CN116042623A - MicroRNA as biomarker and application thereof in skin photoaging - Google Patents
MicroRNA as biomarker and application thereof in skin photoaging Download PDFInfo
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Abstract
The invention provides a micro RNA as a biomarker and application thereof in skin photoaging. By collecting skin samples irradiated by solar ultraviolet rays of a donor and protected from the solar ultraviolet rays, separating single melanocytes, detecting the expression of miR-656-3p and knocking down by using an inhibitor, detecting the therapeutic effect of miR-656-3p inhibitor on an animal model of photoaging, the expression of miR-656-3p is closely related to the aging of the melanocytes and the occurrence and development of the photoaging of the skin, and the bioinformatic analysis shows that LMNB2 is a target gene of miR-656-3p, and miR-656-3p regulates the occurrence and development of the photoaging of the skin by targeting the LMNB2 in the melanocytes. The invention provides a new potential marker for diagnosing skin photoaging; provides a new target point for preventing and treating skin photoaging.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a micro RNA serving as a biomarker and application thereof in skin photoaging.
Background
Photoaging (photoaging) is skin aging caused by long-term irradiation of ultraviolet rays, is an external manifestation of aging, and not only causes various aesthetic appearance changes such as wrinkles, slacks, color spots, darkness and the like, but also reduces the skin barrier function and even can induce skin lesions such as skin cancer and the like. The skin aging is reflected at the cellular level, namely cell aging, and recent researches show that the melanocyte is the only expression of an aging marker p16 INK4a It affects the function of surrounding cells, thereby initiating the skin aging process. Since the mechanisms of cellular senescence are not completely understood, there are few truly effective interventions at present. At the same time, the diagnosis of skin photoaging is currently only based on appearance and partial scale, and lacks a molecular level diagnostic marker. Thus, finding new markers and potential targets for intervention for skin photoaging is currently a more urgent need.
Micrornas (micrornas), which are a class of non-coding single-stranded small-molecule RNAs, are approximately 22nt (nucleotides) in length, recognize and bind to mRNA of a target gene in a complementary form, and regulate expression of the gene at post-transcriptional levels by cleaving or inhibiting its translational process.
The naming of microRNAs is based on the temporal order in which they were found, exemplified by hsa-miR-29b-1-5 p: hsa indicates that the species to which the molecule belongs is human, miR is the identification of the mature microRNA, 29 is a serial number given in sequence when the family member to which the microRNA belongs is found, "b", "-1", "-5p" respectively refer to the number of the precursor sequence, genomic position and 5' arm of the precursor for generating the mature microRNA.
Micrornas have been shown to play an important role in skin aging. Whereas the function of miR-656-3p in skin photoaging is not yet known. This study was aimed at exploring that miR-656-3p can affect the development and progression of skin photoaging by modulating melanocyte senescence.
Disclosure of Invention
It is an object of the present invention to provide a microRNA as biomarker, which is miR-656-3p.
The sequence of miR-656-3p is shown in SEQ ID NO. 1:
Hsa-miR-656-3p:tctccaactgacatattataa。
the miR-656-3p can be obtained from a human tissue specimen and isolated cells or can be obtained by an artificial synthesis mode.
It is another object of the invention to provide the use of said micrornas as biomarkers in the preparation of a reagent for diagnosing skin photoaging. The skin photoaging includes melanocyte aging.
It is still another object of the present invention to provide the use of said micrornas as biomarkers in the manufacture of a medicament for the prevention and treatment of skin photoaging.
The miR-656-3p provided by the invention is obtained through screening by the following technical means:
collecting a normal human skin sample irradiated by sunlight Ultraviolet (UV) and prevented from being irradiated by sunlight ultraviolet, and detecting a micro RNA expression profile by high-throughput sequencing. Comparing the difference of the skin microRNA expression profiles under different irradiation conditions, and screening and summarizing to obtain the skin photoaging microRNA marker miR-656-3p with the expression level obviously increased in the skin of the UV irradiation group.
RT-PCR test shows that 60mJ/cm 2 UVB at the irradiation intensity promotes human epidermal melanocyte senescence, while the marker miR-656-3p is significantly upregulated in cell content. After miR-656-3p inhibitor is applied to knock down content of miR-656-3p in melanocyte, cell cycle arrest and beta-galactosidase positive rate are obviously relieved
The cell cycle arrest and the positive rate of the beta-galactosidase represent the cell aging level.
According to the bioinformatics analysis result, the target gene of the marker miR-656-3p comprises LMNB2.
After the Western blot experiment proves that the expression level of the target gene LMNB2 is obviously increased after the marker miR-656-3p in the melanocyte is knocked down. The target relation between miR-656-3p and LMNB2 in melanocytes is verified by a double-luciferase reporter gene experiment.
The coded product of the LMNB2 gene (Entrez ID: 84823) is nuclear lamin B2 (LMNB 2) which is the main component of nuclear membranes, and the expression level of the LMNB2 gene is inversely related to cell senescence.
By injecting the inhibitor of miR-656-3p into the skin of the back molding area of the photo-aging model of a nude mouse in a multi-point manner, photo-aging manifestations such as skin wrinkles and color sinking of the molding area are obviously reduced, and pathological sections show that the dermis thickness is increased, the epidermis tissue structure is more complete, and collagen fibers are in a tidy wavy arrangement.
The invention reveals that the expression of miR-656-3p is closely related to skin photoaging, and the downregulation of miR-656-3p effectively upregulates the content of LMNB2 protein in melanocytes, thereby relieving cell aging; and miR-656-3p inhibitor can effectively improve skin photoaging. Therefore, miR-656-3p is a key micro RNA in the occurrence and development of skin photoaging for the first time, and provides a new marker for diagnosing skin photoaging; provides a new target point for preventing and treating skin photoaging.
The invention provides micro RNA (miR-656-3 p) and application thereof in preparation of a reagent for diagnosing skin photoaging and an anti-skin photoaging medicament. The reagent for detecting the miR-656-3p content can be used as a novel marker for diagnosing skin photoaging, and the dilemma that clinical skin photoaging diagnosis only depends on appearance and partial scale is solved. The medicine containing the inhibitor component can prevent and treat skin photoaging from the aspect of improving cell aging, and solves the problem that the clinical medicine for resisting skin photoaging is less to a certain extent.
Drawings
FIG. 1 is a cluster heatmap of normal human skin sample differential microRNAs screened by high throughput sequencing under solar UV irradiation and protected from solar UV irradiation.
FIG. 2 is a result of RT-PCR detection of miR-656-3p content in UVB irradiated melanocytes.
FIG. 3 is the effect on melanocyte cycle and intracellular beta-galactosidase content following knockdown of miR-656-3p in melanocytes.
FIG. 4 is a graph showing the effect of UVB irradiation and miR-656-3p content on the content of LMNB2 in melanocytes.
FIG. 5 is a validation of the relationship of the dual luciferase reporter gene system to miR-656-3p and LMNB2 targets in melanocytes.
FIG. 6 is a verification of the anti-photoaging effect of miR-656-3p inhibitor on skin in the back molding area of a photoaging model of a nude mouse.
Detailed Description
The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.
The experimental procedure, in which no specific conditions are noted in the examples, is generally followed by conventional conditions, for example as described in Sambrook et al, molecular cloning, A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.
Example 1: high throughput microRNA expression profiling for detection of solar UV-irradiated and solar UV-irradiated normal human skin samples
1. Obtaining skin samples
Fresh skin samples were obtained from the outpatient clinic of this unit (Zhejiang one-time plastic surgery), and the skin samples were taken from healthy females between 30 and 40 years old, excluding the donors who had stopped menstruation, had pregnant, had smoked, had drunk, and were taken in 3 cases, each case providing 1 skin sample exposed to solar ultraviolet rays and each skin sample exposed to solar ultraviolet rays, respectively.
Wherein the skin irradiated with solar ultraviolet light is obtained from eyelid skin obtained by eyelid cosmetic surgery, wherein the skin protected from solar ultraviolet light is obtained from abdominal or chest skin obtained by abdominal or chest plastic surgery.
Specifically, the skin tissue is collected after the regular plastic surgery, and the skin tissue is treated as soon as possible. Shearing skin tissue, wherein the thickness of the skin is not more than 0.5cm, shearing the skin into a plurality of small tissue blocks with the thickness of 0.5cm multiplied by 1cm (thickness multiplied by length multiplied by width), placing the sheared small tissue blocks into sterile freezing tubes, and adding 2ml RNAlater (Sigma company) into each tube to immerse the small tissue blocks for preservation; and placing 1 piece of cut skin tissue into each freezing tube, and storing in a deep low temperature refrigerator at-80 ℃.
2. Total RNA extraction of skin samples
MirVana Using MirRNA specifically extracted from general tissues and cellular microRNAs TM miRNA Isolation Kit without phenol reagent (Ambion company), extracting total RNA of the sample according to the standard operation flow provided by the manufacturer, and detecting the total RNA obtained by extraction by Agilent Bioanalyzer 2100 (Agilent technologies company) electrophoresis quality for later use.
3. High throughput microRNA sequencing
Library construction was performed with a Total RNA/. Gtoreq.50 ng small RNA starting amount of 1. Mu.g, the main steps were: a) 3' adapter ligation; b) A 5' adapter linkage; c) First strand cDNA synthesis; d) Performing PCR amplification; e) Fragment size selection and library quality inspection with Agilent2200tape station; sample preparation by quality inspection library according to the method described in HiSeq 2500User Guide; hiSeq 2500 was run on-machine using Single Read Flow Cell and single-end (1X 50) standard sequencing procedure was run to map the heat, resulting in microRNA expression profiles of skin exposed to solar UV light and skin protected from solar UV light. The results show that miR-656-3p is significantly up-regulated in skin tissue exposed to solar uv light (see figure 1).
Wherein miR-656-3p is expressed with a significant difference between skin irradiated with solar ultraviolet light and skin samples protected from solar ultraviolet light, see in particular table 1.
TABLE 1 sequencing experiments screening miR-656-3p has significant differences in skin exposed to solar UV and skin protected from solar UV
Example 2: results of RT-PCR detection of content of miR-656-3p in UVB-irradiated melanocytes
1. Primary human skin melanocyte isolation and identification
Taking the foreskin of a healthy infantTissue (foreskin is one of the most abundant and active sites of human melanocytes, with a density of about 2400/mm 2 ) The solution of chloramphenicol (0.25%) was added and each rinse was continued for 5min 3 times. The peeled fat was trimmed with an ophthalmic scissors and rinsed 3 times with PBS. Placed in 0.6U/mL neutral protease (Dispase II) and digested at 4℃for 12h. The epidermis was separated from the dermis and digested with 0.05% trypsin plus 0.53mmol/mL EDTA at 37℃for 30min, and the pancreatin was stopped by adding 10% fetal bovine serum in DMEM. After filtering out the cell suspension with a 100-mesh filter, the cells were centrifuged at 1000r/min×5min to obtain epidermal cells, which were inoculated at 15 ten thousand/well into a six-well plate and cultured in a melanocyte medium (# 2201), and the culture medium was changed once for 3 days. And (3) inhibiting the growth of other cells of the epidermis in a melanocyte culture medium, carrying out passage when the melanocyte grows to 90% fusion, continuously purifying the melanocyte with the increase of passage times, selecting 4-10 passages of cells for test, and identifying by using a melanoma antibody Melan-A (titer 1:50) and exogenous levodopa color detection.
2. UVB irradiation of melanocytes
UVB lamp at 30mJ/cm 2 Or 60mJ/cm 2 Melanocytes were irradiated at 24-hour intervals for 3 consecutive days, followed by culturing for 48 hours.
3. Real-time fluorescent quantitative qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detection of expression level of miRNAs in melanocyte
Melanocytes were digested with 0.25% pancreatin at 2000r/min×15min (4 ℃), washed with PBS, centrifuged 3 times, and the cell pellet was taken, total RNA was extracted with Trizol reagent (Invitrogen), and the concentration was determined by UV spectrophotometry. The total RNA is used as a template, a reverse transcription kit (TaKaRa) and a SYBR Premix Ex TaqTM fluorescent quantitative PCR kit (TaKaRa) and RT-PCR primers (PCR detection of an upstream Primer sequence SEQ ID NO.2: cgcgcgcaatattatacagtcaacctc of miR-656-3p, a downstream Primer is a universal post Primer Takara mRQ 3' Primer, a reverse transcription Primer is a Random Primer (dN) 6, and quantitative analysis of miR-656-3p in melanocytes, a delta Ct model and REST2005 software is carried out by reverse transcription with a poly (A) tailing method. The differences in expression of miRNAs in melanocytes with/without UVB irradiation were compared. The results show that miR-656-3p is significantly upregulated in melanocytes with UVB irradiation (see figure 2).
Example 3: knocking down miR-656-3p in melanocyte, and detecting influence on melanocyte cycle and intracellular beta-galactosidase content
miR-656-3p inhibitor (SEQ ID NO.3: agaggttgactgtataatatt) is transfected into melanocytes after UVB irradiation, and cell cycle and beta-galactosidase content are detected.
1. Cell cycle distribution determination of melanocyte cycle
Cells were resuspended in 100 μl PBS and gradually added 75% ethanol to 3mL to fix the cells. The fixed cells were washed with PBS, then stained with a cell cycle kit (MultiSciences, hangzhou, china), and analyzed using a flow cytometer (BD Biosciences, usa).
2. Aging-related beta-galactosidase (SA-beta-gal) staining for detecting melanocyte aging level
SA- β -gal staining was used to detect cell senescence. SA-beta-gal staining kit was purchased from Beyotime Biotechnology institute (Shanghai, china). Cells were first fixed, washed 3 times with PBS, and then incubated overnight with SA- β -gal staining solution in the dark at 37 ℃. After washing with PBS, blue stained cells were scored under an inverted bright field microscope.
The results show that miR-656-3p inhibitor can reverse the melanocyte cycle block and the beta-galactosidase content increase caused by UVB irradiation (see FIG. 3).
Example 4: RT-PCR and Western blot detection of influence of UVB irradiation and miR-656-3p content on LMNB2 content in melanocytes
1. qRT-PCR detection of intracellular LMNB2 expression level after UVB irradiation
Melanocytes were digested with 0.25% pancreatin at 2000r/min×15min (4 ℃), washed with PBS, centrifuged 3 times, and the cell pellet was taken, total RNA was extracted with Trizol reagent (Invitrogen), and the concentration was determined by UV spectrophotometry. And (3) taking total RNA as a template, adopting a reverse transcription kit (TaKaRa) and a SYBR Premix Ex TaqTM fluorescent quantitative PCR kit (TaKaRa) and RT-PCR primers to detect miR-656-3p in melanocytes, a delta Ct model and REST2005 software for quantitative analysis of qRT-PCR data. Differences in expression of LMNB2 mRNA in melanocytes irradiated with UVB at different intensities were compared.
2. Western blot detection of reversion of LMNB2 expression in cells after UVB irradiation by miR-656-3p inhibitor
The cell culture solution is discarded, the cell culture solution is washed 2 times by PBS precooled at 4 ℃, cells are lysed by RIPA lysate containing 1% of phenylmethylsulfonyl fluoride (PMSF) on ice for 30min, the cell culture solution is centrifuged for 5000r/min multiplied by 15min (4 ℃), protein is quantified after supernatant is taken, loading buffer solution is added according to the ratio of 4:1, and the cell culture solution is boiled for 10min. Proteins were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyvinylidene fluoride (PVDF) membrane transfer was performed for 100min, and 5% BSA was blocked for 1.5h at room temperature by shaking. Adding diluted primary antibody according to the antibody specification, incubating overnight at 4 ℃, washing a membrane by using TBS-T buffer solution, incubating the secondary antibody for 1h at normal temperature, dripping a chemiluminescent reagent on a PVDF membrane, detecting the LMNB2 protein expression in melanocytes, observing the strips on an imaging analyzer, developing and photographing.
The results showed that the expression level of LMNB2 in melanocytes decreased with increasing UVB irradiation intensity, and miR-656-3 pininhibitor reversed UVB-induced down-regulation of LMNB2 (see fig. 4).
Example 5: verification of target relation between miR-656-3p and LMNB2 in melanocytes by using dual-luciferase reporter gene system
By bioinformatics method and literature search, the binding site between LMNB2 'UTR and miR-656-3p is predicted by using Targetscan, miRDB, miRTarBase and MiRWalk database, and primers are designed to PCR amplify LMNB2 (WT LMNB 2) and mutant LMNB2 (MUT LMNB 2) 3' UTR sequences or directly perform gene synthesis and then insert into a dual-luciferase reporter gene vector. Synthesizing corresponding miR-656-3p and miR-656-3p negative control, co-transfecting a double-luciferase reporter gene vector of LMNB 2' UTR and miR-656-3p or miR-656-3p negative control into a melanocyte, detecting double-luciferase after 48h, and determining that a target gene of miR-656-3p is LMNB2. The results show that the target gene of miR-656-3p in melanocytes is LMNB2 (see FIG. 5).
Example 6: verification of anti-photoaging effect of miR-656-3p inhibitor on skin of back molding area of photoaging model of nude mice
The miR-656-3p inhibitor is injected into the skin of the back molding area of the photo-aging model of the nude mice at multiple points, photo-aging manifestations such as skin wrinkles and color sinking of the molding area are obviously reduced, pathological sections show that the dermis thickness is increased, the epidermis tissue structure is more complete, and collagen fibers are arranged in a tidy wavy manner.
1. Construction of photoaging animal models
Female BALB/c nude mice were raised to 8 weeks of age and then began UVB irradiation. A UVB ultraviolet lamp tube (Philips TL 20W/12) of 15W was placed on top of a 40cm high carton (well ventilated) and the experimental group mice were placed in the carton. Once daily, each UVB irradiation was performed for 2 hours, followed by 8 weeks. The rough degree of the skin, the elastic state of the skin, the appearance of wrinkles, no pigmentation and the like are observed by naked eyes. The photoaged histopathological changes were observed using HE staining and Masson staining.
2. Subcutaneous injection of miR-656-3p inhibitor
The nude mice were injected subcutaneously into the back molding area at 4 injection sites for a total of 1OD miR-7704 antagonists twice a week for a total of two weeks.
The results showed that skin wrinkles and color sinks in the molded area showed significantly reduced photoaging, and HE and Masson stained pathological sections showed increased dermis thickness, more complete epidermal tissue structure, and a neat wavy arrangement of collagen fibers (see fig. 6).
Claims (6)
1. A microrna as a biomarker, wherein the microrna is miR-656-3p, and the nucleotide sequence of miR-656-3p is shown in SEQ ID No. 1: tctccaactgacatattataa.
2. The microrna of claim 1, wherein the miR-656-3p is derived from human tissue specimens and isolated cells or is obtained synthetically.
3. The microrna of claim 1, wherein the target gene of the marker miR-656-3p comprises LMNB2.
4. Use of the microrna of claim 1 as a biomarker in the preparation of a reagent for diagnosing photoaging of skin.
5. Use of the microrna of claim 1 as a biomarker in the preparation of a medicament for the prevention and treatment of skin photoaging.
6. The use according to claim 4 or 5, wherein the skin photoaging is melanocyte aging.
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