CN111154867A

CN111154867A - Long-chain non-coding RNA LOC100909675 and application thereof

Info

Publication number: CN111154867A
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: 2020-05-15
Anticipated expiration: 2040-03-06
Also published as: CN111154867B

Abstract

The invention belongs to the technical field of medical research, and particularly relates to a 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 as a molecular intervention target in preparation of medicines for treating diseases related to cell proliferation and migration. Wherein the diseases related to cell proliferation and migration include nerve injury, cancer, atherosclerosis or arthritis. The invention takes LncRNA LOC100909675 as a molecular intervention target, down-regulates or inhibits the LncRNA LOC100909675, obviously inhibits the proliferation, migration and wound healing capacity of astrocytes, and promotes the recovery of spinal cord injury function.

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 a long-chain non-coding RNA LOC100909675 and application thereof.

Background

Spinal cord injury leads to permanent dysfunction in movement and sensation, which severely affects people's quality of life. The pathophysiology process in the spinal cord injury process is systematically and comprehensively known, and the exploration of the cell molecular mechanism is of great importance for the formulation of a new treatment strategy. Astrocytes are the most abundant glial cells and play an important role in the central nervous system. Following spinal cord injury, activation of astrocytes and reactive glial cell 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 (lncrnas) are a class of non-coding RNAs of more than 200 nucleotides. LncRNA mainly realizes the regulation and control of gene expression through epigenetics, 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 technical problem to be solved by the invention is 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 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 provides application of the long-chain non-coding RNA LOC100909675 as a molecular intervention target in preparation of medicines 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 associated with cell proliferation and migration is nerve injury.

Preferably, the nerve injury is spinal cord injury.

The embodiment of the invention also provides a medicament for treating diseases related to cell proliferation and migration, which is characterized by comprising at least long-chain non-coding RNA LOC 100909675.

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

(1) the invention takes LncRNA LOC100909675 as a molecular intervention target spot, down-regulates or inhibits LncRNALOC100909675, obviously inhibits the proliferation, migration and callus capacity of astrocytes, and promotes the recovery of spinal cord injury function.

(2) The embodiment of the invention utilizes a sequencing technology to find that a novel long-chain non-coding RNA LOC100909675 is up-regulated after rat spinal cord injury; qRT-PCR results showed that LncRNA LOC100909675 was increased in expression at various time periods after injury compared to 0 d. The full length of LncRNA LOC100909675 was obtained by RACE experiments, and the length of LncRNALOC100909675 transcript was determined to be 1270 nt. And designing siRNA aiming at the full-length sequence of LncRNA LOC100909675, and carrying out siRNA interference experiments. Edu staining results showed that interfering with the expression of LOC100909675 clearly inhibited astrocyte proliferation. Transwell migration and scratch test results show that interference LOC100909675 significantly inhibited astrocyte migration compared to NC.

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

Description of the figures

FIG. 1 is a graph showing the expression change of LOC100909675 at different time points in spinal cord tissue after spinal cord injury in rats (with internal reference to GAPDH) in example 1 of the present invention;

FIG. 2 is a graph of the effect of siRNA1-3 of LOC100909675 on LOC100909675 expression in primary astrocytes after 24 h and 48 h interference (referenced against GAPDH) in example 2 of the present invention;

FIG. 3 is a graph of the effect of siRNA1 of LOC100909675 (siLOC 100909675) interfering with LOC100909675 on astrocyte proliferation described in example 2 of this invention;

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

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

wherein, FIG. 4A is a graph showing the results of detecting astrocyte migration in a Transwell experiment after treatment of siLOC 100909675; FIG. 4B is a graph of a statistical analysis of FIG. 4A; FIG. 4C is a graph of the results of a scratch experiment detecting astrocyte migration after treatment with siLOC 100909675; FIG. 4D is a graph of the statistical analysis of FIG. 4C.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

Example 1

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

Detecting expression of LOC100909675 and obtaining sequence thereof

(1-1) establishing a rat spinal cord hemisection injury model

(1-1-1) healthy adult male SPF grade SD rats 396, weighing 250 g. + -.10 g, randomized into 2 groups: sham group (sham group) and hemisection group. Experiments were repeated 3 times in total, with 11 time points of 0 h, 0.5 h, 3 h, 6 h, 12 h, 1 d, 3 d, 7 d, 14 d, 21 d, 28 d, 6 per time point.

(1-1-2) after anesthesia, depilation and 70% alcohol disinfection with a combination anesthetic (diluted 1:3, i.p. at 10. mu.l/g body weight), an incision (incision length less than 1 cm) was made in the midline skin of the rat back.

(1-1-3) subcutaneous tissues from T8 to T10 and muscular tissues on the spine were peeled off, muscles were distracted using a distractor, the T9 vertebral plate was exposed, a small number of vertebrae were removed with a pair of fine rongeurs, and the spinal cord was exposed.

(1-1-4) A right side half cut was then carefully made on T9 with an ophthalmic iris knife. The muscle layer is sewed, the skin is fixed by a wound clamp, and the operation is routine. The sham group performed laminectomy only according to the above procedure, exposing the spinal cord without making a half-cut.

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

(1-2-1) animals were deeply anesthetized by intraperitoneal injection of a compound anesthetic containing sodium pentobarbital (0.35 ml/100 g).

(1-2-2) collecting proximal tissues within 5 mm of the injured part of the spinal cord T9 at each time point after the spinal cord hemisection injury of the rat, preserving the proximal tissues in liquid nitrogen, and using dry ice to send the tissues to Shanghai Ohio Biotech limited for long-chain non-coding RNA sequencing.

(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 lncrnas, namely LOC100909675 (NR _ 110709.1), was selected for further analysis and study by performing work such as pre-bioinformatics analysis and qRT-PCR verification screening on 30 lncrnas with higher basal expression abundance (FPKM value).

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

(1-3-1) extracting tissue RNA from proximal tissues within 5 mm of a spinal cord T9 injury part at each time point after rat spinal cord hemisection injury according to TRIZOL washer (Invitrogen) instructions, carrying out reverse transcription according to a reverse transcription Kit (HiScript III 1stStrand cDNA Synthesis Kit (+ gDNA wiper)) (Norrespect), carrying out qRT-PCR on a Stepone PCR (ABI) instrument by adopting an AceQ YBqPCRSR Green Master Mix (High ROX Premix) Kit (Norrespect), and carrying out operation according to the instructions of the Kit (taking GAPDH as an internal reference);

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

The qRT-PCR results are shown in figure 1, showing that LOC100909675 expression was consistently up-regulated in injured spinal cord nerve tissue compared to 0 d.

(1-4) Rapid Amplification of CDNA Ends (RACE)

(1-4-1) SD rat spinal cord nerve tissue was extracted with high quality RNA using RNeasy Mini Kit (Qiagen) strictly according to SMARTer^TMRACE (Clotech) kit, performing First-strand cDNA synthesis using 5 '-CDS primer and 3' -CDS primer, respectively, to obtain the corresponding 5 '-cDNA and 3' -cDNA.

(1-4-2) according to the specification requirements, a primer LOC100909675-5-GSP sequence of 5 '-GSP and a primer LOC100909675-3-GSP of 3' -GSP are designed aiming at LOC100909675, wherein the primer LOC100909675-3-GSP is shown as SEQ ID No: 4-5, the LOC100909675-5-GSP sequence is as follows: 5-ctaacactggcattctcctgcttc-3; LOC100909675-3-GSP is: 5-gcagtgtgggaagatggtcatgtac-3; 5 '-RACE and 3' -RACE were then performed using touchdown PCR to obtain the 5 'and 3' ends of LOC 100909675.

(1-4-3) after the sequencing is finished, 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 as a molecular intervention target in preparation of medicines for treating diseases related to cell proliferation and migration. In further embodiments, the disease associated with cell proliferation or migration includes nerve injury, cancer, atherosclerosis, or arthritis. Preferably, the disease associated with cell proliferation and migration is nerve injury. Preferably, the nerve injury is spinal cord injury.

A medicament for treating diseases associated with cell proliferation and migration, the medicament at least comprising long chain non-coding RNALOC 100909675.

The method for verifying the influence of LOC100909675 on proliferation and migration of astrocytes comprises the following steps:

(2-1) culture of Primary astrocytes

(2-1-1) the spinal cords of the rats, which were collected from the rat at day 1 after birth, were separated and transferred to a petri dish containing a separation buffer (DMEM). Spinal cords were isolated by dissecting the spinal membrane under a scope and transferred to another dish containing isolation buffer (DMEM).

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

(2-1-2) the digestion was stopped by adding complete medium (DMEM/F12 + 10% FBS + 1% L-glutamine + 1% streptomycin) and centrifugation at 1000 rpm for 5 min, and the supernatant was discarded.

(2-1-3) resuspend the cells by adding complete medium. An appropriate amount of cells were inoculated into a flask, cultured in a 5% CO2 constant temperature cell incubator (37 ℃), and after 1 d, the medium was replaced with fresh medium, and thereafter every 3 d, the growth of the cells was observed under a microscope.

(2-1-4) when the astrocytes grow to 95% density, adding sufficient complete medium, placing in a constant temperature shaking table at 37 ℃, shaking 200 g overnight (16 h), removing supernatant (removing microglia and part of oligodendrocytes), and carrying out passage by using 0.25% pancreatin (ratio of 1: 2-1: 3). Astrocytes passaged to P2 passage were subjected to subsequent experiments.

(2-2) astrocyte electrotransformation

(2-2-1) digesting the astrocytes cultured until generation P2 with 0.25% pancreatin, adding complete medium to stop digestion, and washing with PBS for 2 times; 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 100 mu l of mixed liquor per tube⁶Astrocytes + 10 μ l siRNA (concentration 40 μ M), with a cell volume of 90 μ l and an siRNA volume of 10 μ l.

(2-2-3) then, electrotransfer was performed according to the procedure of the electrotransfection of primary nerve cells by NEPA21 (NEPAGENE Co.).

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

(2-3-1) after primary astrocytes are transfected respectively and specifically for LOC100909675 siRNA and its Control siRNAgenetic Control (siCtrl), 24 hours or 48 hours, cells are harvested, astrocyte RNA is extracted according to TRIZOL Reagent (Invitrogen) instructions, reverse transcription is performed, and qRT-PCR is performed, wherein the specific method is the same as the qRT-PCR steps in example 1.

The results are shown in fig. 2, LOC100909675 siRNA1-3 can significantly interfere the expression of LOC100909675 in astrocytes after siRNA treatment for 24 h or 48 h.

(2-4) Edu cell proliferation assay

(2-4-1) transfection of siRNA1 (siLOC 100909675, shown in SEQ ID Nos: 6-7) specific to LOC100909675 and its control (siCtrl) into primary astrocytes, respectively, inoculated with 5X 10 cells⁴After culturing the cells In a 24-well plate for 22 h, Edu Cell proliferation experiments were performed according to Cell-Light EdU Apollo 567 In Vitro Kit (C10310-1, Sharp Biotech, Guangzhou).

(2-4-2) marking by Edu, fixing cells, staining, and counting after photographing.

Edu results of the experiment are shown in FIG. 3, the bar graph is the proliferation rate of astrocytes after siRNA interference of LOC 100909675. Astrocyte proliferation decreased after siLOC100909675 treatment compared to control; edu staining results showed that interfering with the expression of LOC100909675 clearly inhibited astrocyte proliferation.

(2-5) Transwell migration test

(2-5-1) in the superclean bench, the astrocytes in the cell plate to be examined were digested, resuspended in DMEM, counted and adjusted to 2.5X 10⁵and/mL. 200 μ l of resuspended cells from different groups were added to each Transwell chamber (Costar, 8 μm pore size), after which the transwells were gently placed into wells of a 24-well plate containing 700 μ l of complete medium and incubated in an incubator.

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

(2-5-3) observing under an inverted microscope, randomly selecting 5-10 visual fields, and taking pictures. The experiment was repeated 3 times more for statistical analysis.

The results are shown in fig. 4A, and the Transwell results indicate that siLOC100909675 interferes with LOC100909675 and significantly inhibits astrocyte migration compared to the control, and the right-hand bar chart in fig. 4B is the astrocyte migration after siRNA interferes with LOC 100909675.

(2-6) cell migration ability measurement by cell scratching method

(2-6-1) transfecting siRNA1 (siLOC 100909675) specific to LOC100909675 and a control (siCtrl) of the primary astrocytes respectively, digesting the cells by pancreatin after 24 h, adding a proper amount of complete medium to stop digestion, washing 1-2 times by PBS, using the complete medium to resuspend the cells, counting, inoculating 5 x 10⁴Cells were plated into ibidi cell scratch test inserts and incubation was continued for 12 h.

(2-6-2) after the cells were fully adherent, the ibidi insert was carefully removed, washed 2 times with PBS, and DMEM/F12 medium (which would produce a 500 μm cell space) containing 0.15 mg/ml mitomycin C and 0.5% FBS was added.

(2-6-3) taking pictures of multiple points at 0 h, 6 h, 12 h and 24 h time points and counting the distance of cell migration.

The results are shown in FIGS. 4C and 4D, where the interstitial width of the cells decreased from 500 μm to 161.5 μm 24 h after the treatment with SiLOC100909675, compared to 18.3 μm for the control group. Indicating that the interference with LOC100909675 significantly inhibits the migration of astrocytes.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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

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Claims

1. A long-chain non-coding RNA LOC100909675 is characterized in that a sequence cDNA is SEQ NO. 1.

2. An application of long-chain non-coding RNA LOC100909675 as a molecular intervention target in preparing medicines for treating diseases related to cell proliferation and migration.

3. The use of long-chain non-coding RNA LOC100909675 as a target for molecular intervention in the manufacture of a medicament for treating diseases associated with cell proliferation and migration according to claim 2, wherein the diseases associated with cell proliferation and migration include nerve injury, cancer, atherosclerosis, and arthritis.

4. The use of long-chain non-coding RNA LOC100909675 as a molecular intervention target in the preparation of a medicament for treating diseases related to cell proliferation and migration according to claim 3, wherein the diseases related to cell proliferation and migration are nerve injuries.

5. The use of long-chain non-coding RNA LOC100909675 as a molecular intervention target in the preparation of drugs for treating diseases related to cell proliferation and migration according to claim 4, wherein the nerve injury is spinal cord injury.

6. A medicament for treating diseases related to cell proliferation and migration, which at least comprises long-chain non-coding RNA LOC 100909675.