CN110760583B - Long-chain non-coding RNA BC088259 and application thereof - Google Patents

Long-chain non-coding RNA BC088259 and application thereof Download PDF

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CN110760583B
CN110760583B CN201910998562.2A CN201910998562A CN110760583B CN 110760583 B CN110760583 B CN 110760583B CN 201910998562 A CN201910998562 A CN 201910998562A CN 110760583 B CN110760583 B CN 110760583B
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姚淳
于彬
陈燕萍
王靖
顾晓松
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Abstract

The invention discloses a long-chain non-coding RNA BC088259. The research of the invention shows that the expression of the long-chain non-coding RNA BC088259 is up-regulated after the sciatic nerve of a rat is injured, and the further research result shows that the down-regulation of the Lnc RNA BC088259 can inhibit the migration of Schwann cells, and the over-expression of the Lnc RNA BC088259 can promote the migration of the Schwann cells. Suggesting that Lnc RNA BC088259 can be used as a new molecular intervention target for repairing peripheral nerve injury.

Description

Long-chain non-coding RNA BC088259 and application thereof
Technical Field
The invention belongs to the field of biomedicine specialties, and relates to a long-chain non-coding RNA BC088259 and application thereof.
Technical Field
Long non-coding RNAs (lncrnas) are currently generally accepted as non-protein-coding RNAs of greater than 200 nucleotides. Are widely found in the nucleus or cytoplasm, and most exhibit tissue and spatial specificity. The research finds that the expression or function abnormality of the long-chain non-coding RNA is closely related to the occurrence of human diseases.
Repair and functional reconstruction of peripheral nerve injury are one of the most concerned problems in neuroscience research nowadays, and are involved and regulated by various cytokines, cytoskeletal components and intercellular signal molecules. As a glial cell specific to the peripheral nervous system, schwann cells play an irreplaceable role in the repair of morphology and function of the peripheral nerve regeneration process. How to apply and regulate the activity of Schwann cells in the peripheral nerve injury repair process becomes a research hotspot in the peripheral nerve field.
Disclosure of Invention
The invention discloses a long-chain non-coding RNA BC088259, which is up-regulated after peripheral nerve injury, is closely related to Schwann cell migration and has the potential of becoming a new target point for repairing peripheral nerve injury.
The specific technical scheme of the invention is as follows:
a long-chain non-coding RNA BC088259 has a cDNA sequence shown in SEQ ID No:1 is shown.
The invention also aims to provide the application of the long-chain non-coding RNA serving as a molecular intervention target in preparing a medicament for treating diseases related to cell migration.
The diseases related to cell migration comprise nerve injury, cancer, atherosclerosis or arthritis and the like.
Preferably, the disease associated with cell migration is nerve damage, and further preferably peripheral nerve damage.
The LncRNA BC088259 is used as a molecular interference target to regulate or inhibit the LncRNA BC088259 and inhibit the Schwann cell migration, or the LncRNA BC088259 is over-expressed to promote the Schwann cell migration.
The invention utilizes a gene chip to find that the expression of a long-chain non-coding RNA BC088259 is up-regulated after rat sciatic nerve injury. qRT-PCR results showed that LncRNA BC088259 increased expression at 1d, 4d and 7d after injury compared to 0 d. The full length of LncRNA BC088259 was obtained by RACE experiments and it was determined that the LncRNA BC088259 transcript length was 1262nt, mapping to 10q24. siRNA is designed aiming at the full-length sequence of LncRNA BC088259, and siRNA interference experiments are carried out. Transwell migration results showed that interfering LncRNA BC088259 significantly inhibited Schwann cell migration compared to NC. Full-length overexpressed lentiviruses of LncRNA BC088259 were customized and the results indicated that overexpression of LncRNA BC088259 facilitated Schwann cell migration. The scratch experiment further confirmed the regulatory effect of LncRNA BC088259 on Schwann cell migration. The LncRNA BC088259 can regulate Schwann cell migration in the peripheral nerve injury and regeneration process, is favorable for better understanding the important role of LncRNA in the nerve injury repair process, and provides a new target for treatment after nerve injury.
Drawings
Fig. 1 shows that LncRNA BC088259 disclosed by the present invention is up-regulated in sciatic nerve tissues after sciatic nerve injury in rats.
FIG. 2 is a Transwell migration experiment demonstrating the effect of LncRNA BC088259 of the present invention on Schwann cell migration. (FIG. 2A is BC088259siRNA1, siRNA2 interferes with the results of qRT-PCR of RNA levels of LncRNA BC088259 in Schwann cells FIG. 2B is BC088259siRNA1, results of Transwell of Schwann cells after siRNA2 interferes with LncRNA BC088259, results of right histogram of Schwann cells after siRNA interferes with LncRNA BC088259. FIG. 2C is the results of qRT-PCR of LV-BC088259 increasing the RNA levels of LncRNA BC088259 in Schwann cells. FIG. 2D is the results of Transwell of LV-BC088259 overexpressing LncRNA BC 820882088259 promoting the migration of Schwann cells. The right histogram is LV-BC088259 overexpressing LncRNA BC088259. The migration of Schwann cells. P < 0.0001. P.001. The results of P < 0.001).
Fig. 3 is a scratch test to verify the effect of LncRNA BC088259 according to the present invention on Schwann cell migration. (FIG. 3A shows the results of scoring BC088259siRNA1, siRNA2 after interfering with LncRNA BC088259 in Schwann cells. FIG. 3B shows the results of scoring BC088259 in Schwann cells. SiRNA2 shows the results of scoring after interfering with LncRNA BC088259 in Schwann cells. FIG. 3C shows the results of scoring BC088259 in Schwann cells after overexpressing LncRNA BC088259. FIG. 3D shows the results of scoring BC088259 in Schwann cells after overexpressing LncRNA BC088259. P < 0.0001. P < 0.001. P < 0.05.).
Detailed Description
The following examples are provided to illustrate specific steps of the present invention, but are not intended to limit the scope of the invention.
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that these examples are for the purpose of illustrating the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
The present invention is further illustrated by the following specific examples.
Example 1 expression and sequence of LncRNA BC088259
1. Extraction of sciatic nerve tissue RNA and qRT-PCR
Healthy adult male SPF grade SD rats, 24, weighing 160-200g, were randomly divided into 4 groups of 6 animals each, and left sciatic nerve was clipped. 0.5cm sciatic nerve tissue of the proximal sciatic nerve of rat at four time points of 0d, 1d, 4d and 7d after operation is taken respectively
Figure BDA0002240550680000031
Reagent (Invitrogen) instructions for tissue RNA extraction, reverse transcription, and subsequent use
Figure BDA0002240550680000032
PrimeScript RT-PCR Kit (Takara) performed qRT-PCR according to the Kit instructions (GAPDH as internal reference), PCR protocol: stage 1: 2min at 95 ℃, stage 2 (Cycle: 40): 15s at 95 ℃ and 30s at 60 ℃; stage 3:95 ℃ 15s,60 ℃ 1min,95 ℃ 15s. The qRT-PCR primer sequence of LncRNA BC088259 is shown as SEQ ID No: 2-3. The qRT-PCR results are shown in figure 1 and show that LncRNA BC088259 in injured rat sciatic nerve tissue increased expression at 1d, 4d and 7d after injury (referenced as GAPDH) compared to 0 d.
2. Rapid Amplification of CDNA Ends (RACE)
SD rat sciatic nerve tissue is taken, high-quality RNA is extracted by using RNeasy Mini Kit (Qiagen), and the synthesis of First-strand cDNA by using 5'-CDS primer and 3' -CDS primer is respectively carried out according to the instruction system of SMARTERTM RACE (Clotech) Kit to obtain corresponding 5'-cDNA and 3' -cDNA. According to the requirements of the specification, a primer BC088259-5-GSP sequence of 5'-GSP and a primer BC088259-3-GSP of 3' -GSP are designed aiming at LncRNA BC088259, and are shown as SEQ ID No:4-5, followed by 5'-RACE and 3' -RACE using touchdown PCR, the 5 'and 3' ends of BC088259 were obtained. After sequencing is completed, the result is spliced with the original known sequence to obtain the cDNA full-length sequence (shown as SEQ ID No: 1) of BC088259 and UCSC: (http://genome.ucsc.edu/) The rat sequences of the database were aligned and analyzed, indicating that BC088259 is located at 10q24.
Example 2 Effect of LncRNA BC088259 on Schwann cell migration
1. Culture of Primary Schwann cells
Taking a plurality of sciatic nerves of newborn 1d SD rats, adding 1ml of 3mg/ml collagenase, shearing tissues, and digesting for 30min at 37 ℃; centrifuging at 1200rpm at room temperature for 5min, removing collagenase, adding 1ml pancreatin, and digesting at 37 deg.C for 10min; adding 3ml ofTerminating the digestion of the whole culture medium, filtering a screen, centrifuging to remove supernatant (1200rpm, 5min), and then adding 3ml of the whole culture medium to wash for 2 times; the cell seed was cultured in a PLL-coated culture dish (5% CO) 2 37 ℃); the next day, the medium was changed to complete medium containing Arac (10 μ M) to inhibit rapid proliferation of fibroblasts; on day four, the complete medium containing HRG (50 ng/ml) and Forskolin (2. Mu.M) was changed to stimulate the rapid growth of Schwann cells. After cell fusion, the cells were purified. Cells are digested by trypsinization and transferred into a 5ml centrifuge tube, and the supernatant is removed by centrifugation (1200rpm, 5min); suspending the cells in complete medium containing anti-thy1.1 (1: 1000), and incubating on ice for 2h; centrifuging at 1200rpm for 5min to remove supernatant, adding complement (250. Mu.l Rabbit complement + 750. Mu.l DMEM), and incubating at 37 deg.C for 0.5h; washing with 3ml DMEM for three times; planting the seeds in a culture dish coated by PLL; and (3) changing the culture solution for 8-12h (containing HRG and Forskolin), and using the culture solution for subsequent experiments when the cells are fused and the purity reaches more than 95%.
2. Schwann cell transfection
Small molecule RNA transfection: in this experiment, lipofectamine was used TM RNAIMAX siRNA (final concentration: 100 nM), miRNA minic (purchased from Ribobio-technologies, inc., guangzhou, at a final concentration: 20 nM) were transferred to Schwa nn cells, changed to complete medium the next day, and follow-up experiments were performed as needed. The sequences of siRNA 259-si-1 and 259-si-2 aiming at LncRNA BC088259 are shown as SEQ ID No: 6-9. siRNA 259-si-1: sense strand 5 '-UUACAGAGAUUUCAAGCC dTdT-3' (SEQ ID NO: 6). The antisense strand is 5'-GGCUUGAAAUCAUCUGUAAdTdT-3' (SEQ ID NO: 7). siRNA 259-si-2: sense strand 5 '-AAGAGAGUGUUCAGCUCCC dTdT-3' (SEQ ID NO: 8). The antisense strand is 5 '-GGAGAGCUGAAACUCUCUCUU dTdT-3' (SEQ ID NO: 9).
Infection with lentivirus: according to the experiment, a certain number of cells are inoculated, the culture solution is changed to Enhanced infection solution (Eni.S) the next day, the Eni.S is diluted and packaged with full-length BC088259 lentivirus (LV-BC 088259) and Control virus (LV-Control), schwann cells are infected according to MOI =20, and the culture medium is changed back to the conventional culture medium after 12h for continuous culture for subsequent experiments (the virus is packaged by Kjeka gene).
3. Schwann cell RNA extraction, reverse transcription and qRT-PCR
Respectively transfecting siRNA1 and siRNA2 specific to BC088259 and Control siRNA Negative Control (NC) of the siRNA or infecting full-length lentivirus (LV-BC 088259) and Control virus (LV-Control) of the BC088259 by primary Schwann cells, collecting the cells after 48 hours, and performing cell selection according to the formula
Figure BDA0002240550680000041
Reagent (Invitrogen) instructions Schwann cellular RNA was extracted, reverse transcribed, and qRT-PCR was performed as described in example 1. qRT-PCR results are shown in FIG. 2A, BC088259siRNA1 and BC088259siRNA2 can significantly interfere the expression of BC088259 in Schwann cells; as shown in fig. 2C, LV-BC088259 can significantly increase expression of BC088259 in Schwann cells.
4. Transwell migration experiment
In a clean bench, transwell chambers (Corning Co.) were coated 3 times with Fibronectin (10. Mu.g/ml) and air-dried. Schwann cells in the cell plates to be examined were digested, resuspended in DMEM, counted and adjusted to 3X 10 5 and/mL. After 100. Mu.l of each of the resuspended cells from the different groups was added to each of the transwells, the transwells were gently placed into wells of a 24-well plate containing 500. Mu.l of complete medium and cultured in an incubator. After 24h incubation, the Transwell was removed, fixed in 4% paraformaldehyde/PBS for 0.5h, stained with 0.1% crystal violet for 0.5h, washed three times with water, cells that did not cross the membrane were gently wiped off with a cotton swab and allowed to air dry. Observing under an inverted microscope, randomly selecting more than 5 visual fields, and taking a picture. The experiment was repeated 3 times more for statistical analysis. The results are shown in fig. 2B, and the Transwell results indicate that BC088259siRNA1, siRNA2 interference LncRNA BC088259 significantly inhibited Schwann cell migration compared to NC, and the right histogram is the migration rate of Schwann cells after siRNA interference BC088259. As shown in fig. 2D, the Transwell results indicate that LV-BC088259 overexpresses LncRNA BC088259 promotes Schwann cell migration, and the right histogram is the migration rate of Schwann cells after LV-BC088259 overexpresses LncRNA BC088259.
5. Scratch test
The scratching experiment was carried out in 12-well plates, cells were plated in 12-well plates, schwann cell transfection was carried out as in example 2, and after 24h, it was changed to DMEM medium containing 0.5% FBS and 0.15. Mu.g/mL mitomycin C, cultured for 12h, scratched with a 200. Mu.l pipette tip, and the cell migration was observed microscopically every hour and photographed; the area results of the scratched area in the photograph were analyzed by Image-Pro Plus software (Media Cybernetics, maryland). Results as shown in fig. 3A, the scratch results after the interference of the LncRNA BC088259siRNA1 and siRNA2 to the Schwann cells indicate that the interference of the LncRNA BC088259 inhibits the migration of the Schwann cells. Fig. 3B is the scoring statistics of LncRNA BC088259siRNA1, siRNA2 after interfering Schwann cells. As shown in fig. 3C, the scratch results after overexpression of Schwann cells by LV-BC088259 showed that overexpression of LncRNA BC088259 promoted migration of Schwann cells. FIG. 3D is the statistics of the scratch results after the overexpression of Schwann cells by LV-BC 088259. The results indicate that LncRNA BC088259 can regulate Schwann cell migration.
Sequence listing
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ctggtcagca cgtctgtata ggcagcagcc cctctgagct ccagtgttgt tcgggctgga 180
ggcagaagga ccaagaatgc accatcccca tctgtgaggg gcctgatgcc tgcaagaaag 240
atgaggtgtg tgtgaagccg ggcctctgtc gatgtaaacc tggattcttc ggagcccatt 300
gcagctcccg ctgtccaggc cagtactggg gccccgactg tcgtgagacc tgcccttgcc 360
atccacgtgg ccagtgcgaa ccagccacgg gtgtgtgcca gtgccaaccc aactactggg 420
gcaagctctg tgagttcccc tgcacctgcg gcccccacgg aaaatgtgac ccaaagacgg 480
gcttgtgcca ctgcgacccc ggctggtggt cacctacgtg tcgtcgcccc tgccaatgca 540
acccattagc gcgctgtgac caggtcactg gagcctgcat atgcccgacg ggctggtggg 600
gccaccgctg cagcttcagt tgcaactgcc atacctcgcc ctgccttcag gactccggcc 660
gttgcacctg cctgcagggc tggtggggtc cagagtgtag tcgcaggtgc cagtgtgtgc 720
gaggacaatg cagcgttact tccggccact gctcctgccc tcctggcttc caaggagccc 780
tctgtgagct gccttgcaag cccggccgct atggagcgca gtgcaaagaa agacagaatc 840
ctggatggct ggagaggtgg ctcagcagtg aagaggactg actgctcttg cagagatcca 900
gggttcgatt ctcagcacgc acacttgatg gcttgaaatc atctgtaact ccagtttcag 960
gagagctgaa actctcttct ggcttccatg gtcacaaggc actcaagtgg tgtacagaca 1020
tacatgcaag caaaatacac atacacgtta ataaaaagaa aaagaaagaa agggtttcta 1080
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Claims (2)

1. A long non-coding RNA is characterized in that the cDNA sequence is shown as SEQ ID No: 1. as shown.
2. Use of the long non-coding RNA over-expressed according to claim 1 for the preparation of a medicament for the treatment of peripheral nerve injury.
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CN109897853A (en) * 2019-03-13 2019-06-18 南通大学 A kind of long-chain non-coding RNA and its application
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CN110141578A (en) * 2019-06-10 2019-08-20 南通大学 Circular rna circ-Ankib1 promotes the application in nerve regneration and repairing nerve damage drug in preparation

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