Maintenance of cell type identity is crucial for health, yet little is known of the regulation that sustains the long-term stability of differentiated phenotypes. To investigate the roles that key transcriptional regulators play in adult differentiated cells, we examined the effects of depletion of the developmental master regulator PTF1A on the specialized phenotype of the adult pancreatic acinar cell in vivo. Transcriptome sequencing and chromatin immunoprecipitation sequencing results showed that PTF1A maintains the expression of genes for all cellular processes dedicated to the production of the secretory digestive enzymes, a highly attuned surveillance of unfolded proteins, and a heightened unfolded protein response (UPR). Control by PTF1A is direct on target genes and indirect through a ten-member transcription factor network. Depletion of PTF1A causes an imbalance that overwhelms the UPR, induces cellular injury, and provokes acinar metaplasia. Compromised cellular identity occurs by derepression of characteristic stomach genes, some of which are also associated with pancreatic ductal cells. The loss of acinar cell homeostasis, differentiation, and identity is directly relevant to the pathologies of pancreatitis and pancreatic adenocarcinoma. Loss of cellular identity has long been associated with tissue injury and a first step in cancer progression (for examples, see references 1 and 2). Maintenance of a specific cellular phenotype depends on the continued transcription of cell-type-specific genes, largely through open chromatin architecture (3, 4) maintained by a small group of lineage-restricted DNA-binding transcription factors (TFs) (5, 6) that establish a unique transcriptional regulatory network (7). Many physiologic or pathophysiologic perturbations can affect the differentiated state of a cell quantitatively, but fewer affect the state of differentiation qualitatively. Qualitative changes involve the acquisition of characteristics of another cell type (or types), often defined by one or a few cell-specific markers, in addition to the diminution of the original phenotype. Despite progress with cellular reprogramming (for example, see reference 8), the molecular and genetic mechanisms that maintain cellular identity within the context of adult organs remain incompletely understood. In this report, we show that inactivation of the transcriptional regulatory gene Ptf1a in adult pancreatic acinar cells has pleiotropic effects on gene expression that cause quantitative and multigene qualitative changes of acinar differentiation.The acinar cell of the pancreas has been an informative model of terminal cellular differentiation (9). Common cellular processes are greatly exaggerated in support of the prodigious synthesis, processing, storage, and exocytosis of secretory proteins. The pancreatic acinar cell has the most ribosomes (10) and the highest rate of protein synthesis (11) of any mammalian somatic cell; it synthesizes, stores, and secretes its weight in protein daily. Specialized cellular functions ...
MicroRNA are small noncoding RNAs that translationally repress their target messenger RNAs. Many microRNAs are expressed at reduced levels in tumors. microRNAs with reduced expression in cancer often regulate oncogenes, resulting in enhanced tumor growth. One therapeutic option is to restore microRNA levels in the tumor to that of the non-diseased tissue. This is possible by delivering microRNA to the tumor in the form of an oligonucleotide mimic or by expressing the microRNA in the cancer using a gene vector. This article surveys the field of oligonucleotide mimics and gene vector approaches to restore microRNA levels in tumors and reviews the literature on experimental and pre-clinical studies that have used these approaches to treat cancer.
Identifying targets of dysregulated microRNAs (miRNAs) will enhance our understanding of how altered miRNA expression contributes to the malignant phenotype of breast cancer. The expression of miR-205 was reduced in four breast cancer cell lines compared to the normal-like epithelial cell line MCF10A and in tumor and metastatic tissues compared to adjacent benign breast tissue. Two predicted binding sites for miR-205 were identified in the 3’ untranslated region of the high mobility group box 3 gene, HMGB3. Both dual-luciferase reporter assay and Western blotting confirmed that miR-205 binds to and regulates HMGB3. To further explore miR-205 targeting of HMGB3, WST-1 proliferation and in vitro invasion assays were performed in MDA-MB-231 and BT549 cells transiently transfected with precursor miR-205 oligonucleotide or HMGB3 small interfering RNA (siRNA). Both treatments reduced the proliferation and invasion of the cancer cells. The mRNA and protein levels of HMGB3 were higher in the tumor compared to adjacent benign specimens and there was an indirect correlation between the expression of HMGB3 mRNA and patient survival. Treatment of breast cancer cells with 5-Aza/TSA derepressed miR-205 and reduced HMGB3 mRNA while knockdown of the transcriptional repressor NRSF/REST, reduced miR-205 and increased HMGB3. In conclusion, regulation of HMGB3 by miR-205 reduced both proliferation and invasion of breast cancer cells. Our findings suggest that modulating miR-205 and/or targeting HMGB3 are potential therapies for advanced breast cancer.
Extracellular vesicles (EVs) hold great potential as novel systems for nucleic acid delivery due to their natural composition. Our goal was to load EVs with microRNA that are synthesized by the cells that produce the EVs. HEK293T cells were engineered to produce EVs expressing a lysosomal associated membrane, Lamp2a fusion protein. The gene encoding pre-miR-199a was inserted into an artificial intron of the Lamp2a fusion protein. The TAT peptide/HIV-1 transactivation response (TAR) RNA interacting peptide was exploited to enhance the EV loading of the pre-miR-199a containing a modified TAR RNA loop. Computational modeling demonstrated a stable interaction between the modified pre-miR-199a loop and TAT peptide. EMSA gel shift, recombinant Dicer processing and luciferase binding assays confirmed the binding, processing and functionality of the modified pre-miR-199a. The TAT-TAR interaction enhanced the loading of the miR-199a into EVs by 65-fold. Endogenously loaded EVs were ineffective at delivering active miR-199a-3p therapeutic to recipient SK-Hep1 cells. While the low degree of miRNA loading into EVs through this approach resulted in inefficient distribution of RNA cargo into recipient cells, the TAT TAR strategy to load miRNA into EVs may be valuable in other drug delivery approaches involving miRNA mimics or other hairpin containing RNAs.
bMuch remains unknown regarding the regulatory networks formed by transcription factors in mature, differentiated mammalian cells in vivo, despite many studies of individual DNA-binding transcription factors. We report a constellation of feed-forward loops formed by the pancreatic transcription factors MIST1 and PTF1 that govern the differentiated phenotype of the adult pancreatic acinar cell. PTF1 is an atypical basic helix-loop-helix transcription factor complex of pancreatic acinar cells and is critical to acinar cell fate specification and differentiation. MIST1, also a basic helix-loop-helix transcription factor, enhances the formation and maintenance of the specialized phenotype of professional secretory cells. The MIST1 and PTF1 collaboration controls a wide range of specialized cellular processes, including secretory protein synthesis and processing, exocytosis, and homeostasis of the endoplasmic reticulum. PTF1 drives Mist1 transcription, and MIST1 and PTF1 bind and drive the transcription of over 100 downstream acinar genes. PTF1 binds two canonical bipartite sites within a 0.7-kb transcriptional enhancer upstream of Mist1 that are essential for the activity of the enhancer in vivo. MIST1 and PTF1 coregulate target genes synergistically or additively, depending on the target transcriptional enhancer. The frequent close binding proximity of PTF1 and MIST1 in pancreatic acinar cell chromatin implies extensive collaboration although the collaboration is not dependent on a stable physical interaction.T he mammalian pancreas is a mixed exocrine and endocrine gland consisting of acini, ducts, and islets. Approximately 90% of the mass of the adult pancreas is exocrine acinar tissue. Pancreatic acinar cells are highly specialized for the synthesis and secretion of digestive enzymes that are flushed via ducts to the intestine for digestion of complex nutrients (1). Abundant rough endoplasmic reticulum (ER) supports an extraordinary level of secretory protein synthesis (2). Maintenance of ER homeostasis without the accumulation of misfolded and unfolded proteins is especially important for acinar function and viability (3). Acinar cells are polarized, with basal rough ER and an extensive supranuclear Golgi apparatus for sorting and condensing newly synthesized secretory proteins into secretory vesicles (zymogen granules) that fill the apical cellular domain nearest the luminal plasma membrane. Secretion is regulated to ensure an appropriate surge of digestive enzyme release in response to feeding (1).Several transcription factors, including PTF1 and MIST1, are known to play crucial roles in the specification, differentiation, and maturation of pancreatic acinar cells (4-6). PTF1 is a complex of three tightly associated DNA-binding subunits: the cell-typerestricted basic helix-loop-helix (bHLH) protein PTF1A, one of the common bHLH E proteins (e.g., E47) (7), and RBPJ or its paralog RBPJL (8, 9). All three subunits contribute to the recognition of an extended bipartite binding sequence consisting of an E box boun...
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