CN117925610A - LncRNA (ribonucleic acid) as marker for regulating lipid-forming differentiation capacity of human mesenchymal stem cells and application thereof - Google Patents
LncRNA (ribonucleic acid) as marker for regulating lipid-forming differentiation capacity of human mesenchymal stem cells and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to lncRNA (ribonucleic acid) serving as a marker for regulating and controlling adipogenic differentiation capacity of human mesenchymal stem cells and application thereof. The invention also provides a preparation for regulating and controlling the adipogenic differentiation capacity of the mesenchymal stem cells and a regulating and controlling method. The invention discovers that the lncRNA can play an important role in regulating and controlling the adipogenic differentiation capacity of human adipose-derived mesenchymal stem cells for the first time, has higher application potential, is expected to become an effective target for clinically preventing overweight or treating obesity, and provides a new research direction for developing a weight-losing medicament with little or no side effect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to lncRNA (ribonucleic acid) serving as a marker for regulating and controlling adipogenic differentiation capacity of human mesenchymal stem cells and application thereof.
Background
As one of the major risk factors for non-infectious diseases, the world health organization defines overweight and obesity as abnormal or excessive fat accumulation that can impair health. The data provided by the global health observation station showed a significant rise in the proportion of obese people over the last 40 years. More and more data alert us that obesity has become a chronic disease of global pandemic and that the obese population is progressively younger. At present, the fast rise of obesity incidence is caused by the interaction of inheritance, epigenetic, endocrine and other intrinsic factors, unhealthy dietary structures, sedentary life patterns, socioeconomic status and other factors.
The human body suffers from various damages in an obese state. First, obese individuals are in a chronic inflammatory state for a long period of time. Secondly, metabolism and endocrine of obese individuals are in a disturbed state, which leads to increased prevalence of metabolic-related diseases such as insulin resistance, type II diabetes, non-alcoholic fatty liver, sleep apnea, cardiovascular disease, and the like; on the other hand, malignant tumors related to digestive organs and female hormone secretion, which lead to the increase of risks of various cancers, are proved to have strong relevance to obesity. Furthermore, studies have shown that COVID-19 may be closely related to an obese pandemic. However, many challenges remain in the overall diagnosis, prevention, and effective and safe treatment of obesity. For example, many obese populations (especially elderly and children) are not suitable for weight loss by exercise; most weight-losing medicines such as rimonabant, sibutramine and lorcaserin are continuously withdrawn from the international market due to side effects such as easy addiction, easy headache or blood pressure rise and the like. Therefore, the disease mechanism behind obesity needs to be deeply explored, a new thought is provided for the clinical problem of obesity, and the social medical resource pressure is relieved.
Obesity is mainly caused by the pathological expansion of adipocytes in number and volume. It is currently believed that adipocytes are developed primarily by progressive differentiation of mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs). MSCs are an important class of pluripotent stem cells whose complex adipogenic differentiation process is precisely regulated. MSCs are differentiated into preadipocytes and then mature adipocytes, and the two stages have a plurality of key transcription factors such as C/EBP beta, C/EBP delta, C/EBP alpha, PPARgamma and the like, and are regulated and controlled by complex signal paths such as Wnt/beta-catein, TGF beta/BMP, IGF, notch and the like. Mature adipocytes store triglycerides, fatty acids, phospholipids, cholesterol, and the like as energy stores, secrete fat-specific cytokines, and mediate the regulation of the insulin sensitivity mechanism. Abnormal adipogenic differentiation processes are closely related to the occurrence of obesity, but little is known about this process. In particular, in the field of epigenetic science such as non-coding RNAs, such heritable epigenetic mechanisms may be important factors in determining the heterogeneity of obesity susceptibility, body mass index BMI, etc. in the context of obesity pandemic.
Long non-coding RNAs (lncRNAs) refer to RNAs in the transcriptome that are more than 200nt long and mostly exist in secondary structure. In the genome of mammals, 4% -9% of the sequences produce transcripts that are lncRNAs. The above features enable lncRNAs to target chromatin, mRNA, proteins, etc. in a variety of mechanisms to regulate delicate biological processes such as cell lineage differentiation, immunomodulation, cancer progression. Interestingly, most lncRNAs are less species-conservative, are less abundant in expression, and have a highly specific spatiotemporal expression pattern. This means that the discovery of lncRNAs with regulatory functions for adipogenesis and/or adipogenic differentiation and the elucidation of their regulatory mechanisms would not only be very potential as human-specific biomarkers, but would also be expected to enable clinical transformation by non-coding RNA therapies for the prevention and treatment of obesity-related diseases. However, because of the poor conservation of lncRNAs among species, most of the lncRNAs found in recent years to be related to adipogenic differentiation, fat metabolism and the like are based on mice or cell lines, and the guiding value of the studies on human obesity prevention and treatment is remarkably reduced. Therefore, there is an urgent need to find lncRNAs associated with adipogenic differentiation, fat metabolism, obesity, etc. of human MSCs and to make intensive studies on their functions and mechanisms.
Disclosure of Invention
The invention aims to overcome the problems in the prior art, and provides a lncRNA (ribonucleic acid) as a marker for regulating and controlling the adipogenic differentiation capacity of mesenchymal stem cells and application thereof, which have important significance in research and development of medicines for treating obesity and treatment of obesity.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
The first aspect of the invention provides an application of lncRNA as a marker for regulating the adipogenic differentiation capacity of human mesenchymal stem cells.
Preferably, the lncRNA is LNCRNA ENST00000585537.1, is positioned on human chromosome Chu 17q24, and has a nucleotide sequence shown as SEQ ID NO. 1.
The second aspect of the invention provides an application of lncRNA in preparing a human mesenchymal stem cell adipogenic differentiation product.
Preferably, the product comprises an inhibitor or promoter of lncRNA expression.
The inhibitor or the promoter for expressing the lncRNA can obviously change the expression of the adipogenic differentiation marker of the human mesenchymal stem cells by knocking down or promoting LNCRNA ENST00000585537.1, thereby affecting the adipogenic differentiation capacity of the human mesenchymal stem cells. Preferably, the adipogenic differentiation marker comprises cebpα, pparγ, FABP4, LPL, PLIN1; more preferably, the adipogenic differentiation marker comprises pparγ, FABP4, PLIN1.
The inhibitor or promoter of lncRNA expression described above affects free fatty acid content by knocking down or promoting LNCRNA ENST00000585537.1 to significantly alter the expression of some pathway genes associated with fatty acid metabolism. Preferably, the pathway may be a pathway related to fatty acid metabolism, including but not limited to an unsaturated fatty acid synthesis pathway (hsa 01040), a fatty acid metabolism pathway (hsa 01212), a pyruvate metabolism pathway (hsa 00620), etc.; the free fatty acids may be fatty acids in the body in a free state, including but not limited to caprylic acid, capric acid, undecanoic acid, lauric acid, tridecylic acid, cinnamic acid, myristic acid, palmitic acid, oleic acid, palmitoleic acid, gamma-linolenic acid, cis-5, 8,11,14, 17-eicosapentaenoic acid, docosatetraenoic acid, cis-docosahexaenoic acid, and the like.
In a third aspect, the invention provides a preparation for regulating the adipogenic differentiation capacity of human mesenchymal stem cells, wherein the preparation takes an inhibitor of lncRNA or an promoter of lncRNA as an active ingredient.
Preferably, the lncRNA is LNCRNA ENST00000585537.1, is positioned on human chromosome Chu 17q24, and has a nucleotide sequence shown as SEQ ID NO. 1.
Preferably, the lncRNA inhibitor or promoter affects the lipid-forming differentiation capacity of human mesenchymal stem cells by knocking down or promoting lncRNA expression such that expression of lipid-forming differentiation markers is significantly changed.
Preferably, the lncRNA inhibitor is an agent that knocks down the expression level of lncRNA.
Preferably, the inhibitors are siRNA-1 and siRNA-2, and the sequence of the siRNA-1 is: sense strand 5'GCUGUGACCAAUGAACAAUTT 3', antisense strand 5'AUUGUUCAUUGGUCACAGCTT 3'; the sequence of the siRNA-2 is as follows: sense strand 5'GUGGUUCCCAUCUCAAGAATT 3', antisense strand 5'UUCUUGAGAUGGGAACCACTT 3'.
Preferably, the lncRNA promoter is an agent that promotes the expression level of lncRNA.
Preferably, the promoter is a vector plasmid over-expressing lncRNA, the vector type of the plasmid is LV3-H1-GFP-Puro, wherein LV3 is a lentivirus type used for packaging the plasmid, GFP is green fluorescent protein which indicates transfection efficiency, puro is puromycin, and resistance marker is used for screening positive cells.
In a fourth aspect, the present invention provides a method for regulating the adipogenic differentiation capacity of human mesenchymal stem cells, which comprises knocking down or promoting lncRNA expression by using the preparation, thereby inhibiting or promoting the adipogenic differentiation capacity of human mesenchymal stem cells.
Preferably, the lipid-forming differentiation of human mesenchymal stem cells is inhibited by knocking down lncRNA in the above method; more preferably, the human mesenchymal stem cells are inhibited from adipogenic differentiation by transfecting the human mesenchymal stem cells with a lncRNA inhibitor to knock down lncRNA expression; the lncRNA inhibitor is siRNA-1 and siRNA-2, and the sequence of the siRNA-1 is: sense strand 5'GCUGUGACCAAUGAACAAUTT 3', antisense strand 5'AUUGUUCAUUGGUCACAGCTT 3'; the sequence of the siRNA-2 is as follows: sense strand 5'GUGGUUCCCAUCUCAAGAATT 3', antisense strand 5'UUCUUGAGAUGGGAACCACTT 3'.
The specific method for knocking down the lncRNA comprises the following steps: wrapping two pairs of small interfering RNAs (siRNA-1 and siRNA-2) by using liposome (Lipofectamine 3000), inoculating the small interfering RNAs into a third-generation human fat-derived MSC for transfection, and collecting hAMSCs after 24 hours of transfection to detect knock-down efficiency; adipogenic differentiation induction was performed when the cell growth density reached about 90%, and the hAMSCs were harvested at day 6 to examine adipogenic differentiation key markers PPARγ, FABP4, PLIN1, and examined for adipogenic droplet formation at day 12.
Preferably, in the above method, the adipogenic differentiation of human mesenchymal stem cells is promoted by over-expressing lncRNA; more preferably, the lncRNA is overexpressed by transfection of an lncRNA promoter into human mesenchymal stem cells, thereby promoting adipogenic differentiation of human mesenchymal stem cells; the promoter is a carrier plasmid for over-expressing lncRNA, the carrier type of the plasmid is LV3-H1-GFP-Puro, wherein LV3 is a slow virus type used for packaging the plasmid, GFP is green fluorescent protein which can indicate transfection efficiency, puro is puromycin, and the Puro is a resistance marker which can be used for screening positive cells.
The specific method for over-expressing lncRNA comprises the following steps: after packaging over-expression plasmid (LV 3-H1-GFP-Puro) with slow virus, infecting the over-expression plasmid into third generation human fat source MSC by slow virus infection method, wherein the used slow virus titer (MOI) is 10, observing infection efficiency through GFP (green fluorescent protein) after 48 hours of infection, screening with puromycin, collecting hAMSCs after screening, detecting over-expression efficiency, performing adipogenic differentiation induction when the cell growth density reaches about 90%, collecting hAMSCs at the 6 th day, detecting adipogenic differentiation key markers PPARgamma, FABP4 and PLIN1, and detecting lipid drop generation at the 12 th day.
The fifth aspect of the invention provides an application of a preparation for regulating and controlling the adipogenic differentiation capacity of human mesenchymal stem cells in preparing a product for inhibiting or promoting adipogenic differentiation of human mesenchymal stem cells.
The sixth aspect of the invention provides an application of a preparation for regulating the adipogenic differentiation capacity of human mesenchymal stem cells in preparing a medicament for treating obesity by regulating the adipogenic differentiation capacity of human mesenchymal stem cells.
By means of the technical scheme, the invention has at least the following advantages: the LNCRNA ENST00000585537.1 provided by the invention has the full length of 584bp, has extremely high species conservation, is only expressed in certain tissues and organs of human beings, and is distributed in cytoplasm and nucleus of human mesenchymal stem cells. The LNCRNA ENST00000585537.1 can obviously promote the adipogenic differentiation of the mesenchymal stem cells in vitro and promote the adipogenesis of the mesenchymal stem cells in vivo; the LNCRNA ENST00000585537.1 of the invention has positive correlation with the adipogenesis key gene expression in the human fat sample, namely, the knock-down LNCRNA ENST00000585537.1 can inhibit the adipogenesis and differentiation capacity of the human mesenchymal stem cells, or the over-expression LNCRNA ENST00000585537.1 can promote the adipogenesis and differentiation capacity of the human mesenchymal stem cells. The result suggests that the compound can play an important role in regulating and controlling human adipogenesis and differentiation, has higher application potential, is expected to become an effective target for clinically preventing overweight or treating obesity, and provides a new research direction for developing a weight-losing medicament with little or no side effect.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Drawings
FIG. 1 is a diagram of a map of the position and species conservation analysis of LNCRNA ENST00000585537.1 of the present invention in the human genome;
FIG. 2 shows the full length 584bp amplified fragment obtained by passing LNCRNA ENST00000585537.1 'RACE and 3' RACE according to the present invention; wherein A is agarose gel electrophoresis of the amplified fragment; b is a nucleotide sequence diagram;
FIG. 3 is a graph showing the distribution of LNCRNA ENST00000585537.1 of the present invention in human adipose-derived mesenchymal stem cells;
FIG. 4 is a fluorescent in situ hybridization diagram of LNCRNA ENST00000585537.1 of the present invention in human adipose-derived mesenchymal stem cells;
FIG. 5 is a graph showing the expression trend of LNCRNA ENST00000585537.1 of the present invention during in vitro adipogenic differentiation of human adipose-derived mesenchymal stem cells;
FIG. 6 is a graph showing in vitro adipogenic differentiation of human adipose-derived mesenchymal stem cells after LNCRNA ENST00000585537.1 knockdown using siRNA; wherein, A is the knock-down efficiency map of siRNA pair LNCRNA ENST 00000585537.1; b is the mRNA expression level of key markers PPARgamma, FABP4 and PLIN1 for adipogenic differentiation; c is the protein expression level of key markers PPARgamma, FABP4 and PLIN1 for adipogenic differentiation; d is an oil red O staining chart of each group of cell lipid drops;
FIG. 7 is a graph showing in vitro adipogenic differentiation of human adipose-derived mesenchymal stem cells after using lentivirus over-expression LNCRNA ENST 00000585537.1; wherein, an over-expression efficiency map of lentivirus pair LNCRNA ENST 00000585537.1; b is the mRNA expression level of key markers PPARgamma, FABP4 and PLIN1 for adipogenic differentiation; c is the protein expression level of key markers PPARgamma, FABP4 and PLIN1 for adipogenic differentiation; d is an oil red O staining chart of each group of cell lipid drops;
FIG. 8 is a graph showing the results of transcriptomic sequencing of human adipose-derived mesenchymal stem cells after LNCRNA ENST00000585537.1 has been knocked down using siRNA; wherein A is a cluster heat map of differentially expressed genes after LNCRNA ENST00000585537.1 knockdown, red represents up-regulation of gene expression, and green represents down-regulation of gene expression; b is the enrichment analysis of the signal path of the significantly down-regulated gene;
FIG. 9 is a graph showing the change in free fatty acid content during in vitro adipogenic differentiation of human adipose-derived mesenchymal stem cells after LNCRNA ENST00000585537.1 knockdown using siRNA; wherein A is the relative content of 39 free fatty acids after LNCRNA ENST00000585537.1 is knocked down; b is a significantly reduced relative content of 9 free fatty acids;
FIG. 10 is a graph showing the change in free fatty acid content during in vitro adipogenic differentiation of human adipose-derived mesenchymal stem cells after using lentivirus over-expression LNCRNA ENST 00000585537.1; wherein A is the relative content of 39 free fatty acids after LNCRNA ENST00000585537.1 is over-expressed; b is the relative content of 6 free fatty acids that rises significantly;
FIG. 11 is a graph showing the in vivo adipogenic capacity of human adipose-derived mesenchymal stem cells after LNCRNA ENST00000585537.1 knockdown using shRNA; wherein A is a schematic diagram of an animal experiment scheme; b is a staining chart of the subcutaneous fat generation condition of hAMSCs in nude mice;
FIG. 12 is a graph showing the in vivo adipogenic capacity of human adipose-derived mesenchymal stem cells after using lentivirus over-expression LNCRNA ENST 00000585537.1; wherein A is a schematic diagram of an animal experiment scheme; b is a staining chart of the subcutaneous fat generation condition of hAMSCs in nude mice;
FIG. 13 is an analysis of the correlation of expression levels LNCRNA ENST00000585537.1 in human adipose tissue, LNCRNA ENST00000585537.1 with the expression of key marker genes in human adipose tissue; wherein A is the expression level of LNCRNA ENST00000585537.1 in human adipose tissue; b is LNCRNA ENST00000585537.1 and the expression correlation analysis of key marker genes in human adipose tissues.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purposes and the effects of the present invention easy to understand, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, 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.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. Reagents and instruments used herein are commercially available, and reference to characterization means is made to the relevant description of the prior art and will not be repeated herein.
The present examples are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.
The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
All data were statistically compared between the two groups using t-test using GRAPHPAD PRISM (GRAPHPAD PRISM, san Diego, CA) software. The comparison between the groups adopts single-factor analysis of variance. The differences are statistically significant, P <0.05, < P <0.01, < P <0.001, < P <0.0001, respectively.
For a further understanding of the present invention, the present invention will be described in further detail with reference to the following preferred embodiments.
In the following examples, the main sources of materials used are: medium DMEM/F12 was purchased from Gibco; fetal bovine serum was purchased from Gibco; penicillin, streptomycin were purchased from Yu Saiwei moles of organism (Servicebio), dexamethasone, ascorbic acid from Sigma-Aldrich.
Example 1: culture and induction of human adipose-derived mesenchymal stem cells
Isolation of primary human adipose-derived mesenchymal stem cells from adipose tissue (the adipose tissue is isolated from liposuction operation, liposuction fat is normally medical waste, and is taken from orthopedics department of China medical science, and the informed consent of the patient is obtained without causing additional damage to the patient and leakage of patient privacy due to the material taking mode, and the approval is applied by ethical review Committee of basic medical institute of China medical science), and the cell isolation step can be described as follows: washing (liposuction adipose tissue is split into 50mL centrifuge tube, adding PBS solution containing double penicillin and streptomycin, centrifuging at 800rpm for 3min, repeating for 2 times), digestion (adding filtered 0.2% collagenase P, sealing the centrifuge tube by sealing membrane, shaking table digestion for 20-30 min at 37 ℃ until no lump is formed), filtering (adding proper PBS solution for dilution, filtering by 100 μm screen to remove undigested part of tissue, etc.), centrifuging (centrifuging at 1500rpm for 10min, washing blood cells and collagenase P, taking care of replacing new centrifuge tube during washing, reducing oily residue, repeating for 2 times), inoculating (inoculating T75 flask density of resuspended cells according to about 10-20 mL fat, re-suspending 100IU/mL of total medium used in DMEM/F12+2% fetal bovine serum+100 IU/mL of penicillin, culturing cells in incubator at 37 ℃ and 5% CO 2% for 24-48 h, replacing fresh medium, centrifuging at 1200rpm, inoculating flask density of cells according to 5min, avoiding waste of primary flask weight of 1) inoculating according to 1 min. The primary human fat source mesenchymal stem cells successfully extracted are cultured in a incubator with 5% CO 2 at 37 ℃ by using the complete culture medium, the culture medium is replaced every 2 days, and when the cell growth density reaches 80% -90%, the cells are passaged.
Human adipose-derived mesenchymal stem cells (human adipose-DERIVED MESENCHYMAL STEM CELLS, abbreviated as hAMSCs) of passage 3 were induced using adipogenic differentiation induction medium (DMEM+10% fetal bovine serum+penicillin 100 IU/mL+streptomycin 100. Mu.g/mL+dexamethasone 10-6M+IBMX 100. Mu.g/mL+ascorbic acid 50. Mu.g/mL) and used for each experiment.
It should be noted that, in theory, any generation of human adipose-derived mesenchymal stem cells can be induced using adipogenic differentiation induction medium before aging and used for each experiment, and it is preferred in the art to use the cells cultured in the 1 st to 7 th generation, and based on the most preferred manner, the cells cultured to the 3 rd generation are selected in the examples of the present invention.
Example 2: quick amplification experiment of cDNA EncRNA (ENST 00000585537.1)
The cDNA ends (RACE) were rapidly amplified from the normally cultured third generation hAMSCs using Thermo Fisher FirstChoice TM RLM-RACE kit. RACE is a polymerase chain reaction based technique that facilitates cloning of full-length cDNA sequences in cases where only partial cDNA sequences are available. After 5 'end RACE and 3' end RACE and nucleotide sequencing, the obtained LNCRNA ENST00000585537.1 cDNA has total length of 584bp, the nucleotide sequence is shown as SEQ ID NO. 1, and is consistent with the sequence provided in UCSC and other biological information databases, which shows that LNCRNA ENST00000585537.1 is actually present in hAMSCs and can be used for designing primers, vectors and the like of subsequent experiments.
SEQ ID NO:1
GTAGATTTCTGCAGATCGCAACAAGCCTCGACTGCTTATTGGCACAGTCTGCAAAGGGCCTCCTCCGTTCCAAAGTTGCTCATCCTAAAACCTGAAAAAAAAAATGACTCAGAAGTGGTTCCCATCTCAAGAACTACCCACGAGTCTCAGTGGACACATGTGAGTATGAGATTCCCCGATGCAGCCTTCCAATCCAGCAGAGGTTCCAACCAACTTCCAAACAGGTGCAACTTGATGGCACCTCATTTTTGGCTGTACCAGATGCCTTCTATCTCTGGACTCCTGCCCTCAGACTTCTCCGACACCTGAAGAAAGCTTCTGGGACAGCTGACATGCACAACCTGGGAGCTAAAACATGTGGGACCAGCTGTGACCAATGAACAATGATGGACAGGAGACGATAACCCCATTAGTTCCTTACAGTGCTGAGATAAATTTCATGAGTCTCCTAGAAGATCTGATGGGATTGAACATCAGTTGTACCTAGCATGGCCGCTGTACTGGCTTTCCTTCCTTCCCTATTTCATCCCCCCTCGTCCCTCCTACTCCTTGGGATCACATTGACAAATAAGTCTCTTGCATGC.
Agarose gel electrophoresis and sequencing (electrophoresis and sequencing are completed by Shanghai Shuo biotechnology Co., ltd.) are carried out on the obtained fragments, and the result is shown in FIG. 2, wherein A is an agarose gel electrophoresis diagram of the 5 '-end RACE and the 3' -end RACE amplified fragments, and both fragments are about 300bp according to DNA MARKER; b is a sequencing result obtained after cutting and recovering the two fragments, and the total length of the final spliced sequence is 584bp. And (3) injection: the green base is a kit 5'RACE joint sequence, the red bases are splice point sequences, and the blue bases are 3' terminal poly A tail.
Example 3: LNCRNA ENST00000585537.1 cell positioning experiment
Firstly, using Thermo Fisher to obtain the third generation hAMSCs which are normally culturedNuclear and Cytoplasmic Extraction Reagents kit, according to the instruction, separating cell nucleus and cell cytoplasmic components, respectively extracting RNA, making reverse transcription into cDNA, then using qRT-PCR to detect relative content of LNCRNA ENST00000585537.1 in cell nucleus and cytoplasmic components, and using beta-actin and U1 as cell cytoplasm and cell nucleus components respectively so as to make control. Then taking the normally cultured third generation hAMSCs for carrying out RAN fluorescence in situ hybridization (RNA FISH) experiments, wherein the RNA FISH kit, LNCRNA ENST00000585537.1 probes, control U6 (cell nucleus control) probes and 18S RNA (cytoplasm control) probes are purchased from Guangzhou Ruibo biological company.
In the above process, the RNA extraction and reverse transcription into cDNA adopts a conventional general method in the laboratory, specifically: RNA extraction is performed with reference to Trizol (Invitrogen) instructions; the reverse transcription system was 3. Mu.g RNA+2. Mu.L MLV reverse transcriptase (Takara) +8. Mu.L MLV reverse transcriptase buffer (Takara) +1. Mu.L RNase inhibitor (Takara) +3. Mu.L ddH 2 O (Induster); the PCR program parameters were: 42 ℃,60 minutes, 70 ℃,15 minutes and 4 ℃ cooling, and the product can be immediately used for qRT-PCR or stored in a refrigerator at the temperature of minus 20 ℃ for a long time.
In the above procedure, qRT-PCR experiments were performed using conventional general methods in the laboratory: the reaction system was 5. Mu.L SYBR Green (Yeasen) +4. Mu.L ddH 2 O (Instructor) +0.5. Mu.L cDNA template+0.5. Mu.L primer (Instructor); the PCR program parameters were: pre-denaturation at 5℃for 3 min, denaturation at 95℃for 10 sec, annealing at 60℃for 40 sec (40 cycles), and collection of melting curves; the primer sequences used were:
the GAPDH forward primer sequences were: GGTCACCAGGGCTGCTTTTA A
The GAPDH reverse primer sequences were: GGATCTCGCTCCTGGAAGATG A
The forward primer sequence of the beta-actin is as follows: CATGTACGTTGCTATCCAGGC A
The beta-actin reverse primer sequence is as follows: CTCCTTAATGTCACGCACGAT A
The sequence of the U1 forward primer is as follows: GGGAGATACCATGATCACGAAGGT A
The sequence of the U1 reverse primer is as follows: CCACAAATTATGCAGTCGAGTTTCCC A
LNCRNA ENST00000585537.1 forward primer sequences are: GTGAGTATGAGATTCCCCGATG A
LNCRNA ENST00000585537.1 reverse primer sequences were: AGGAGTCCAGAGATAGAAGGC.
Analysis of results: taking GAPDH gene as an internal reference, making 3 compound holes for each sample, calculating the relative expression quantity of the gene by using a2 -ΔΔCt method, and carrying out statistical mapping by GRAPHPAD PRISM software.
The experimental results are shown in fig. 3 and 4. FIG. 3 shows the results of a nucleoplasm separation experiment, qRT-PCR, showing that most LNCRNA ENST00000585537.1 is distributed in the cytoplasm and a small amount in the nucleus. FIG. 4 shows the results of RNA FISH experiments, consistent with the results obtained in FIG. 3. And (3) injection: the scale bar is 20 μm.
Example 4: LNCRNA ENST00000585537.1 expression trend analysis experiment
When the cell growth density reached about 90%, the adipogenic differentiation induction was performed by taking the third generation hAMSCs of normal culture, changing the induction medium (described in example 1) every 3 days, collecting hAMSCs on days 0, 3, 6, 9, respectively, and extracting RNA by conventional procedure to perform qRT-PCR experiment, the specific procedure was completely the same as in example 3, and the primer sequences of GAPDH, LNCRNA ENST00000585537.1, etc. were the same as in example 3.
The experimental results are shown in FIG. 5, and the qRT-PCR experiment shows that LNCRNA ENST00000585537.1 expression level is in an ascending trend along with the time extension in a certain time range, which shows that the expression level is obviously increased in the adipogenic differentiation process of hAMSCs.
Example 5: experiment of the Effect of knock-down LNCRNA ENST00000585537.1 on in vitro adipogenic differentiation of hAMSCs
Taking the normally cultured third generation hAMSCs, effectively knocking down LNCRNA ENST00000585537.1 by using two pairs of small interfering RNAs (siRNA-1 and siRNA-2), wherein the nucleotide sequences of the two pairs of siRNAs are respectively as follows: siRNA-1 (sense strand: GCUGUGACCAAUGAACAAUTT, antisense strand: AUUGUUCAUUGGUCACAGCTT); siRNA-2 (sense strand: GUGGUUCCCAUCUCAAGAATT, antisense strand: UUCUUGAGAUGGGAACCACTT). Both siRNA-1 and siRNA-2 were purchased from Ji Ma Biolabs, suzhou, and both siRNA-1 and siRNA-2 were transfected using the liposome transfection method commonly used in the laboratory, taking 1 well of 16 well plate as an example, the transfection system was: mu.L of siRNA+500. Mu.L of OPTI serum-free medium (Gibco) +5. Mu.L of Lipofectamine 3000 (Invitrogen), after mixing, the mixture was added dropwise to a 6-well plate with cells spread thereon, gently mixed, and incubated at a constant temperature of 5% CO 2 in an incubator at 37 ℃. The specific procedure for harvesting hAMSCs 24 hours after transfection, extracting RNA in conventional manner and performing qRT-PCR experiments to examine knock-down efficiency was exactly as described in example 3. And (3) performing adipogenic differentiation induction when the cell growth density reaches about 90%, collecting hAMSCs at the 6 th day, extracting RNA and protein according to conventional steps, respectively performing qRT-PCR experiments and Western Blot (WB) experiments to detect the expression levels of mRNA and protein of key markers PPARgamma, FABP4 and PLIN1 for adipogenic differentiation, and performing oil red O staining on the hAMSCs at the 12 th day to detect the generation of lipid droplets.
In the above procedure, the specific procedures for RNA extraction and qRT-PCR experiments were exactly as described in example 3. The primer sequences used are:
the GAPDH forward primer sequences were: GGTCACCAGGGCTGCTTTTA A
The GAPDH reverse primer sequences were: GGATCTCGCTCCTGGAAGATG A
LNCRNA ENST00000585537.1 forward primer sequences are: GTGAGTATGAGATTCCCCGATG A
LNCRNA ENST00000585537.1 reverse primer sequences were: AGGAGTCCAGAGATAGAAGGC A
The PPARgamma forward primer sequences are: TGAACGTGAAGCCCATCGAG A
The pparγ reverse primer sequences were: CTTGGCGAACAGCTGAGAGG A
The FABP4 forward primer sequence is as follows: ACTGGGCCAGGAATTTGACG A
The FABP4 reverse primer sequence is as follows: CTCGTGGAAGTGACGCCTT A
The PLIN1 forward primer sequence was: CCATGTCCCTATCAGATGCCC A
The PLIN1 reverse primer sequence was CTGGTGGGTTGTCGATGTC.
In the process, the experimental process of extracting protein and WB is as follows: the main reagent consumables are RIPA protein lysate (Biyun day), phenylmethylsulfonyl fluoride PMSF (Biyun day), BCA protein concentration determination kit (Biyun day), BSA protein standard (Biyun day), protein loading buffer (Biyun day), SDS-PAGE precast gel (yase), PVDF membrane (Millipore), skimmed milk powder (illi), methanol (Beijing chemical reagent factory), bovine serum albumin BSA (Cunninghamia sinensis), tris-HCl (pririley), tween (Sigma-Aldrich), HRP-labeled goat anti-rabbit secondary antibody (Xinbo), PPARgamma primary antibody (proteintech), FABP4 (proteintech), PLIN1 (CST), GAPDH (proteintech) and the like; BCA assay for protein concentration: cells were lysed on RIPA ice and centrifuged at 12000rpm for 30min, and the resulting supernatant was aspirated in small amounts for protein concentration determination; vertical protein gel electrophoresis: taking out the gel carefully after electrophoresis for 1 hour at 120V according to the measured concentration of the sample; wet transfer: transferring 300mA to a membrane for 2 hours; closing: sealing at room temperature for 1 hour; antibody hybridization reaction: primary antibodies (pparγ, FABP4, PLIN1, GAPDH) were hybridized overnight and secondary antibodies were hybridized for 1 hour; ECL color reaction.
In the process, the oil red O dyeing experiment process is as follows: after discarding the culture medium, PBS is washed for 3 times, and residual liquid is sucked as much as possible; fixing: 4% paraformaldehyde, fixing at room temperature for 10 min, washing with PBS for 3 times, and sucking to dryness; dyeing: the filtered oil red O dye was added to the wells, incubated at about 300. Mu.L/well for 30 minutes at room temperature, washed 3 times with PBS, added to the wells, observed under a microscope and photographed.
The experimental results are shown in fig. 6, wherein in fig. 6, a is the knockdown efficiency of siRNA pair LNCRNA ENST 00000585537.1; B. c is mRNA of a key marker of adipogenic differentiation, and the change of protein level is in a decreasing trend; d is an oil red O staining chart of each group of cell lipid drops, and the formation of hAMSCs lipid drops is obviously reduced after siRNA-1 and siRNA-2 knockdown. All the experiments show that after the lncRNAENST00000585577.1 is knocked down, the in vitro adipogenic differentiation of hAMSCs is inhibited. And (3) injection: siRNA-NC is nonsensical control sequence, siRNA-1, siRNA-2 is specific sequence designed for ENST 00000585537.1. The scale bar is 100 μm.
Example 6: experiment of the Effect of over-expression LNCRNA ENST00000585537.1 on in vitro adipogenic differentiation of hAMSCs
And taking the third-generation hAMSCs cultured normally, and using a lentiviral vector to effectively overexpress LNCRNA ENST 00000585537.1. Both the over-expression plasmid and the lentiviral vector were purchased from Ji Ma biological company, su, and the vector type of this plasmid was LV3-H1-GFP-Puro, LV3 being the lentivirus type used to package the plasmid, GFP (green fluorescent protein) being indicative of transfection efficiency, puro (puromycin) being a resistance marker useful for screening positive cells. Infection is carried out by using a lentivirus infection method commonly used in laboratories, and the specific process is as follows: cell densities of about 60% -70% may be ready for transfection; discarding the old culture medium, replacing the fresh culture medium, dropwise adding virus suspension by using a special virus operation pipettor, and using a gun head with a filter element; shaking slightly, placing into incubator, culturing at 37deg.C with 5% CO 2 at constant temperature; after 24 hours, the culture medium is changed into a fresh culture medium, and the transfection efficiency is observed under a lens; passaging is carried out when the cells grow to about 90%, and puromycin is added after the cells are attached to the wall) and screening is carried out; hAMSCs were harvested after screening and RNA was extracted according to conventional procedures and tested for overexpression efficiency by qRT-PCR. The adipogenic differentiation induction is carried out when the effective over-expressed cell growth density reaches about 90%, RNA and protein are extracted from hAMSCs by the conventional steps at the 6 th day, the expression levels of mRNA and protein of key adipogenic differentiation markers PPARgamma, FABP4 and PLIN1 are detected by respectively carrying out qRT-PCR experiments and Western Blot (WB) experiments, and the hAMSCs are subjected to oil red O staining at the 12 th day to detect the lipid drop generation condition. The procedure for RNA extraction and qRT-PCR experiments, protein extraction and WB experiments, and oil red O staining experiments were exactly as described in example 5.
The experimental results are shown in FIG. 7, wherein A is LNCRNA ENST00000585537.1 for the over-expression efficiency detection; B. c is RNA of key markers of adipogenic differentiation, and the change of protein level is in an ascending trend; d is the oil red O staining of each group of cell lipid droplets, and the formation of hAMSCs lipid droplets is significantly increased after overexpression. The experimental detection results show that after LNCRNA ENST00000585537.1 is over-expressed, the in vitro adipogenic differentiation of hAMSCs is promoted. And (3) injection: lenti-NC is a nonsensical control sequence and Lenti-lncRNA is a specific sequence designed for ENST 00000585537.1. The scale bar is 100 μm.
Example 7: LNCRNA ENST00000585537.1 experiments on the Effect of free fatty acid content levels during adipogenic differentiation of hAMSCs
The knockdown procedure of LNCRNA ENST00000585537.1 in this example was exactly the same as that of example 5, i.e., using two pairs of small interfering RNAs (siRNA-1 and siRNA-2) to effectively knockdown LNCRNA ENST00000585537.1, taking the third generation hAMSCs that were normally cultured. sirnas were purchased from Ji Ma biosystems, su, and transfected using liposome transfection methods commonly used in the laboratory. hAMSCs were harvested 24 hours after transfection, and RNA was extracted according to conventional procedures for qRT-PCR experiments to detect knock-down efficiency. Adipogenic differentiation induction was performed when the cell growth density reached about 90%, and after harvest of hAMSCs at day 6, polyA transcriptome sequencing was performed by Guangzhou Ruibo biological company. Screening differential significant genes with a threshold value of 1.5 times of difference and p-value less than or equal to 0.05, and performing KEGG pathway enrichment analysis on the differential significant genes.
In addition, after LNCRNA ENST00000585537.1 was knocked down or overexpressed, hAMSCs were harvested on day 6 of adipogenic differentiation induction and snap frozen with liquid nitrogen and tested for 39 free fatty acid contents at Kahler Biotech, st.Johnson, according to the methods described in examples 5 and 6.
The experimental results are shown in FIGS. 8-10. In FIG. 8, A is a cluster heat map of differentially expressed genes after LNCRNA ENST00000585537.1 knockdown, red represents up-regulation of gene expression, and green represents down-regulation of gene expression; b is a signaling pathway enrichment analysis of significantly down-regulated genes. In FIG. 9, A is the relative content of 39 free fatty acids after LNCRNA ENST00000585537.1 knockdown; b is a significantly reduced relative content of 6 free fatty acids. FIG. 10A is the relative amounts of 39 free fatty acids after over-expression LNCRNA ENST 00000585537.1; b is the relative content of 6 free fatty acids that is significantly elevated.
The results in FIG. 8 show that, following the knock down LNCRNA ENST00000585537.1, the significantly down-regulated genes are mainly enriched in some pathways associated with fatty acid metabolism, such as the unsaturated fatty acid synthesis pathway (hsa 01040), the fatty acid metabolism pathway (hsa 01212), the pyruvate metabolism pathway (hsa 00620), etc. The results in fig. 9 show that the levels of various free fatty acids, such as caprylic acid, capric acid, etc., are significantly reduced after the knock down LNCRNA ENST 00000585537.1. The results in FIG. 10 show that the levels of various free fatty acids are significantly up-regulated after LNCRNA ENST00000585537.1 over-expression, such as cis-5, 8,11,14, 17-eicosapentaenoic acid, docosatetraenoic acid, and the like. The results indicated that LNCRNA ENST00000585537.1 had a significant effect on fatty acid metabolism in hAMSCs.
Example 8: experiment of the Effect of knock-down LNCRNA ENST00000585537.1 on the in vivo adipogenic Capacity of hAMSCs
And taking the third-generation hAMSCs which are normally cultured, and effectively knocking down LNCRNA ENST00000585537.1 by using a slow virus vector. Knockdown lentiviral vectors were purchased from Ji Ma Bio Inc. in Suzhou, the vector type of this plasmid was LV3-H1-GFP-Puro, LV3 was the lentivirus type used to package the plasmid, GFP (green fluorescent protein) was indicative of transfection efficiency, puro (puromycin) was a resistance marker was used to screen positive cells, and the knockdown sequence was GTGCTGAGATAAATTTCATGA (5 '-3'). Infection was performed using a lentivirus infection method commonly used in the laboratory, and the infection process was exactly the same as in example 6. Adipogenic differentiation induction was performed for 3 days when the cell growth density reached about 90%, and hAMSCs were harvested and mixed on Matrigel (Corning) ice and injected subcutaneously into the back of nude mice. Nude mice (MDL company) were 8 week old females, the number of injected cells was 4X 10 6, the animals were sacrificed 2 weeks after injection, and the tissues were sectioned by paraffin embedding and then HE-stained and IHC-stained (marked by adipogenic differentiation key protein PLIN 1) from Sivel company.
The experimental results are shown in FIG. 11. In fig. 11, a is a schematic diagram of an animal experiment scheme, and 5 mice are injected in total; b is the detection of the subcutaneous fat formation of hAMSCs in nude mice. The results showed that, compared to the control group, the ability of hAMSCs to differentiate into adipocytes in vivo was significantly reduced after the knock down LNCRNA ENST00000585537.1, and the expression of PLIN1 was significantly reduced, indicating that lipid droplet formation was also significantly reduced. And (3) injection: sh-NC is nonsensical control sequence, sh-lncRNA is specific sequence designed for ENST00000585537.1, and the specific sequence is GTGCTGAGATAAATTTCATGA (5 '-3'); the scale bar is 100 μm.
Example 9: experiment of the Effect of over-expression LNCRNA ENST00000585537.1 on the in vivo adipogenic Capacity of hAMSCs
And taking the third-generation hAMSCs cultured normally, and using a lentiviral vector to effectively overexpress LNCRNA ENST 00000585537.1. Over-expression lentiviruses were purchased from Ji Ma Biotechnology, suzhou, the vector type of this plasmid was LV3-H1-GFP-Puro, LV3 was the lentivirus type used to package the plasmid, GFP (green fluorescent protein) indicated transfection efficiency, puro (puromycin) was a resistance marker used to screen positive cells, and knockdown sequence was GTGCTGAGATAAATTTCATGA (5 '-3'). Infection was performed using a lentivirus infection method commonly used in the laboratory, and the infection process was exactly the same as in example 6. Adipogenic differentiation induction was performed for 3 days when the cell growth density reached about 90%, and hAMSCs were harvested and mixed on Matrigel (Corning) ice and injected subcutaneously into the back of nude mice. Nude mice (MDL company) were 8 week old females, the number of injected cells was 4X 10 6, the animals were sacrificed 2 weeks after injection, and the tissues were sectioned by paraffin embedding and then HE-stained and IHC-stained (marked by adipogenic differentiation key protein PLIN 1) from Sivel company.
The experimental results are shown in FIG. 12. FIG. 12 shows the detection of subcutaneous fat formation of hAMSCs in nude mice, wherein A is a schematic diagram of an animal experiment scheme, and 5 mice are injected in total; b is a staining chart of the subcutaneous fat formation of hAMSCs in nude mice. The results show that, after over-expression LNCRNA ENST00000585537.1, the ability of hAMSCs to differentiate into adipocytes in vivo is significantly enhanced, and PLIN1 expression is significantly increased, compared to the control group, indicating that lipid droplet formation is also significantly increased. And (3) injection: lenti-NC is a nonsensical control sequence; the scale bar is 100 μm.
Example 10: LNCRNA ENST00000585537.1 analysis of correlation of expression of key marker genes in human adipose tissue
The fat extraction fat is normally medical waste and is obtained from the orthopedic surgery of the national academy of medical science, the material taking mode does not cause extra damage to the patient and does not reveal the privacy of the patient, the informed consent of the patient is obtained, and the application is applied to the ethical review board of the basic medical institute of the national academy of medical science and the approval is obtained. A total of 30 human adipose tissue samples were obtained and grouped according to Body Mass Index (BMI) into BMI <25 groups (i.e., normal body weight) and BMI > 25 groups (i.e., overweight or obese). 30 human adipose tissue samples were all added Trizol (Invitrogen) to extract RNA for reverse transcription and qRT-PCR experiments were performed, which were exactly the same as in example 3. The primer sequences used were:
the GAPDH forward primer sequences were: GGTCACCAGGGCTGCTTTTA A
The GAPDH reverse primer sequences were: GGATCTCGCTCCTGGAAGATG A
LNCRNA ENST00000585537.1 forward primer sequences are: GTGAGTATGAGATTCCCCGATG A
LNCRNA ENST00000585537.1 reverse primer sequences were: AGGAGTCCAGAGATAGAAGGC A
The LPL forward primer sequences were: TCATTCCCGGAGTAGCAGAGT A
The LPL reverse primer sequences were: GGCCACAAGTTTTGGCACC A
The PLIN1 forward primer sequence was: CCATGTCCCTATCAGATGCCC A
The PLIN1 reverse primer sequence was: CTGGTGGGTTGTCGATGTC.
Expression correlation analysis was performed by GRAPHPAD PRISM software correlation function, and correlation coefficients r and p-value were automatically analyzed.
The experimental results are shown in FIG. 13. FIG. 13 is an analysis of expression in human adipose tissue LNCRNA ENST00000585537.1, wherein A is a sample with LNCRNA ENST00000585537.1 expression significantly higher than BMI <25 in human adipose tissue samples with BMI.gtoreq.25; b is in a human adipose tissue sample with the BMI more than or equal to 25; the results show that LNCRNA ENST00000585537.1 has forward expression correlation with key marker genes PLIN1 and LPL in human adipose tissue, and further show that LNCRNA ENST00000585537.1 has great correlation with human adipose tissue steady state, and has application value in preparing medicines for treating obesity.
While the invention has been described with respect to preferred embodiments, 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, and that any such changes and modifications as described in the above embodiments are intended to be within the scope of the invention.
Claims (10)
1. The application of the lncRNA as a marker for regulating the adipogenic differentiation capacity of human mesenchymal stem cells is characterized in that the lncRNA is LNCRNA ENST00000585537.1 and is positioned on human chromosome Chr17q24, and the nucleotide sequence of the lncRNA is shown as SEQ ID NO. 1.
2. A preparation for regulating and controlling the adipogenic differentiation capacity of human mesenchymal stem cells is characterized in that the preparation takes an inhibitor of lncRNA or an accelerator of lncRNA as an active ingredient, wherein the lncRNA is LNCRNA ENST00000585537.1 and is positioned on human chromosome Chr17q24, and the nucleotide sequence of the preparation is shown as SEQ ID NO. 1.
3. The formulation of claim 2, wherein the lncRNA inhibitor or promoter affects human mesenchymal stem cell adipogenic differentiation capacity by knocking down or promoting lncRNA expression such that expression of adipogenic differentiation markers is significantly altered.
4. The formulation of claim 3, wherein the lncRNA inhibitor is an agent that knocks down the expression level of lncRNA; the inhibitors are siRNA-1 and siRNA-2, and the sequence of the siRNA-1 is as follows: sense strand 5'GCUGUGACCAAUGAACAAUTT 3', antisense strand 5'AUUGUUCAUUGGUCACAGCTT 3'; the sequence of the siRNA-2 is as follows: sense strand 5'GUGGUUCCCAUCUCAAGAATT 3', antisense strand 5'UUCUUGAGAUGGGAACCACTT 3'.
5. The formulation of claim 3, wherein the lncRNA promoter is an agent that promotes lncRNA expression levels; the promoter is a carrier plasmid for over-expressing lncRNA, the carrier type of the plasmid is LV3-H1-GFP-Puro, wherein LV3 is a lentivirus type used for packaging the plasmid, GFP is green fluorescent protein, and Puro is puromycin.
6. A method for regulating the adipogenic differentiation capacity of human mesenchymal stem cells, characterized in that the preparation of any one of claims 2 to 5 is used to knock down or promote lncRNA expression, thereby inhibiting or promoting the adipogenic differentiation capacity of human mesenchymal stem cells.
7. The method of claim 6, wherein the human mesenchymal stem cells are inhibited from adipogenic differentiation by transfecting the human mesenchymal stem cells with a lncRNA inhibitor to knock down lncRNA expression; the lncRNA inhibitor is siRNA-1 and siRNA-2, and the sequence of the siRNA-1 is: sense strand 5'GCUGUGACCAAUGAACAAUTT 3', antisense strand 5'AUUGUUCAUUGGUCACAGCTT 3', the sequence of said siRNA-2 being: sense strand 5'GUGGUUCCCAUCUCAAGAATT 3', antisense strand 5'UUCUUGAGAUGGGAACCACTT 3'.
8. The method of claim 6, wherein the human mesenchymal stem cells are promoted for adipogenic differentiation by over-expressing lncRNA by transfection of lncRNA promoter into human mesenchymal stem cells; the promoter is a carrier plasmid for over-expressing lncRNA, the carrier type of the plasmid is LV3-H1-GFP-Puro, wherein LV3 is a lentivirus type used for packaging the plasmid, GFP is green fluorescent protein, and Puro is puromycin.
9. Use of the agent for regulating the adipogenic differentiation capacity of human mesenchymal stem cells according to any one of claims 2 to 5 for preparing a product for inhibiting or promoting adipogenic differentiation of human mesenchymal stem cells.
10. Use of the agent for regulating the adipogenic differentiation capacity of human mesenchymal stem cells according to any one of claims 2 to 5 for the preparation of a medicament for treating obesity by regulating the adipogenic differentiation capacity of human mesenchymal stem cells.
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