CN113122536B - Long-chain non-coding RNA for promoting differentiation of neural stem cells into neurons and screening method thereof - Google Patents
Long-chain non-coding RNA for promoting differentiation of neural stem cells into neurons and screening method thereof Download PDFInfo
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Abstract
The invention provides a long-chain non-coding RNA (lncRNA) for promoting differentiation of neural stem cells to neurons and a screening method thereof, comprising the following steps: preparing a rat model of central nervous system injury, namely cutting rat fornix hippocampus, and extracting a hippocampus exosome; comparing the method of co-culturing exosomes and neural stem cells with a normal control group, and verifying whether the hippocampal exosomes in the nerve injury group can promote the differentiation of the neural stem cells to neurons; performing high-throughput full transcriptome sequencing on exosomes of the nerve injury group and the normal control group, and constructing a differential expression profile of long-chain non-coding RNA; selecting upregulated lncRNA with differential expression multiple greater than 2 and P <0.05 for verification; re-extracting the hippocampal exosomes of the nerve injury rat model, and carrying out differential lncRNA verification; the lncRNA which can promote the differentiation of the neural stem cells to neurons is obtained through analysis and experimental screening, thereby providing a new direction for the cell replacement therapy of the nervous system degenerative diseases.
Description
Technical Field
The invention belongs to the technical field of neurobiology, and particularly relates to long-chain non-coding RNA for promoting differentiation of neural stem cells into neurons, a screening method thereof and a validation method thereof.
Background
With the acceleration of the aging speed of the global population, the degenerative diseases of the central nervous system with cognitive dysfunction and motor dysfunction as main symptoms are in a rapid rise, so that the health of human beings is seriously threatened, the life quality of the human beings is influenced, and a heavy burden is brought to society and families of patients.
These neurodegenerative diseases are all diseases characterized by neuronal degeneration death, such as Alzheimer's Disease (AD), which is a disease characterized mainly by hippocampal and cortical neuronal degeneration death in the brain of the central nervous system; parkinson's Disease (PD) is a disease characterized primarily by degeneration and death of brain substantia nigra dopamine neurons in the central nervous system. The degeneration and death of the hippocampal neurons of AD can cause the abrupt decline of cognitive functions such as learning and memory of patients; however, the degeneration and death of the midbrain substantia nigra dopamine neurons of PD will lead to the reduction of the neurotransmitter dopamine delivered to the striatum, thereby causing dysfunction of extrapyramidal functions and seriously affecting the motor coordination function of patients. At present, no effective treatment means exists for the diseases, and the used medicines can only relieve symptoms, but cannot fundamentally control the progress of the diseases. In order to effectively treat degenerative diseases of the central nervous system, new neurons must be fundamentally used to replace degenerated and dead neurons, and neural networks should be re-established to restore neural function.
What are new neurons coming from? Past belief is that once the brain and spinal cord of the central nervous system are damaged, it is impossible to regenerate the nerve cells after degeneration and death, and the damaged part is formed by gliosis. But since the beginning of the 90 s of the last century, the view of the inability of neurons to regenerate in the central nervous system was broken through as neuroscience research continued. This is mainly due to the finding that neural stem cells exist mainly around the central nervous system central axis lumen, which proliferate, migrate and differentiate into cells in the central nervous system such as neurons, astrocytes and oligodendrocytes (which are not differentiated from neural stem cells because microglia belong to phagocytes of the immune system), but which differentiate into neurons only in small amounts and glial cells in large amounts under the induction of disease microenvironment, and the differentiated neurons are far from being able to adapt to the need for nerve injury repair and restoration of nerve function. Over the past twenty years, neurosciences have sought methods such as induction of neurotrophic factors, transfection of transcription factors and non-coding RNAs into neural stem cells by viruses to promote differentiation of neural stem cells into neurons, but the proportion of differentiated neurons obtained is still low, although it is partially effective.
In research on Long non-coding RNAs (lncRNA) to promote differentiation of neural stem cells into neurons, how to find efficient lncRNA is very important and a problem to be solved by the present invention is also a urgent need.
Disclosure of Invention
The invention aims to: aiming at the problems or the defects in the prior art, the invention provides long-chain non-coding RNA for promoting the differentiation of neural stem cells to neurons and a screening method thereof.
To achieve the above object, an embodiment of the present invention provides a long non-coding RNA that promotes differentiation of neural stem cells into neurons, which includes at least lncRNA Loc102556004 and lncRNA Loc102549726.
Furthermore, the lncRNA Loc102556004 and the lncRNA Loc102549726 can obviously promote the expression of neuron markers Tuj1 and MAP2 proteins in differentiated neural stem cells by transfecting the neural stem cells with lentivirus.
The embodiment of the invention also provides a screening method of long-chain non-coding RNA for promoting the differentiation of neural stem cells to neurons, which is characterized by comprising the following steps of:
step (1), preparing a rat model with central nervous system injury, namely cutting a rat fornix hippocampus, separating the rat hippocampus of the model and normal rat hippocampus tissues 1 week after operation, and extracting the exosomes of the hippocampus;
step (2), comparing the normal control group with the exosome and nerve stem cell co-culture method, and verifying whether the hippocampal exosome of the nerve injury group can promote the differentiation of the nerve stem cell to the neuron;
step (3), performing high-throughput full transcriptome sequencing on exosomes of the nerve injury group and the normal control group, and constructing a differential expression profile of long-chain non-coding RNA;
step (4), up-regulating lncRNA with the differential expression multiple larger than 2 and P <0.05 is selected for verification;
step (5), re-extracting hippocampal exosomes of the nerve injury rat model, and verifying the difference lncRNA of the bioinformatics analysis by using a Real-time PCR method;
step (6), detecting the difference lncRNA of bioinformatics analysis in embryo brain tissues by using Real-time PCR technology;
and (7) selecting lncRNA which is consistent with the whole transcriptome sequencing trend and is also highly expressed in the central nervous system of the embryo, and carrying out an experiment for promoting the differentiation of the neural stem cells into neurons.
Further, in the step (7), long non-coding RNAs that promote differentiation of neural stem cells into neurons, including lncRNA Loc102556004 and lncRNA Loc102549726, are selected for the experiment.
The embodiment of the invention additionally provides a method for verifying the validity of the screened long-chain non-coding RNA, which comprises the following steps:
s1, in-vitro neural stem cell culture, transfecting selected lncRNA Loc102556004 and lncRNA Loc102549726 into the neural stem cells through lentivirus, and performing relevant detection after 1 week of culture;
s2, detecting the expression level of the mRNA of the neuron markers Tuj1 and MAP2 by using Real-time PCR technology, and comparing the expression level with the expression level of the mRNA of the normal control Tuj1 and MAP 2;
s3, observing the expression levels of the neuron markers Tuj1 and MAP2 proteins by using a Western blot method, and comparing the expression levels with the expression levels of the Tuj1 and MAP2 proteins of a normal control group;
s4, detecting the proportion of Tuj1 positive neurons in the differentiated neural stem cells by using a flow technology, and comparing the proportion with the proportion of Tuj1 positive neurons in the differentiated cells in a normal control group;
s5, detecting the percentage of the neuron markers Tuj1 and MAP2 positive neurons in the differentiated cells by using an immunofluorescence technology, and comparing the percentage of the neuron markers Tuj1 and MAP2 positive neurons in the differentiated cells with the percentage of the neuron markers Tuj1 and MAP2 positive neurons in a normal control group;
s6, carrying out statistical analysis on the index data.
The technical scheme of the invention has the following beneficial effects:
(1) According to the invention, through the extraction of the exosomes of the brain tissue with the damaged central nerve and the normal brain tissue, a high-flux gene detection method is applied, some differentially expressed lncRNAs are detected in the exosomes of the brain with the damaged central nerve, and the lncRNAs which can promote the differentiation of the neural stem cells into neurons are screened through analysis and experiments, so that the lncRNAs can effectively promote the differentiation of the neural stem cells into neurons, and a new direction is provided for the cell replacement treatment of the degenerative diseases of the nervous system.
(2) In the embodiment of the invention, lncRNA Loc102556004 (lncRNA Loc102556004, abbreviated as lncRNA 004) and lncRNA Loc102549726 (lncRNA Loc102549726, abbreviated as lncRNA 726) are taken as examples, and Real-time PCR technology is used for proving that lncRNA 004 and lncRNA726 can obviously promote the expression of neuron markers Tuj1 and MAP2 mRNA in differentiated neural stem cells; western blot technology proves that lncRNA 004 and lncRNA726 can obviously promote the expression of neuron markers Tuj1 and MAP2 proteins in differentiated neural stem cells to be obviously increased; after 1 week of transfection of lncRNA 004 and lncRNA726 into neural stem cells by lentivirus, the proportion of neurons positive for the neuronal marker Tuj1 was significantly increased as demonstrated by flow techniques; after 1 week of transfection of lncRNA 004 and lncRNA726 into neural stem cells by lentivirus, a significant increase in the proportion of neurons positive for the neuronal markers Tuj1 and MAP2 was detected by immunofluorescence techniques, thereby verifying the effectiveness of the screening method of the present invention.
Drawings
FIG. 1 is an exosome and identification chart extracted from nerve damaged hippocampus and normal hippocampus in the examples; wherein, FIG. 1A shows that exosome specific protein bands are detected by Western blot; FIG. 1B is an exosome electron micrograph; FIG. 1C is a graph showing the particle size distribution of exosome samples; FIG. 1D is a bar graph of exosome sample particle size.
FIG. 2 is a graph showing the effect of hippocampus exosomes on proliferation of Neural Stem Cells (NSCs) in the examples; wherein fig. 2A shows that CM-Dil marked hippocampal exosomes are present in NSCs, scale = 200 μm; FIG. 2B shows the detection of cell proliferation by MTS assay; FIG. 2C shows the cell cycle distribution of NSCs detected by flow cytometry; fig. 2D shows immunofluorescence detection of cell proliferation, cell nuclei counterstained with Hoechst, scale = 200 μm. * P <0.01, P <0.001.
FIG. 3 is a graph showing the effect of hippocampus exosomes on the differentiation of NSCs into neurons in the examples; wherein, fig. 3A: western blot detection of Tuj1 protein levels; FIG. 3B shows the ratio of Tuj1 positive cells detected by cell flow technology; fig. 3C shows Tuj1 and GFAP positive immunofluorescence assays, nuclei counterstained with Hoechst, scale = 200 μm. * P <0.05, P <0.01, P <0.001.
FIG. 4 is a schematic representation of Real-time PCR verification of upregulation of lncRNA in hippocampal exosomes and expression in embryos in the nerve injury group in the examples; wherein, fig. 4A shows that 4 lncRNA are significantly upregulated compared to normal group hippocampal exosomes, P <0.01, P <0.001. Fig. 4B shows that 4 upregulated lncRNA were only upregulated during embryonic central nervous development, P <0.05, P <0.01, P <0.001.
FIG. 5 is a diagram showing the expression of the neuronal markers Tuj1 mRNA and MAP2 mRNA after transfection of neural stem cells with lncRNA 004 and lncRNA726 by Real-time PCR in the examples; after lncRNA 004 and lncRNA726 are transfected into the neural stem cells for 1 week, the up-regulation of the expression of neuron markers Tuj1 mRNA and MAP2 mRNA in the neural stem cells can be obviously promoted, wherein P is less than 0.05, and P is less than 0.01.
FIG. 6 is an expression diagram of neuronal markers Tuj1 and MAP2 proteins after transfection of neural stem cells with lncRNA 004 and lncRNA726 by Western blot detection in the examples; after lncRNA 004 and lncRNA726 are transfected into the neural stem cells for 1 week, the expression of neuron markers Tuj1 and MAP2 proteins in the neural stem cells can be obviously promoted, and P is less than 0.05.
FIG. 7 is a graph showing the differentiation ratio of Tuj1 positive neurons after transfection of neural stem cells with lncRNA 004 and lncRNA726 by flow assay in the examples; after transfection of lncRNA 004 and lncRNA726 into neural stem cells for 1 week, the proportion of Tuj1 positive neurons differentiated significantly. * P < 0.01.
FIG. 8 is a graph of neurons differentiated after transfection of neural stem cells with lncRNA 004 and lncRNA726 detected by immunofluorescence techniques in the examples; after transfection of lncRNA 004 and lncRNA726 into neural stem cells for 1 week, the ratio of differentiation of neural stem cells into Tuj 1-positive neurons and MAP 2-positive neurons was significantly increased, with P <0.01 and P <0.001.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention more apparent, the following detailed description will be made with reference to specific embodiments.
The embodiment of the invention provides a screening method of long-chain non-coding RNA for promoting neural stem cells to differentiate into neurons, which comprises the following steps:
(1) Preparation of a rat model of CNS injury-cutting of rat dome Hippocampus umbrella, separation of rat hippocampus from normal rat hippocampal tissue of model rat 1 week after operation, and extraction of hippocampal exosomes. As shown in fig. 1, exosomes have been successfully extracted from hippocampal tissue and normal hippocampal tissue of a nerve-injured rat model.
(2) By means of the co-culture of exosomes and neural stem cells, compared with a normal control group, it is confirmed that the hippocampal exosomes in the nerve injury group can promote the differentiation of the neural stem cells to neurons. These exosomes were co-cultured with neural stem cells, and the hippocampal exosomes of the nerve injury group significantly inhibited the proliferation of neural stem cells, as shown in fig. 2, and simultaneously promoted the differentiation of neural stem cells into neurons, as shown in fig. 3.
(3) And (3) performing high-throughput whole transcriptome sequencing on exosomes of the nerve injury group and the normal control group, and constructing a differential expression profile of long-chain non-coding RNA.
High-throughput RNA-seq assays found that 11 lncRNA with more than 2-fold and P <0.05 expression differences in hippocampal exosomes in the nerve-injured group compared to the normal control group, 7 up-regulated and 4 down-regulated, as shown in table 1 below:
TABLE 1 lncRNA differential expression profile
(4) Upregulated lncRNA with fold difference greater than 2 and P <0.05 were selected for study.
(5) Re-extracting the sea horse exosome of the nerve injury rat model, and verifying the difference lncRNA of the bioinformatics analysis by using a Real-time PCR method.
(6) The differential lncRNA of the bioinformatics analysis in embryonic brain tissue was detected using Real-time PCR technology.
(7) An experiment for promoting the differentiation of the neural stem cells into neurons is carried out by selecting lncRNA which is consistent with the whole transcriptome sequencing trend and is highly expressed in an embryo central nervous system:
as detected by Real-time PCR technique, 4 lncRNAs were significantly up-regulated in the nerve injury group compared to the normal control group (FIG. 4-A), while only 3 of these 4 up-regulated lncRNAs were up-regulated in the embryonic CNS (FIG. 4-B), indicating that the 3 lncRNAs were highly likely to be involved in neuronal development and regeneration. Since lncRNA GAS5 is often on an ascending trend at the time of tumorigenesis, two other lncrnas, lncRNA Loc102556004 (abbreviated as lncRNA 004) and lncRNA Loc102549726 (abbreviated as lncRNA 726), were selected for verification.
By verifying lncRNA Loc102556004 (abbreviated as lncRNA 004) and lncRNA Loc102549726 (abbreviated as lncRNA 726)
S1, in-vitro neural stem cell culture, transfecting selected lncRNA 004 and lncRNA726 into the neural stem cells through lentivirus, and performing relevant detection after culturing for 1 week;
s2, detecting the expression level of the mRNA of the neuron markers Tuj1 and MAP2 by using Real-time PCR technology, and comparing the expression level with the expression level of the mRNA of the normal control Tuj1 and MAP 2; real-time PCR techniques demonstrated that lncRNA 004 and lncRNA726 significantly promoted expression of neuronal markers Tuj1 and MAP2 mRNA in differentiated neural stem cells after 1 week of transfection into neural stem cells by lentiviruses, as shown in fig. 5.
S3, observing the expression levels of the neuron markers Tuj1 and MAP2 proteins by using a Western blot method, and comparing the expression levels with the expression levels of the Tuj1 and MAP2 proteins of a normal control group; western blot technique shows that lncRNA 004 and lncRNA726 can obviously promote the expression of neuron markers Tuj1 and MAP2 protein in differentiated neural stem cells to be obviously increased after being transfected into the neural stem cells for 1 week through lentivirus, as shown in FIG. 6.
S4, detecting the proportion of Tuj1 positive neurons to differentiated neural stem cells by using a streaming technology, and comparing the proportion with the proportion of Tuj1 positive neurons to differentiated cells in a normal control group (because MAP2 antibodies for streaming are not sold, only Tuj1 can be used as an index); after 1 week of transfection of lncRNA 004 and lncRNA726 into neural stem cells by lentivirus, the proportion of neurons positive for the neuronal marker Tuj1 was significantly increased as demonstrated by flow techniques, as shown in fig. 7.
S5, detecting the percentage of the neuron markers Tuj1 and MAP2 positive neurons in the differentiated cells by using an immunofluorescence technology, and comparing the percentage of the Tuj1 and MAP2 positive neurons in the normal control group in the differentiated cells; after 1 week of transfection of lncRNA 004 and lncRNA726 into neural stem cells by lentivirus, a significant increase in the proportion of neurons positive for the neuronal markers Tuj1 and MAP2 was detected by immunofluorescence techniques, as shown in fig. 8.
S6, carrying out statistical analysis on the index data.
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.
Claims (3)
1. The application of long-chain non-coding RNA in preparing a medicament for promoting the differentiation of neural stem cells to neurons is characterized in that the long-chain non-coding RNA is lncRNA Loc102556004 or lncRNA Loc102549726, and the lncRNA Loc102556004 or lncRNA Loc102549726 can obviously promote the expression of neuron markers Tuj1 and MAP2 proteins in the differentiated neural stem cells through slow virus transfection to the neural stem cells.
2. A method for screening long non-coding RNAs that promote differentiation of neural stem cells into neurons, comprising the steps of:
step (1), preparing a rat model with central nervous system injury, namely cutting a rat fornix hippocampus, separating the rat hippocampus of the model and normal rat hippocampus tissues 1 week after operation, and extracting the exosomes of the hippocampus;
step (2), comparing the normal control group with the exosome and nerve stem cell co-culture method, and verifying whether the hippocampal exosome of the nerve injury group can promote the differentiation of the nerve stem cell to the neuron;
step (3), performing high-throughput full transcriptome sequencing on exosomes of the nerve injury group and the normal control group, and constructing a differential expression profile of long-chain non-coding RNA;
step (4), up-regulating lncRNA with the differential expression multiple larger than 2 and P <0.05 is selected for verification;
step (5), re-extracting hippocampal exosomes of the nerve injury rat model, and verifying the difference lncRNA of the bioinformatics analysis by using a Real-time PCR method;
step (6), detecting the difference lncRNA of bioinformatics analysis in embryo brain tissues by using Real-time PCR technology;
step (7), selecting lncRNA which is consistent with the sequencing trend of the whole transcriptome and is also highly expressed in the central nervous system of the embryo, and carrying out an experiment for promoting the differentiation of the neural stem cells into neurons; long non-coding RNAs, including lncRNA Loc102556004 and lncRNA Loc102549726, were selected to promote differentiation of neural stem cells into neurons for experiments.
3. A method of validating the effectiveness of long non-coding RNAs screened by the screening method of claim 2, comprising the steps of:
s1, in-vitro neural stem cell culture, transfecting selected lncRNA Loc102556004 and lncRNA Loc102549726 into the neural stem cells through lentivirus, and performing relevant detection after 1 week of culture;
s2, detecting the expression level of the mRNA of the neuron markers Tuj1 and MAP2 by using Real-time PCR technology, and comparing the expression level with the expression level of the mRNA of the normal control Tuj1 and MAP 2;
s3, observing the expression levels of the neuron markers Tuj1 and MAP2 proteins by using a Western blot method, and comparing the expression levels with the expression levels of the Tuj1 and MAP2 proteins of a normal control group;
s4, detecting the proportion of Tuj1 positive neurons in the differentiated neural stem cells by using a flow technology, and comparing the proportion with the proportion of Tuj1 positive neurons in the differentiated cells in a normal control group;
s5, detecting the percentage of the neuron markers Tuj1 and MAP2 positive neurons in the differentiated cells by using an immunofluorescence technology, and comparing the percentage of the neuron markers Tuj1 and MAP2 positive neurons in the differentiated cells with the percentage of the neuron markers Tuj1 and MAP2 positive neurons in a normal control group;
s6, carrying out statistical analysis on the index data.
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| WO2018125019A2 (en) * | 2016-12-30 | 2018-07-05 | Istanbul Üni̇versi̇tesi̇ | Use of some mirnas for the diagnosis and treatment of diseases associated with insulin |
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| JP2014183768A (en) * | 2013-03-22 | 2014-10-02 | National Institute Of Advanced Industrial & Technology | Monitoring method of nerve and mental disease and diabetes by using adult neural stem cell derived from hippocampus or olfactory bulb |
| CN105400879A (en) * | 2015-12-10 | 2016-03-16 | 深圳市默赛尔生物医学科技发展有限公司 | Screening method for IncRNAs, ADSCs, and induced differentiation method for cartilage cells |
| WO2018125019A2 (en) * | 2016-12-30 | 2018-07-05 | Istanbul Üni̇versi̇tesi̇ | Use of some mirnas for the diagnosis and treatment of diseases associated with insulin |
| CN110885881A (en) * | 2019-11-14 | 2020-03-17 | 南通大学 | Method for researching hippocampal nerve regeneration microenvironment by denervation-free hippocampal exosomes |
| CN111850048A (en) * | 2020-08-07 | 2020-10-30 | 南通大学 | Method for promoting differentiation of hippocampal neural stem cells to cholinergic neurons |
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