VHL suppresses RAPTOR and inhibits mTORC1 signaling in clear cell renal cell carcinoma.

Inactivation of the tumor suppressor von Hippel-Lindau (VHL) gene is a key event in hereditary and sporadic clear cell renal cell carcinomas (ccRCC). The mechanistic target of rapamycin (mTOR) signaling pathway is a fundamental regulator of cell growth and proliferation, and hyperactivation of mTOR signaling is a common finding in VHL-dependent ccRCC. Deregulation of mTOR signaling correlates with tumor progression and poor outcome in patients with ccRCC. Here, we report that the regulatory-associated protein of mTOR (RAPTOR) is strikingly repressed by VHL. VHL interacts with RAPTOR and increases RAPTOR degradation by ubiquitination, thereby inhibiting mTORC1 signaling. Consistent with hyperactivation of mTORC1 signaling in VHL-deficient ccRCC, we observed that loss of vhl-1 function in C. elegans increased mTORC1 activity, supporting an evolutionary conserved mechanism. Our work reveals important new mechanistic insight into deregulation of mTORC1 signaling in ccRCC and links VHL directly to the control of RAPTOR/mTORC1. This may represent a novel mechanism whereby loss of VHL affects organ integrity and tumor behavior.

Pareto optimality between growth-rate and lag-time couples metabolic noise to phenotypic heterogeneity in Escherichia coli.

Despite mounting evidence that in clonal bacterial populations, phenotypic variability originates from stochasticity in gene expression, little is known about noise-shaping evolutionary forces and how expression noise translates to phenotypic differences. Here we developed a high-throughput assay that uses a redox-sensitive dye to couple growth of thousands of bacterial colonies to their respiratory activity and show that in Escherichia coli, noisy regulation of lower glycolysis and citric acid cycle is responsible for large variations in respiratory metabolism. We found that these variations are Pareto optimal to maximization of growth rate and minimization of lag time, two objectives competing between fermentative and respiratory metabolism. Metabolome-based analysis revealed the role of respiratory metabolism in preventing the accumulation of toxic intermediates of branched chain amino acid biosynthesis, thereby supporting early onset of cell growth after carbon starvation. We propose that optimal metabolic tradeoffs play a key role in shaping and preserving phenotypic heterogeneity and adaptation to fluctuating environments.

Progesterone Receptors Promote Quiescence and Ovarian Cancer Cell Phenotypes via DREAM in p53-Mutant Fallopian Tube Models.

CONTEXT: The ability of ovarian steroids to modify ovarian cancer (OC) risk remains controversial. Progesterone is considered to be protective; recent studies indicate no effect or enhanced OC risk. Knowledge of progesterone receptor (PR) signaling during altered physiology that typifies OC development is limited. OBJECTIVE: This study defines PR-driven oncogenic signaling mechanisms in p53-mutant human fallopian tube epithelia (hFTE), a precursor of the most aggressive OC subtype. METHODS: PR expression in clinical samples of serous tubal intraepithelial carcinoma (STIC) lesions and high-grade serous OC (HGSC) tumors was analyzed. Novel PR-A and PR-B isoform-expressing hFTE models were characterized for gene expression and cell cycle progression, emboli formation, and invasion. PR regulation of the DREAM quiescence complex and DYRK1 kinases was established. RESULTS: STICs and HGSC express abundant activated phospho-PR. Progestin promoted reversible hFTE cell cycle arrest, spheroid formation, and invasion. RNAseq/biochemical studies revealed potent ligand-independent/-dependent PR actions, progestin-induced regulation of the DREAM quiescence complex, and cell cycle target genes through enhanced complex formation and chromatin recruitment. Disruption of DREAM/DYRK1s by pharmacological inhibition, HPV E6/E7 expression, or DYRK1A/B depletion blocked progestin-induced cell arrest and attenuated PR-driven gene expression and associated OC phenotypes. CONCLUSION: Activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Our data support an alternative perspective on the tenet that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease; our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting PRs, DREAM, and/or DYRKs.

Disentangling Intrinsic and Extrinsic Gene Expression Noise in Growing Cells.

Gene expression is a stochastic process. Despite the increase of protein numbers in growing cells, the protein concentrations are often found to be confined within small ranges throughout the cell cycle. Generally, the noise in protein concentration can be decomposed into an intrinsic and an extrinsic component, where the former vanishes for high expression levels. Considering the time trajectory of protein concentration as a random walker in the concentration space, an effective restoring force (with a corresponding "spring constant") must exist to prevent the divergence of concentration due to random fluctuations. In this work, we prove that the magnitude of the effective spring constant is directly related to the fraction of intrinsic noise in the total protein concentration noise. We show that one can infer the magnitude of intrinsic, extrinsic, and measurement noises of gene expression solely based on time-resolved data of protein concentration, without any a priori knowledge of the underlying gene expression dynamics. We apply this method to experimental data of single-cell bacterial gene expression. The results allow us to estimate the average copy numbers and the translation burst parameters of the studied proteins.

Neuroepithelial cell competition triggers loss of cellular juvenescence.

Cell competition is a cell-cell interaction mechanism which maintains tissue homeostasis through selective elimination of unfit cells. During early brain development, cells are eliminated through apoptosis. How cells are selected to undergo elimination remains unclear. Here we aimed to identify a role for cell competition in the elimination of suboptimal cells using an in vitro neuroepithelial model. Cell competition was observed when neural progenitor HypoE-N1 cells expressing RASV12 were surrounded by normal cells in the co-culture. The elimination through apoptosis was observed by cellular changes of RASV12 cells with rounding/fragmented morphology, by SYTOX blue-positivity, and by expression of apoptotic markers active caspase-3 and cleaved PARP. In this model, expression of juvenility-associated genes Srsf7 and Ezh2 were suppressed under cell-competitive conditions. Srsf7 depletion led to loss of cellular juvenescence characterized by suppression of Ezh2, cell growth impairment and enhancement of senescence-associated proteins. The cell bodies of eliminated cells were engulfed by the surrounding cells through phagocytosis. Our data indicates that neuroepithelial cell competition may have an important role for maintaining homeostasis in the neuroepithelium by eliminating suboptimal cells through loss of cellular juvenescence.

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells.

Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ∼2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci, and three loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ∼30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

LncRNA GAS5 suppresses CD4+ T cell activation by upregulating E4BP4 via inhibiting miR-92a-3p in systemic lupus erythematosus.

Increasing evidence reveals that long noncoding RNAs (lncRNAs) are associated with autoimmune and inflammatory diseases, such as systemic lupus erythematosus (SLE). In this study, we aimed to explore the role of lncRNA growth arrest specific 5 (GAS5) in the pathogenesis of SLE. We found that lncRNA GAS5 was decreased in CD4+ T cells and plasma from SLE patients. Overepression of GAS5 inhibited activation of normal CD4+ T cells and attenuated the self-reactivity of SLE CD4+ T cells. Additionally, we demonstrated that adenovirus E4 binding protein 4 (E4BP4) was involved in lncRNA GAS5-mediated inhibition of CD4+ T cell activation. GAS5 could upregulate E4BP4 by inhibiting miR-92a-3p. Taken together, our results indicate that the GAS5/miR-92a-3p/E4BP4 pathway plays an important role in inhibiting CD4+ T cell activation in SLE, thus providing a potential therapeutic target for SLE treatment.

Functional annotation of human long noncoding RNAs via molecular phenotyping.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Genome-wide identification of loci associated with growth in rainbow trout.

BACKGROUND: Growth is a major economic production trait in aquaculture. Improvements in growth performance will reduce time and cost for fish to reach market size. However, genes underlying growth have not been fully explored in rainbow trout. RESULTS: A previously developed 50 K gene-transcribed SNP chip, containing ~ 21 K SNPs showing allelic imbalances potentially associated with important aquaculture production traits including body weight, muscle yield, was used for genotyping a total of 789 fish with available phenotypic data for bodyweight gain. Genotyped fish were obtained from two consecutive generations produced in the NCCCWA growth-selection breeding program. Weighted single-step GBLUP (WssGBLUP) was used to perform a genome-wide association (GWA) analysis to identify quantitative trait loci (QTL) associated with bodyweight gain. Using genomic sliding windows of 50 adjacent SNPs, 247 SNPs associated with bodyweight gain were identified. SNP-harboring genes were involved in cell growth, cell proliferation, cell cycle, lipid metabolism, proteolytic activities, chromatin modification, and developmental processes. Chromosome 14 harbored the highest number of SNPs (n = 50). An SNP window explaining the highest additive genetic variance for bodyweight gain (~ 6.4%) included a nonsynonymous SNP in a gene encoding inositol polyphosphate 5-phosphatase OCRL-1. Additionally, based on a single-marker GWA analysis, 33 SNPs were identified in association with bodyweight gain. The highest SNP explaining variation in bodyweight gain was identified in a gene coding for thrombospondin-1 (THBS1) (R2 = 0.09). CONCLUSION: The majority of SNP-harboring genes, including OCRL-1 and THBS1, were involved in developmental processes. Our results suggest that development-related genes are important determinants for growth and could be prioritized and used for genomic selection in breeding programs.

Correlation between cribriform/intraductal prostatic adenocarcinoma and percent Gleason pattern 4 to a 22-gene genomic classifier.

BACKGROUND: The Decipher test measures expression of 22 RNA biomarkers associated with aggressive prostate cancer used to improve risk stratification of patients to help guide management. To date, Decipher's genomic classification has not been extensively correlated with specific histologic growth patterns in prostatic adenocarcinoma. With a growing understanding of the clinical aggressiveness associated with cribriform growth pattern (CF), intraductal carcinoma (IDC), and percent Gleason pattern 4 (G4%), we sought to determine if their presence was associated with an increased genomic risk as measured by the Decipher assay. DESIGN: Clinical use of the Decipher assay was performed on the highest Gleason score (GS) tumor nodule of prostatectomy specimens from a prospective cohort of 48 patients, with GS varying from 7 through 9 to help guide clinical risk stratification. The tumors were reviewed for CF, IDC, and G4%, which were then compared to the Decipher score (0-1) and risk stratification (high vs not high). RESULTS: The presence of CF/IDC was significantly associated with Decipher risk score (P = .007), with a high-risk Decipher score in 22% vs 56% of patients without or with CF/IDC. On binary logistic regression analysis, G4% (odds ratio [OR] 1.04 per percent increase [95% confidence interval [CI], 1.02-1.06]; P = .0004) and CF predominant (OR, 9.60 [95%CI, 1.48-62.16]; P = .02) were significantly associated with a high-risk GC score. IDC did not reach significance (OR, 1.92 [95%CI, 0.65-5.67]; P = .24). CONCLUSIONS: Our findings add to an expanding knowledge base that supports G4% and CF/IDC as molecularly unique and clinically relevant features in prostatic adenocarcinoma. These histologic features should be standardly reported as they are associated with more aggressive prostate cancer. Future work should determine the independent information of these histologic findings that are relative to genomic assessment on long-term outcomes.

A Genetic Screen in Drosophila To Identify Novel Regulation of Cell Growth by Phosphoinositide Signaling.

Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP3, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, we performed a genetic screen to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, we identified and classified additional candidates in the developing Drosophila melanogaster eye that regulate growth either cell autonomously or via cell-cell interactions. We report three genes: Pi3K68D, Vps34 and fwd that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. Our findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins.

Distinct TP53 Mutation Subtypes Differentially Influence Cellular Iron Metabolism.

The most commonly mutated gene in all human cancers is the tumor suppressor gene TP53; however, in addition to the loss of tumor suppressor functions, mutations in TP53 can also promote cancer progression by altering cellular iron acquisition and metabolism. The primary objective of this work was to determine how TP53 mutation status influences the molecular control of iron homeostasis. The effect of TP53 mutation type on cellular iron homeostasis was examined using cell lines with inducible versions of either wild-type TP53 or a representative mutated TP53 gene from exemplary "hotspot" mutations in the DNA binding domain (R248, R273, and R175) as well as H193Y. The introduction of distinct TP53 mutation types alone was sufficient to disrupt cellular iron metabolism. These effects were mediated, at least in part, due to differences in the responsiveness of iron regulatory proteins (IRPs) to cellular iron availability. IRPs are considered the master regulators of intracellular iron homeostasis because they coordinate the expression of iron storage (ferritin) and iron uptake (transferrin receptor) genes. In response to changes in iron availability, cells harboring either a wild-type TP53 or R273H TP53 mutation displayed canonical IRP-mediated responses, but neither IRP1 RNA binding activity nor IRP2 protein levels were affected by changes in iron status in cells harboring the R175H mutation type. However, all mutation types exhibited robust changes in ferritin and transferrin receptor protein expression in response to iron loading and iron chelation, respectively. These findings suggest a novel, IRP-independent mode of iron regulation in cells expressing distinct TP53 mutations. As TP53 is mutated in nearly half of all human cancers, and iron is necessary for cancer cell growth and proliferation, the studies have implications for a wide range of clinically important cancers.

In ovaries with high or low variation in follicle size, granulosa cells of antral follicles exhibit distinct size-related processes.

Antral follicle size might be a valuable additive predictive marker for IVF outcome. To better understand consequences of antral follicle size as a marker for reproductive outcome, we aimed to obtain insight in follicle size-related granulosa cell processes, as granulosa cells play an essential role in follicular development via the production of growth factors, steroids and metabolic intermediates. Using the pig as a model, we compared gene expression in granulosa cells of smaller and larger follicles in the healthy antral follicle pool of sows, which had a high variation versus low variation in follicle size. Selected gene expression was confirmed at the protein level. Granulosa cells of smaller antral follicles showed increased cell proliferation, which was accompanied by a metabolic shift towards aerobic glycolysis (i.e. the Warburg effect), similar to other highly proliferating cells. High granulosa cell proliferation rates in smaller follicles might be regulated via increased granulosa cell expression of the androgen receptor and the epidermal growth factor receptor, which are activated in response to locally produced mitogens. While granulosa cells of smaller follicles in the pool are more proliferative, granulosa cells of larger follicles express more maturation markers such as insulin-like growth factor-1 (IGF1) and angiopoietin 1 (ANGPT1) and are therefore more differentiated. As both higher IGF1 and ANGPT1 have been associated with better IVF outcomes, the results of our study imply that including smaller follicles for oocyte aspiration might have negative consequences for IVF outcome.

Inhibition of MicroRNA-381 Promotes Tumor Cell Growth and Chemoresistance in Clear-Cell Renal Cell Carcinoma.

BACKGROUND MicroRNA-381 (miR-381) is proven to be involved in many human tumors. Bioinformatics prediction suggests that miR-381 is decreased in renal cell carcinoma. However, its biological functions in clear-cell renal cell carcinoma (ccRCC) remain largely unknown. The present research aimed to evaluate miR-381 expression in renal cancer tissues and its effects on cell proliferation, growth, migration, and chemoresistance. MATERIAL AND METHODS Sixty pairs of ccRCC and the adjacent non-tumor specimens were collected during routine therapeutic surgery. Quantitative real-time PCR (qRT-PCR) assay was employed to examine miR-381 expression in the ccRCC tissues and the associated adjacent tissues (the normal tissues adjacent to tumor tissues). Cell transfection assay and Thiazolyl Blue Tetrazolium Bromide (MTT) assay were utilized to observe effects of miR-381 on the cell proliferation, growth, invasion, and chemoresistance in the Caki-1 cell line and 786-O cell line. Flow cytometry was used to assess cell apoptosis. Caki-1 cell and 786-O cell Xenograft BALB/c mouse models were established. RESULTS miR-381 expression was downregulated in ccRCC tissues in vivo and in cell lines in vitro. Downregulation of miR-381 promoted growth of cells and restrained the ccRCC cell apoptosis. Increased miR-381 combined with Ci and Pa suppressed the proliferation and enhanced the anti-tumor effects of Ci and Pa at tolerated concentrations in vitro. miR-381 inhibition promoted chemoresistance in vitro. CONCLUSIONS miR-381 levels were significantly downregulated in renal cancer tissues and miR-381 inhibition promoted tumor cell growth, migration, and chemoresistance.

miR-186-5p promotes cell growth, migration and invasion of lung adenocarcinoma by targeting PTEN.

OBJECTIVE: To explore the expression of miR-186-5p in lung adenocarcinoma (LUAD) and its possible function associated with cancer cell proliferation, migration and invasion. METHODS: MiR-186-5p expression levels in LUAD samples, human LUAD cell lines H1299 and NCI-H1975, and normal human lung epithelial cell line BEAS-IB were assessed by quantitative real-time PCR (qRT-PCR). H1299 and NCI-H1975 cells were transfected with miR-186-5p mimic or miRNA negative control. CCK-8 assay was performed to evaluate the cell proliferation. Transwell assay and transwell-matrigel™ invasion assay were applied to assess the migration and invasion abilities of H1299 and NCI-H1975 cells. RESULTS: miR-186-5p expression was significantly up-regulated in LUAD tumor tissues and LUAD cell lines as compared with tumor-adjacent tissues and normal human lung epithelial cells, respectively. MiR-186-5p overexpression remarkably promoted the proliferation, migration and invasion of LUAD cells. Furthermore, phosphatase and tensin homolog (PTEN) was a direct target of miR-186-5p verified by luciferase reporter assay. Overexpression of PTEN significantly suppressed LUAD cells to proliferate, migrate and invade. MiR-186-5p overexpression-induced LUAD cell phenotype could be partially rescued by co-overexpression of miR-186-5p and PTEN. CONCLUSION: This study demonstrated that miR-186-5p is up-regulated in LUAD, and functionally associated with cell proliferation, migration and invasion. MiR-186-5p promotes the proliferation, migration and invasion of LUAD cells by targeting PTEN. MiR-186-5p may be utilized as a novel molecular marker and therapeutic target of LUAD.

PDCD4 Is an Androgen-Repressed Tumor Suppressor that Regulates Prostate Cancer Growth and Castration Resistance.

Androgen receptor (AR) transcriptional activity contributes to prostate cancer development and castration resistance. The growth and survival pathways driven by AR remain incompletely defined. Here, we found PDCD4 to be a new target of AR signaling and a potent regulator of prostate cancer cell growth, survival, and castration resistance. The 3' untranslated region of PDCD4 is directly targeted by the androgen-induced miRNA, miR-21. Androgen treatment suppressed PDCD4 expression in a dose responsive and miR-21-dependent manner. Correspondingly, AR inhibition dose-responsively induced PDCD4 expression. Using data from prostate cancer tissue samples in The Cancer Genome Atlas (TCGA), we found a significant and inverse correlation between miR-21 and PDCD4 mRNA and protein levels. Higher Gleason grade tumors exhibited significantly higher levels of miR-21 and significantly lower levels of PDCD4 mRNA and protein. PDCD4 knockdown enhanced androgen-dependent cell proliferation and cell-cycle progression, inhibited apoptosis, and was sufficient to drive androgen-independent growth. On the other hand, PDCD4 overexpression inhibited miR-21-mediated growth and androgen independence. The stable knockdown of PDCD4 in androgen-dependent prostate cancer cells enhanced subcutaneous tumor take rate in vivo, accelerated tumor growth, and was sufficient for castration-resistant tumor growth. IMPLICATIONS: This study provides the first evidence that PDCD4 is an androgen-suppressed protein capable of regulating prostate cancer cell proliferation, apoptosis, and castration resistance. These results uncover miR-21 and PDCD4-regulated pathways as potential new targets for castration-resistant prostate cancer.

A size-invariant bud-duration timer enables robustness in yeast cell size control.

Cell populations across nearly all forms of life generally maintain a characteristic cell type-dependent size, but how size control is achieved has been a long-standing question. The G1/S boundary of the cell cycle serves as a major point of size control, and mechanisms operating here restrict passage of cells to Start if they are too small. In contrast, it is less clear how size is regulated post-Start, during S/G2/M. To gain further insight into post-Start size control, we prepared budding yeast that can be reversibly blocked from bud initiation. While blocked, cells continue to grow isotropically, increasing their volume by more than an order of magnitude over unperturbed cells. Upon release from their block, giant mothers reenter the cell cycle and their progeny rapidly return to the original unperturbed size. We found this behavior to be consistent with a size-invariant 'timer' specifying the duration of S/G2/M. These results indicate that yeast use at least two distinct mechanisms at different cell cycle phases to ensure size homeostasis.

Uncovering Natural Longevity Alleles from Intercrossed Pools of Aging Fission Yeast Cells.

Quantitative traits often show large variation caused by multiple genetic factors . One such trait is the chronological lifespan of non-dividing yeast cells, serving as a model for cellular aging. Screens for genetic factors involved in aging typically assay mutants of protein-coding genes. To identify natural genetic variants contributing to cellular aging, we exploited two strains of the fission yeast, Schizosaccharomyces pombe, that differ in chronological lifespan. We generated segregant pools from these strains and subjected them to advanced intercrossing over multiple generations to break up linkage groups. We chronologically aged the intercrossed segregant pool, followed by genome sequencing at different times to detect genetic variants that became reproducibly enriched as a function of age. A region on Chromosome II showed strong positive selection during aging. Based on expected functions, two candidate variants from this region in the long-lived strain were most promising to be causal: small insertions and deletions in the 5'-untranslated regions of ppk31 and SPBC409.08 Ppk31 is an ortholog of Rim15, a conserved kinase controlling cell proliferation in response to nutrients, while SPBC409.08 is a predicted spermine transmembrane transporter. Both Rim15 and the spermine-precursor, spermidine, are implicated in aging as they are involved in autophagy-dependent lifespan extension. Single and double allele replacement suggests that both variants, alone or combined, have subtle effects on cellular longevity. Furthermore, deletion mutants of both ppk31 and SPBC409.08 rescued growth defects caused by spermidine. We propose that Ppk31 and SPBC409.08 may function together to modulate lifespan, thus linking Rim15/Ppk31 with spermidine metabolism.

MicroRNA-365 Inhibits Cell Growth and Promotes Apoptosis in Melanoma by Targeting BCL2 and Cyclin D1 (CCND1).

BACKGROUND MicroRNA-365 (miR-365) is involved in the development of a variety of cancers. However, it remains largely unknown if and how miRNAs-365 plays a role in melanoma development. MATERIAL AND METHODS In this study, we overexpressed miR-365 in melanoma cell lines A375 and A2058, via transfection of miR-365 mimics oligos. We then investigated alterations in a series of cancer-related phenotypes, including cell viability, cell cycle, apoptosis, colony formation, and migration and invasion capacities. We also validated cyclin D1 (CCND1) and BCL2 apoptosis regulator (BCL2) as direct target genes of miR-365 by luciferase reporter assay and investigated their roles in miR-365 caused phenotypic changes. To get a more general view of miR-365's biological functions, candidate target genes of miR-365 were retrieved via searching online databases, which were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for potential biological functions. We then analyzed The Cancer Genome Atlas (TCGA) Skin Cutaneous Melanoma (SKCM) dataset for correlation between miR-365 level and clinicopathological features of patients, and for survival of patients with high and low miR-365 levels. RESULTS We found that miR-365 was downregulated in melanoma cells. Overexpression of miR-365 remarkably suppressed cell proliferation, induced cell cycle arrest and apoptosis, and compromised the migration and invasion capacities in A375 and A2058 cell lines. We also found that the phenotypic alterations by miR-365 were partially due to downregulation of CCND1 and BCL2 oncogenes. The bioinformatics analysis revealed that predicted targets of miR-365 were widely involved in transcriptional regulation and cancer-related signaling pathways. However, analysis of SKCM dataset failed to find differences in miR-365 level among melanoma patients at different clinicopathologic stages. The Kaplan-Meier analysis also failed to discover significant differences in overall survival and disease-free survival between patients with high and low miR-365 levels. CONCLUSIONS Our findings suggested that miR-365 might be an important novel regulator for melanoma formation and development, however, the in vivo roles in melanoma developments need further investigation.

Transcriptional alterations in skin fibroblasts from Parkinson's disease patients with parkin mutations.

Mutations in the parkin gene (PRKN) are the most common cause of autosomal-recessive juvenile Parkinson's disease (PD). PRKN encodes an E3 ubiquitin ligase that is involved in multiple regulatory functions including proteasomal-mediated protein turnover, mitochondrial function, mitophagy, and cell survival. However, the precise molecular events mediated by PRKN mutations in PRKN-associated PD (PRKN-PD) remain unknown. To elucidate the cellular impact of parkin mutations, we performed an RNA sequencing study in skin fibroblasts from PRKN-PD patients carrying different PRKN mutations (n = 4) and genetically unrelated healthy subjects (n = 4). We identified 343 differentially expressed genes in PRKN-PD fibroblasts. Gene ontology and canonical pathway analysis revealed enrichment of differentially expressed genes in processes such as cell adhesion, cell growth, and amino acid and folate metabolism among others. Our findings indicate that PRKN mutations are associated with large global gene expression changes as observed in fibroblasts from PRKN-PD patients and support the view of PD as a systemic disease affecting also non-neural peripheral tissues such as the skin.