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Micro RNAs upregulated in Vitiligo skin play an important role in its aetiopathogenesis by altering TRP1 expression and keratinocyte-melanocytes cross-talk.

ABSTRACT: Translation of genes is regulated by many factors including microRNAs (miRNAs). miRNA profiling of lesional and non-lesional epidermal RNA from 18 vitiligo patients revealed significant upregulation of 29 miRNAs in the lesional epidermis, of which 6 miRNAs were transfected in normal human epidermal keratinocytes (NHEKs) to study their downstream effects using quantitative proteomics. Many proteins involved in oxidative stress, Vesicle trafficking, Cellular apoptosis, Mitochondrial proteins and Keratins were regulated after miRNA transfections in the keratinocytes. However, tyrosinase related protein-1 (TRP1/TYRP1), a melanogenesis protein, was consistently downregulated in NHEKs by all the six miRNAs tested, which was quite intriguing. TRP1 was also downregulated in lesional epidermis compared with non-lesional epidermis. Since melanocytes synthesize and transfer melanosomes to the surrounding keratinocytes, we hypothesized that downregulation of TRP1 in NHEKs may have a role in melanosome transfer, which was confirmed by our co-culture experiments. Downregulation of TRP1 in keratinocytes negatively affected the melanosome transfer from melanocytes to keratinocytes resulting in melanin accumulation which may be leading to melanin induced cytotoxicity in melanocytes. Regulation of key processes involved in aetiopathogenesis of vitiligo along with TRP1 suggests that miRNAs act in an integrated manner which may be detrimental for the loss of melanocytes in vitiligo.

SUBMITTER: Vaish U

PROVIDER: S-EPMC6625998 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Purpose: In order to explore the mechanism of hydroxychloroquine (HCQ) in treating vitiligo. We report the application of transcriptome profiling (RNA-seq) in human vitiligo melanocytes cell line PIG3V after HCQ treatment. Methods: The PIG3V cells were divided randomly in two groups: HCQ treatment (1 μg/mL, 24 hours) and the cells without treatment (only medium). RNA samples extracted from PIG3V cells were quantified by NanoDrop and Bioanalyzer. After 2 rounds of purification Poly A RNA was extracted then from total RNA by using Dynabeads Oligo. Small pieces of Poly A RNA were returned through Magnesium RNA Fragmentation Module under the condition of 94℃ for 5 minutes. Subsequently, SuperScript Reverse Transcriptase was used to reverse-transcribe the cleaved RNA pieces into cDNA. Then, U-labeled second-stranded DNAs was synthesized. After adding of A-base to each strand, preparing for indexed adapters, and heat-labile UDG enzyme treatment, PCR amplification was performed under (1) 95℃, 3 minutes; (2) 98℃, 15 seconds for 8 cycles; (3) 60℃, 15 seconds, 72℃, 5 minutes. Ultimately, RNA sequencing was performed according to protocol of Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China). After calculation, differential expressed genes were screened by fold change (FC)＞2 or FC＜0.5 and p value＜0.05. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample and identified 60612 genes and 229420 transcripts. Approximately 0.3% of the genes showed differential expression between the HCQ treated PIG3V cells and the cells without HCQ, with a fold change＞2 or＜0.5 , p value＜0.05. Conclusions: Our research showed the first detailed transcriptome analysis of human vitiligo melanocytes PIG3V, with or without HCQ treatment. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic mechanisms of HCQ in treating vitiligo.

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Micro RNAs upregulated in Vitiligo skin play an important role in its aetiopathogenesis by altering TRP1 expression and keratinocyte-melanocytes cross-talk.

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