CN114990113B - Long-chain non-coding RNA NONRATT026122.2 and application thereof - Google Patents

Abstract

The invention discloses a long-chain non-coding RNA NONRATT026122.2, the sequence cDNA of which is SEQ ID NO.1. The research of the invention shows that NONRATT026122.2 is down-regulated after spinal cord injury, NONRATT026122.2 is used as a molecular intervention target, and the result shows that the up-regulation of NONRATT026122.2 expression can inhibit proliferation, migration and cell viability of astrocytes, induce the phenotype of the astrocytes to be converted into A2 nutrition, improve the secretion level of neurotrophic factors in the astrocytes, and finally promote the axon growth of neurons.

Description

Long-chain non-coding RNA NONRATT026122.2 and application thereof

Technical Field

The invention belongs to the technical field of biomedical science, and particularly relates to long-chain non-coding RNA NONRATT026122.2 and application thereof in preparation of a medicament for treating nerve injury.

Background

Spinal cord injury is a medical challenge to be addressed. The spinal cord injury often causes the loss of part or all of the sensory and motor functions below the injury plane, and serious patients can cause paraplegia or quadriplegia, and even endanger the lives of patients. Currently, strategies for spinal cord injury repair can be broadly divided into two categories: one is to promote axon regeneration of injured neurons, and the other is to promote sprouting of collateral of undamaged neurons, which innervate the injured side through the normal side. Although some technical means exist at present, the regeneration of injured axons can be promoted to a certain extent, the regeneration is very limited and the requirement of functional recovery is far from being met. The lateral branch sprouting of the residual neurons is promoted with higher feasibility compared with spinal cord regeneration.

Astrocytes are an important component of the central nervous system and represent approximately 30% of the total cellular fraction. After spinal cord injury, astrocytes around the injury activate and proliferate, and resting astrocytes excite "reactive astrocytes" that are characteristic of abnormal behavior. Recent studies have found that "reactive astrocytes" include two different types: type A1 and type A2, which act in opposition, astrocytes of type A1 can cause neuronal and oligodendrocyte death, which can disrupt synapses; the A2 type astrocytes have neuroprotective effect, secrete substances supporting the growth and survival of neurons near the injury, and promote tissue repair and regeneration. More and more researches show that after spinal cord injury, the transformation of A1/A2 type astrocytes is regulated, the proportion of the A2 type astrocytes is increased, and the repair treatment after spinal cord injury is facilitated.

Long non-coding RNAs (lncRNA) are a class of gene transcripts greater than 200 nucleotides in length, but not possessing the function of encoding proteins. Can be used for regulating various cell biological processes at the aspects of epigenetic, transcription, post-transcriptional level and the like through combining with DNA, RNA or protein. A large number of researches show that lncRNA can participate in the processes of survival of neurons, activation of glial cells and the like by regulating the expression of various mRNA, and is a novel regulating factor in the pathological process of spinal cord injury.

Disclosure of Invention

According to the invention, long-chain non-coding RNA which is different in samples at different time points after the spinal cord of the rat is analyzed through full transcriptome sequencing, so that long-chain non-coding RNA NONRATT026122.2 related to astrocyte proliferation, migration and phenotype conversion is obtained, and the long-chain non-coding RNA NONRATT026122.2 has potential to become a new target point for spinal cord injury repair.

The specific technical scheme of the invention is as follows:

a cDNA sequence of the long-chain non-coding RNA NONRATT026122.2 is shown in SEQ ID NO.1.

The invention also aims to provide the application of the long-chain non-coding RNA NONRATT026122.2 in preparing medicines for treating diseases related to cell proliferation, migration and cell phenotype conversion.

The application specifically takes long-chain non-coding RNA NONRATT026122.2 as a main active ingredient of the medicine or takes the main active ingredient as a molecular intervention target point to design a medicine for up-regulating the expression of the long-chain non-coding NONRATT026122.2.

Preferably, the medicament is a vector carrying a nucleic acid sequence expressing long non-coding RNA noneratt 026122.2. The vector is selected from a plasmid, phage, virus or host cell.

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

Preferably, the disease associated with cell proliferation, migration and cell phenotype switching is a nerve injury.

Preferably, the nerve injury is a spinal cord injury.

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

1. according to the invention, NONRATT026122.2 is taken as a molecular intervention target point, the expression of NONRATT026122.2 is up-regulated, proliferation, migration and cell activity of astrocytes can be obviously inhibited, the phenotype of the astrocytes is induced to be converted into A2 nutrition, the level of the neurotrophic factors secreted by the astrocytes is improved, and the growth of neurons is further promoted.

2. The embodiment of the invention discovers that the expression of a novel long-chain non-coding RNA NONRATT026122.2 is down-regulated after rat spinal cord injury through whole transcriptome sequencing, and confirms the accuracy of a sequencing result through qRT-PCR. The full-length sequence of noneratt 026122.2 was further obtained by RACE reaction, and an over-expression vector (AAV-noneratt 026122.2) containing the full-length sequence of noneratt 026122.2 was constructed using adeno-associated virus (AAV), followed by transfection of astrocytes, in which the expression level of noneratt 026122.2 was up-regulated. Cell proliferation detection results show that after the expression of NONRATT026122.2 is up-regulated, proliferation of astrocytes is obviously inhibited; the results of Transwell cells and scratch healing experiments show that after the expression of NONRATT026122.2 is up-regulated, the migration of astrocytes is obviously inhibited; qRT-PCR detects the condition of the expression of the astrocyte A1/A2 type related marker, and discovers that after NONRATT026122.2 expression is up-regulated, the A1 type marker is down-regulated to different degrees, and the A2 type marker is up-regulated to different degrees; neurotrophic factor detection shows that NONRATT026122.2 expression is up-regulated to improve the level of astrocyte secreted trophic factors; results of co-culture experiments with neurons showed that astrocytes up-regulated expression of nonrat 026122.2 promoted growth of neuronal axons.

3. The NONRATT026122.2 long-chain non-coding RNA can regulate astrocyte proliferation, migration and phenotype conversion in the spinal cord injury repair process, is helpful for deeply understanding the important role of the long-chain non-coding RNA in the central nerve injury repair process, and provides a new molecular intervention target for nerve injury repair.

Drawings

FIG. 1 shows that the expression of the long non-coding RNA NONRATT026122.2 is down-regulated after rat spinal cord injury, and the accuracy of the sequencing results is confirmed by qRT-PCR. Wherein, the circular mark represents the sequencing result, and the ordinate is the left Y axis; square marks represent qRT-PCR results, with the ordinate being the right Y-axis. * P <0.05 compared to 1d is shown.

FIG. 2 shows the results of detection of overexpression of NONRATT026122.2 in primary astrocytes. Wherein OE-NC is control, OE-noneratt 026122.2 is over-expressed, and p <0.001 compared to control.

FIG. 3 is a graph showing the effect of upregulation of NONRATT026122.2 expression on astrocyte proliferation in accordance with example 2 of the present invention. FIG. 3A is a representative image of proliferation assay, green for GFAP, astrocytes, red for ki67, proliferating cells, blue for DAPI, showing all nuclei; fig. 3B is a statistical analysis. Wherein OE-NC is control, OE-noneratt 026122.2 is over-expressed, and p <0.001 compared to control.

FIG. 4 is a graph showing the effect of upregulation of NONRATT026122.2 expression on astrocyte migration in accordance with example 2 of the present invention. FIG. 4A shows the lateral migration of astrocytes detected by the scratch test; FIG. 4B shows the longitudinal migration of astrocytes detected by a Transwell assay; FIG. 4C is a statistical analysis of FIG. 4A; fig. 4D is a statistical analysis of fig. 4B. Wherein OE-NC is control, OE-noneratt 026122.2 is over-expressed, p <0.01 is shown compared to control, and p <0.001 is shown compared to control.

FIG. 5 is a graph showing the effect on astrocyte phenotype and neurotrophic factor expression levels after upregulation of NONRATT026122.2 in example 2 of the present invention. FIG. 5A shows the detection of the expression of astrocyte type A1/A2 associated markers; FIG. 5B is a graph showing the detection of neurotrophic factor expression in astrocytes. Wherein OE-NC is control, OE-noneratt 026122.2 is over-expressed, p <0.01 is shown compared to control, and p <0.001 is shown compared to control.

FIG. 6 is the effect on neurite outgrowth of neurons co-cultured therewith after upregulation of NONRATT026122.2 expression in astrocytes according to example 3 of the present invention. FIG. 6A is a representative picture of neuronal growth after 48h and 72h co-culture; FIG. 6B is a plot of the longest neurite statistics of neurons after 48h co-culture; FIG. 6C is a plot of total length of neuronal axons after 48h co-culture; FIG. 6D is a plot of the longest neurite statistics of neurons after 72h co-culture; FIG. 6E is a plot of the total length of neuronal axons after 72h co-culture. Wherein OE-NC is control, OE-noneratt 026122.2 is over-expressed, and p <0.001 compared to control.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 construction of a rat spinal cord semi-transection injury model and spinal cord tissue Whole transcriptome sequencing

Healthy adult male SD rats 54, SPF grade, body weight 220-250g, were randomly divided into two groups: spinal cord semi-transection group and sham surgery group. Experiments were performed at 3 time points 1d,3d,7d, 6 times each, and 3 replicates.

The rats were weighed and injected intraperitoneally with a compound anesthetic (0.3 ml/100 g) and after deep anesthesia the dorsal side was shaved and disinfected. The spinal cord T9 segment is positioned through the osseous mark, skin and muscle are sequentially cut, partial vertebral lamina is removed, the spinal cord is exposed, and the right half-transection is carried out on the spinal cord T9 segment by using an ophthalmic iris cutter. The muscle, fascia and skin are sutured sequentially after operation, and local iodophor is sterilized. The sham group performed laminectomy only as per the above procedure, exposing but not damaging the spinal cord.

After the semi-transection injury of the spinal cord of the rat, the distal tissues within 5mm of the T9 injury site at each time point were collected, stored in liquid nitrogen, and then sent to Shanghai European Biotechnology Co.Ltd for full transcriptome sequencing.

Sequencing results 132 differentially expressed long non-coding RNAs (lncRNA) were obtained according to Q-value less than 0.01. Pre-bioinformatic analysis and qRT-PCR validation were performed on 39 lncRNA with higher basal expression abundance (FPKM value). Subsequently, after screening for oxygen deprivation using an in vitro astrocyte oxygen deprivation model, lncRNA whose expression was significantly altered in astrocytes, 3 lncRNA were finally obtained (NONRATT 026122.2, NONRATT026123.2, NONRATT 003820.2). Then, respectively designing siRNA interference sequences aiming at the 3 lncRNAs, and the detection result shows that after NONRATT026122.2 interference, migration and proliferation capacity of astrocytes are obviously promoted, and activity of the astrocytes is improved; while interfering with noneratt 026123.2 or noneratt 003820.2 has no significant effect on astrocyte behavior. Thus in subsequent studies, lncRNA NONRATT026122.2 was selected for further study.

Total RNA from distal tissues within 5mm of the T9 lesion at each time point was extracted by Trizol method, reverse transcribed by a reverse transcription kit (HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wind)) (Norfirazan), and then qRT-PCR was performed on a StepOne PCR (ABI) apparatus using a AceQ qPCR SYBR Green Master Mix (High ROX Premixed) kit (Norfirazan), the procedure was as described in the kit, and the PCR reaction procedure was as follows: stage 1:95℃for 10min, stage 2 (Cycle: 40): 95 ℃ for 10s and 60 ℃ for 30s; stage 3:95℃15s,60℃1min,95℃15s. The NONRATT026122.2 primer sequences are shown in SEQ ID NO.2 and 3. The qRT-PCR results are shown in figure 1, which shows that expression of noneratt 026122.2 continues to be down-regulated following spinal cord injury.

Example 2 upregulation of NONRATT026122.2 expression on astrocyte proliferation, migration and phenotype

The RACE reaction gave the full-length sequence NONRATT026122.2. The primer sequence in RACE reaction is shown as SEQ ID NO. 4-7.

RACE upstream primer: 5'-CTGCGCAGGAGGAGCAGTGGTGCTC-3', (SEQ ID NO. 4);

RACE downstream primer: 5'-CCCTTGTCCCATGCATGCCACCATCATG-3', (SEQ ID NO. 5);

NEST upstream primer: 5'-ATGCTGGGCACGCACAGGAGCAT-3', (SEQ ID NO. 6);

NEST downstream primer: 5'-TGCCCTATGGAACTGGGGTTCCCTTTTC-3', (SEQ ID NO. 7).

The product obtained by the RACE reaction is sequenced and verified to finally obtain the NONRATT026122.2 full-length sequence (SEQ ID NO. 1). The known sequence length of NONRATT026122.2 on NONCODE (http:// www.noncode.org /) is 1549bp, the unknown sequence of NONRATT026122.2 on the 5 'end and the 3' end is obtained through RACE reaction, the length of the 5 'end is 91bp, and the length of the 3' end is 623bp. An over-expression vector (AAV-noneratt 026122.2) containing the full-length sequence of noneratt 026122.2 was then constructed using adeno-associated virus (AAV).

Primary astrocytes were isolated and cultured. The spinal cord of the red skin mice after 1 day of growth is separated under a dissecting scope, placed in a culture dish containing precooled tissue dissection solution (HBSS), the dura mater is removed cleanly, transferred to clean HBSS solution, and the tissue is sheared as much as possible by sterilizing micro-shears. Adding 2ml of 0.25% pancreatin, digesting at 37deg.C for 15min, shaking for several times every 5min, adding complete medium (DMEM/F12+10% FBS+1% L-glutamine+1% penicillin), stopping digestion, centrifuging at 1000rpm for 5min, and discarding supernatant. Add complete media to resuspend cells, 7.5x10 ⁶ The individual cells were seeded at 75cm ² The flask was filled with complete medium to 15-20ml. After 1d, the culture bottle is changed, before changing the liquid, the culture bottle is rotated and rocked to remove the non-adherent cells, after each 2-3d of liquid change, before each liquid change, the culture bottle is tapped by hands for 5-10 times to remove microglial cells and part of oligodendrocytes. When astrocytes grow to 95% density, a sufficient amount of complete medium is added and placed in a thermostatic shaker at 37℃and 200g is shaken overnight (16 h) after which the supernatant is removed and passaged with 0.25% pancreatin (1:2 to 1:3 ratio). Astrocytes passed to the P2 generation were subjected to subsequent experiments.

NONRATT026122.2 is overexpressed in primary astrocytes. Astrocytes cultured to the P2 generation were digested with 0.25% pancreatin, stopped by adding complete medium, washed 2 times with PBS, and then transfected with AAV-noneratt 026122.2, i.e. noneratt 026122.2 over-expression group (OE-noneratt 026122.2), and control overexpression negative control (OE-NC). After 48h transfection, cells were harvested, astrocyte total RNA was extracted by Trizol method, reverse transcribed, and qRT-PCR was performed in the same manner as in the procedure of qRT-PCR in example 1. The results are shown in figure 2, which significantly up-regulates the expression level of nonrat 026122.2 in astrocytes 48h after transfection.

ki67 proliferation assay. After primary cultured astrocytes overexpressed NONRATT026122.2 (OE-NONRATT 026122.2) or control (OE-NC), the proliferation status of the cells was checked with ki67 antibody and all cells were counterstained with DAPI. Taking a cell microscopic picture by using a fluorescence microscope, counting the number of cells positive for ki67 and the total number of cells, and finally measuring the proliferation rate of the cells, wherein the calculation formula is as follows: cell proliferation rate = ki67 positive number of cells/total number of cells, the experiment was repeated 3 more times. The results of the Ki67 proliferation experiments are shown in fig. 3, and up-regulating the expression of nonrat 026122.2 significantly inhibited astrocyte proliferation compared to the control group.

Scratch healing analysis. Astrocytes overexpressing NONRATT026122.2 (OE-NONRATT 026122.2) or control (OE-NC) were inoculated into culture molds with inserts 1mm wide, and when the cells had fused to 95%, the inserts were removed, leaving a 1mm wide gap at the fusion. Culturing was continued for 12 or 24 hours, photographing was performed under an inverted microscope, and the gap closure was detected. The clean area was calculated using Image-Pro Plus to determine the lateral migration capacity of the cells, and the experiment was repeated 3 more times. The experimental results are shown in fig. 4A, C, and up-regulating the expression of NONRATT026122.2 significantly inhibited the lateral migration ability of astrocytes compared to the control group.

Transwell migration analysis. Astrocyte cells overexpressing NONRATT026122.2 (OE-NONRATT 026122.2) or control (OE-NC), respectively, were suspended in serum-free medium, and then the cell suspension was added to the upper chamber of a transwell chamber having a diameter of 6.5mm and a pore size of 8 μm, and the cells were allowed to migrate toward the bottom chamber of the serum-containing medium, and after culturing for 24 hours, the cells remaining in the upper chamber were removed, and the cells on the bottom surface of the upper chamber were fixed and stained with 0.1% crystal violet. Cell micrographs were taken under an inverted microscope, crystal violet staining areas were observed, the longitudinal migration ability of cells was examined, and the experiment was repeated 3 times or more. The experimental results are shown in fig. 4B, D, and up-regulating the expression of NONRATT026122.2 significantly inhibited the ability of astrocytes to migrate longitudinally compared to the control group.

Upregulation of nonrat 026122.2 expression affects astrocyte phenotype. The method for extracting total RNA from astrocytes by using the Trizol method after 48h and over-expressing NONRATT026122.2 (OE-NONRATT 026122.2) in astrocytes is the same as that of the qRT-PCR in example 1, and the expression of the relevant markers of type A1/A2 of astrocytes (type A1 is toxic astrocytes and type A2 is nutritional astrocytes) is detected by qRT-PCR. As shown in FIG. 5A, the A1-type markers were down-regulated to a different extent and A2-type markers were up-regulated to a different extent after up-regulating NONRATT026122.2 compared to the control (OE-NC).

Upregulation of the effect of nonrat 026122.2 on neurotrophic factor expression in astrocytes. Overexpression of NONRATT026122.2 (OE-NONRATT 026122.2) in astrocytes, cell harvest after 48h, extraction of total RNA by Trizol method, reverse transcription, qRT-PCR detection of neurotrophic factor expression in astrocytes. As shown in FIG. 5B, the expression level of neurotrophic factors NGF, GDNF and bFGF was significantly improved after the expression of NONRATT026122.2 was up-regulated compared to the control group (OE-NC).

Example 3 effects of up-regulating expression of nonrat 026122.2 in astrocytes on neuronal axon growth.

Pretreatment of the glass slide. After the glass slide is cleaned and sterilized, the sterilized paraffin is melted, a small amount of paraffin is dipped in a dropper, and the dropper is rapidly contacted with the glass slide, so that 4 wax dropping feet are formed on the surface of the glass slide, and the size is as follows: about 0.5mm high and 2mm wide. After the wax drop foot is firmly adhered, the slide glass is coated by a PLL for standby.

Spinal cord neurons are isolated and cultured. Pregnant 13-14d rats, after euthanasia, the uterus is removed and placed in sterile petri dishes. The embryo was removed, the fetal mouse brain bag removed, and placed in pre-chilled dissecting solution (HBSS). Under dissecting scope, spinal cord was separated, membrane on spinal cord surface was peeled off, and membraneless spinal cord was placed in freshly pre-chilled HBSS fluid. After collection, the mixture was placed in a 15ml centrifuge tube, HBSS was added to 4.5ml, 2.5% pancreatin was added to 0.5ml, and the mixture was incubated in a 37℃water bath for 15min (allowed to stand without blowing). The pancreatin solution was carefully removed by pipetting, adding 5ml of HBSS, standing at room temperature for 5min, and then carefully removed by pipetting, and repeating the procedure 2 times (this procedure allowed the residual pancreatin solution to diffuse throughout the tissue). Then, HBSS was added to 2-3ml, and the blow was repeated until no tissue mass remained. After counting, the cells were seeded in a 60mm dish (PLL slide with "wax drop") prepared in advance, 15 ten thousand cells/60 mm dish. After the neuron is attached for 3-4 hours, the cell can be used for the experiment of co-culturing with astrocytes.

Astrocytes were co-cultured with neurons. After 3-4h of neuron attachment, the slide was transferred to an astrocyte culture dish over-expressing NONRATT026122.2 (OE-NONRATT 026122.2) or control (OE-NC), and the slide was turned upside down with "wax feet" facing downward, and neurons were co-cultured with astrocytes. After 48h or 72h of co-culture, the slide glass is taken out, PBS is washed for 3 times, 4% PA is fixed, neuron antibody ChAT is stained and observed, experimental results are shown in figure 6, and compared with a control group, the growth of neuron axons is obviously promoted after NONRATT026122.2 is up-regulated in astrocytes.

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

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Claims (3)

1. The application of the long-chain non-coding RNA NONRATT026122.2 in preparing a medicament for treating spinal cord injury is provided, and the cDNA sequence of the long-chain non-coding RNA NONRATT026122.2 is shown in SEQ ID NO.1.

2. The use according to claim 1, characterized in that the medicament is a vector expressing long non-coding RNA nonrat 026122.2.

3. Use according to claim 2, characterized in that the vector is selected from the group consisting of a plasmid, a phage or a virus.

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