CN114921462A - Application of non-coding gene in promoting growth of hair follicle stem cells - Google Patents
Application of non-coding gene in promoting growth of hair follicle stem cells Download PDFInfo
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Abstract
The invention discloses an application of a non-coding gene in promoting hair follicle stem cell growth, and belongs to the technical field of hair follicle stem cells. Experiments prove that the lncRNA-AC079586.1 gene inhibitor can effectively inhibit the protein expression of P21 and promote the protein expression of CDK1, so that the growth of hair follicle stem cells is promoted, and therefore the gene inhibitor can be used for preparing the hair follicle stem cell growth promoter.
Description
The scheme is a divisional application, and the original application name is as follows: the application of a long-chain non-coding RNA gene inhibitor in promoting the proliferation of hair follicle stem cells is as follows: 2021-11-02, the application number of the original application is: CN 202110946626.1.
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to application of a non-coding gene in promoting growth of hair follicle stem cells.
Background
The hair follicle stem cell is a unique stem cell widely distributed in hair follicle structures, has the capability of multidirectional differentiation, and can be differentiated into hair follicles, sebaceous glands, epidermis and the like. Unlike embryonic stem cells, which are generally in a resting state and are activated only in the early anagen phase of the follicle, hair follicle stem cells not only maintain the regeneration of the follicle in normal skin, but also participate in the reconstruction of the follicle, epidermis and sebaceous glands upon skin injury. Thus, hair follicle stem cells are considered to be important seed cells for promoting skin wound repair. However, the proliferation capacity of hair follicle stem cells is not ideal when the cells are expanded in vitro. Therefore, how to increase the proliferation rate of hair follicle stem cells in vitro culture is an urgent problem to be solved.
Long non-coding RNAs are non-coding RNAs greater than 200 nucleotides in length that are not directly involved in the coding of proteins, and were originally thought to be "noise" of genome transcription. However, with the wide application of gene chip technology and RNA sequencing technology, more and more lncrnas have been identified and proved to have complex biological functions through research, and have different degrees of influence on cell proliferation, differentiation, apoptosis, migration and the like. Therefore, the research on the long-chain non-coding RNA playing a role in the proliferation of the hair follicle stem cells has important significance for solving the problem of low proliferation speed of the hair follicle stem cells in vitro culture.
Disclosure of Invention
The invention aims to provide application of long-chain non-coding RNA in promoting hair follicle stem cell proliferation.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a long-chain non-coding RNA lncRNA-AC079586.1, wherein the gene inhibitor of lncRNA-AC079586.1 can promote the proliferation of hair follicle stem cells, the Transcript sequence of lncRNA-AC079586.1 is shown in SEQ ID NO.1, and the External Transcript ID of lncRNA-AC079586.1 is ENST 00000375987.3.
In addition, the invention provides an application of the lncRNA-AC079586.1 gene inhibitor in preparation of the hair follicle stem cell proliferation promoter, wherein the gene inhibitor is siRNA, and the sense strand sequence of the siRNA is 5'-AGUGUAUGUCGCAUAUCAC-3'; the antisense strand sequence of the siRNA is 5'-GUGAUAUGCGACAUACACU-3'.
In addition, the invention provides a gene inhibitor for promoting the proliferation of hair follicle stem cells, wherein the gene inhibitor is siRNA of lncRNA-AC079586.1 gene, and the sense strand sequence of the siRNA is 5'-AGUGUAUGUCGCAUAUCAC-3'; the antisense strand sequence of the siRNA is 5'-GUGAUAUGCGACAUACACU-3'.
Preferably, the siRNA is capable of promoting hair follicle stem cell proliferation
Preferably, the siRNA is capable of reducing the expression of cell cycle gene P21; the siRNA is capable of promoting expression of a cell cycle gene CDK-1.
In addition, the invention provides application of the lncRNA-AC079586.1 gene inhibitor in preparation of a cell cycle gene P21 expression inhibitor of hair follicle stem cells, wherein the gene inhibitor is siRNA, and the sense strand sequence of the siRNA is 5'-AGUGUAUGUCGCAUAUCAC-3'; the antisense strand sequence of the siRNA is 5'-GUGAUAUGCGACAUACACU-3'.
In addition, the invention provides application of the lncRNA-AC079586.1 gene inhibitor in preparation of an expression promoter of a cell cycle gene CDK1 of hair follicle stem cells, and is characterized in that the gene inhibitor is siRNA, and the sense strand sequence of the siRNA is 5'-AGUGUAUGUCGCAUAUCAC-3'; the antisense strand sequence of the siRNA is 5'-GUGAUAUGCGACAUACACU-3'.
The invention has the beneficial effects that
The invention proves that the inhibition of the expression of lncRNA-AC079586.1 in hair follicle stem cells can obviously promote the proliferation of the hair follicle stem cells, and can inhibit the expression of cell cycle gene P21 and promote the expression of cell cycle gene CDK1, thereby providing a basis for further preparing a novel hair follicle stem cell proliferation promoter.
Drawings
FIG. 1 detection of inhibitory effect of si-lncRNA-AC079586.1 on lncRNA-AC079586.1 expression;
FIG. 2 CCK-8 measures the effect of si-lncRNA-AC079586.1 on hair follicle stem cell proliferation;
FIG. 3 fluorescent quantitative PCR detection of mRNA expression of P21 and CDK-1 gene;
FIG. 4 Wsetren Blot detects protein expression of P21 and CDK-1 genes.
Detailed Description
Example 1
(1) Using sterile scissors and forceps to extract several hairs including intact hair follicles, and rinsing the hairs 3 times with sterile PBS containing double antibody;
(2) remaining intact hair follicles, cutting off the rest parts, and placing 2 hair follicles in a 24-pore plate respectively;
(3) adding 50 mu L DMEM/F-12 culture medium to soak hair follicles to ensure good adherence, and placing the hair follicles in a cell culture box;
(4) after 24h of culture, 200. mu.L of medium was slowly added, and fresh medium was replaced every 3 days and cell-climbing was observed.
(5) After culturing for 10-14 days, after observing that the long spindle-shaped cells climb out of hair follicles, changing DMEM/F-12 medium containing 10% FBS to continue culturing;
(6) when the cell fusion degree reaches 80-90%, removing the culture medium, washing with PBS, adding 0.25% pancreatin, and placing in a cell culture box;
(7) after 1-2min, gently tapping the cell culture plate, stopping digestion after 80% of cells float, collecting cells, centrifuging, and paving the plate for conventional culture after the culture medium is resuspended.
Example 2
siRNA of lncRNA-AC079586.1 was designed and its inhibitory effect was confirmed
1. Transfection of si-lncRNA-AC079586.1
(1) The siRNA sequence of lncRNA-AC079586.1 is as follows:
5′- AGUGUAUGUCGCAUAUCAC-3′（SEQ ID NO.2）
5′- GUGAUAUGCGACAUACACU-3′（SEQ ID NO.3）
transfection with siRNA
(2) P3 hair follicle stem cells were seeded in 6-well plates and si-lncRNA-AC079586.1 and si-NC were transfected into cells according to lip2000 instructions, control group was left untreated;
(3) after 48h of transfection, RNA was extracted.
RNA extraction
(1) Collecting cells of a control group, a si-NC group and a si-lncRNA-AC079586.1 group;
(2) adding 1ml of Trizol, slightly blowing and beating the cells, transferring the cells into a 2ml centrifuge tube, and standing for 10min at room temperature;
(3) placing the centrifugal tube in a low-temperature high-speed centrifuge, centrifuging for 5min at 12000 r, and taking the supernatant;
(4) adding 200 μ l chloroform into the supernatant, mixing, and standing at room temperature for 15 min;
(5) placing the centrifuge tube in a low-temperature high-speed centrifuge, centrifuging for 5min at 12000 r, and taking 750 mu l of supernatant to a new centrifuge tube;
(6) adding 750 μ l of precooled isopropanol, mixing, and standing for 10 min;
(7) placing the centrifuge tube in a low-temperature high-speed centrifuge, centrifuging at 12000 rpm for 10min, and carefully removing the supernatant;
(8) adding 1ml of 75% ethanol into the precipitate, reversing, mixing, placing the centrifuge tube in a low-temperature high-speed centrifuge, centrifuging at 8000 rpm and 4 ℃ for 5 min;
(9) carefully removing the supernatant, standing for 5-10min until the ethanol is completely evaporated;
(10) DEPC water was added to dissolve the precipitate and RNA concentration and purity were determined for subsequent experiments.
2. Reverse transcription reaction
The reverse transcription reaction was performed according to TAKARA reverse transcription kit.
Reverse transcription reaction system: 5 XPrimeScript RT master mix 2. mu.l; RNA template 0.5. mu.g; RNase Free water to 10. mu.l.
Reverse transcription reaction conditions: 15min at 37 ℃; 5s at 85 ℃; cooling at 4 ℃.
3. Real-time fluorescent quantitative PCR
Primer sequences
lncRNA-AC079586.1
Forward primer 5'-TCCCTGTGCTGACATCCCTA-3'（SEQ ID NO.4）
Reverse primer 5'-GAAGGGTCTTGTCCAGCAGG-3'（SEQ ID NO.5）
GAPDH
Forward primer 5'- GAAGGTGAAGGTCGGAGTC-3'（SEQ ID NO.6）
Reverse primer 5'-GAAGATGGTGATGGGATTTC-3'（SEQ ID NO.7）
Amplifying the real-time fluorescent quantitative PCR according to a TAKARA qPCR (SYBR) kit under the experimental condition of 95 ℃ for 120 s; 95 ℃ 30 s, 60 ℃ 40 s 35 cycles.
Results of the experiment
The experimental result is shown in fig. 1, and it can be seen that the relative expression amount of the si-lncRNA-AC079586.1 group is 0.147 ± 0.044, and the inhibition rate is 85.3%, which indicates that the si-lncRNA-AC079586.1 can effectively inhibit the expression of lncRNA-AC079586.1 in hair follicle stem cells, and has an excellent inhibition effect.
Example 3
CCK-8 tests on the influence of si-lncRNA-AC079586.1 on the proliferative capacity of hair follicle stem cells
(1) Experimental grouping si-NC group (P3 hair follicle stem cell transfection si-NC) and si-lncRNA-AC079586.1 group (P3 hair follicle stem cell transfection si-lncRNA-AC 079586.1);
(2) according to the grouping, 1000 cells/well were seeded in 96-well plates at 100. mu.l per well and transfected using lip 2000;
(3) after 48h, 10. mu.l of CCK-8 reagent is added, the mixture is incubated in an incubator for 2h, and the 0D value is detected in a 450nm microplate reader.
As shown in FIG. 2, it can be seen that the OD value of si-lncRNA-AC079586.1 group was 0.587. + -. 0.031, and the OD value of si-NC group was 0.391. + -. 0.034, and the difference was statistically significant. The results show that the inhibition of lncRNA-AC079586.1 can remarkably promote the proliferation of hair follicle stem cells.
Example 4
Fluorescent quantitative PCR detection of influence of si-lncRNA-AC079586.1 on hair follicle stem cell proliferation related genes CDK-1 and P21
(1) Experimental grouping si-NC group (P3 hair follicle stem cell transfection si-NC) and si-lncRNA-AC079586.1 group (P3 hair follicle stem cell transfection si-lncRNA-AC 079586.1);
(2) after transfection for 48h, RNA extraction, reverse transcription reaction and fluorescent quantitative PCR reaction were carried out in the same manner as in example 2;
(3) the sequence of the P21 primer is
Forward primer 5′- CTGGGGATGTCCGTCAGAAC-3'（SEQ ID NO.8）
Reverse primer 5′- TCGACCCTGAGAGTCTCCAG-3'（SEQ ID NO.9）
CDK-1 primer sequence is
Forward primer 5′-CAGACTAGAAAGTGAAGAGGAAGG-3'（SEQ ID NO.10）
Reverse primer 5′-ACTGACCAGGAGGGATAGAATC-3'（SEQ ID NO.11）
The experimental conditions are 95 ℃ for 100 s; 35 cycles of 95 ℃ for 35 s and 60 ℃ for 40 s.
As shown in FIG. 3, it can be seen that the inhibition of IncRNA-AC 079586.1 can effectively inhibit the proliferation-suppressing gene P21 (the relative expression of si-IncRNA-AC 079586.1 group is 0.351 + -0.059, the difference is statistically significant), and the inhibition of IncRNA-AC 079586.1 can effectively promote the periodic gene CDK-1 (1.977 + -0.218, the difference is statistically significant).
Example 5
Western Blot to test the influence of si-lncRNA-AC079586.1 on the proliferation-related proteins CDK-1 and P21 of hair follicle stem cells
(1) Experimental grouping si-NC group (P3 hair follicle stem cell transfection si-NC) and si-lncRNA-AC079586.1 group (P3 hair follicle stem cell transfection si-lncRNA-AC 079586.1);
(2) according to experimental grouping, hair follicle stem cells are inoculated in a 6-well plate 24h before transfection, and then transfection is carried out according to lipo2000 instructions;
(3) after culturing for 48 hours, adding 100 mu L of PIPA lysate into each hole, scraping the cells by using a cell scraper after the cells are fully lysed, and collecting the cells into a centrifuge tube;
(4) placing on ice for 30min to completely lyse cells;
(5) centrifuging at 12000g for 20 min at 4 deg.C, sucking supernatant, detecting protein concentration by BCA method, adjusting protein concentration to 2 μ g/ml, and decocting at 100 deg.C for 5min to obtain protein sample;
(6) preparing 10% SDS-PAGR gel, and adding 10 uL protein sample into each hole;
(7) performing constant-voltage 90V electrophoresis, adjusting the voltage to 130V when the electrophoresis strip completely runs out of the concentrated gel, and stopping electrophoresis when the bromophenol blue part runs out of the separation gel to prevent P21 from running out of the concentrated gel;
(8) placing a membrane rotating clamp according to a model of sponge-filter paper-separation gel-NC membrane-filter paper-sponge, and rotating for 1.5h at 250 mA;
(9) after the electrotransformation is finished, taking out the NC membrane, placing the NC membrane in 5% skimmed milk powder, and sealing the shaking table for 1 h;
(10) according to the protein size, cutting off the P21 and CDK-1 protein bands, incubating corresponding primary antibodies, and sealing the shaker at 4 ℃ overnight;
(11) washing the membrane with TBST for 3 times, each time for 10min, incubating the secondary antibody, and incubating for 60min in a shaking table at room temperature;
(12) the film was washed 3 times with TBST for 10min, and then exposed to light, and the results are shown in FIG. 4.
As can be seen from the figure, inhibition of IncRNA-AC 079586.1 effectively inhibited protein expression of the proliferation suppressor gene P21 and promoted protein expression of the periodic gene CDK-1.
SEQUENCE LISTING
<110> Jinan Shengtang Biotech GmbH
Application of non-coding gene in promoting growth of hair follicle stem cells
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 2213
<212> DNA
<213> Human source (Human)
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aggaactttc ctactcagtg gtgccttatt gaaatggagc atattcctgc cacccatagg 180
ttggagcacc cacccctgct ggacaagacc cttcctgggg gggccacctt ggaactctga 240
atagagactt gaagagtgag gaagactaag aggctgctgc catttgaaga tgaagtgtac 300
aatacaacag ggaatcaagt gaacagaaag tgttgatggg gatcccagca gatagccaac 360
aaaagggaac ctcacttcta cagcagcaag gaactgcatt ctgccaaaaa cctaatgaac 420
ttggaagcag attattttct ggagtctgta gagagatgag caggaagagg gatattagaa 480
gagagaaagt gaaaactgga ctgaaacatt gctagctggg gatcttagcc tctctttagg 540
caactcactc ctggtcctgt tctctgcaca tgtgggaatt ataatgagca ccaagcagaa 600
tgaatgaaaa taaatccaga ccaagaacca ctgaagaaac tgcaacccac aagagaaaac 660
tagccagagt atctgcacag gaaggacaag gagacttctc tttataataa cagaacccag 720
aagacgacct acaaggttct gcccacaggg gagattgtga cctattgctg gccccaacac 780
caagtcgaag ttacacattt gccttggcct tgccctgaga aggtgctgtg acatattgct 840
gggcccagca ccaaggtgat gggactctcc tctttctcct ggaacctgtc cacagtgggg 900
attgtgacat attgctgggc ccagcaccac tgtgatgtga ctctcctctc gtgcctgggg 960
ccagcccact ggggtaattg tgacatataa ctggacctag cccctaggtt atgtgactta 1020
cctctccttc ctgagccctg cccacaaggg ggcctcatga catatctctg gacccctcac 1080
ctaggtgatg tgagtctctt gcctgggccc acccctcagg gtgtatagtg acatattgct 1140
ggacccaaca cctaggtgat gtgactcttt tctactgctt ggctctgccc aaggagaaat 1200
tgtgatgtat cactgggccg agcacctagg tgatgtgact ctcctctcct gcctggctcc 1260
agccaaaagg ggggattgtg acatatcact ggacccaaca cctaggtgat gggactctcc 1320
tcttttgcct gggcccacat attttgggta ttgtgacctg ggtgatggga gcctactgct 1380
tgaaccctac ccagagggag ctttgtgaca tatctctgca tccatcacct aggagatgtg 1440
actctcctct cgtgcctggc cccagccaaa aggggggatc gtgacatgtc actggattca 1500
tcacctaggt gatgggactc ctcttttgtc ttggtcccac atactgtggg tattgtgaca 1560
taatgctggg cccaacacct gggtgatgga aggctcctgc ctgggccctg ctcacagaga 1620
gccttgtgac agatctctgc atccatcatc acctagatga tgtgactctc ctgttctgcc 1680
tgcaccctgc ccatggggaa gattgtaaca tatccctggg tgctgttccc agtcttgcct 1740
catggccttt gccccagctg tgtcctctgc cctgcaggat gcacctctgc tccgtgtggc 1800
tccctcccac tctcactctc caaatgcccc gaacccctga gccccactgc agccccactg 1860
taccctggct ctgcagcgct gcttcttcat gatgtgtgtc ttccaacagc agacggggcc 1920
atgaatgtgg atgcctcatt cactgctgtg tgccggcatc cagagcagga ctggagccca 1980
gagcaggcac ctgactgata gttgttgaat gagtacagta atgaatgaca atgaaagcac 2040
tgtgtagaaa cactggctga atagtaccta gaaagagaat gtctcaaata cctacattag 2100
caatgaaaaa aacactgcta attaatgaac taagcatcta attcaagaaa caaagcaaca 2160
catctaactt atagaaaaag tgaaaaaaat aaaaaacata agatcaaaaa ctg 2213
<210> 2
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aguguauguc gcauaucac 19
<210> 3
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gugauaugcg acauacacu 19
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tccctgtgct gacatcccta 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaagggtctt gtccagcagg 20
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gaaggtgaag gtcggagtc 19
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaagatggtg atgggatttc 20
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctggggatgtccgtcagaac 20
<210> 9
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tcgaccctgagagtctccag 20
<210> 10
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cagactagaa agtgaagagg aagg 24
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
actgaccagg agggatagaa tc 22
Claims (3)
- The application of the lncRNA-AC079586.1 gene inhibitor in the preparation of the hair follicle stem cell growth promoter is characterized in that the transcript sequence of lncRNA-AC079586.1 is shown as SEQ ID NO.1, the gene inhibitor is siRNA, and the sense strand sequence of the siRNA is shown as SEQ ID NO. 2; the antisense strand sequence of the siRNA is shown in SEQ ID NO. 3.
- 2. The application of the lncRNA-AC079586.1 gene inhibitor in preparing a cell cycle gene P21 protein expression inhibitor of hair follicle stem cells is characterized in that the transcript sequence of lncRNA-AC079586.1 is shown as SEQ ID No.1, the gene inhibitor is siRNA, and the sense strand sequence of the siRNA is shown as SEQ ID No. 2; the antisense strand sequence of the siRNA is shown in SEQ ID NO. 3.
- 3. The application of the lncRNA-AC079586.1 gene inhibitor in preparing a cell cycle gene CDK1 protein expression promoter of hair follicle stem cells is disclosed, wherein the transcript sequence of lncRNA-AC079586.1 is shown as SEQ ID NO.1, the gene inhibitor is siRNA, and the sense strand sequence of the siRNA is shown as SEQ ID NO. 2; the antisense strand sequence of the siRNA is shown in SEQ ID NO. 3.
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| CN202110946626.1A CN113584031B (en) | 2021-08-18 | 2021-08-18 | Application of long-chain non-coding RNA gene inhibitor in promotion of hair follicle stem cell proliferation |
| CN202210318054.7A CN114921462A (en) | 2021-08-18 | 2021-08-18 | Application of non-coding gene in promoting growth of hair follicle stem cells |
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Citations (3)
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| US20140329704A1 (en) * | 2013-03-28 | 2014-11-06 | President And Fellows Of Harvard College | Markers for mature beta-cells and methods of using the same |
| CN112301032A (en) * | 2020-11-12 | 2021-02-02 | 济南生发堂生物科技股份有限公司 | Gene inhibitor for inhibiting hair follicle stem cell aging |
| CN113069548A (en) * | 2021-04-23 | 2021-07-06 | 青岛市中医医院(青岛市海慈医院、青岛市康复医学研究所) | Reagent for cardiac revascularization |
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2021
- 2021-08-18 CN CN202210318054.7A patent/CN114921462A/en active Pending
- 2021-08-18 CN CN202110946626.1A patent/CN113584031B/en active Active
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| US20140329704A1 (en) * | 2013-03-28 | 2014-11-06 | President And Fellows Of Harvard College | Markers for mature beta-cells and methods of using the same |
| CN112301032A (en) * | 2020-11-12 | 2021-02-02 | 济南生发堂生物科技股份有限公司 | Gene inhibitor for inhibiting hair follicle stem cell aging |
| CN113069548A (en) * | 2021-04-23 | 2021-07-06 | 青岛市中医医院(青岛市海慈医院、青岛市康复医学研究所) | Reagent for cardiac revascularization |
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| CN113584031B (en) | 2022-05-13 |
| CN113584031A (en) | 2021-11-02 |
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