CN114134146B - A kind of long-chain non-coding RNA and its application - Google Patents

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

A kind of long-chain non-coding RNA and its application

technical field

The invention belongs to the field of rheumatism molecular biology, and relates to a long-chain non-coding RNA and an application thereof.

Background technique

Long noncoding RNA (lncNA) is a regulatory noncoding RNA with a length of >200 nt, which was considered to be a kind of "noise" and "dark matter" in the past, and has no ability to encode proteins. In recent years, many studies have shown that lncRNAs can play a sponge-like role at the epigenetic, transcriptional, and post-transcriptional levels, competitively bind to microRNAs (miRNAs), affect the expression of target genes, regulate downstream pathways, and participate in various disease progression.

Fibroblast-like synovial cells (FLS) are a kind of inflammatory cells, which are the main components of synovial cells and secrete a large number of inflammatory cytokines. Long-term inflammatory stimulation can lead to joint swelling, pain, deformity, loss of function, etc. The stimulation of inflammatory signals can cause abnormal proliferation and insufficient apoptosis of FLS, thereby producing a variety of inflammatory cytokines, which in turn continuously activate FLS, and the two promote each other and form a vicious circle. Insufficient immune inflammatory response and apoptosis are associated with the pathogenesis of various rheumatic diseases, such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis and gouty arthritis. Therefore, the study found that lncRNAs related to cellular inflammation and apoptosis have great clinical application value.

SUMMARY OF THE INVENTION

The present invention utilizes full transcription high-throughput sequencing technology to analyze the lncRNAs of RA patients, and obtains a lncRNA MAPKAPK5-AS1 related to cellular inflammatory response and insufficient apoptosis, which has the potential to inhibit the synovial inflammatory response and promote the apoptosis of synovial cells in patients with rheumatism. A new target for death.

The specific technical solution adopted in the present invention is as follows: a long-chain non-coding RNA is lncRNA MAPKAPK5-AS1, and its cDNA sequence is shown in SEQ ID No: 1.

Another object of the present invention is to provide the above-mentioned long-chain non-coding RNA and the application of miR-146a-3p as molecular intervention targets in regulating cellular inflammatory response and insufficient apoptosis. The sequence of miR-146a-3p is shown in SEQ ID No: 2 Show.

The long-chain non-coding RNA of the present invention specifically binds to miR-146a-3p as a miRNA molecular sponge, thereby inhibiting the function of miRNA; after the function of miR-146a-3p is inhibited, it can promote the expression of SIRT1 gene, thereby reducing cellular inflammation and Inadequate regulation of apoptosis.

Another object of the present invention is to provide the application of the long-chain non-coding RNA and miR-146a-3p as molecular intervention targets in the preparation of drugs for treating rheumatism related to cellular inflammatory response and insufficient apoptosis.

The rheumatic diseases associated with insufficient cellular inflammatory response and apoptosis include rheumatoid arthritis, ankylosing spondylitis, osteoarthritis or gouty arthritis.

A specific research application of the present invention is the application of long-chain non-coding RNA as a molecular intervention target in a drug for inhibiting the inflammatory response of fibroblast-like synoviocytes and promoting apoptosis of fibroblast-like synoviocytes.

In the present invention, 30 differentially expressed lncRNAs are obtained by analyzing RA patients by using full-transcription high-throughput sequencing technology, wherein the lncRNA MAPKAPK5-AS1 of the present invention is down-regulated in RA patients. RT-qPCR demonstrated that the expression changes of lncRNA MAPKAPK5-AS1 were consistent with the high-throughput sequencing results, and the expression trend in fibroblast-like synoviocytes (FLS) was consistent with that in PBMCs. The full-length sequence of lncRNA MAPKAPK5-AS1 was obtained by RACE reaction, located at 12q24.12-q24.13. Subcellular localization analysis The lncRNA MAPKAPK5-AS1 was mainly expressed in the cytoplasm of FLS.

The research of the present invention shows that overexpression of lncRNA MAPKAPK5-AS1 in vitro significantly inhibits the inflammatory response of FLS and promotes cell apoptosis, and in vitro interference with lncRNA MAPKAPK5-AS1 significantly promotes the inflammatory response of FLS and inhibits cell apoptosis. Analysis of sequencing results and ceRNA prediction combined with Luciferase detection indicated that lncRNA MAPKAPK5-AS1 combined with miR-146a-3p to regulate the expression of SIRT1, a target gene related to inflammation and insufficient apoptosis.

The study of the present invention suggests that lncRNA MAPKAPK5-AS1 may affect the inflammation and apoptosis of FLS by regulating the expression of genes related to cellular inflammatory response and insufficient apoptosis, and has the potential to become a new target for inhibiting inflammatory response and promoting apoptosis.

Description of drawings

Figure 1 shows the expression trend and distribution of the lncRNA MAPKAPK5-AS1 of the present invention (MK5-AS1 in the figure represents lncRNA MAPKAPK5-AS1, the same below). Figure 1A shows the expression changes of lncRNA MAPKAPK5-AS1 in PBMCs of 30 RA patients and 30 normal people by RT-qPCR (with β-actin as the internal reference). Compared with normal people, the expression of lncRNA MAPKAPK5-AS1 in PBMCs of RA patients was decreased ( ^*** p<0.001), consistent with the sequencing results. Figure 1B shows RT-qPCR detection of the expression of lncRNA MAPKAPK5-AS1 in RA-FLS (with β-actin as the internal reference). After stimulation with TNF-ɑ, the expression of lncRNA MAPKAPK5-AS1 was decreased ( ^*** p<0.001), which was similar to that in PBMCs The expression results were consistent. Figure 1C shows the results of FISH experiments showing that lncRNA MAPKAPK5-AS1 is located in the cytoplasm of RA-FLS.

Figure 2 shows the effects of interference and overexpression of lncRNA MAPKAPK5-AS1 according to the present invention on inflammation and apoptosis in RA-FLS. Figure 2A shows the effects of interference and overexpression of lncRNA MAPKAPK5-AS1 on RA-FLS inflammation by ELISA assay, which are the inflammatory cytokines IL-8 and IL-10, respectively. Figure 2B shows the effects of interference and overexpression of lncRNA MAPKAPK5-AS1 on RA-FLS apoptotic proteins, Bax and Bcl-2, respectively, by Western Blotting assay. Figure 2C shows Annexin V-TITC/PI staining, and flow cytometry detects the effect of interfering with lncRNA MAPKAPK5-AS1 on the apoptosis of RA-FLS cells. Figure 2D shows the histogram of the RA-FLS cell apoptosis rate in different experimental groups. picture. ( ^*** p<0.001 vs. control group, ^### p<0.001 vs. si-NC group, ^▲▲▲ p<0.001 vs. OV-NC group, ns means no statistical significance) .

Figure 3 shows that the lncRNA MAPKAPK5-AS1 of the present invention affects the expression of the gene SIRT1 by regulating miR-146a-3p. Figure 3A shows the effect of interference and overexpression of lncRNA MAPKAPK5-AS1 in RA-FLS on the expression of miR-146a-3p by RT-qPCR. Figure 3B shows RT-qPCR detection of the expression of the gene SIRT1 affected by interference and overexpression of lncRNA MAPKAPK5-AS1 in RA-FLS. Figure 3C shows the binding of lncRNA MAPKAPK5-AS14 to miR-146a-3p detected by Luciferase reporter assays. Figure 3D shows the binding of miR-146a-3p to the 3'UTR of SIRT1 detected by Luciferase reporter assays. Figure 3E shows RT-qPCR detection of SRIT1 expression in RA-FLS after transfection of miR-146a-3p. ( ^*** p<0.001 compared to the control group, ^### p<0.001 compared to the si-NC group, ^▲▲▲ p<0.001 compared to the OV-NC group, ns means no statistical significance) .

Figure 4 shows that the lncRNA MAPKAPK5-AS1 of the present invention regulates RA-FLS inflammation and apoptosis through miR-146a-3p. Figure 4A shows the effect of overexpression of miR-146a-3p on the regulation of RA-FLS inflammation by overexpression of lncRNA MAPKAPK5-AS1, which are the inflammatory cytokines IL-8 and IL-10, respectively, by ELISA assay. Figure 4B shows the effect of overexpression of miR-146a-3p on overexpression of lncRNA MAPKAPK5-AS1 on the regulation of RA-FLS apoptotic proteins, Bax and Bcl-2, respectively, by Western Blotting assay. Figure 4C shows Annexin V-TITC/PI staining, and flow cytometry detects the effect of overexpression of miR-146a-3p on the regulation of RA-FLS cell apoptosis by overexpression of lncRNA MAPKAPK5-AS1, and the histogram in Figure 4D shows different experiments of flow cytometry Statistical chart of the apoptosis rate of RA-FLS cells in the group. ( ^*** p<0.001 vs. control group, ^### p<0.001 vs. OV-NC group, ^▲▲▲ p<0.001 vs. mimic-NC group, ns means no statistical significance) .

Figure 5 shows that the lncRNA MAPKAPK5-AS1 of the present invention regulates RA-FLS inflammation and apoptosis through SIRT1. Figure 5A shows the effect of interfering with SIRT1 on the regulation of RA-FLS inflammation by overexpressing lncRNA MAPKAPK5-AS1 by ELISA assay, which are the inflammatory cytokines IL-8 and IL-10, respectively. Figure 5B shows the effect of interfering SIRT1 on the overexpression of lncRNA MAPKAPK5-AS1 to regulate RA-FLS apoptotic proteins, the apoptotic proteins Bax and Bcl-2, respectively, by Western Blotting assay. Figure 5C shows Annexin V-TITC/PI staining, and flow cytometry detects the effect of interfering with SIRT1 on the overexpression of lncRNA MAPKAPK5-AS1 in regulating the apoptosis of RA-FLS cells. Figure 5D shows the histogram of RA-FLS cells in different experimental groups. Apoptosis rate statistics. ( ^*** p<0.001 compared with the control group, ^### p<0.001 compared with the OV-NC group, ^▲▲▲ p<0.001 compared with the si-NC group, ns means no statistical significance) .

Detailed ways

Example 1

To investigate the expression and distribution characteristics of lncRNA MAPKAPK5-AS1

1. RNA extraction and RT-qPCR from PBMCs of RA patients

The peripheral blood of RA patients was collected with an EDTA anticoagulant tube, PBMCs were extracted, and RNA from PBMCs was extracted according to the instructions of TRIzol Reagent (Invitrogen). After reverse transcription, RT-qPCR was performed using PrimeScript RT-PCR Kit (Takara). (with β-actin as the internal reference), the PCR instrument reaction program: Stage 1: 95°C for 1 min, Stage 2 (Cycle: 40): 95°C for 20s, 60°C for 1 min. The sequence of the lncRNA MAPKAPK5-AS1 primer is shown in SEQ ID 1. The RT-qPCR results are shown in Figure 1A, and the expression of lncRNA MAPKAPK5-AS1 in PBMCs of RA patients was decreased compared with normal people.

2. Isolation, identification, preparation and culture of RA-FLS

Take the knee joint tissue of patients with RA surgery, soak it in 75% alcohol for about 2 minutes, put it in PBS containing P/S, cut the tissue block into squares with a side length of about 0.1cm, wash repeatedly, discard the supernatant, and place it in a In a petri dish with complete medium, incubate at 37°C for 30-60 min, incubate upside down in a 5% CO ₂ incubator for 2 h, add 2 ml of fibroblast complete medium, infiltrate tissue pieces, and place in a 5% CO ₂ cell incubator, The medium was changed every 3 days. After the cells growing around the tissue block fused into a sheet, the tissue block was removed and the cells were digested with trypsin. After the cells climbed, aspirate the culture medium, add 4% PFA and fix it at 4°C for 30min, take 50uL of membrane-breaking sealing droplets on the waterproof membrane, cover the side with cells on the glass slide for 2h, after the membrane-breaking sealing, take 50uL Resist on the waterproof membrane, incubate the secondary antibody (secondary antibody: PBS=1:500) for 2 h at room temperature, wash with PBS for 3×5 min/time, stain with DAPI (DAPI:PBS=1:1000) for 5 min, wash with PBS for 3× 5 min/time, drop 1 drop of Fluoromount-G on each glass slide, cover the side with cells, and identify the cells as P1 generation cells. The cells were seeded in a 6-well plate, and the number of cells in each well was about 1×10 ⁵ . After the cells adhered, 1 mL of complete medium was added, and 20 μL of SV40 overexpressing lentivirus was added. When the cells covered the bottom of the plate, passage to T25 In the culture flask, fresh medium containing different concentrations of puromycin (1ug/mL, 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL, 7ug/mL) was added to the plated cells. 24-well plate, the minimum concentration of puromycin used is the lowest concentration that kills all cells within 1-4 days from the start of puromycin selection. The result is that the concentration of puromycin used is 1ug/mL, the action time is 2d, RA- The FLS immortalized cell line was prepared, and after cell fusion, the purity was over 95%, which was used for subsequent experiments. RA-FLS were cultured in high glucose DMEM medium supplemented with 15% (V/V) heat-inactivated fetal bovine serum (FBS) and penicillin-streptomycin solution in cell culture flasks at 37°C, 5% CO ₂ .

3. RNA extraction and RT-qPCR in RA-FLS

RNA from RA-FLS was extracted according to the instructions of TRIzol Reagent (Invitrogen), and after reverse transcription, RT-qPCR was performed using PrimeScript RT-PCR Kit (Takara). Program: Stage 1: 95°C for 1 min, Stage 2 (Cycle: 40): 95°C for 20s, 60°C for 1 min. The RT-qPCR results are shown in Figure 1B. After stimulation with TNF-ɑ, the expression of lncRNA MAPKAPK5-AS1 in RA-FLS was decreased, which was consistent with the expression trend in PBMCs of RA patients.

4. FISH assay to detect the subcellular localization of lncRNAs

Fixation of cell slides: The cell slides were fixed in 4% paraformaldehyde (DEPC) for 20 minutes, and washed 3 times in PBS (PH7.4) on a decolorizing shaker for 5 minutes each time; digestion: gene stroke circles, according to different Tissues with different index characteristics were digested with proteinase K (20ug/ml) dropwise for 5 min, washed with pure water and washed with PBS for 3 times for 5 min; pre-hybridization: drop-wise added pre-hybridization solution in a 37°C incubator for 1 h; hybridization: poured out the pre-hybridization solution, Add hybridization solution (containing probe MAPKAPK5-AS1 concentration 500nM), hybridize overnight at 42 degrees; wash after hybridization: wash off the hybridization solution, 2×SSC, wash at 37°C for 10min, 1×SSC, wash at 37°C for 2×5min, 0.5 ×SSC was washed at 37°C for 10min. If there are too many non-specific hybrids, formamide washing can be added; secondary label incubation: dropwise add hybridization solution containing secondary label probes at a dilution ratio of 1:400. Incubate at 42°C for 3h, then wash with 2×SSC at 37°C for 10min, 1× SSC, washed at 37°C for 2×5min, 0.5×SSC at 37°C for 10min; dropwise addition of blocking solution: dropwise addition of blocking serum normal rabbit serum, room temperature for 30min; dropwise addition of mouse anti-digoxigenin-labeled peroxidase (anti-DIG- HRP): Pour off the blocking solution, add anti-DIG-HRP dropwise, incubate at 37°C for 50 min, then wash 3 times with PBS for 5 min; dropwise add CY3-TSA: add CY3-TSA reagent dropwise, react at room temperature in the dark for 5 min, then wash with PBS Washed 3 times for 5 min; DAPI counterstained nucleus: DAPI staining solution was added dropwise to the section, incubated in the dark for 8 min, after washing, anti-fluorescence quenching mounting medium was added dropwise to mount; Microscopic examination and photographing: Sections were observed under a Nikon upright fluorescence microscope And collect images (CY3 red light excitation wavelength 510-560, emission wavelength 590nm, red light); interpretation of the results of the DAPI fluorescence in situ hybridization experiment: the nuclei stained by DAPI are blue under the excitation of ultraviolet, Positive expression is the corresponding fluorescein (CY3)-labeled red light. The results are shown in Figure 1C, which indicated that the lncRNA MAPKAPK5-AS1 was mainly expressed in the cytoplasm

Example 2

To investigate the effects of interference and overexpression of lncRNA MAPKAPK5-AS1 on inflammation and apoptosis in RA-FLS

1. RA-FLS transfection

Small molecule RNA transfection: In this experiment, LipofectamineTM RNAiMAX was used to transfer siRNA (final concentration: 100nM) and miR-146a-3p mimic (purchased from Guangzhou Ribo Biotechnology Co., Ltd., final concentration: 20nM) into RA-FLS medium, change to complete medium the next day, and perform subsequent experiments as needed. The siRNA si-1, si-2 and si-3 sequences for LncRNA MAPKAPK5-AS1 are shown below, and the siRNA si-1, si-2 and si-3 sequences for SIRT1 are shown below:

Plasmid DNA transfection: firstly inoculate cells as required, and when the density of RA-FLS reaches more than 85%, use LipofectamineTM2000 to transfer pcDNA3.1-NC and pcDNA3.1-MAPKAPK5-AS1 according to the concentration ratio of lip3000:plasmid=2μl:1μg , dropwise evenly into the cell culture medium, mix gently, change to a new complete medium after 4 to 6 hours, and change it to a new complete medium again the next day, and carry out follow-up experiments as needed.

2. ELISA experiment

Take the cell supernatant and centrifuge at 4000rpm for 20min to remove cell pellets and polymers. Store the supernatant below -20°C to avoid repeated freezing and thawing; take out the required strips from the aluminum foil bag: set standard wells and 0 For wells, blank wells and sample wells, add 50 μL of standards with different concentrations to each of the standard wells, add 50 μL of sample diluent to the 0-value wells, add 50 μL of the sample to be tested to the blank wells, and add 50 μL of the sample to be tested to the sample wells; except for the blank wells, the standard wells , 0 value wells and sample wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody; cover the reaction plate with a sealing film, and incubate for 60 min at 37°C in a water bath or incubator; remove the sealing film, Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 20 s, shake off the washing solution, pat dry on absorbent paper, repeat 5 times; mix substrates A and B well at 1:1 volume , add 100 μL of the substrate mixture to all wells, cover the reaction plate with a sealing film, and incubate in a water bath or incubator at 37°C for 15 min; add 50 μL of stop solution to all wells, read the absorbance (OD) of each well on a microplate reader value). RA-FLS was transfected with lncRNA MAPKAPK5-AS1 with the highest interference efficiency siRNA (si-2) and siRNA NegativeControl (NC) and overexpression plasmids pcDNA3.1-MAPKAPK5-AS1 and pcDNA3.1-NC, 48h after transfection , according to the kit method, the ELISA experimental results are shown in Figure 2A. The results showed that compared with the control group, the expression of IL-8 was increased and the expression of IL-10 was decreased in the model group. Compared with the pcDNA3.1-NC group, the overexpression of lncRNA MAPKAPK5-AS1 decreased the expression of IL-8 and increased the expression of IL-10.

3. Western blot experiment

After collecting cells, add 150uL of RIPA cell lysate (containing 1mM PMSF) to each well of a 6-well plate for lysis, centrifuge at 12,000rpm for 10min, collect the supernatant, which contains total cell protein; prepare SDS-PAGE gel; Add 5X SDS-PAGE protein loading buffer to the protein sample at a ratio of 1:4, and heat it in a boiling water bath for 10 minutes to fully denature the protein; after the sample is cooled to room temperature, directly load the protein sample into the SDS-PAGE gel loading well Inside, add 30ug of sample to each well; pre-cut filter paper and PVDF membrane of the same size as the adhesive strip (soaked in methanol for 3 minutes in advance), immerse it in the transfer buffer for 5 minutes, and press the membrane transfer device from top to bottom. The anode plate, 3 layers of filter paper, PVDF membrane, gel, 3 layers of filter paper, and cathode plate are placed in order, the filter paper, gel, and PVDF membrane are precisely aligned, and the membrane is transferred at a constant current of 300 mA; In the pre-prepared Western washing solution, rinse for 5 minutes to wash off the transfer solution on the membrane, add Western blocking solution (5% nonfat dry milk), shake slowly on a shaker, and block at room temperature for 2 hours; according to β-actin ( 1: 1000), Bax (1: 1000) and Bcl-2 (1: 1000) primary antibody diluents were diluted and incubated overnight at 4°C with slow shaking; and Bcl-2 (1:3000) diluted horseradish peroxidase (HRP)-labeled secondary antibody with secondary antibody diluent; in the dark room, the two reagents of ECL A solution and ECL B solution were 1:1 in a centrifuge tube After mixing, place the protein side of the PVDF membrane on the central position of the exposure plate of the automatic exposure apparatus, add the mixed ECL solution to fully react, and analyze the film strips with Image J software. RA-FLS was transfected with lncRNA MAPKAPK5-AS1 with the highest interference efficiency siRNA (si-2) and siRNA-NC and the overexpression plasmids pcDNA3.1-MAPKAPK5-AS1 and pcDNA3.1-NC, 48h after transfection, according to the kit The method was operated, and the results of the ELISA experiment were shown in Figure 2A. The results showed that compared with the control group, the expression of Bcl-2 was increased and the expression of Bax was decreased in the model group. Compared with -NC group, Bcl-2 decreased and Bax expression increased after overexpression of lncRNA MAPKAPK5-AS1.

4. Apoptosis detection of Annexin V-FITC cells

Digest the RA-FLS with trypsin and transfer it to 5ml centrifuge; centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, resuspend the cells with an appropriate amount of PBS and count; take 50,000-100,000 resuspended cells and centrifuge at 1000g for 5 minutes, discard the supernatant, add 195 μl Annexin V-FITC binding solution to gently resuspend the cells; add 5 μl Annexin V-FITC, and mix gently. Add 10 μl of PI (propidium iodide) staining solution and mix gently; incubate at room temperature (20-25°C) for 10-20 minutes in the dark, and then place in an ice bath. You can use aluminum foil to protect from light. Resuspend the cells 2-3 times to improve the staining effect; then perform flow cytometry detection, Annexin V-FITC is green fluorescence, propidium iodide (PI) is red fluorescence. RA-FLS was transfected by lncRNA MAPKAPK5-AS1 with the highest interference efficiency siRNA (si-2) and siRNA-NC and overexpression plasmids pcDNA3.1-MAPKAPK5-AS1 and pcDNA3.1-NC. The above method was used to detect the apoptosis of RA-FLS by flow cytometry. The flow cytometry results are shown in Figure 2C. Compared with the control group, the apoptosis rate of cells in the model group was reduced. Compared with the si-NC group, the interference of lncRNA MAPKAPK5 Compared with the pcDNA3.1-NC group, the apoptosis rate of cells overexpressing lncRNA was increased after -AS1 (Fig. 2D).

Example 3

To investigate the effect of lncRNA MAPKAPK5-AS1 on the expression of gene SIRT1 by regulating miR-146a-3p

1. Target gene screening

For lncRNAs obtained by high-throughput sequencing, 341 genes down-regulated by more than 2 times were screened, and a total of 16 lncRNAs related to inflammation and apoptosis were obtained by GO and KEGG analysis. According to the correlation between the full-length sequence of lncRNA MAPKAPK5-AS1 and the gene co-expression network, Tay Y et al. (Tay Y, Kats L, Salmena L, et al. Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs[J]. Cell, 2011, 147(2):344-357.) established a bioinformatics prediction method for ceRNA, selected 15 miRNAs that lncRNA MAPKAPK5-AS1 may be regulated by ceRNA, combined with three public databases miRBase, TargetScan, RNAhybrid, The 5 miRNAs that intersected the two were further verified as miR-146a-3p by RT-qPCR and dual luciferase reporter; induced acute lung injury via up-regulating SIRT1 and mediating NF-κB pathway[J].J Drug Target,2021,29(4):420-429.) The downstream target gene of miRNA was predicted to be SIRT1, and the target gene was predicted to be SIRT1 by RT-qPCR and dual fluorescence Validation of nephelase reports.

2. RT-qPCR detection of miR-146a-3p and SIRT1 expression after interference and overexpression of lncRNA MAPKAPK5-AS1

RA-FLS was transfected with specific siRNA (si-2) of lncRNA MAPKAPK5-AS1 and pcDNA3.1-MAPKAPK5-AS1. 48h after transfection, the cells were harvested, RNA was extracted, and miR-146a-3p and miR-146a-3p were detected by RT-qPCR. For the expression of SIRT1, the primer sequences for the gene SIRT1 are shown. The results are shown in Figure 3A and 3B, the results showed that after interfering with lncRNA MAPKAPK5-AS1, the expression of miR-146a-3p was increased and the expression of SIRT1 was decreased. high. This indicated that lncRNA MAPKAPK5-AS1 could regulate the expression of miR-146a-3p and SIRT1.

3. Dual-luciferase reporter assay

Detection was performed using the Dual-Luciferase Reporter Assay System (Promega) kit. 293T cells were cultured for 24 hours after transfection, observed the cell status, washed once with pre-cooled PBS; added 100 μl 1×PLB, shaken the plate vigorously at room temperature for 15 min; pipetted each well 5 times, and transferred the lysate to an EP tube Centrifuge at 4°C, 13000rpm, 5min; add 20μl LABII to 96-well plate, then add 20μl cell lysate, pipette 5 times per well, the operation is uniform, put it on a microplate reader and use the software Gene5 to detect Firefly luciferase (Firefly) 20 μl Stop&Glo was added, and the fluorescence intensity of Renilla luciferase (Renilla) was detected by a microplate reader; the two fluorescences were compared (relativeluciferase). Three replicate wells were made in each experiment, and the experiment was repeated more than three times, and the results were statistically analyzed. The full-length lncRNAMAPKAPK5-AS1 was constructed into pmirGLO vector, and pmirGLO-lncRNAMAPKAPK5-AS1-full was co-transfected with miR-146a-3p mimic and mimic NC in 293T cells. The enzyme reporter gene system detects the fluorescence values of Firefly and Renilla, and calculates Relative luciferase (Firefly/Renilla). The results are shown in Figure 3C. The results showed that compared with the mimic NC, the normalized luciferase activity was significantly reduced in the experimental group transfected with miR-146a-3pmimic, indicating that the lncRNA MAPKAPK5-AS1 may have a certain degree of binding to miR-146a-3p. Then, the luciferase experiment was used to detect the binding of miR-146a-3p to the 3'-UTR of its corresponding target gene SIRT1. The results are shown in Figure 3D. The results showed that luciferase activity was significantly down-regulated after transfection of miR-146a-3p mimic compared with mimic NC, and miR-146a-3p could bind to SIRT1.

4. RT-qPCR to detect the effect of miRNAs on the expression of target gene SIRT1

In RA-FLS, miR-146a-3p mimic, mimic NC, miR-146a-3p inhibitor and inhibitor NC were transfected. 48h after transfection of miRNA, cells were harvested, RNA was extracted, reverse transcribed, and the expression of gene SIRT1 was detected by RT-qPCR with the same method as above. RT-qPCR detection results are shown in Figure 3E, the results showed that the expression of SIRT1 was significantly decreased after transfection of miR-146a-3p mimic, and the expression of SIRT1 was significantly increased after transfection of miR-146a-3p inhibitor. This indicates that miR-146a-3p can inhibit the expression of SIRT1 by binding to the 3'-UTR of SIRT1, and the lncRNA MAPKAPK5-AS1 can competitively bind to miR-146a-3p, thereby antagonizing the inhibition of SIRT1 expression by miR-146a-3p.

Example 4

Investigating lncRNA MAPKAPK5-AS1 regulates RA-FLS inflammation and apoptosis via miR-146a-3p

RA-FLS was transfected with overexpressing lncRNA MAPKAPK5-AS1. After 12 h, the medium was changed back to normal medium and the culture was continued. Then, RA-FLS overexpressing lncRNA MAPKAPK5-AS1 was transfected with miR-146a-3p mimic and Negative control (NC). ), and ELISA, Western blot and Annexin V-FITC apoptosis experiments were performed according to the above methods after 48 h. The results of ELISA experiments are shown in Figure 4A. After overexpression of lncRNA MAPKAPK5-AS1, the expression of IL-8 decreased and the expression of IL-10 increased, while overexpression of miR-146a-3p could antagonize the effect of lncRNA MAPKAPK5-AS on RA-FLS. Influence of inflammatory response; Western blot and apoptosis assay results are shown in Figure 4B and Figure 4C, after overexpression of lncRNA MAPKAPK5-AS1, Bcl-2 expression decreased, Bax expression increased, and apoptosis rate increased, while overexpression of miR After -146a-3p, lncRNA MAPKAPK5-AS could antagonize the effect of lncRNA MAPKAPK5-AS on RA-FLS cell apoptosis, indicating that lncRNA MAPKAPK5-AS1 regulates RA-FLS inflammatory response and apoptosis through miR-146a-3p (Fig. 4D).

Example 5

Investigating lncRNA MAPKAPK5-AS1 regulates RA-FLS inflammation and apoptosis via SIRT1

RA-FLS was transfected with overexpressing lncRNA MAPKAPK5-AS1, changed back to conventional medium after 12 h, continued to culture, and then transfected si-SIRT1 and Negative control (NC) in RA-FLS overexpressing lncRNA MAPKAPK5-AS1 for 48 h Then, ELISA, Western blot and Annexin V-FITC apoptosis experiments were carried out according to the above methods. The results of the ELISA experiment are shown in Figure 5A. After overexpression of lncRNA MAPKAPK5-AS1, the expression of IL-8 decreased and the expression of IL-10 increased. After interfering with SIRT1, it could antagonize the effect of lncRNA MAPKAPK5-AS1 on the inflammatory response of RA-FLS; The results of Western blot and apoptosis experiments are shown in Figure 5B and Figure 5C. After overexpression of lncRNA MAPKAPK5-AS1, the expression of Bcl-2 was decreased, the expression of Bax was increased, and the apoptosis rate was increased, while interfering with SIRT1 could antagonize lncRNA The effect of MAPKAPK5-AS1 on RA-FLS apoptosis indicated that the lncRNA MAPKAPK5-AS1 regulates RA-FLS inflammatory response and apoptosis through SIRT1 (Fig. 5D).

The above content is only an example and description of the concept of the present invention. Those skilled in the art can make various modifications or supplements to the described specific embodiments or replace them in a similar manner, as long as they do not deviate from the concept of the invention. Or beyond the scope defined by the claims, all belong to the protection scope of the present invention.

sequence listing

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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

<400> 2

ccgatgtgta tcctcagctt tgagaactga attccatggg ttgtgtcagt gtcagacctc 60

tgaaattcag ttcttcagct gggatatctc tgtcatcgt 99

Claims (1)

1. A long-chain non-coding RNA whose sequence is as shown in SEQ ID No: 1 is used as a miRNA molecular sponge in regulating the non-therapeutic purpose of miR-146a-3p function inhibition as shown in SEQ ID No: 2 in a specific binding sequence. The application is characterized in that after the function of miR-146a-3p is inhibited, it can promote the expression of SIRT1 gene, thereby regulating cell inflammatory response and insufficient apoptosis.

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