Pluripotency Factor Lin28 Research Articles

Multi-Level Regulatory Interactions between NF-κB and the Pluripotency Factor Lin28.

An appreciation for the complex interactions between the NF-κB transcription factor and the Lin28 RNA binding protein/let-7 microRNA pathways has grown substantially over the past decade. Both the NF-κB and Lin28/let-7 pathways are master regulators impacting cell survival, growth and proliferation, and an understanding of how interfaces between these pathways participate in governing pluripotency, progenitor differentiation, and neuroplastic responses remains an emerging area of research. In this review, we provide a concise summary of the respective pathways and focus on the function of signaling interactions at both the transcriptional and post-transcriptional levels. Regulatory loops capable of providing both reinforcing and extinguishing feedback have been described. We highlight convergent findings in disparate biological systems and indicate future directions for investigation.

Comprehensive Mapping of Pluripotent Stem Cell Metabolism Using Dynamic Genome-Scale Network Modeling

Metabolism is an emerging stem cell hallmark tied tocell fate, pluripotency, and self-renewal, yet systems-level understanding of stem cell metabolism has been limited by the lack of genome-scale network models. Here, we develop a systems approach to integrate time-course metabolomics data with a computational model of metabolism to analyze the metabolic state of naive and primed murine pluripotent stem cells. Using this approach, we find that one-carbon metabolism involving phosphoglycerate dehydrogenase, folate synthesis, and nucleotide synthesis is a key pathway that differs between the two states, resulting in differential sensitivity to anti-folates. The model also predicts that the pluripotency factor Lin28 regulates this one-carbon metabolic pathway, which we validate using metabolomics data from Lin28-deficient cells. Moreover, we identify and validate metabolic reactions related to S-adenosyl-methionine production that can differentially impact histone methylation in naive and primed cells. Our network-based approach provides a framework for characterizing metabolic changes influencing pluripotency and cell fate.

Coupled Caspase and N-End Rule Ligase Activities Allow Recognition and Degradation of Pluripotency Factor LIN-28 during Non-Apoptotic Development

Recent findings suggest that components of the classical cell death machinery also have important non-cell-death (non-apoptotic) functions in flies, nematodes, and mammals. However, the mechanisms for non-canonical caspase substrate recognition and proteolysis, and the direct roles for caspases in gene expression regulation, remain largely unclear. Here we report that CED-3 caspase and the Arg/N-end rule pathway cooperate to inactivate the LIN-28 pluripotency factor in seam cells, a stem-like cell type in Caenorhabditis elegans, thereby ensuring proper temporal cell fate patterning. Importantly, the caspase and the E3 ligase execute this function in anon-additive manner. We show that CED-3 caspase and the E3 ubiquitin ligase UBR-1 form a complex that couples their invivo activities, allowing for recognition and rapid degradation of LIN-28 and thus facilitating a switch in developmental programs. The interdependence of these proteolytic activities provides a paradigm for non-apoptotic caspase-mediated protein inactivation.

H19/let-7/LIN28 reciprocal negative regulatory circuit promotes breast cancer stem cell maintenance

Long noncoding RNA-H19 (H19), an imprinted oncofetal gene, has a central role in carcinogenesis. Hitherto, the mechanism by which H19 regulates cancer stem cells, remains elusive. Here we show that breast cancer stem cells (BCSCs) express high levels of H19, and ectopic overexpression of H19 significantly promotes breast cancer cell clonogenicity, migration and mammosphere-forming ability. Conversely, silencing of H19 represses these BCSC properties. In concordance, knockdown of H19 markedly inhibits tumor growth and suppresses tumorigenesis in nude mice. Mechanistically, we found that H19 functions as a competing endogenous RNA to sponge miRNA let-7, leading to an increase in expression of a let-7 target, the core pluripotency factor LIN28, which is enriched in BCSC populations and breast patient samples. Intriguingly, this gain of LIN28 expression can also feedback to reverse the H19 loss-mediated suppression of BCSC properties. Our data also reveal that LIN28 blocks mature let-7 production and, thereby, de-represses H19 expression in breast cancer cells. Appropriately, H19 and LIN28 expression exhibits strong correlations in primary breast carcinomas. Collectively, these findings reveal that lncRNA H19, miRNA let-7 and transcriptional factor LIN28 form a double-negative feedback loop, which has a critical role in the maintenance of BCSCs. Consequently, disrupting this pathway provides a novel therapeutic strategy for breast cancer.

Abstract B52: The epithelial-mesenchymal transition factor Snail represses the tumor suppressor microRNA let-7 and contributes to invasiveness of ovarian cancer cells.

Abstract Introduction: Cell fate choices are made throughout development, and in adult organisms whenever stem cells divide. We study the interrelation of transcription factors (TF) and microRNAs in cell fate decisions. Metastasis of ovarian and other solid tumors involves epithelial-mesenchymal transition (EMT), brought about by TF such as Snail, Twist, Slug, and Zeb1. Cells undergoing reprogramming paradoxically upregulate the EMT TF Snail and downregulate let-7, and Snail binds the promoters of several let-7 family members consistent with direct regulation. Let-7 is a microRNA highly expressed in differentiated cells, and absent in pluripotent cells. Its targets include the oncogenes Myc, Ras, and HMGA2. Many aspects of somatic cell reprogramming are seen as analogous to the acquisition of stem cell phenotypes observed in cancer. Let-7 levels are decreased in many ovarian tumors, and low let-7 correlates with poor prognosis. In breast cancer, let-7 downregulation is associated with metastasis. Results: Let-7 levels are known to be regulated by control of processing by the RNA binding protein and pluripotency factor Lin28, but transcriptional regulators of let-7 have not been described. We show that Snail represses let-7 transcription in cancer-related EMTs. ChIP assays demonstrate binding of Snail to let-7 promoters, and luciferase assays demonstrate direct regulation of Let-7i by Snail. Thus we are exploring two avenues for metastasis inhibition in OC: decreasing Snail and increasing let-7. We have characterized several OC cell lines for their phenotype with regard to EMT and cancer stem cell markers, in order to select agents to advance to testing in our orthotopic patient-derived xenograft model. We have examined the role of Snail in OC metastasis by manipulating expression levels and carrying out invasiveness assays. In epithelial and cancer cell lines, stimulation with TGF-beta or EGF caused morphological and gene expression changes consistent with EMT. The OC line OVSAHO underwent EMT when stimulated with EGF, but not with TGF-b. In breast, pancreatic, and OC cell lines, increases in Snail expression correlate with decreased let-7 expression. Conclusions: We propose that the inverse relationship between Snail and let-7 plays an important role at decision points crucial for cell fate decisions. In cancer metastasis, derepression of let-7 targets (many of them oncofetal genes) leads to dedifferentiation and a more aggressive phenotype. In addition to its role in directly causing EMT and affording cells with the migratory and invasive phenotype necessary for metastasis, our model places Snail at an important control point for initiation of dedifferentiation events normally held in check by let-7. Citation Format: Yevgeniya Ioffe, Alyse Hill, Michael McCarthy, Linda Sanderman, Hugo Campos, Tise Suzuki, Andrew Caster, Juli Unternaehrer-Hamm. The epithelial-mesenchymal transition factor Snail represses the tumor suppressor microRNA let-7 and contributes to invasiveness of ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B52.

Abstract 2975: The pluripotency factor LIN28 promotes colorectal tumorigenesis and tumor progression

Abstract LIN28A and LIN28B are RNA-binding protein paralogs expressed in embryonic stem cells and in early developing tissues. Through blocking the terminal processing of let-7 miRNAs and regulating the translation of a number of mRNA binding targets, LIN28 controls an array of targets that mediate proliferation, pluripotency, differentiation and metabolism. While highly expressed in the early embryos, the levels of LIN28A/B are downregulated as differentiation and development proceed. However, aberrant overexpression of LIN28 is often observed in human malignancies especially those with poor prognosis. We have demonstrated that LIN28 promotes cellular transformation in vitro, yet it is unknown how LIN28 regulates tumorigenesis in vivo. Here, we investigate the role of LIN28 in colorectal cancer (CRC) in humans and in mouse models. We first investigated the status of LIN28A/B in 1000 CRC patients and observed 35-60% of the cancer samples express LIN28A and/or LIN28B. Furthermore, we generated intestinal-specific LIN28 overexpressing mice, and observed intestinal adenoma as well as invasive adenocarcinoma that highly resemble the histology of human CRCs. Moreover, in Apcmin/+ animals, additional LIN28 expression significantly increased tumor incidence and accelerated disease onset, as their tumors are poorly differentiated and more invasive compared with Apcmin/+ tumors. Together, our study shows that LIN28 drives intestinal tumorigenesis and further promotes tumor progression in vivo. CRC is one of the major contributors to cancer-related mortality; however, current mouse models seldom progress to later stages of the disease, which greatly impedes efforts to developing more effective therapies. Based on the knowledge gained from this research, we hope to provide novel insights into designing therapeutics targeting late-stage CRC. Citation Format: Sarah Schwitalla, Ho-Chou Tu, Srinivas Viswanathan, Hao Zhu, Zhirong Qian, Samar Shah, Shuji Ogino, George Q. Daley. The pluripotency factor LIN28 promotes colorectal tumorigenesis and tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2975. doi:10.1158/1538-7445.AM2014-2975

Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway.

Summary paragraphThe pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs1–4. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development5, glucose metabolism6 and tissue regeneration7. Alternatively, Lin28 overexpression is correlated with the onset of numerous cancers8, while let-7, a tumor suppressor, silences several human oncogenes5. Lin28 binds to precursor let-7 (pre-let-7) hairpins9, triggering the 3' oligo-uridylation activity of TUT4/710–12. The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3L213,14, a homolog of the catalytic subunit of the RNA exosome. The molecular basis of Lin28 mediated recruitment of TUT4/7 to pre-let-7 and its subsequent degradation by Dis3L2 is largely unknown. To examine the mechanism of Dis3L2 substrate recognition we determined the structure of mouse Dis3L2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first twelve nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3L2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28/let-7 pathway.

Polyamines are oncometabolites that regulate the LIN28/let‐7 pathway in colorectal cancer cells

Polyamine metabolism is a highly coordinated process that is essential for normal development and neoplastic growth in mammals. Although polyamine metabolism is a validated pathway for prevention of carcinogenesis, the mechanisms by which polyamines elicit their tumorigenic effects are poorly understood. In this study, we investigated the role of polyamine metabolism in colon cancer by screening a non-coding RNA (ncRNA) platform to identify polyamine responsive signaling nodes. We report that multiple non-coding RNAs are altered by polyamine depletion including induction of microRNA (miRNA) let-7i, a member of the tumor suppressive let-7 family. The let-7 family targets several RNAs for translational repression, including the growth-associated transcription factor HMGA2 and is negatively regulated by the pluripotency factor LIN28. Depletion of polyamines using difluoromethylornithine (DFMO) or genetic knockdown of the polyamine-modified eukaryotic translation initiation factor 5A isoforms 1 and 2 (eIF5A1/2) resulted in robust reduction of both HMGA2 and LIN28. Locked nucleic acid (LNA) oligonucleotides targeting the seed region of the let-7 family rescued the expression of HMGA2, but not LIN28, in both DFMO-treated and eIF5A1/2 knockdown cultures. Our findings suggest that polyamines are oncometabolites that influence specific aspects of tumorigenesis by regulating pluripotency associated factors, such as LIN28, via an eIF5A-dependent but let-7-independent mechanism while the expression of proliferation-related genes regulated by let-7, such as HMGA2, is mediated through microRNA mediated repression. Therefore, manipulating polyamine metabolism may be a novel method of targeting the LIN28/let-7 pathway in specific disease states.

DIS3L2, the final player in let-7 degradation

The pluripotency factor LIN28 maintains stem cells in an undifferentiated state by blocking the expression of let-7 microRNAs (miRNAs). LIN28 achieves this by recruiting 3′ terminal uridylyl transferases, which add a terminal oligouridine tail to pre-let-7 that inhibits its processing and promotes miRNA decay. This study identifies the 3′-5′ endonuclease DIS3L2 as the enzyme responsible for promoting pre-let-7 degradation in mouse embryonic stem (ES) cells. DIS3L2 was found to preferentially degrade uridylated pre-let-7 over unmodified pre-let-7 or unrelated miRNAs in vitro. Moreover, siRNA-mediated depletion of DIS3L2 in mouse ES cells resulted in the accumulation of uridylated pre-let-7, whereas knockdown of the exosome 3′-5′ endonucleases EXOSC10 or RRP44 had no effect, confirming that DIS3L2 is the nuclease responsible for carrying out pre-let-7 decay in the LIN28 pathway.

A role for the Perlman syndrome exonuclease Dis3l2 in the Lin28–let-7 pathway

The pluripotency factor Lin28 blocks the expression of let-7 microRNAs (miRNAs) in undifferentiated cells during development and functions as an oncogene in a subset of cancers1. Lin28 binds to let-7 precursor RNAs and recruits 3′ terminal uridylyl transferases (TUTases) to selectively inhibit let-7 biogenesis2–4. Uridylated pre-let-7 is refractory to processing by Dicer and is rapidly degraded by an unknown ribonuclease5. Here we identify Dis3l2 as the 3′-5′ exonuclease responsible for the decay of uridylated pre-let-7. Biochemical reconstitution assays reveal that 3′ oligouridylation stimulates Dis3l2 activity in vitro, and knockdown of Dis3l2 in mouse embryonic stem cells leads to the stabilization of pre-let-7. Our study establishes 3′ oligouridylation as an RNA decay signal for Dis3l2 and identifies the first physiological RNA substrate of this novel exonuclease that is mutated in the Perlman syndrome of fetal overgrowth and predisposition to Wilms’ tumor6.

Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development

The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7)

The pluripotency factor Lin28 recruits a 3' terminal uridylyl transferase (TUTase) to selectively block let-7 microRNA biogenesis in undifferentiated cells. Zcchc11 (TUTase4/TUT4) was previously identified as an enzyme responsible for Lin28-mediated pre-let-7 uridylation and control of let-7 expression. Here we investigate the protein and RNA determinants for this interaction. Biochemical dissection and reconstitution assays reveal the TUTase domains necessary and sufficient for Lin28-enhanced pre-let-7 uridylation. A single C2H2-type zinc finger domain of Zcchc11 was found to be responsible for the functional interaction with Lin28. We identify Zcchc6 (TUTase7) as an alternative TUTase that functions with Lin28 in vitro, and accordingly, we find Zcchc11 and Zcchc6 redundantly control let-7 biogenesis in embryonic stem cells. Our study indicates that Lin28 uses two different TUTases to control let-7 expression and has important implications for stem cell biology as well as cancer.

The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells?

Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT–PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.

Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28

Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5'-AGGAGAU-3'. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5'-NGNNG-3' consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.

Importance of the NCp7-like domain in the recognition of pre-let-7g by the pluripotency factor Lin28

The pluripotency factor Lin28 is a highly conserved protein comprising a unique combination of RNA-binding motifs, an N-terminal cold-shock domain and a C-terminal region containing two retroviral-type CCHC zinc-binding domains. An important function of Lin28 is to inhibit the biogenesis of the let-7 family of microRNAs through a direct interaction with let-7 precursors. Here, we systematically characterize the determinants of the interaction between Lin28 and pre-let-7g by investigating the effect of protein and RNA mutations on in vitro binding. We determine that Lin28 binds with high affinity to the extended loop of pre-let-7g and that its C-terminal domain contributes predominantly to the affinity of this interaction. We uncover remarkable similarities between this C-terminal domain and the NCp7 protein of HIV-1, not only in terms of primary structure but also in their modes of RNA binding. This NCp7-like domain of Lin28 recognizes a G-rich bulge within pre-let-7g, which is adjacent to one of the Dicer cleavage sites. We hypothesize that the NCp7-like domain initiates RNA binding and partially unfolds the RNA. This partial unfolding would then enable multiple copies of Lin28 to bind the extended loop of pre-let-7g and protect the RNA from cleavage by the pre-microRNA processing enzyme Dicer.

Abstract 3454: LIN28 is expressed in glioblastomas and promotes KRAS-mediated transformation of human neural stem cells

Abstract The pluripotency factor LIN28 is activated in many cancers arising outside the central nervous system, but its role in brain tumors is poorly understood. LIN28 inhibits the maturation of let-7, which in turn represses KRAS, c-myc, HMGA2 and other oncogenic factors, many of which are known to be activated in glioblastoma (GBM). Because malignant gliomas are thought to arise from normal human neural stem cells (hNSC), we hypothesized that the stem cell factor LIN28 plays a role in the development of high grade gliomas. Using immunohistochemistry, we detected LIN28 protein in a subset of gliomas, with higher expression in GBMs than in lower grade tumors. To examine if LIN28 would facilitate transformation of hNSC, we infected these cells with lentivirus encoding LIN28 together with dominant negative R248W p53 (DN-p53), constitutively active KRAS (CA-KRAS) and hTERT (hNSC-LIN28/DN-p53/CA-KRAS/hTERT), which are all commonly dysregulated in GBM. As controls, hNSC were also infected with DN-p53/CA-KRAS/hTERT, LIN28/DN-p53/hTERT, DN-p53/hTERT, or with GFP. The lentivirus transduced hNSCs continued to express the stem cell markers CD133, SOX2, Nestin and OLIG2, and retained the potential to differentiate along neuronal and glial lineages. The LIN28/DN-p53/CA-KRAS/hTERT expressing cells showed increased proliferation by BrdU assay compared to normal hNSCs. To test their tumorigenecity in vivo, hNSC-LIN28/DN-p53/CA-KRAS/hTERT were injected into the brains of immunocompromised mice. Eight weeks after injection, 66.7% (12 of 18) of the mice developed invasive brain tumors resembling GBMs which were positive for Nestin and GFAP, and negative for synaptophysin. In contrast, control injections including hNSC-DN-p53/hTERT (5 mice), hNSC-LIN28/DN-p53/hTERT (5 mice) and hNSC-GFP (10 mice) did not generate tumors over 6 months. Interestingly, hNSC-DN-p53/CA-KRAS/hTERT cultures proliferated very poorly, suggesting that both LIN28 and activated KRAS may play key roles in transformation. In support of this possibility, we found using quantitative RT-PCR and western blotting that hNSC-LIN28/DN-p53/CA-KRAS/hTERT expressed higher levels of KRAS and phospho-MAPK than hNSC-DN-p53/CA-KRAS/hTERT lacking LIN28, suggesting this stem cell factor is required to facilitate MAPK activation and complete transformation of hNSCs. To further confirm the role of LIN28 in the tumorigenicity of GBMs, a tumor-derived neurosphere line (JHH-GBM14) was infected with LIN28 (GBM14-LIN28) or GFP control (GBM14-GFP) and injected as orthotopic xenografts into immunocompromised mice. In addition to forming brain tumors larger than GBM14-GFP controls, GBM14-LIN28 tumors significantly invaded into normal tissues including the corpus callosum and the contralateral brain. Our data show that LIN28 is expressed in human GBMs and that LIN28 can facilitate tumorigenicity in orthotopic GBM mouse models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3454. doi:10.1158/1538-7445.AM2011-3454

The pluripotency factor LIN28 is targeted by neuronal microRNAs participating in myofibroblastic differentiation

The pluripotency factor LIN28 is targeted by neuronal microRNAs participating in myofibroblastic differentiation

Neuronal microRNA participate in myofibroblastic differentiation by targeting the pluripotency factor Lin-28

Hepatic Stellate Cells (HSC) transdifferentiate into myofibroblastic cells, which are the main producers of extracellular matrix components in hepatic fibrogenesis. Myofibroblastic transition is associated with an altered profile of growth factors and mediators of inter- and intracellular cell communication. Small non-coding, so called microRNAs (miRNA) participate in regulation of gene expression by interaction with the untranslated region (UTR) of many transcripts and subsequent inhibition of translation. Thus, they are involved in many cellular processes such as differentiation, carcinogenesis and apoptosis. Since during myofibroblastic transition HSC express a wide spectrum of neuronal signal transducers we studied the expression of miRNAs known to be involved in neuronal differentiation. Primary HSCs were isolated from male Wistar rats and cultured for up to 10 days. The RNA was extracted by the Trizol method. Polyadenylation combined reverse transcription were performed and neuronal miR-9, miR-125b, miR-128 were quantified by Real Time PCR. All three neuronal miRNA were upregulated during myofibroblastic HSC differentiation. Screening of the databases revealed putative binding sites of the neuronal miRNAs in transcripts encoding for several pluripotency factors such as Lin-28.. For validation of the target, the putative binding site in the respective 3-UTR was cloned in fusion to the coding sequence of the luciferase reporter. By reporter assays of the cloned wild type in comparison to corresponding mutated sequences carrying two point mutations in the binding consensus we identified Lin28, triggering pluripotency in embryonic stem cells as target for miR-125b and miR-9. In conclusion, the neuronal miRNAs miR-9, miR-125b and miR-128 are suggested to participate in myofibroblastic transition and in expression of neuronal features of HSC by targeting Lin28 transcripts.

The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2

The let-7 miRNA and its target gene Lin-28 interact in a regulatory circuit controlling pluripotency. We investigated an additional let-7 target, mLin41 (mouse homologue of lin-41), as a potential contributor to this circuit. We demonstrate the presence of mLin41 protein in several stem cell niches, including the embryonic ectoderm, epidermis and male germ line. mLin41 colocalized to cytoplasmic foci with P-body markers and the miRNA pathway proteins Ago2, Mov10 and Tnrc6b. In co-precipitation assays, mLin41 interacted with Dicer and the Argonaute proteins Ago1, Ago2 and Ago4. Moreover, we show that mLin41 acts as an E3 ubiquitin ligase in an auto-ubiquitylation assay and that mLin41 mediates ubiquitylation of Ago2 in vitro and in vivo. Overexpression and depletion of mLin41 led to inverse changes in the level of Ago2 protein, implicating mLin41 in the regulation of Ago2 turnover. mLin41 interfered with silencing of target mRNAs for let-7 and miR-124, at least in part by antagonizing Ago2. Furthermore, mLin41 cooperated with the pluripotency factor Lin-28 in suppressing let-7 activity, revealing a dual control mechanism regulating let-7 in stem cells.

The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse

BackgroundLife-long production of spermatozoa depends on spermatogonial stem cells. Spermatogonial stem cells exist among the most primitive population of germ cells – undifferentiated spermatogonia. Transplantation experiments have demonstrated the functional heterogeneity of undifferentiated spermatogonia. Although the undifferentiated spermatogonia can be topographically divided into As (single), Apr (paired), and Aal (aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions.ResultsIn the present study, we show that LIN28, a pluripotency factor, is specifically expressed in undifferentiated spermatogonia (As, Apr, and Aal) in mouse. Ngn3 also specifically labels undifferentiated spermatogonia. We used Ngn3-GFP knockin mice, in which GFP expression is under the control of all Ngn3 transcription regulatory elements. Remarkably, Ngn3-GFP is only expressed in ~40% of LIN28-positive As (single) cells. The percentage of Ngn3-GFP-positive clusters increases dramatically with the chain length of interconnected spermatogonia.ConclusionOur study demonstrates that LIN28 specifically marks undifferentiated spermatogonia in mice. These data, together with previous studies, suggest that the LIN28-expressing undifferentiated spermatogonia exist as two subpopulations: Ngn3-GFP-negative (high stem cell potential) and Ngn3-GFP-positive (high differentiation commitment). Furthermore, Ngn3-GFP-negative cells are found in chains of Ngn3-GFP-positive spermatogonia, suggesting that cells in the Aal spermatogonia could revert to a more primitive state.