Comparison of miRNA-101a-3p and miRNA-144a-3p regulation with the key genes of alpaca melanocyte pigmentation
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Many miRNA functions have been revealed to date. Single miRNAs can participate in life processes by regulating more than one target gene, and more than one miRNA can also simultaneously act on one target mRNA. Thus, a complex regulatory network involved in many processes can be formed. Herein, the pigmentation regulation mechanism of miR-101a-3p and miR-144a-3p was studied at the cellular level by the overexpression and equal overexpression of miR-101a-3p and miR-144a-3p.
Results revealed that miR-101a-3p and miR-144a-3p directly regulated the expression of microphthalmia-associated transcription factor, thereby affecting melanin synthesis.
The two miRNAs with the same binding site in the same gene independently excreted each other’s function. However, the inhibitory effect of miR-144a-3p was stronger than that of miR-101-3p in alpaca melanocytes, although both decreased melanin production.
KeywordsMelanocyte miRNA-101a-3p miRNA-144a-3p Melanin
microphthalmia-associated transcription factor
tyrosinase-related proteins 1
tyrosinase-related proteins 2
As agricultural and companion animals, alpacas (Vicugna pacos) have a great variety of natural coat colors. The alpaca’s hair, known as soft gold, has considerable economic value, and it has become a good model for studying animal hair color genes . Melanocytes are present in mammalian hair, skin, and irises. In these tissues, the distribution, proliferation, survival, and number and type of melanocytes, as well as the production of melanin, are regulated by various factors [2, 3]. At present, more than 400 genes have been found to regulate animal hair color. A series of genes can regulate melanocyte development and melanin [4, 5]. Microphthalmia-associated transcription factor (MITF) is an important regulator of melanocyte survival and development, and MITF controls their transcription by binding to tyrosinase (TYR) and TYR-related proteins 1 (TYRP1) and 2 (TYRP2) promoter regions, which are predominantly involved in melanin synthesis [6, 7]. In animals, miRNAs inhibit target mRNA translation by pairing with the specific bases of the target mRNA gene; miRNAs are involved in regulating gene expression and a range of biological functions, such as cell differentiation and tissue and tumor development [8, 9]. Numerous miRNAs have been discovered with the development of sequencing technology. Single miRNAs can participate in life processes by regulating more than one target gene, and more than one miRNA can also simultaneously act on one target mRNA [10, 11]. Thus, a complex regulatory network involved in many processes, such as skin tissue development, melanocyte formation, pigment cell migration, and melanin formation, is formed [12, 13].
In the present study, TargetScan software demonstrated that miR-101-3p and miR-144-3p exhibited the same binding site on the 3′UTR of MITF. The functions of miR-101-3p and miR-144-3p have been verified in many tumors, but no study on pigmentation in melanocytes has been conducted. Can miR-101-3p and miR-144-3p regulate hair color? Can both of them inhibit the same target gene?
Materials and methods
Alpaca melanocytes were established and maintained at the Alpaca Biological Engineering Laboratory (Shanxi Agricultural University, Taigu, Jinzhong, Shanxi. China) [14, 15]. Alpacas were for research animals from Alpaca Farm (Zhuangzi, Yuci, Jinzhong, Shanxi, China). Housing and care of the alpacas and collection of skin samples were approved by the Animal Experimentation Ethics Committee of Shanxi Agricultural University, Taigu, China (SXAU-EAW-2019-L013003). Punch skin biopsies (4 × 8 mm) were obtained from alpacas under local anesthesia . After the skin tissues were taken, debridement therapy was performed for the alpacas’ injury. The sixth-passage alpaca melanocytes from the Alpaca Biological Engineering Laboratory were used in this experiment.
The frozen melanocytes were resuscitated in a water bath at 37 °C, and an equal amount of Dulbecco’s minimum essential medium (DMEM; Gibco, New York, NY, USA) with 10% fetal bovine serum was added and centrifuged at 1000g/min for 10 min. The supernatant was discarded, and the cell pellet was suspended in melanocyte medium (MelM; ScienCell, Carlsbad, CA, USA). The cells were seeded into six-well plates and maintained at 37 °C and 5% CO2. Some cells were used to isolate total RNA, and some were further cultured for transfection experiments.
HEK 293T cells were purchased from Ribobio (Guangzhou, China), seeded into six-well plates, and maintained (37 °C, 5% CO2) in DMEM (Gibco, New York, USA) supplemented with 10% fetal bovine serum. HEK 293T cells were prepared for luciferase assays.
miR-101a-3p and miR-144a-3p bioinformatics analysis
The homology of miR-101a-3p, miR-144a-3p, and MITF 3′UTR in human, bovine, chicken, mouse, camel, and other species was analyzed using DNAMAN (LynnonBiosoft, USA). The target relationship among miR-101a-3p, miR-144a-3p, and MITF was predicted using TargetScan (TargetScan 5.2, https://www.targetscan.org).
To examine the MITF 3′UTR as a target of miR-101a-3p and miR-144a-3p in vitro, luciferase assays were performed using a pmiRGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Fitchburg, WI, USA), containing both the coding sequences of firefly luciferase and Renilla luciferase (internal control). The miR-144a-3p and miR-101a-3p binding site or mismatched site of MITF 3′UTR (NW 005882703.1) was cloned into the pmiRGLO vector system. HEK 293T cells were transfected (48 h) with either wild type (WT) or mutant pmirGLO dual-luciferase vector (100 ng) or miRNA mimics (50 nM) by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. A dual-luciferase reporter assay system (Promega) was then used to stimulate a luminescent signal that was measured by a luminometer (Glomax; Promega). Luminescence values were normalized to those produced by co-transfected Renilla luciferase constructs . An empty pmiRGLO vector was used as a background control. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared with that of the empty pmiRGLO vector control. For each transfection, luciferase activity was averaged from three replicates.
miR-101a-3p and miR-144a-3p mimics and inhibitors were synthesized by Ribobio (Ribobio, Guangzhou, China). Melanocyte transfections were performed using riboFECT™ CP (Ribobio) in accordance with the manufacturer’s protocol. Approximately 5 μL of 20 μM miRNA mimic was diluted with 120 μL of 1× riboFECT™ CP buffer and added with 12 μL of riboFECT™ CP reagent. The mixture was incubated for 15 min at room temperature. The prepared riboFECT™ CP mixture was added to 1863 μL of cell culture medium. The melanocyte density reached 50% during transfection. The melanocytes were confluent in six-well plates. The culture plate was placed in a CO2 incubator at 37 °C for 48 h, and the cells were then collected.
RNA isolation and reverse transcription
The melanocytes were confluent in the six-well plate. The medium in the culture plate was discarded, and the cells were gently washed three times with PBS (Ca2+-free, Mg2+-free) at 37 °C. PBS was then discarded. Total RNA was isolated from the cells by using a Trizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration was assessed using a NanoDrop 2000c spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and the quality was confirmed by 1% agarose gel electrophoresis. The isolated mRNAs and miRNAs were then reverse transcribed to cDNA using a SYBR Green I qRT-PCR (TaKaRa, Dalian, China) or miRcute miRNA SYBR Green I qRT-PCR kit (Tiangen, Beijing, China) in accordance with the manufacturer’s instructions, respectively.
Quantitative real-time PCR detection of the expression of miR-101a-3p, miR-144a-3p, MITF, TYR, TYRP1, and TYRP2
Primers used in this study
Western blot analysis
Whole-cell protein was extracted from melanocytes using a protein extraction kit (Solarbio, Beijing, China), and the concentrations of the isolated cell lysates were determined spectrophotometrically by using a NanoDrop 2000c spectrophotometer (NanoDrop Technologies). The protein samples were added to 1% SDS and 5× protein loading buffer and boiled for 10 min for protein denaturation. Heat-denatured protein samples (200 ng per lane) were resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Boster, Wuhan, China). The membranes were placed in blocking solution (5% non-fat dehydrated milk) and blocked at room temperature on a shaker (1 h, 80 r/min). The membranes were then placed in the diluted primary antibody at 4 °C overnight with mouse monoclonal MITF (Thermo Fisher Scientific) or rabbit monoclonal TYR, TYRP1, or TYRP2 (Abcam, Cambridge, UK) antibodies (1:2000 in Tris-buffered saline supplemented with Tween-20 [TBST]). A mouse monoclonal antibody to β-actin (1:3000 in TBST; TransGen Biotech, Beijing, China) was used as the control. The membranes were washed three times with TBST for 10 min each time. The membranes were placed in the diluted secondary antibody with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (1:1700 in TBST; Cwbio, Beijing, China), incubated at 37 °C for 1 h, and washed six times with TBST for 5 min each time. Finally, the membranes were detected by using a Superstar ECL Plus ready-to-use kit (Boster) according to the manufacturer’s instructions. The membranes were scanned on a ChemiDoc XRS + imager (Bio-Rad, Hercules, CA, USA), and the intensities of the generated protein signals were quantified using Image-Pro Plus software (Media Cybernetics, Inc., Georgia, MD, USA). The methodology has been published previously, because those protein detection conditions were same .
The glass slides were placed in a 24-well culture plate. The frozen melanocytes were resuscitated in a 37 °C water bath, and an equal amount of DMEM and 10% fetal bovine serum was added. The mixture was centrifuged at 1000g/min for 10 min, and the supernatant was discarded. The cell pellet was suspended in melanocyte culture medium, dispensed in a 24-well plate, and maintained at a cell incubator (37 °C, 5% CO2).
The cells on the glass slides were fixed with 4% paraformaldehyde (4 °C, 30 min), incubated with 3% hydrogen peroxide (25 °C, 20 min), and blocked with blocking solution (37 °C, 35 min). The primary antibody (1:100) was added and reacted at 4 °C overnight, and the secondary antibody (1:200) was added and reacted at 37 °C for 30 min. The cells were stained with DAB and hematoxylin for 6 min and 30 s, respectively. The cells were then dehydrated with 70%, 80%, 90%, 95% ethanol; 100% ethanol I, and 100% ethanol II for 2, 2, 4, 4, 4 and 4 min, respectively. The cells were made transparent by using xylene I and II for 10 and 10 min, respectively. Finally, the glass slides were sealed with neutral resin.
Melanocytes were collected, washed three times with PBS, and subjected to cell counting. The cells were lysed using 0.2 mol/L NaOH (106 cells/mL) and dispensed in a 96-well plate. The absorbance of the samples was measured at a wavelength of 475 nm [16, 17] by using a Multiskan Spectrum microplate reader (Thermo Fisher Scientific). Each group was repeated three times.
The results of real-time quantitative PCR were quantified using the comparative threshold cycle method established by Livak and Schmittgen , and gene expression levels were normalized to those of U6 and 18S rRNA. Immunohistochemistry was used to analyze the optical density by using Image pro plus. Data were presented as the mean ± standard error. Significant (P < 0.05 or P < 0.01) or non-significant (P > 0.05) differences were evaluated via ANOVA using SPSS (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software (MacStats, UCI Graduate School of Management).
Bioinformatics analysis of miR-101a-3p and miR-144a-3p
Confirmation of the MITF 3′UTR as a target of miR-101a-3p and miR-144a-3p by luciferase reporter assays
miR-101a-3p and miR-144a-3p-modulated MITF and its downstream gene expression
In the miRNA inhibitor groups, the expression levels of TYR, TYRP1, and TYRP2 mRNA in the transfected miR-144a-3p mimic, miR-101a-3p, and miR-101a-3p & miR-144a-3p (1:1) groups were significantly higher than those in the miRNA mimic group (P < 0.001, P < 0.001, and P < 0.001; Fig. 4b–d).
miR-101a-3p and miR-144a-3p-modulated MITF and its downstream gene protein expression
Protein localization and expression of miR-101-3p and miR-144-3p on MITF and its downstream gene
Effects of miR-101-3p and miR-144-3p on melanin synthesis
Melanin is synthesized by melanocytes, and melanin deposited in hair determines its color. MITF regulates many pigment-related enzymes and is a critical transcription factor. TYR, TYRP1, and TYRP2 are important downstream genes of MITF and key enzymes for pigmentation. Therefore, many different external signals affect melanin production by regulating the expression of MITF and its downstream genes [19, 20]. miRNAs are highly conserved non-coding RNAs that inhibit post-transcriptional regulation of target genes by binding to the target genes.
miR-203 was first identified miRNA in skin, which is involved in the regulation of melanocyte pigmentation . The expression level of miR-203 is increased in the skin of patients with psoriasis compared with that in normal skin, and miR-203 plays an important role in mouse epidermal differentiation [22, 23]. miR-137 acts on MITF by binding to the specific bases of MITF mRNA and affects the expression of its downstream genes TYR, TYRP1, and TYRP2, thereby regulating pigmentation . The role of miR-340 and miR-25 in the formation of coat color has been reported in the literature [25, 26]. miR-101a-3p and miR-144a-3p play important roles in cell apoptosis, immunity, and tumor suppression [27, 28]. However, the miR-101-3p and miR-144-3p hair color pigmentation mechanisms remain largely unknown. Only one study has reported that miR-101-3p inhibits melanoma proliferation and expansion by regulating MITF in melanoma .
miRNA regulation of target genes is at the post-transcriptional level, and cleavage is the mechanism of target gene mRNA; its translational inhibition depends mainly on its degree of complementation with the target gene mRNA sequence . In our study, software predictions and analysis demonstrated that miR-101a-3p and miR-144a-3p targeted the same site of MITF 3′UTR, and the degree of miR-144a-3p regulating MITF was stronger than that of miR-101a-3p. After the overexpression of miR-101a-3p and miR-144a-3p, the expression level of MITF mRNA remained the same, and the MITF protein expression decreased. The mRNA and protein expression levels of TYR, TYRP1, and TYRP2 significantly decreased, and the melanin content also decreased. The regulation of miR-144a-3p on the expression of MITF protein was stronger than that of miR-101-3p, and the melanin content was lower than the miR-101a-3p group. The two miRNAs regulated the expression of MITF, which further affected downstream genes (TYR, TYRP1, and TYRP2) to fulfill the regulation of melanocyte pigmentation.
miR-101-3p and miR-144a-3p exhibited the same binding site on the 3′UTR of MITF. The inhibitory effect of miR-144a-3p was stronger than that of miR-101-3p in alpaca melanocytes, which decreased melanin production.
We would like to thank Prof. Changsheng Dong (College of Animal Science and Technology, Shanxi Agricultural University) for assistance, and the members of the Alpaca Biological Engineering Laboratory for providing alpaca melanocytes.
ZZ and PL designed the experiments. ZZ, YM and YL performed the experiments and wrote the manuscript; ZC performed the data analyses and contributed reagents/materials; HL, LZ and DX helped perform the analysis with constructive discussions. All authors read and approved the final manuscript.
This study was supported by a grant from the Key Research and Development Project of Shanxi Province (Grant No. 201803D31062) for some regents of the study, the Program for the Top Young Innovative Talents of Shanxi Agricultural University (Grant No. TYIT201403) for analysis, and interpretation of data and in writing the manuscript and the National Natural Science Foundation of China (Grant Nos. 31402156 and 31873002) for design of the study.
Ethics approval and consent to participate
Housing and care of the alpacas and collection of skin samples were approved by the Animal Experimentation Ethics Committee of Shanxi Agricultural University, Taigu, China (SXAU-EAW-2019-L013003).
Consent for publication
The authors declare that they have no competing interests.
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