Understanding the role of non-coding RNAs in skin homeostasis and cancer

The epidermis is a stratified epithelium with continuous self-renewing capacity. As the outermost layer of our body, it provides a protective barrier against external trauma, produces pigmentation, and keeps the skin hydrated. Keratinocytes are the primary constituent cells type within the epidermis. A fine balance is maintained between keratinocyte cell proliferation and differentiation to sustain a functional epidermis. The interplay between multiple signaling pathways, transcription factors, epigenetic modulators, and non-coding RNAs is the key to maintaining this balance. Disrupted epidermal homeostasis can cause various diseases, including cancer. Cutaneous squamous cell carcinoma (cSCC) is one such keratinocyte-derived cancer that begins with the accumulation of somatic mutations and genetic abnormalities. The pigment-producing melanocytes within the epidermis can undergo oncogenic transformation due to numerous genetic and environmental factors, and give rise to malignant melanoma. In this thesis, we have explored the role of non-coding RNAs in epidermal homeostasis and the development of skin cancers. Paper I: In this study, we investigated the role of miR-203 in cSCC, and found that its expression was negatively correlated with the differentiation grade of the tumors. Functionally, miR-203 inhibited cell cycle progression, self-renewability, motility and proangiogenic-activity of cSCC cells in vitro, and reduced xenograft tumor growth and angiogenesis in vivo. We identified c-MYC as a potential upstream regulator of the transcriptomic changes caused by miR-203 overexpression, and subsequently demonstrated that c-MYC is a direct target of miR203 in cSCC. In line with these findings, overexpression of c-Myc rescued the growthinhibitory effect of miR-203 in cSCC cell lines. Paper II: In this study, we analyzed the small RNA-seq data from the skin cutaneous melanoma (TCGA SKCM-cohort) and found that miR-203 is the most downregulated miRNA in metastatic melanoma. Moreover, high miR-203 abundance seems to confer longer overall survival to patients with metastatic melanoma. Methylome data from patient samples, together with results from in vitro experiments, suggested that promoter hypermethylation could suppress miR-203 expression in metastatic tumors. Functionally, miR-203 acted as a tumor suppressor by inhibiting cancer/metastatic hallmarks such as cell migration, invasion, selfrenewal, and angiogenesis. SLUG, an essential regulator of epithelial-mesenchymal transition, was found to be a direct target of miR-203. In vivo, miR-203 effectively suppressed melanoma metastasis to the inguinal lymph nodes and the lungs. Paper III: In this study, we investigated the changes in the coding and non-coding landscape in cSCC using RNA-seq. We identified a large number of differentially expressed coding transcripts linear lncRNAs and circRNAs. Representative transcripts from each group were validated using an extended cohort. We found that several transcription factors regulating skin development and cSCC oncogenesis were altered at mRNA level. In addition to various lncRNAs with potential oncogenic function, we identified a set of skin-specific lncRNAs, which were mostly downregulated in cSCC. We observed a global downregulation of circRNA abundance in cSCC. Apart from previously annotated circRNAs, novel skin-enriched circRNAs were also identified and validated. Paper IV: In this study, we characterized a skin-specific lncRNA located at the Epidermal Differentiation Complex (EDC) on human chromosome 1. It is highly induced during the late stages of keratinocyte differentiation and localized to the granular layer of the human epidermis. We showed that transcription factor YY1 suppresses its expression in the progenitor keratinocytes. CRISPR-mediated activation of this lncRNA locus led to an increased expression of late differentiation marker genes. In contrast, loss-of-function experiment in a 3D organotypic skin model resulted in impaired terminal differentiation program and formation of thinner cornified envelope. Due to its functional requirement in late differentiation, we have renamed this lncRNA as ELDAR (Epidermal Late Differentiation Associated RNA)

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project “Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing”, we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside- enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis

MiR-130a Acts as a Tumor Suppressor MicroRNA in Cutaneous Squamous Cell Carcinoma and Regulates the Activity of the BMP/SMAD Pathway by Suppressing ACVR1

Cutaneous Squamous Cell Carcinoma (cSCC) is a malignant neoplasm of the skin resulting from the accumulation of somatic mutations due to solar radiation. It is one of the fastest increasing malignancies and it represents a particular problem among immunosuppressed individuals. MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein-coding genes at the posttranscriptional level. Here we identify miR-130a to be downregulated in cSCC compared with healthy skin and with precancerous lesions (actinic keratosis) and demonstrate that it is regulated at the transcriptional level by HRAS and MAPK-signaling. We report that miR-130a suppresses the growth of cSCC xenografts in mice. We demonstrate that overexpression of miR-130a suppresses long-term capacity of growth, cell motility and invasion ability in human cSCC cell lines. Mechanistically, miR-130a directly targets Activin A receptor, type I (ACVR1/ALK2) and changes in miR-130a levels result in the diminished activity of BMP/SMAD1 pathway via ACVR1. These data reveal a link between activated MAPK-signaling and decreased expression of miR-130a, which acts as a tumor suppressor miRNA in cSCC and contributes to a better understanding of molecular processes in malignant transformation of epidermal keratinocytes

The Keratinocyte Transcriptome in Psoriasis: Pathways Related to Immune Responses, Cell Cycle and Keratinization

Psoriasis is a common immune-mediated disease resulting from altered cross-talk between keratinocytes and immune cells. Previous transcriptomic studies have identified thousands of deregulated genes in psoriasis skin; however, the transcriptomic changes confined to the epidermal compartment remained poorly characterized. The aim of this study was to characterize the transcriptomic landscape of psoriatic keratinocytes, using sorted CD45neg epidermal cells. Genes with functions in innate immunity, type I interferon response, cell cycle and keratinization were enriched among deregulated genes in psoriatic keratinocytes. Gene set enrichment analysis indicated the dominance of interleukin (IL)-22/IL-17A signatures in the epidermal psoriasis-signature. A set of deregulated genes overlapped with psoriasis-associated genetic regions, suggesting that genetic variations affecting gene expression in keratinocytes contribute to susceptibility to psoriasis. Several psoriasis-susceptibility genes, which were previously believed to be expressed preferentially or exclusively in immune cells, were identified as having altered expression in psoriatic keratinocytes. These results highlight the role of keratinocytes in the pathogenesis of psoriasis, and indicate that both genetic factors and an inflammatory microenvironment contribute to epidermal alterations in psoriasis

'We pledge to improve the health of our entire community': Improving health worker motivation and performance in Bihar, India through teamwork, recognition, and non-financial incentives.

BACKGROUND:Motivation is critical to health worker performance and work quality. In Bihar, India, frontline health workers provide essential health services for the state's poorest citizens. Yet, there is a shortfall of motivated and skilled providers and a lack of coordination between two cadres of frontline health workers and their supervisors. CARE India developed an approach aimed at improving health workers' performance by shifting work culture and strengthening teamwork and motivation. The intervention-"Team-Based Goals and Incentives"-supported health workers to work as teams towards collective goals and rewarded success with public recognition and non-financial incentives. METHODS:Thirty months after initiating the intervention, 885 health workers and 98 supervisors completed an interviewer-administered questionnaire in 38 intervention and 38 control health sub-centers in one district. The questionnaire included measures of social cohesion, teamwork attitudes, self-efficacy, job satisfaction, teamwork behaviors, equitable service delivery, taking initiative, and supervisory support. We conducted bivariate analyses to examine the impact of the intervention on these psychosocial and behavioral outcomes. RESULTS:Results show statistically significant differences across several measures between intervention and control frontline health workers, including improved teamwork (mean = 8.8 vs. 7.3), empowerment (8.5 vs. 7.4), job satisfaction (7.1 vs. 5.99) and equitable service delivery (6.7 vs. 4.99). While fewer significant differences were found for supervisors, they reported improved teamwork (8.4 vs. 5.3), and frontline health workers reported improved fulfillment of supervisory duties by their supervisors (8.9 vs. 7.6). Both frontline health workers and supervisors found public recognition and enhanced teamwork more motivating than the non-financial incentives. CONCLUSIONS:The Team-Based Goals and Incentives model reinforces intrinsic motivation and supports improvements in the teamwork, motivation, and performance of health workers. It offers an approach to practitioners and governments for improving the work environment in a resource-constrained setting and where there are multiple cadres of health workers

Additional file 1: of Evaluation of the mobile nurse training (MNT) intervention – a step towards improvement in intrapartum practices in Bihar, India

Direct Observation of Delivery tool. (DOCX 86 kb

Genome-Wide Screen for MicroRNAs Reveals a Role for miR-203 in Melanoma Metastasis

Melanoma is one of the deadliest human cancers with limited therapeutic options. MicroRNAs are a class of short noncoding RNAs regulating gene expression at the post-transcriptional level. To identify important miRNAs in melanoma, we compared the miRNome of primary and metastatic melanomas in The Cancer Genome Atlas dataset and found lower miR-203 abundance in metastatic melanoma. Lower level of miR-203 was associated with poor overall survival in metastatic disease. We found that the methylation levels of several CpGs in the MIR203 promoter negatively correlated with miR-203 expression and that treatment with the demethylating agent 5-aza-2-deoxycytidine induced miR-203 expression, which was associated with demethylation of the promoter CpGs, in melanoma cell lines. In vitro, there was a decreased expression of miR-203 in melanoma cell lines in comparison with primary melanocytes. Ectopic overexpression of miR-203 suppressed cell motility, colony formation, and sphere formation as well as the angiogenesis-inducing capacity of melanoma cells. In vivo, miR-203 inhibited xenograft tumor growth and reduced lymph node and lung metastasis. SLUG was shown as a target of miR-203, and knockdown of SLUG recapitulated the effects of miR-203, whereas its restoration was able to reverse the miR-203-mediated suppression of cell motility. These results establish a role for miR-203 as a tumor suppressor in melanoma which suppresses both early and late steps of metastasis. Hence, restoration of miR-203 has therapeutic potential in melanoma.WoSScopu

A comprehensive analysis of coding and non-coding transcriptomic changes in cutaneous squamous cell carcinoma

Cutaneous Squamous Cell Carcinoma (cSCC) is the most common and fastest-increasing cancer with metastatic potential. Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are novel regulators of gene expression. To identify mRNAs, lncRNAs and circRNAs, which can be involved in cSCC, RNA-seq was performed on nine cSCCs and seven healthy skin samples. Representative transcripts were validated by NanoString nCounter assays using an extended cohort, which also included samples from pre-cancerous skin lesions (actinic keratosis). 5,352 protein-coding genes, 908 lncRNAs and 55 circular RNAs were identified to be differentially expressed in cSCC. Targets of 519 transcription factors were enriched among differentially expressed genes, 105 of which displayed altered level in cSCCs, including fundamental regulators of skin development (MYC, RELA, ETS1, TP63). Pathways related to cell cycle, apoptosis, inflammation and epidermal differentiation were enriched. In addition to known oncogenic lncRNAs (PVT1, LUCAT1, CASC9), a set of skin-specific lncRNAs were were identified to be dysregulated. A global downregulation of circRNAs was observed in cSCC, and novel skin-enriched circRNAs, circ_IFFO2 and circ_POF1B, were identified and validated. In conclusion, a reference set of coding and non-coding transcripts were identified in cSCC, which may become potential therapeutic targets or biomarkers

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project “Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing”, we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twentytwo miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3KAkt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis