A Preliminary Study Of The Role Of MicroRNA In The Pathogenesis Of Early Mycosis Fungoides And Clinicopathologic Analyses Of 6 Patients With Granulomatous Slack Skin

Mycosis fungoides (MF) is the most common type of cutaneous T cell lymphoma (CTCL). Its pathogenesis is unclear. The conventional Alibert-Bazin type MF is a slowly progressive disease, wihich characterized clinically by an evolution of patches, plaques to tumors. MF has various clinical manifestations and may be easily misdiagnosed or improperly treated. microRNAs (miRNAs) are small nonprotein-encoding RNA molecules, of about 22 nucleotides in length. They play a pivotal role in many cellular processes, including cell growth and proliferation, differentiation,’metabolism and apoptosis. miRNAs may also play essential role in the pathogenesis of malignancies. Over the lastfive years, several reports analyzing the role of miRNAs in pathogenesis, diagnose, and prognosis of MF have been published, which brings potential new tool for treating MF. This preliminary study is the first research in China to analyze the relationship between early MF and miRNAs. Our research emphasizes on the role of miRNAs in the pathogenesis of MF.Part1:A preliminary study of the role of miRNA in the pathogenesis of early mycosis fungoidesChapter 1:Screening microRNAs related to early mycosis fungoidesObjective To screen microRNAs(miRNAs) related to early mycosis fungoides (MF). Methods Differentially expressed miRNAs between tissue samples from 6 patients with early MF and 6 control patients with eczema or lichen planus were screened with a high throughout miRNA PCR array. The differentially expressed miRNAs were then verified by real-time fluorescent quantitative PCR (RT-qPCR) with tissue samples, Myla and Hut-78 cell lines. Results Three differentially expressed miRNAs were showed in the array, which were hsa-miR-378a-5p、hsa-miR-302c-3p、hsa-miR-107. All of them were statistically significantly up-regulated. RT-qPCR results of tissue samples were in accordance with miRNA array. However, the RT-qPCR results of Myla cell line showed significantly up-regulated hsa-miR-378a-5p and hsa-miR-107, compared with peripheral T lymph cell from a healthy volunteer, hsa-miR-302c-3p was not significantly different. 3 miRNAs were not significantly differentiated expressed in Hut-78 cell line.Conclusion There are different expression profiles of miRNAs between early MF and benign inflammatory dermatoses.Chapter 2:Prediction of target genes and pathways by the bioinformatics analysis of hsa-miR-378a-5pObjective The bioinformatics analysis of hsa-miR-378a-5p was performed, including prediction of target genes and GO pathway analysis. Methods Online databases such as TargetScan, was used for predicting target genes. Based on the literature and the prediction result, we chose SUFU and IGF1R as possible target genes, classical MAPK and p38 MAPK as possible pathways for analysis and validation with RT-qPCR in CTCL cell lines. Results The predicted target genes were enriched in cellular proliferation, T cell differentiation, apoptosis, immune regulation, angiogenesis, and so on. GO pathway analysis showed the target genes were mainly involved in TGF-β signaling pathway, p53 pathway, Wnt pathway, p38 MAPK pathway. RT-qPCR of SUFU and IGF1R were performed in Myla Hut-78 and T lymph cells, which showed SUFU and IGF1R were significantly highly expressed in Myla cell line (P<0.05), but not significant in Hut-78 cell line. ERK1、ERK2、p38, as the key genes in MAPK and p38 MAPK pathways, were also significantly highly expressed in Myla and Hut-78 cells (P<0.05). Conclusion The analysis of target genes of hsa-miR-378a-5p lies the foundation for subsequent research about its function and regulatory mechanisms in MF.Chapter 3:The influence of hsa-miR-378a-5p siRNA to Myla cell lineObjective To investigate the influence of hsa-miR-378a-5p siRNA on Myla cell line.Methods miR-378a-5p siRNA and negative control (NC) was transfected to Myla cells through liposomes. CCK-8 was performed to explore the proliferation of different groups. Western blot was performed to study the protein expression level of SUFU. Results Myla/miR-378a-5p-siRNA, Myla/NC-siRNA and Myla groups were established. The expression of miR-378a-5p was inhibited in Myla/miR-378a-5p-siRNA. CCK-8 results revealed the proliferation of Myla/miR-378a-5p-siRNA groups at 24h was inhibited, comparing with the other groups.48h,72h, and 96h showed negative results. SUFU protein was highly expressed at 24h,48h, and 72h. Conclusion miR-378a-5p expression was interfered successfully. SUFU may not be relative to miR-378a-5p in MF.Part 2:Clinicopathologic analyses of 6 patients with granulomatous slack skinObjective To study the clinicopathological, diagnostic and therapeutic features of granulomatous slack skin (GSS). Methods 6 cases were diagnosed in our hospital in recent 7 years. Their clinical and pathological data were analyzed retrospectively.Results The mean age of onset was 29 years. All 6 patients presented with pendulous skin folds.5 of them mainly or only affected the flexure areas, and the other patient involved the right chest. One patient was accompanied with classical MF. One patient suffered from itching while the others had no clinical symptoms. The histology demonstrates non-caseating granulomas with macrophages and medium-sized lymphoid cells. Atypical lymphoid cells were seen in 1 patient. Epidermotropism was observed in another patient. The lymphoid tumor cells have a T helper cell phenotype with expression of CD3 and CD4. Elastin stain was performed in 3 patients, which showed fracture, destruction, and even engulfment of elastic fibers by multiclear giant cells. There were monoclonal rearrangement of TCR y genes in 3 patients. The lesions in 2 patients were excised completely after radiotherapy with electron beam or superficial X-ray, and recurrence was not reported. The lesions in other 4 patients were improved after treatment with intramuscular administration of interferon α -2b, external use of mechlorethamine solution (0.02%) and (or) partial excision. Conclusion GSS is rarely seen. The diagnosis should be based on the characteristic skin lesions, histology, and immunohistolochemistry. GSS has a slowly progressive course and over treatment was not recommended. Surgical excision could be the first choice for GSS located to the flexure areas.