CN111154867B

CN111154867B - Long-chain non-coding RNA LOC100909675 and application thereof

Info

Publication number: CN111154867B
Application number: CN202010149235.2A
Authority: CN
Inventors: 巫荣华; 姚淳; 于彬; 刘梅; 刘炎; 顾晓松
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-03-06
Filing date: 2020-03-06
Publication date: 2023-06-02
Anticipated expiration: 2040-03-06
Also published as: CN111154867A

Abstract

The invention belongs to the technical field of medical research, and particularly relates to long-chain non-coding RNA LOC100909675 and application thereof. The invention discloses a long-chain non-coding RNA LOC100909675, which is characterized in that the sequence cDNA is SEQ NO.1; the embodiment of the invention also discloses application of the long-chain non-coding RNA LOC100909675 serving as a molecular intervention target in preparing medicines for treating diseases related to cell proliferation and migration. Wherein the diseases related to cell proliferation and migration comprise nerve injury, cancer, atherosclerosis or arthritis. According to the invention, lncRNA LOC100909675 is used as a molecular intervention target, lncRNA LOC100909675 is down-regulated or inhibited, proliferation, migration and callus capacity of astrocytes are obviously inhibited, and spinal cord injury function recovery is promoted.

Description

Long-chain non-coding RNA LOC100909675 and application thereof

Technical Field

The invention belongs to the technical field of medical research, and particularly relates to long-chain non-coding RNA LOC100909675 and application thereof.

Background

Spinal cord injury results in permanent dysfunction of movement and sensation, severely affecting people's quality of life. The pathophysiological process in the spinal cord injury process is systematically and comprehensively known, and the exploration of the cellular molecular mechanism in the pathophysiological process is vital to the establishment of a new treatment strategy. Astrocytes are the most abundant glial cells and play an important role in the central nervous system. After spinal cord injury, activation of astrocytes and reactive glioblast proliferation are involved in various aspects of neuroplasticity and CNS regeneration. The research on the regulation and molecular mechanism of astrocytes after spinal cord injury opens up a new way for the treatment of spinal cord injury, and has important significance and value.

Long non-coding RNAs (LncRNA) are a class of non-coding RNAs that exceed 200 nucleotides. The LncRNA realizes the regulation and control of gene expression mainly through the aspects of epigenetic science, transcriptional regulation, post-transcriptional regulation and the like, thereby playing an important role in various biological processes and being closely related to the occurrence of human diseases.

Disclosure of Invention

The invention aims to provide a long-chain non-coding RNA LOC100909675 and application thereof, so as to solve the problems in the background art.

The embodiment of the invention provides long-chain non-coding RNA LOC100909675, which is characterized in that the sequence cDNA is SEQ NO.1.

The embodiment of the invention also provides an application of siRNA for inhibiting long-chain non-coding RNA LOC100909675 in preparing a medicament for treating diseases related to cell proliferation and migration.

Further, the diseases related to cell proliferation and migration include nerve injury, cancer, atherosclerosis or arthritis.

Preferably, the disease related to cell proliferation and migration is nerve injury.

Preferably, the nerve injury is a spinal cord injury.

The embodiment of the invention further provides a medicine for treating diseases related to cell proliferation and migration, which is characterized by at least comprising siRNA for inhibiting long-chain non-coding RNA LOC100909675.

The technical scheme of the invention has the following beneficial effects:

(1) According to the invention, lncRNA LOC100909675 is used as a molecular intervention target, lncRNA LOC100909675 is down-regulated or inhibited, proliferation, migration and callus capacity of astrocytes are obviously inhibited, and spinal cord injury function recovery is promoted.

(2) The embodiment of the invention discovers that the expression of a novel long-chain non-coding RNA LOC100909675 is up-regulated after rat spinal cord injury by utilizing a sequencing technology; qRT-PCR results showed increased expression of LncRNA LOC100909675 at each time period after injury compared to 0 d. The full length of LncRNA LOC100909675 was obtained by RACE experiments, and the length of LncRNA LOC100909675 transcript was determined to be 1270 nt. And (3) designing siRNA aiming at the LncRNA LOC100909675 full-length sequence, and carrying out siRNA interference experiments. Edu staining results showed that interfering with LOC100909675 expression significantly inhibited astrocyte proliferation. Transwell migration and scratch assay results show that interfering LOC100909675 significantly inhibits astrocyte migration compared to NC.

(3) The long-chain non-coding RNA LOC100909675 can regulate proliferation and migration of astrocytes in the spinal cord injury repair process, is beneficial to better understand the important role of LncRNA in the central nerve injury repair process, and provides a new target point for treatment after spinal cord nerve injury.

Description of the drawings

FIG. 1 is a graph showing the variation of LOC100909675 expression in spinal cord tissue at various time points after spinal cord injury in rats (GAPDH as an internal reference) according to example 1 of the present invention;

FIG. 2 is a graph showing the effect of siRNA1-3 of LOC100909675 on expression of LOC100909675 in primary astrocytes (GAPDH as an internal reference) after interfering with 24 h and 48 h described in example 2 of the present invention.

FIG. 3 is a graph showing the effect of siRNA1 (siLOC 100909675) of LOC100909675 on interference of LOC100909675 on astrocyte proliferation as described in example 2 of the present invention;

wherein, FIG. 3A is a representative image of EdU; FIG. 3B is a graph of statistical analysis;

FIG. 4 is a graph showing the effect of siRNA1 (siLOC 100909675) of LOC100909675 on interference of LOC100909675 on astrocyte migration as described in example 2 of the present invention;

FIG. 4A is a graph showing the results of the astrocyte migration detection by the Transwell test after the treatment of SiLOC 100909675; FIG. 4B is a graph of statistical analysis of FIG. 4A; FIG. 4C is a graph showing the results of the scratch test for astrocyte migration after treatment with SiLOC 100909675; fig. 4D is a graph of statistical analysis of fig. 4C.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.

Example 1

The embodiment of the invention provides long-chain non-coding RNA LOC100909675, and the sequence cDNA is SEQ NO.1.

Detection of LOC100909675 expression and acquisition of its sequence

(1-1) establishing a rat spinal cord half-cut injury model

(1-1-1) healthy adult male SPF-grade SD rats 396, 250: 250 g.+ -. 10: 10 g, were randomized into 2 groups: sham group (sham group) and half cut group. Experiment 0 h,0.5 h,3 h,6 h,12 h,1 d,3 d,7 d,14 d,21 d,28 d was set up at 11 time points, 6 per time point, and the experiment was repeated 3 times in total.

(1-1-2) after anesthesia, dehairing and 70% alcohol sterilization using a compound anesthetic (1:3 dilution, intraperitoneal injection at 10 μl/g body weight), an incision (incision length less than 1 cm) was made in the dorsal midline skin of the rat.

(1-1-3) subcutaneous tissues of T8 to T10 and muscular tissues on the spinal column were peeled off, the muscles were distracted using a distractor to reveal the T9 lamina, a small number of vertebrae were removed with a fine-meshed rongeur, and the spinal cord was exposed.

(1-1-4) then right half cut was carefully performed on T9 with an ophthalmic iris blade. The muscle layers were sutured, the skin was secured with wound clips, and the animals were routinely fed post-operatively. The sham group performed laminectomy only as above, exposing spinal cord, without half-cutting.

(1-2), spinal tissue sampling and RNA sequencing analysis

(1-2-1) deep anesthesia of animals was performed by intraperitoneal injection of a complex anesthetic containing sodium pentobarbital (0.35 ml/100 g).

(1-2-2) the proximal tissues in spinal cord T9 injury site 5 mm at each time point after the above rat spinal cord half-cut injury were collected, stored in liquid nitrogen, and sent to Shanghai European Biotechnology Co., ltd for long-chain non-coding RNA sequencing using dry ice.

(1-2-3) sequencing results 1125 differentially expressed LncRNAs were obtained according to the condition that the Q-value was less than 0.01. One of the LncRNA, LOC100909675 (NR 110709.1), was selected for further analytical study by performing pre-bioinformatic analysis and qRT-PCR validation screening of 30 LncRNA, where basal expression abundance (FPKM value) was high.

(1-3), spinal cord tissue RNA extraction and qRT-PCR

(1-3-1) tissue RNA was extracted from proximal tissue in spinal cord T9 injury site 5 mm at each time point after half-cut injury of the spinal cord of a rat according to TRIZOL Reagent (Invitrogen), reverse transcribed according to the reverse transcription kit (HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wind)) (Norweizan), qRT-PCR was performed on a Stepone PCR (ABI) apparatus using AceQ qPCR SYBR Green Master Mix (High ROX Premixed) kit (Norweizan) and the procedure was performed according to the kit instructions (GAPDH as an internal reference);

wherein, the PCR instrument reaction program: stage 1:95 ℃ for 5 min, stage 2 (Cycle: 40): 95 ℃ for 10 s,60 ℃ for 30 s; stage 3:95℃for 15 s,60℃for 1 min,95℃for 15 s. LOC100909675 primer sequence is shown as SEQ ID No: 2-3.

The qRT-PCR results are shown in fig. 1, which shows that LOC100909675 expression in injured spinal nerve tissue is continuously up-regulated compared to 0 d.

(1-4), rapid Amplification of CDNA Ends (RACE)

(1-4-1) SD rat spinal cord nerve tissue was taken, and high quality RNA was extracted using RNeasy Mini Kit (Qiagen) in strict accordance with SMART ^TM The manual system of RACE (Clotech) kit, the synthesis of First-strand cDNA was completed by 5'-CDS primer and 3' -CDS primer, respectively, to obtain phases5'-cDNA and 3' -cDNA, respectively.

(1-4-2) according to the requirements of the specification, a primer LOC100909675-5-GSP sequence of 5'-GSP and a primer LOC100909675-3-GSP of 3' -GSP are designed for LOC100909675, and the primer LOC100909675-3-GSP is shown as SEQ ID No:4-5, LOC100909675-5-GSP sequence is: 5-ctaacactggcattctcctgcttc-3; LOC100909675-3-GSP is: 5-gcagtgtgggaagatggtcatgtac-3; subsequently, 5'-RACE and 3' -RACE were performed using touchdown PCR to obtain the 5 'and 3' ends of LOC100909675.

After sequencing (1-4-3), splicing the result with the original known sequence to obtain the full-length cDNA sequence of LOC100909675 (shown as SEQ ID No. 1).

Example 2

The embodiment of the invention also provides application of the long-chain non-coding RNA LOC100909675 serving as a molecular intervention target in preparing medicines for treating diseases related to cell proliferation and migration. In further embodiments, the cell proliferation, migration-related disease comprises nerve injury, cancer, atherosclerosis, or arthritis. Preferably, the disease related to cell proliferation and migration is nerve injury. Preferably, the nerve injury is a spinal cord injury.

A medicament for treating diseases related to cell proliferation and migration, which comprises at least long-chain non-coding RNA LOC100909675.

A method for verifying the effect of LOC100909675 on astrocyte proliferation and migration comprising the steps of:

(2-1) culture of primary astrocytes

(2-1-1) spinal cord of the red-skin mice 1 day after birth was separated and transferred to a petri dish containing a separation buffer (DMEM). Spinal cord was isolated by subscopic stripping of the spinal film and transferred to another petri dish with separation buffer (DMEM).

(2-1-1) tissue was washed with separation buffer (DMEM/F12), and the tissue was minced with surgical scissors. Adding 0.25% pancreatin 2 ml into the digestion solution, and digesting at 37deg.C for 15 min, and shaking several times every 5 min.

(2-1-2) complete medium (DMEM/F12+10% FBS+1% L-glutamine+1% Streptomyces griseus) was added to terminate digestion, and the supernatant was discarded after centrifugation at 1000 rpm for 5 min.

(2-1-3) cells were resuspended by addition of complete medium. Inoculating appropriate amount of cells into a culture flask, culturing in a 5% CO2 constant temperature cell incubator (37 ℃), replacing fresh culture medium after 1 d, replacing liquid every 3 d, and observing the growth of cells under a microscope.

(2-1-4) when astrocytes grow to 95% density, a sufficient amount of complete medium was added and placed in a constant temperature shaker at 37℃and after 200 g shaking overnight (16 h), the supernatant was removed (microglial and part of oligodendrocytes were removed) and passaging was performed using 0.25% pancreatin (1:2 to 1:3 ratio). Astrocytes passaged to the P2 generation were subjected to subsequent experiments.

(2-2), astrocyte electrotransformation

(2-2-1) astrocytes cultured to the P2 generation were digested with 0.25% pancreatin, and the digestion was stopped by adding complete medium, and washed 2 times with PBS; cells were resuspended in opti-MEM medium.

(2-2-2) mixing a certain amount of cells with siRNA, and fully and uniformly mixing to ensure that the final concentration of the mixture reaches 10 in each 100 mu l of mixed solution ⁶ Astrocytes+10 μl siRNA (concentration 40 μM), wherein the cell volume is 90 μl and the siRNA volume is 10 μl.

(2-2-3) electrotransfection was then performed with reference to the NEPA21 (NEPAGENE Inc.) primary neural cell electrotransfection procedure.

(2-3), astrocyte RNA extraction, reverse transcription and qRT-PCR

(2-3-1) Primary astrocytes were transfected with siRNA specific for LOC100909675 and control siRNA Negative Control (SiCtrl), 24. 24 h or 48. 48 h, respectively, and after harvesting, astrocyte RNA was extracted according to TRIZOL Reagent (Invitrogen), reverse transcribed, and qRT-PCR was performed in the same manner as in the procedure of qRT-PCR in example 1.

As shown in fig. 2, LOC100909675 siRNA1-3 could significantly interfere with LOC100909675 expression in astrocytes after

siRNA treatment

24 h or 48 h.

(2-4), edu cell proliferation assay

(2-4-1) Primary astrocytes were transfected with siRNA1 (siLOC 100909675 as shown in SEQ ID No. 6-7) specific to LOC100909675 and their control (siCtrl), respectively, and inoculated with 5X 10 ⁴ After further culturing 22 h cells In 24 well plates, edu Cell proliferation experiments were performed according to Cell-Light ™ EdU Apollo [ 567 In Vitro Kit (C10310-1, sharp Biotechnology Co., guangzhou, inc.).

(2-4-2) after Edu labeling, cell fixation, staining, photographing and counting.

Edu the experimental results are shown in FIG. 3, and the bar graph shows the proliferation rate of astrocytes after siRNA interference with LOC100909675. Astrocyte proliferation decreased after siroc 100909675 treatment compared to the control group; edu staining results showed that interfering with LOC100909675 expression significantly inhibited astrocyte proliferation.

(2-5), transwell migration experiments

(2-5-1) in an ultra clean bench, astrocytes in the cell plate to be detected were digested, resuspended in DMEM, counted and adjusted to 2.5X10 ⁵ /mL. 200. Mu.l of the different groups of resuspended cells were added to each Transwell chamber (Costar, 8 μm pore size), after which the Transwell was gently placed into wells of a 24-well plate containing 700. Mu.l of complete medium and incubated in an incubator.

(2-5-2) after 22-h culture, taking out Transwell, fixing with 4% paraformaldehyde/PBS for 15 min, dyeing with 0.1% crystal violet for 10 min, rinsing with distilled water for 2 times each for 2min; washing with water to appropriate color, gently wiping off cells which do not pass through the membrane with cotton swab, and air drying.

(2-5-3) observation under an inverted microscope, randomly selecting 5-10 visual fields, and photographing. The experiment was repeated 3 more times and statistical analysis was performed.

The results are shown in fig. 4A, which shows that siroc 100909675 interferes with LOC100909675 and significantly inhibits astrocyte migration compared to the control, and the histogram of fig. 4B on the right shows that siRNA interferes with the mobility of astrocytes after LOC100909675.

(2-6) detection of cell migration Capacity by cell scratch method

(2-6-1) Primary astrocytes were transfected with siRNA1 (SiLOC 100909675) specific for LOC100909675 and its control (SiCtrl), respectively, cells were digested with pancreatin after 24. 24 h, the digestion was stopped by adding an appropriate amount of complete medium, washed 1-2 times with PBS, cells were resuspended and counted in complete medium, inoculated with 5X 10 ⁴ Individual cells were grown into the ibidi cell scratch test insert and continued to culture 12 h.

(2-6-2) after the cells have completely adhered, the ibidi insert was carefully removed, washed 2 times with PBS, and DMEM/F12 medium containing 0.15. 0.15 mg/ml mitomycin C and 0.5% FBS (which resulted in a 500 μm cell gap) was added.

(2-6-3) Multi-point photographs were taken at

time points

0 h,6 h,12 h, 24 h and the distance of cell migration was counted.

The results are shown in FIGS. 4C and 4D, with 24. 24 h cell gap width reduced from 500 μm to 161.5 μm after the SiLOC100909675 treatment, and 18.3 μm in the control. Indicating significant inhibition of astrocyte migration following LOC100909675 interference.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> university of Nantong

<120> a long non-coding RNA LOC100909675 and application thereof

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Claims

1. An application of siRNA for inhibiting long-chain non-coding RNA LOC100909675 in preparing a medicament for treating spinal cord injury diseases is characterized in that: the siRNA sequence of the long-chain non-coding RNA LOC100909675 is shown as SEQ ID N0: 6-7.

2. A medicament for treating spinal cord injury diseases, which is characterized by at least comprising siRNA for inhibiting long-chain non-coding RNA LOC100909675, wherein the sequence of the siRNA of the long-chain non-coding RNA LOC100909675 is shown as SEQ ID N0: 6-7.