CN113637671B - LncRNA related to epidermal stem cell differentiation and application thereof - Google Patents
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Abstract
The invention relates to LncRNA related to the differentiation of epidermal stem cells, wherein LncRNA is RP4-784A16.2, and the LncRNA sequence is SEQ ID No.1. And the application of LncRNA in preparing a medicament for promoting healing of skin wounds. And a method for identifying LncRNA, comprising the following steps: isolated culture and induced differentiation of human epidermal stem cells; detecting cell microarray chips before and after differentiation; real-time fluorescent quantitative PCR detection proves that RP4-784A16.2 is up-regulated in the differentiation of the epidermal stem cells. The RP4-784A16.2 can promote cell differentiation, provides a detailed foundation and a new thought for subsequent research on regulation and control of epidermal stem cell differentiation, and can be applied to the technical field of preparing medicaments for promoting healing of skin wounds.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to identification and application of LncRNA RP4-784A16.2 differentiated from epidermal stem cells.
Background
The skin is the largest organ of the human body, covers the surface of the human body, and has important functions in resisting radiation, preventing infection, excreting metabolites, regulating body temperature balance, sensing external stimulus and the like due to the direct contact of the outside. The epidermis and dermis two-layer are formed into the skin, and the epidermis is the superficial layer structure of the skin and consists of two parts, namely a stratum corneum and a germinal layer. The horny layer has no life activity, water cannot easily pass through, and the functions of resisting abrasion, infection and the like are better.
Epidermal stem cells (epidermal stem cells, ESCs) are important for maintaining continuous renewal of skin, in which the ectoderm derived from embryos has a strong potential for self-renewal and bi-directional differentiation: can migrate downwards to differentiate into basal epidermis layer or migrate upwards to differentiate into various epidermis cells, which is the basis for ensuring the stable state of skin tissue. At present, organ transplantation is the main treatment method for serious injury of organs and tissues, and autologous transplantation is mostly adopted aiming at skin. However, autologous skin is limited in number, the skin source is insufficient, and aesthetic appearance may be affected, and skin wound repair is difficult and heavy. The research on the differentiation of the epidermal stem cells provides a new idea for the treatment of skin wounds.
Long non-coding RNAs (lncrnas) are greater than 200nt long, often do not have the function of translation into proteins, are generally low in expression abundance, and have a conserved secondary structure. Although initially LncRNA was considered to be "noise" of the transcription process, mRNA-like transcripts not encoding proteins, more and more studies have shown that LncRNA has a specific function. As the LncRNA was studied in depth, it was found to be involved in a number of important biological functions: regulate transcription, participate in translation, protein stabilization process, etc. In the field of epidermal stem cell differentiation, the regulation of LncRNA is not clear.
Disclosure of Invention
The invention aims to:
the invention aims to provide LncRNA related to the differentiation of epidermal stem cells and application thereof, wherein the name of LncRNA related to the differentiation of epidermal stem cells is RP4-784A16.2; the regulatory mechanism of LncRNA RP4-784A16.2 is obtained by analyzing the expression change of LncRNA in the differentiation process of the epidermal stem cells with high flux, so that the LncRNA RP4-784A16.2 differentiated by the epidermal stem cells is disclosed, and the LncRNA RP4-784A16.2 is applied to the preparation of related medicaments for treating skin wounds and promoting healing, and has wide application prospect in the field of promoting the healing of the wounds.
The technical scheme is as follows:
LncRNA related to the differentiation of the epidermal stem cells is RP4-784A16.2, and the LncRNA sequence is SEQ ID No.1.
Use of LncRNA for the preparation of a medicament for promoting healing of skin wounds.
An identification method of LncRNA comprises the following steps:
step one, separating and culturing human epidermal stem cells and inducing differentiation;
step two, detecting cell microarray chips before and after differentiation;
and step three, carrying out real-time fluorescence quantitative PCR detection to prove that the expression of RP4-784A16.2 is up-regulated in the differentiation of the epidermal stem cells.
In the third step, products capable of detecting the expression and/or transcription of the LncRNA are primer sequences capable of amplifying full-length sequences or partial sequences of the LncRNA, wherein the primer sequences are as follows:
RP4-784A16.2 specific forward primer:
5’-ATGGAGGAGCTGGCTGACTG-3’；
RP4-784A16.2 specific reverse primer:
5’-CACGCCCACTTGTGACTTTC-3’。
the primer sequences used to detect GAPDH internal reference in step three were as follows:
internal reference GAPDH-specific forward primer: 5'-GGGAAACTGTGGCGTGAT-3';
reference GAPDH specific reverse primer: 5'-GAGTGGGTGTCGCTGTTGA-3'.
The beneficial effects are that:
the invention identifies the transcriptome of mRNA and LncRNA before and after the differentiation of the epidermal stem cells, and analyzes and integrates the results, and further analyzes the differential expression genes of the mRNA and LncRNA. Screening out positive factors RP4-784A16.2 for epithelial differentiation, and researching to find that RP4-784A16.2 can promote cell differentiation, provides a detailed foundation and a new thought for subsequent research on regulating and controlling epidermal stem cell differentiation, and can be applied to the technical field of preparing medicaments for promoting healing of skin wounds.
Drawings
FIG. 1 shows the cell differentiation under high calcium induction by isolated culture of epidermal stem cells (A is epidermal stem cells, B is 1.5mM Cacl) 2 Induced differentiated cells);
FIG. 2 is a thermal diagram of microarray analysis of mRNA and LncRNA differentially expressed before and after differentiation (A is mRNA and B is LncRNA);
FIG. 3 is a graph of GO enrichment results for differentially expressed mRNA (A is up-regulated expression, B is down-regulated expression);
FIG. 4 is a relationship between differentially expressed LncRNA and adjacent coding genes (A is up-regulated expression, B is down-regulated expression);
FIG. 5 is a graph showing the detection of RP4-784A16.2 expression levels in differentiated cells using real-time fluorescent quantitative PCR;
FIG. 6 siRNA of RP4-784A16.2 is capable of silencing the expression of RP4-784A16.2 in epidermal stem cells;
FIG. 7 at 1.5mM Cacl 2 Under induction, knock-down of RP4-784A16.2 reduced expression of the differentiation markers CK10 and Involucrin.
Detailed Description
The invention is described in more detail below with reference to the drawings accompanying the specification.
The invention starts from the separation and purification of human epidermal stem cells, and adds 1.5mM Cacl 2 And inducing the differentiation of the epidermal stem cells, performing differential analysis on the cells before and after the differentiation through an LncRNA expression chip, wherein the differential standard is fold change more than 2 and P less than 0.05, and screening LncRNA RP4-784A16.2 with the expression change in the differentiation process. The invention relates to the deep research and clinical transformation application of stem cells and regenerative medicine and non-coding RNA in the field of basic medicine, and is mainly applied to the basic research of LncRNA in promoting epidermal differentiation and the development of wound healing technology.
The identification method of LncRNA comprises the following steps:
example 1
Isolated culture and induced differentiation of human epidermal stem cells
Materials and methods:
neutral proteolytic enzyme (Roch)
Pancreatin (trypsin) BI
Epilife medium (Thermo)
FBS（Hyclone）
Taking waste skin pieces after foreskin operation, washing with PBS to remove blood cells, removing subcutaneous fat cells, shearing into tissue blocks with the size of 2X 1cm to 1X 1cm, digesting with 2mg/mL neutral proteolytic enzyme at 4 ℃ for 10-12h, continuing digestion at 37 ℃ for 3h, and separating to obtain epidermis. Cutting epidermis, digesting with 0.25% pancreatin-EDTA, adding DMEM/F12 culture solution containing 10% FBS at 37deg.C for 20min, stopping digestion, blowing into single cell suspension, and grinding with 200 mesh screen. Cells were collected by centrifugation at 1000rpm for 10min. The supernatant was removed, washed 1 time with PBS, and the cells were suspended in Epilife. At 3X 10 6 Is inoculated in NUNC plastic culture flask, and incubated for 20min at 37 ℃. Removing suspended cells and impurities by changing liquid, and stickingThe cells attached to the bottom surface of the culture flask are epidermal stem cells. 37 ℃ 5% CO 2 The incubator continues to incubate and cultivate. Cells are grown to 70% -80%, and pancreatin-EDTA digestion and subculture are performed. The second or third generation cells grew to 20% -30% and were changed to contain 1.5mM Cacl 2 The culture of the Epilife culture broth of (C) was continued for 72 hours as shown in FIG. 1, and the cells became multi-layered, indicating that the cells had differentiated. After 72h, cells were washed 3 times with PBS and total RNA was extracted using Trizol.
Cell microarray chip detection before and after differentiation:
microarray analysis was performed by using "Arraystar Human LncRNA Microarray" by Shanghai Kangshen Bioengineering Co., ltd, mRNA and LncRNA differentially expressed before and after differentiation were selected, and co-expression and association studies were performed between the two.
(1) The total RNA before and after fractionation was detected using the ArrayStar LncRNA chip product (Human LncRNA Microarray) from ArrayStar corporation of Shanghai Karaoka biological engineering, and the thermal map of the mRNA and LncRNA differentially expressed by the microarray chip analysis was as shown in FIG. 2, each row of the thermal map represented one gene, each column represented one sample, red represented an increase in expression level, and green represented a decrease. The brightness of a color represents the degree of increase or decrease.
(2) Differential analysis of mRNA and LncRNA expression before and after differentiation: and (3) comparing the expression results obtained by statistics in the step (1), performing differential analysis, wherein the differential standard is fold change > 2, P is less than 0.05, screening out mRNA and LncRNA which are obviously differential expressed, and recording the differential expression result. Functional enrichment of differentially expressed mRNA and LncRNA was performed.
Results:
comparing mRNA expression profiles of the differentiated group and the undifferentiated group, 6665 differential expression genes are detected, wherein 3129 differential expression of the genes are up-regulated, 3536 differential expression of the genes are down-regulated, the result of the differential gene GO enrichment is shown in figure 3, and the expression of a large number of genes in the differential genes is related to epidermis differentiation, epidermis development and epidermis keratinization.
Comparing the expression profile of the differentiated group with that of the undifferentiated group of LncRNA, 6750 of the differential expression of LncRNA was detected, wherein 3217 of the differential expression of LncRNA was up-regulated, 3533 of the differential expression of LncRNA was down-regulated, classification of these LncRNAs is shown in FIG. 4, panel A shows LncRNA whose expression was up-regulated after differentiation, and panel B shows LncRNA whose expression was down-regulated after differentiation. The LncRNA whose expression was up-regulated was classified as follows, bidirectory 8%, exon senes-overlapping 11%, intersystemic 46%, intersystemic 3%, intersystemic 17%, natural antisense%. The LncRNA whose expression was down-regulated was classified as follows, bidirectory 7%, exon senes-overlapping 13%, intersystemic 44%, intersystemic 5%, intersystemic 14%, natural antisense%.
From the analysis in FIG. 3, it was found that the expression of Wnt pathway gene is generally down-regulated after differentiation, which suggests that the Wnt pathway is an important differentiation regulation network, and from LncRNA co-expression and association studies, RP4-784A16.2 is found to be located in Chromosome 1: 53,288,024-53,289,706 and is natural antisense sequence of Wnt pathway gene LRP8 (low-density lipoprotein receptor-related protein 8), so we speculate that it can bind with mRNA of LRP8 structurally and play an important role in regulating differentiation of epidermal stem cells.
Example 2
Real-time fluorescent quantitative PCR detection proves that RP4-784A16.2 is up-regulated in the differentiation of the epidermal stem cells.
Materials and methods:
2× PCR master mix（Superarray）
the primer sequences are as follows:
RP4-784A16.2 specific forward primer: 5'-ATGGAGGAGCTGGCTGACTG-3' (SEQ ID NO. 2)
RP4-784A16.2 specific reverse primer: 5'-CACGCCCACTTGTGACTTTC-3' (SEQ ID NO. 3)
For convenient detection, the invention can use GAPDH as an internal reference, and a primer for detecting the GAPDH internal reference has the following sequence:
internal reference GAPDH-specific forward primer: 5'-GGGAAACTGTGGCGTGAT-3' (SEQ ID NO. 8)
Reference GAPDH specific reverse primer: 5'-GAGTGGGTGTCGCTGTTGA-3' (SEQ ID NO. 9)
All cDNA samples were separately configured into a RealtimePC reaction system. The system configuration is as follows:
2× Master Mix 5 µL
10uM PCR specific primer F0.5 mu L
10uM PCR specific primer R0.5 mu L
cDNA 2 µL
Adding water to a total volume of 10 mu L
The solution was mixed at the bottom of the flick tube, centrifuged briefly at 5000 rpm, and the PCR reaction was set up: 95 ℃ for 10min;40 PCR cycles (95 ℃,10 seconds; 60 ℃,60 seconds). To establish a melting curve of the PCR product, after the amplification reaction is completed, the PCR product is subjected to the amplification reaction (95 ℃,10 seconds; 60 ℃,60 seconds; 95 ℃ 15 seconds); and slowly heated from 60 ℃ to 99 ℃. After the reaction, the P value was calculated by T test based on the gene CT value and the reference Gene (GAPDH) normalized.
Results:
as shown in FIG. 5, the real-time fluorescence quantitative PCR detection shows that the expression of RP4-784A16.2 in the differentiated cells is up-regulated, and the expression quantity is 3.156 times (P < 0.05) of that before differentiation, so that the expression quantity is in line with expectations.
Example 3
Effect of RP4-784A16.2 on epidermal differentiation
Materials and methods:
cells were plated at 4X 10 1 day prior to transfection 5 Wells were seeded in 6-well plates. The transfection solution was prepared with the following amounts per well:
A. 75pmol siRNA (SEQ ID NO. 10) was diluted with 125. Mu.L Opti-MEM medium and gently mixed;
RP4-784A16.2 siRNA sequence 5'-GGCGUGGCAGGGCUUUGUUTT-3' (SEQ ID NO. 10)
B. 7.5uL Lipofectamine 3000 was diluted with 125. Mu.L of Opti-MEM medium;
C. mixing the DNA diluted in the first two steps with Lipofectamine 3000 (to make the total volume 250 mu L), gently mixing, and standing at room temperature for 15 minutes;
mu.L of the transformation solution was added to each well of cells, and gently mixed. After 24 hours of incubation at 37℃1.5mM Cacl was added 2 After 48 hoursAnd detecting gene expression.
2× PCR master mix（Superarray）
The primer sequences are as follows:
CK10 specific forward primer: 5'-AGGAGGAGTGTCATCCCTAAG-3' (SEQ ID NO. 4)
CK10 specific reverse primer: 5'-AAGCTGCCTCCATAACTCCC-3' (SEQ ID NO. 5)
Involurin-specific forward primer: 5'-TCCTCCAGTCAATACCCATCAG-3' (SEQ ID NO. 6)
Involurin-specific reverse primer: 5'-CAGCAGTCATGTGCTTTTCCT-3' (SEQ ID NO. 7)
Internal reference GAPDH-specific forward primer: 5'-GGGAAACTGTGGCGTGAT-3' (SEQ ID NO. 8)
Reference GAPDH specific reverse primer: 5'-GAGTGGGTGTCGCTGTTGA-3' (SEQ ID NO. 9)
All cDNA samples were separately configured into a Realtime PCR reaction system. The system configuration is as follows:
2× Master Mix 5 µL
10uM PCR specific primer F0.5 mu L
10uM PCR specific primer R0.5 mu L
cDNA 2 µL
Adding water to a total volume of 10 mu L
The solution was mixed at the bottom of the flick tube, centrifuged briefly at 5000 rpm, and the PCR reaction was set up: 95 ℃ for 10min;40 PCR cycles (95 ℃,10 seconds; 60 ℃,60 seconds). To establish a melting curve of the PCR product, after the amplification reaction is completed, the PCR product is subjected to the amplification reaction (95 ℃,10 seconds; 60 ℃,60 seconds; 95 ℃ 15 seconds); and slowly heated from 60 ℃ to 99 ℃. After the reaction, the P value was calculated by T test based on the gene CT value and the reference Gene (GAPDH) normalized.
Results:
as shown in FIG. 6, the real-time fluorescence quantitative PCR detection shows that the siRNA of RP4-784A16.2 can obviously reduce the expression of RP4-784A16.2 in the epidermal stem cells, and the expression quantity is 56.2 percent (P < 0.05) of the control.
As shown in FIG. 7, at 1.5mM Cacl 2 Under induction, knock-down of RP4-784A16.2 can significantly reduce the expression of differentiation markers CK10 and Involurin, the expression levels are 37.9% and 45% of that of the control (P < 0.05), respectively. This suggests that RP4-784A16.2 acts as a differentiation promoting factor, and if silenced during differentiation, inhibits epidermal differentiation.
The foregoing description of the preferred embodiments of the present invention is not intended to be limiting in any way or nature, but rather to be construed as embodying the scope of the present invention.
Sequence listing
<110> Liaoning Aimi Stem cells, a company of national institute of sciences
<120> LncRNA related to epidermal stem cell differentiation and use thereof
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gagggaaaag ctccctgtgg ccacaaaccc tccccaagca cacactccag gctgcacacg 180
actctgcctc ttgttagctg ggcagccttg ggcaaggggg agaactgact ccagcctcag 240
tttctttctc tctacatggg gatcctcctc cttccttagg ttgccaagag gatgaaagtc 300
acaagtgggc gtggcagggc tttgttcctt ctgaagctca ggacaaagca ctgggcttcc 360
ctcctgcctg ggagccttca ggacggcatg gagtttggcc acgtggtggg agcgccctga 420
gtgcgtggca cagaatgttc aactggttac ctctcctgag cctcacagcc tctcaaggaa 480
gatctgagga ggaaactgag gcccaccccc acccagactg aggggcagga ctcactgcaa 540
gtggcctcgg actcatcgga gccatcagga cactcctcct cgccgtcaca cttccaccgt 600
tcgtggatgc agtggccgtt gtcacaggtg aagtcactgt ctgcacaggt cttcttggct 660
gcagggcaag gaaga 675
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Claims (2)
1. A method for identifying up-regulation of expression of RP4-784A16.2 in epidermal stem cell differentiation, comprising:
the method comprises the following steps:
step one, taking waste skin sheets after foreskin operation to obtain human epidermal stem cells, and separating and culturing the human epidermal stem cells in 1.5mM Cacl 2 Inducing differentiation in the Epilife culture solution;
step two, detecting cell microarray chips before and after differentiation;
and step three, carrying out real-time fluorescence quantitative PCR detection to prove that the expression of RP4-784A16.2 is up-regulated in the differentiation of the epidermal stem cells, wherein the sequence of the RP4-784A16.2 is SEQ ID No.1.
2. The method according to claim 1, characterized in that: the primer sequence for the real-time fluorescence quantitative PCR specific detection of RP4-784A16.2 in the third step is as follows:
RP4-784A16.2 specific forward primer: 5'-ATGGAGGAGCTGGCTGACTG-3';
RP4-784A16.2 specific reverse primer: 5'-CACGCCCACTTGTGACTTTC-3'.
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