain non-coding RNA and application thereof

Publications (2)

Publication Number Publication Date
CN114134146A CN114134146A (en) 2022-03-04
CN114134146B true CN114134146B (en) 2022-06-14

Family

ID=80389808

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111358287.1A Active CN114134146B (en) 2021-11-15 2021-11-15 Long-chain non-coding RNA and application thereof

Country Status (1)

Country Link
CN (1) CN114134146B (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113481297A (en) * 2021-05-29 2021-10-08 杭州医学院 Long non-coding RNA and application thereof in diagnosis and treatment of liver cancer

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010001632A1 (en) * 2008-07-03 2010-01-07 国立大学法人神戸大学 Micro-rna associated with rheumatoid arthritis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113481297A (en) * 2021-05-29 2021-10-08 杭州医学院 Long non-coding RNA and application thereof in diagnosis and treatment of liver cancer

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Homo sapiens MAPKAPK5 antisense RNA 1 (MAPKAPK5-AS1), transcript variant 2, long non-coding RNA;NR_152605.1;《Genbank》;20211109;参见核苷酸序列 *
lncRNA expression profiles related to apoptosis and autophagy in peripheral blood mononuclear cells of patients with rheumatoid arthritis;Jianting Wen等;《FEBS Open Bio》;20200722;第10卷;1642-1654页 *
miR-146a对类风湿关节炎患者滑膜成纤维细胞的抑制作用研究;李茹 等;《全国临床免疫检验研讨会暨第六届全国临床免疫学术会议论文汇编》;20090731;216-217页 *
新风胶囊通过调控lncRNA MAPKAPK5-AS1对类风湿关节炎滑膜成纤维细胞凋亡与炎症的影响;文建庭 等;《中国中药杂志》;20211215;第46卷(第24期);6542-6548页 *

Also Published As

Publication number Publication date
CN114134146A (en) 2022-03-04

Similar Documents

Publication Publication Date Title
CN110129447A (en) Application of the PQBP1 in Diagnosis of Ovarian Cancer
CN108949988A (en) A kind of application of long-chain non-coding RNA SNHG6 in breast cancer diagnosis or treatment
CN108660212B (en) Application of WDR1 gene in preparation of non-small cell lung cancer treatment and detection products
CN111743912B (en) Gene inhibitor for promoting colon cancer cell apoptosis and inhibiting colon cancer cell migration
CN114032237A (en) Circular non-coding RNA circSTK39 and application thereof in preventing and treating atherosclerosis
CN114134146B (en) Long-chain non-coding RNA and application thereof
CN102558336B (en) The albumen and application of PRR11 genes and its coding
Chen et al. The elevated circ_0067835 could accelerate cell proliferation and metastasis via miR-1236-3p/Twist2 axis in hepatocellular carcinoma
CN111096962A (en) Application of fatty acid oxidation inhibitor in preparation of medicine for treating colorectal cancer
CN108913695B (en) Application of ZEB1 in human heart fibroblast
CN113278696B (en) Molecular marker RAD51B-AS1 and application thereof
CN111575384B (en) Application of human GLT8D1 gene in clinical diagnosis and treatment of tumor
CN109097358A (en) A kind of lncRNA is preventing or is treating the application in hypertension
CN110607368B (en) Application of miRNA3926-1 gene as pancreatic cancer diagnosis and curative effect marker
CN109529018B (en) Application of ATXN2L in preparation of drug for reversing oxaliplatin resistance of gastric cancer
CN115058517B (en) Application of PRKD1 gene as non-small cell lung cancer diagnosis or prognosis marker
CN112760377A (en) Application of lncRNA068 in diagnosis or treatment of malignant melanoma
CN112501303A (en) Molecular marker COL11A1 and application thereof in breast cancer drugs
CN114561466B (en) Application of FTO in diagnosis and treatment of osteosarcoma
CN113293208A (en) Molecular marker related to lung cancer proliferation and metastasis and application thereof
CN115287359B (en) Application of MCOLN2 in colorectal cancer
CN113913523B (en) Application of BUD31 as ovarian cancer prevention, diagnosis or prognosis marker
CN115807077B (en) Use of ETS1 in the preparation of a product for diagnosing or treating ciliated disorders
CN108330193B (en) Application of IQUB gene or protein in preparing breast cancer diagnosis product and treatment medicine
CN117159719B (en) Application of MFSD12 in preparing medicament for treating lung adenocarcinoma

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant