CN114134146A

CN114134146A - Long-chain non-coding RNA and application thereof

Info

Publication number: CN114134146A
Application number: CN202111358287.1A
Authority: CN
Inventors: 刘健; 文建庭; 万磊; 王馨; 王杰
Original assignee: First Affiliated Hospital of AHUTCM
Current assignee: First Affiliated Hospital of AHUTCM
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2022-03-04
Anticipated expiration: 2041-11-15
Also published as: CN114134146B

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 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 insufficient apoptosis are associated with the onset of various 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 inflammation reaction and apoptosis deficiency, wherein the miR-146a-3p sequence is shown as 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 over-expressed in vitro, the inflammatory reaction of FLS is obviously inhibited, the apoptosis of cells is promoted, the lncRNA MAPKAPK5-AS1 is interfered in vitro, the inflammatory reaction of FLS is obviously promoted, and the apoptosis of cells is inhibited. The analysis of the sequencing result and the prediction of the ceRNA combined with the Luciferase detection suggest that the lncRNA MAPKAPK5-AS1 is combined with the 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 can influence the inflammation and apoptosis of FLS by regulating the gene expression related to the cell inflammation reaction and apoptosis deficiency, and can potentially become a new target for inhibiting the inflammation reaction and promoting the cell apoptosis.

Drawings

FIG. 1 shows the expression trend and distribution of lncRNA MAPKAPK5-AS1 (MK 5-AS1 represents lncRNA MAPKAPK5-AS1, the same below) according to the present invention. FIG. 1A shows that RT-qPCR detects the expression change of lncRNA MAPKAPK5-AS1 (referred to AS beta-actin internal reference) in 30 RA patients and 30 normal human PBMCs, and the expression of lncRNA MAPKAPK5-AS1 in the RA patients PBMCs is reduced compared with that in the normal human (see the description of beta-actin)^***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 internal 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 IncRNA MAPKAPK5-AS1 on inflammation and apoptosis of RA-FLS according to the present invention. FIG. 2A shows ELISA assays to determine the effect of interference and overexpression of IncRNA MAPKAPK5-AS1 on the inflammation of RA-FLS, inflammatory cytokines IL-8 and IL-10, respectively. FIG. 2B is a Western Blotting experiment to examine the effect of interference and overexpression of lncRNA MAPKAPK5-AS1 on the apoptotic proteins of RA-FLS, i.e., the apoptotic proteins Bax and Bcl-2, respectively. 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 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. 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. 3DThe binding of miR-146a-3p to the 3' UTR of SIRT1 was examined for the Luciferase reporter assays. 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 inflammation and apoptosis of RA-FLS through miR-146a-3 p. FIG. 4A is an ELISA experiment to detect the effect of miR-146a-3p overexpression on the regulation of RA-FLS inflammation by lncRNA MAPKAPK5-AS1 overexpression, which is 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 the regulation of RA-FLS apoptotic proteins by lncRNA MAPKAPK5-AS1, and the apoptotic proteins Bax and Bcl-2 are respectively. FIG. 4C is Annexin V-TITC/PI staining, flow cytometry is used for detecting the influence of over-expressed miR-146a-3p on the regulation of RA-FLS apoptosis by over-expressed lncRNA MAPKAPK5-AS1, and FIG. 4D 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 with the mimic-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 RT-qPCR by using PrimeScript RT-PCR Kit (Takara) after reverse transcription, operating according to Kit instructions (taking beta-actin as an internal reference), and performing reaction procedures by using 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.

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% CO₂In the cell culture box, the liquid is changed every 3 days, when the cells growing around the tissue block are fused into pieces, the tissue block can be removed, and the cells are digested by trypsin. After cell climbing, the culture medium is sucked out, 4% PFA is added to be fixed at 4 ℃ for 30min, 50uL of broken membrane sealing liquid is dripped on a waterproof membrane, one surface with cells on a glass slide is covered for 2h, after the broken membrane sealing, 50uL of primary antibody is dripped on the waterproof membrane, after the secondary antibody (PBS: 1:500) is incubated for 2h in a dark place at room temperature, PBS is washed for 3 multiplied by 5 min/time, DAPI (DAPI: PBS: 1:1000) is stained for 5min, PBS is washed for 3 multiplied by 5 min/time, 1 drop of fluorocount-G is dripped on each glass slide, one surface with cells is covered, and the cells are identified as P1 generation cells. Cells were seeded into 6-well plates at approximately 1X 10 cells per well⁵After the cells are attached to the wall, 1mL of complete medium is added with 20 muL SV40 overexpression lentivirus, after the cells grow to the bottom of a plate, the cells are passed to a T25 culture bottle, fresh culture medium containing puromycin with different concentrations (1ug/mL, 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL, 7ug/mL) is added into a 24-well plate paved with the cells, the minimum puromycin use concentration is the lowest screening concentration for killing all the cells within 1-4d from puromycin screening, the result is that the puromycin use concentration is 1ug/mL, the action time is 2d, the preparation of the RA-FLS immortalized cell line is completed, the cells are fused, the purity is more than 95%, and the immortalized cell line is used for subsequent experiments. RA-FLS at 37 ℃ with 5% CO₂The 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-transcribed, then RT-qPCR was performed using PrimeScript RT-PCR Kit (Takara), the procedure was performed according to the Kit instructions (beta-actin was used 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 results are shown in FIG. 1B, and after TNF-alpha stimulation, the expression of lncRNA MAPKAPK5-AS1 in RA-FLS is reduced, which is consistent with the expression trend in PBMCs of RA patients.

4. FISH experiment detection of subcellular localization of lncRNA

Fixing the cell slide: fixing the cell slide in 4% paraformaldehyde (DEPC) for 20min, and washing in PBS (pH7.4) for 5min on a decolorizing shaker for 3 times; 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:

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 for 20min at 4000rpm, removing cell particles and polymers, and storing the supernatant 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 ℃; stop solution (50. mu.L) was added to all wells, 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 the control group, the model group has increased IL-8 expression and reduced IL-10 expression, compared with the si-NC group, the IL-8 is increased and the IL-10 expression is reduced after the lncRNA MAPKAPK5-AS1 is interfered, and compared with the pcDNA3.1-NC group, the IL-8 is reduced and the IL-10 expression is increased after the lncRNA MAPKPK 5-AS1 is over-expressed.

3. Western blot experiment

After collecting cells, adding RIPA cell lysate (containing 1mM PMSF) into each well of a 6-well plate at 150uL per well for lysis, centrifuging at 12,000rpm for 10min, and collecting supernatant, namely containing total cell proteins; preparing SDS-PAGE gel; adding 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 the protein; after the sample is cooled to room temperature, directly loading the protein sample into SDS-PAGE gel loading holes, and adding 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 transfer is finished, immediately placing the protein membrane into a prepared Western washing solution, rinsing for 5 minutes to wash off the membrane transfer solution on the membrane, adding a Western confining liquid (5 percent of skimmed milk powder), slowly shaking on a shaking table, and sealing for 2 hours at room temperature; diluting according to beta-actin (1: 1000), Bax (1: 1000) and Bcl-2 (1: 1000) primary anti-dilution solution, and incubating overnight at 4 ℃ with 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 siRNA-NC with 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 performed according to the 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 with pancreatin, transferring to 5ml centrifuge; centrifuging for 5 minutes at 1000g, discarding the 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 highest interference efficiency of lncRNA MAPKAPK5-AS1 for RA-FLS, the flow cytometry was performed to detect the apoptosis of RA-FLS by the above method after transfection for 48h, and the flow results are shown in FIG. 2C, compared with the control group, the apoptosis rate was decreased in the model group, compared with the si-NC group, the apoptosis rate after interference of lncRNA MAPKPK 5-AS1 was increased, and compared with the pcDNA3.1-NC group, the apoptosis rate of over-expression lncRNA was increased (FIG. 2D).

Example 3

Investigation of lncRNA MAPKAPK5-AS1 affecting expression of gene SIRT1 by regulating miR-146a-3p

1. Target Gene screening

For lncRNAs obtained by high-throughput sequencing, 341 genes with the down-regulation of more than 2 times are screened, and 16 lncRNAs related to inflammation and apoptosis are obtained by GO and KEGG analysis. According to the correlation of the full-length sequence and the gene co-expression network of lncRNA MAPKAPK5-AS1, using a bioinformatics prediction method established by Tay Y and the like (Tay Y, Kats L, Salmena L, et al.coding-independent regulation of the molecular deletion PTEN by synthesizing endogenous mRNAs [ J ] Cell,2011,147(2):344 and 357.), selecting 15 miRNAs which can be regulated in a cerRNA mode by the lncRNA MAPKAPK5-AS1, combining three public databases of miRBase, targetScan and RNAhybrid, taking 5 miRNAs which intersect, and further verifying through RT-qPCR and double-luciferase report to determine the miR-146a-3 p; next, refer to Yang Y et al (Yang Y, Li L. rejection microRNA-146a-3p inhibitors lipopolysaccharides-induced nucleic acid luminescence in vitro via 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 verify by RT-qPCR and dual luciferase report.

2. RT-qPCR detection of miR-146a-3p and SIRT1 expression after interference and 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 performed to detect the expression of miR-146a-3p and SIRT1, and the primer sequence of gene SIRT1 was AS 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, 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.

3. Dual luciferase reporter assay

Detection was performed 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 MAPKPK 5-AS1-full, miR-146a-3p mix and mix NC were co-transfected in 293T cells, after 24h cell culture, fluorescence values of Firefly and Renilla were detected and Relative luciferase enzyme (Firefly/Renilla) was calculated using the dual luciferase reporter gene 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 micid, micid 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 SIRT1 expression by miR-146a-3 p.

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 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 the IncRNA 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 IncRNA MAPKPK 5-AS on RA-FLS apoptosis can be antagonized, so that the IncRNA 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 MAPKAPK5-AS1 on RA-FLS apoptosis can be antagonized, so that the IncRNA MAPKPK 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> Anhui TCM university first subsidiary hospital (Anhui province TCM college)

<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. A long non-coding RNA, is lncRNA MAPKAPAK 5-AS1, and the cDNA sequence is shown in SEQ ID No: 1 is shown.

2. The use of the long non-coding RNA and miR-146a-3p as molecular intervention targets in modulating cellular inflammatory responses and apoptosis deficiency as claimed in claim 1, wherein the sequence of miR-146a-3p is as set forth in SEQ ID No: 2, respectively.

3. The use of claim 2, wherein the long non-coding RNA specifically binds to miR-146a-3p as a miRNA molecule sponge, thereby inhibiting miRNA function.

4. The use of claim 3, wherein the inhibition of miR-146a-3p function promotes SIRT1 gene expression, thereby modulating cellular inflammatory responses and apoptosis deficiencies.

5. The use of claim 2, wherein the long non-coding RNA and miR-146a-3p are targets for molecular intervention in the manufacture of a medicament for the treatment of rheumatic diseases associated with a cellular inflammatory response and insufficient apoptosis.

6. The use according to claim 5 wherein the rheumatic disease associated with a cellular inflammatory response and insufficient apoptosis comprises rheumatoid arthritis, ankylosing spondylitis, osteoarthritis or gouty arthritis.

7. The use of claim 6, wherein the long non-coding RNA is used as a target for molecular intervention in drugs that inhibit inflammatory responses of fibroblast-like synoviocytes and promote apoptosis of fibroblast-like synoviocytes.