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PDF file - 858KB, Supplementary Methods, Supplementary References, Figure S1-6, Table S1-5. Figure S1. Identification of a new miR in FHIT intron. Figure S2. The SNP variant around the Pri-C2-miR region in FHIT did not affect the C2-miR expression. Figure S3. C2-miR and FHIT are under the same transcriptional control. Figure S4. C2-miR inhibiting human tumor cell growth was not through promoting apoptosis. Figure S5. C2-miR inhibits human tumor cell growth through targeting CCND1, ERBB2, DNMT3A or DNMT3B. Figure S6. Multiple members of the miR-548 family could target the same oncogenes. Table S1. The documented miR-548 family members. Table S2. Quantities of FHIT or C2-miR in total cell RNA (20 ng). Table S3. Expression comparison of pri-miRs or mature miRs in 293FT cells. Table S4. Primers used in this study. Table S5. NanoString probes for detecting 29 members of the miR-548 family.

Loss or attenuated expression of the tumor-suppressor gene FHIT is associated paradoxically with poor progression of human tumors. Fhit promotes apoptosis and regulates reactive oxygen species; however, the mechanism by which Fhit inhibits tumor growth in animals remains unclear. In this study, we used a multidisciplinary approach based on bioinformatics, small RNA library screening, human tissue analysis, and a xenograft mouse model to identify a novel member of the miR-548 family in the fourth intron of the human FHIT gene. Characterization of this human-specific microRNA illustrates the importance of this class of microRNAs in tumor suppression and may influence interpretation of Fhit action in human cancer. Cancer Res; 74(8); 2283–94. ©2014 AACR.

Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ~9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.

Loss or attenuated expression of the tumor-suppressor gene FHIT is associated paradoxically with poor progression of human tumors. Fhit promotes apoptosis and regulates reactive oxygen species; however, the mechanism by which Fhit inhibits tumor growth in animals remains unclear. In this study, we used a multidisciplinary approach based on bioinformatics, small RNA library screening, human tissue analysis, and a xenograft mouse model to identify a novel member of the miR-548 family in the fourth intron of the human FHIT gene. Characterization of this human-specific microRNA illustrates the importance of this class of microRNAs in tumor suppression and may influence interpretation of Fhit action in human cancer. Cancer Res; 74(8); 2283–94. ©2014 AACR.

MicroRNAs (miRNAs) are a class of endogenous molecules that post-transcriptionally regulate target gene expression and play an important role in many developmental processes. Matrix extracellular phosphoglycoprotein (MEPE) is related to bone metabolism. We recently reported that MEPE protects cells from DNA damage-induced killing. The purpose of this study is to investigate whether miRNAs targeting MEPE play an important role in DNA damage response. We report in this study that miR-376a directly targets MEPE, and overexpression of miR-376a reduces the G2 arrest of the cells and sensitizes the cells to DNA damage-induced killing. These results indicate an association of MEPE gene inactivation with decreased survival after DNA damage and also provide useful information for miRNA-based drug development: a new target for sensitizing human tumor cells to radiotherapy or chemotherapy.

Previously, we discovered that ZFP57 is a maternal-zygotic effect gene, and it maintains DNA methylation genomic imprint at multiple imprinted regions in mouse embryos. Despite these findings, it remains elusive how DNA methyltransferases are targeted to the imprinting control regions to initiate and maintain DNA methylation imprint. To gain insights into these essential processes in genomic imprinting, we examined how ZFP57 maintains genomic DNA methylation imprint in mouse embryonic stem (ES) cells. Here we demonstrate that the loss of ZFP57 in mouse ES cells led to a complete loss of genomic DNA methylation imprint at multiple imprinted regions, similar to its role in mouse embryos. However, reintroduction of ZFP57 into Zfp57-null ES cells did not result in reacquisition of DNA methylation imprint, suggesting that the memory for genomic imprinting had been lost or altered in Zfp57-null ES cells in culture. Interestingly, ZFP57 and DNA methyltransferases could form complexes in the presence of KAP1/TRIM28/TIF1β when co-expressed in COS cells. We also found that the wild-type exogenous ZFP57 but not the mutant ZFP57 lacking the KRAB box that interacts with its co-factor KAP1/TRIM28/TIF1β could substitute for the endogenous ZFP57 in maintaining the DNA methylation imprint in ES cells. These results suggest that ZFP57 may recruit DNA methyltransferases to its target regions to maintain DNA methylation imprint, and this interaction is likely facilitated by KAP1/TRIM28/TIF1β.

We have previously reported that resistin induces hypertrophy and impairs contractility in isolated rat cardiomyocytes. To examine the long-term cardiovascular effects of resistin, we induced in vivo overexpression of resistin using adeno-associated virus serotype 9 injected by tail vein in rats and compared to control animals. Ten weeks after viral injection, overexpression of resistin was associated with increased ratio of left ventricular (LV) weight/body weight, increased end-systolic LV volume and significant decrease in LV contractility, measured by the end-systolic pressure volume relationship slope in LV pressure volume loops, compared to controls. At the molecular level, mRNA expression of ANF and β-MHC, and protein levels of phospholamban were increased in the resistin group without a change in the level of SERCA2a protein expression. Increased fibrosis by histology, associated with increased mRNA levels of collagen, fibronectin and connective tissue growth factor were observed in the resistin-overexpressing hearts. Resistin overexpression was also associated with increased apoptosis in vivo, along with an apoptotic molecular phenotype in vivo and in vitro. Resistin-overexpressing LV tissue had higher levels of TNF-α receptor 1 and iNOS, and reduced levels of eNOS. Cardiomyocytes overexpressing resistin in vitro produced larger amounts of TNFα in the medium, had increased phosphorylation of IκBα and displayed increased intracellular reactive oxygen species (ROS) content with increased expression and activity of ROS-producing NADPH oxidases compared to controls. Long-term resistin overexpression is associated with a complex phenotype of oxidative stress, inflammation, fibrosis, apoptosis and myocardial remodeling and dysfunction in rats. This phenotype recapitulates key features of diabetic cardiomyopathy. This article is part of Special Issue Item Group entitled "Possible Editorial".

MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression and play an important role in many developmental processes. Recent studies suggest roles of miRNAs in carcinogenesis. Fragile histidine triad (FHIT) gene deletion, methylation, and reduced Fhit protein expression occur in about 70% of human epithelial tumors and are clearly associated with tumor progression. Although it has been previously reported that Fhit(-/-)cells exhibit more resistance to multi-DNA damage inducers, including ionizing radiation, it remains unclear how miRNAs targeting FHIT in DNA damage response play the role. This study reports that miR-143 directly targets FHIT and that overexpression of miR-143 results in significant G2-phase arrest and protects cells from DNA damage-induced killing. These results indicate an association of FHIT gene inactivation with increased survival after DNA damage and also provide useful information for miRNA-based drug development in two directions: protect cells from DNA damage-induced killing and sensitize cells to radiation therapy.

RAS activation is implicated in physiologic and pathologic cardiac hypertrophy. Cross-talk between the Ras and calcineurin pathways, the latter also having been implicated in cardiac hypertrophy, has been suspected for pathologic hypertrophy. Our recent discovery that germ-line mutations in RAF1, which encodes a downstream RAS effector, cause Noonan and LEOPARD syndromes with a high prevalence of hypertrophic cardiomyopathy provided an opportunity to elaborate the role of RAF1 in cardiomyocyte biology. Here, we characterize the role of RAF1 signaling in cardiomyocyte hypertrophy with an aim of identifying potential therapeutic targets. We modeled hypertrophic cardiomyopathy by infecting neonatal and adult rat cardiomyocytes (NRCMs and ARCMs, respectively) with adenoviruses encoding wild-type RAF1 and three Noonan/LEOPARD syndrome-associated RAF1 mutants (S257L, D486N or L613V). These RAF1 proteins, except D486N, engendered cardiomyocyte hypertrophy. Surprisingly, these effects were independent and dependent of mitogen activated protein kinases in NRCMs and ARCMs, respectively. Inhibiting Mek1/2 in RAF1 overexpressing cells blocked hypertrophy in ARCMs but not in NRCMs. Further, we found that endogenous and heterologously expressed RAF1 complexed with calcineurin, and RAF1 mutants causing hypertrophy signaled via nuclear factor of activated T cells (Nfat) in both cell types. The involvement of calcineurin was also reflected by down regulation of Serca2a and dysregulation of calcium signaling in NRCMs. Furthermore, treatment with the calcineurin inhibitor cyclosporine blocked hypertrophy in NRCMs and ARCMs overexpressing RAF1. Thus, we have identified calcineurin as a novel interaction partner for RAF1 and established a mechanistic link and possible therapeutic target for pathological cardiomyocyte hypertrophy induced by mutant RAF1. This article is part of a Special Issue entitled 'Possible Editorial'.