Protein Lin28 Research Articles

LIN28-Targeting Chromenopyrazoles and Tetrahydroquinolines Induced Cellular Morphological Changes and Showed High Biosimilarity with BRD PROTACs.

The probing of small molecules with heterocyclic scaffolds covering unexplored chemical space and the evaluation of their biological relevance are essential parts of forward chemical genetics approaches and for the development of potential small-molecule therapeutics. In this study, we profiled sets of chromenopyrazoles (CMPs) and tetrahydroquinolines (THQs), originally developed to target the protein-RNA interaction of LIN28-let-7, in a cell painting assay (CPA) measuring cellular morphological changes. Selected LIN28-inactive CMPs and THQs induced cellular morphological changes to different extents. The most CPA-active CMPs 2 and 3 exhibited high bio-similarity with the LCH and BET clusters, while the most CPA-active THQs 13 and 20 indicated a mechanism of action beyond the currently established biosimilarity clusters. Overall, this work demonstrated that CPA is useful in revealing "hidden" biological targets and mechanisms of action for biologically inactive small molecules, which are CMPs and THQs targeting the RNA-binding protein LIN28 in this case, evaluated in target-based strategies. When compared with annotated reference compounds, CMP 3 exhibited a high biosimilarity with the dual BRD7/9 degrading PROTAC VZ185, suggesting that CPA could potentially function as a new phenotypic approach to identify degrader molecules.

Liver cancer initiation requires translational activation by an oncofetal regulon involving LIN28 proteins.

It is unknown which posttranscriptional regulatory mechanisms are required for oncogenic competence. Here, we show that the LIN28 family of RNA-binding proteins (RBPs), which facilitate posttranscriptional RNA metabolism within ribonucleoprotein networks, is essential for the initiation of diverse oncotypes of hepatocellular carcinoma (HCC). In HCC models driven by NRASG12V/Tp53, CTNNB1/YAP/Tp53, or AKT/Tp53, mice without Lin28a and Lin28b were markedly impaired in cancer initiation. We biochemically defined an oncofetal regulon of 15 factors connected to LIN28 through direct mRNA and protein interactions. Interestingly, all were RBPs and only 1 of 15 was a Let-7 target. Polysome profiling and reporter assays showed that LIN28B directly increased the translation of 8 of these 15 RBPs. As expected, overexpression of LIN28B and IGFBP1-3 was able to genetically rescue cancer initiation. Using this platform to probe components downstream of LIN28, we found that 8 target RBPs were able to restore NRASG12V/Tp53 cancer formation in Lin28a/Lin28b-deficient mice. Furthermore, these LIN28B targets promote cancer initiation through an increase in protein synthesis. LIN28B, central to an RNP regulon that increases translation of RBPs, is important for tumor initiation in the liver.

Abstract 1815: Optimization of Lin28 inhibitors: A promising therapeutic approach for the suppression of cancer cell proliferation, stemness properties, and spheroid growth

Abstract Background: The pluripotency factor known as Lin28 plays a crucial role in the regulation of cancer cell stem-like characteristics, hence facilitating the formation of cancer and the evolution of therapy-resistant tumors. The protein exerts its function by utilizing its cold shock domain and zinc knuckle domain (ZKD) to engage with the Let-7 pre-microRNA and impede the process of Let-7 production. The implementation of chemical agents to hinder the interaction between Lin28 and Let-7 has been proven as a therapeutic approach for treating aggressive tumors. Methods: Computer-aided drug design techniques were employed in the development of a novel series of small molecule inhibitors, which are derivatives of our previously identified lead compounds Ln115, Ln15, and Ln7. Fluorescent Polarization Assay (FP), and Electrophoretic Mobility Shift Assay (EMSA) were used to screen the chemicals as Lin28-Let7 interaction disruptors using Lin28A/B ZKD domain. To further screen the chemicals, the Incucyte cell proliferation assay was performed using two Lin28A and Lin28B positive cells, IGROV-1, and DuNE cells, respectively. The inhibition of Lin28 activity was subsequently evaluated by measuring Let-7 micro-RNA and Lin28 protein levels using RT-PCR and Western Blot. Then, the metabolism profile was explored using Microsomal Stability Assay. Incucyte proliferation assay and MTS assay determined the IC50 of lead compound in Lin28 positive cells. The anti-stemness properties of the lead compound were subsequently evaluated through assays for stem gene expression levels and colony formation. To evaluate the potential of the lead compound to further move into animal studies, we tested it on a newly developed 3D Neuroendocrine Prostate Cancer (NEPC) model. Results: In this study, we provide a novel set of derivatives, within which, Ln268 was chosen as the lead compound. Ln268 shows higher metabolic stability with a half-life time of 462 min and clearance rate of 5 ul/min/mg, compared to its father compound at 347 min and 46 ul/min/mg, respectively. Ln268 treatment resulted in a time-dependent inhibition of cell growth with an IC50 of 2.5 uM at 72 hrs. Ln268 reduces mRNA levels of stemness and neurodifferentiation markers such as CHGA, CHG, SYP, SCGN, OCT3/4, NANOG, ID4, FOXC1, ALDH1A2, and FOXO3. In the 3D NEPC model, Ln268 suppresses spheroid growth in a dose- and time-dependent manner. Ln268 achieved 100% spheroid growth inhibition at the concentration of 100 uM, while the current commercially available C1632 only reached 50%. Conclusion: We discovered a novel Ln15 derivative named Ln268, which has a better metabolism profile and increased inhibitory activity to distrust Lin28-Let7 interaction and cancer cell proliferation. Ln268 has the potential to be improved and used as an anti-cancer treatment for a variety of malignancies, such as prostate cancer. Citation Format: Victor M. Matias-Barrios, Mariia Radaeva, Graciella Rosellinny, Artem Cherkasov, Xuesen Dong. Optimization of Lin28 inhibitors: A promising therapeutic approach for the suppression of cancer cell proliferation, stemness properties, and spheroid growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1815.

Lin28 affects the proliferation and osteogenic differentiation of human dental pulp stem cells by directly inhibiting let-7b maturation

ObjectiveActivation of Lin28 gene under certain conditions promotes tissue damage repair. However, it remains unknown whether conditional expression of Lin28 facilitates the recovery of damaged pulp tissue. In the study, we focus on exploring the effects and possible regulatory mechanisms of Lin28 on the proliferation and differentiation of human dental pulp stem cells (hDPSCs).Materials and methodsWe adopted techniques such as the ethynyl-2ʹ-deoxyuridine (EdU) incorporation assay, RNA-protein immunoprecipitation (RIP) analysis, and luciferase assays to study the regulation of hDPSCs by Lin28. Furthermore, gain-of-function and loss-of-function analyses were also used in explored factors regulating hDPSCs activation.ResultsThe results show that Lin28 inhibited osteogenic differentiation by directly targets pre-let-7b. Through bioinformatics sequencing and dual luciferase experiments we learned that let-7b directly targets the IGF2BP2 3’UTR. Silencing of IGF2BP2 showed a similar biological effect as overexpression of let-7b. Overexpression of IGF2BP2 counteracted the differentiation-promoting effects produced by let-7b overexpression.Discussion/conclusionsIn conclusion, the RNA-binding protein Lin28 regulates osteogenic differentiation of hDPSCs by inhibiting let-7 miRNA maturation. And mature let-7b directly regulated the expression of IGF2BP2 by targeting the 3’UTR region of IGF2BP2 mRNA thus further inhibiting the differentiation of hDPSCs.

Dynamics of miRNA accumulation during C. elegans larval development.

Temporally and spatially controlled accumulation underlies the functions of microRNAs (miRNAs) in various developmental processes. In Caenorhabditis elegans, this is exemplified by the temporal patterning miRNAs lin-4 and let-7, but for most miRNAs, developmental expression patterns remain poorly resolved. Indeed, experimentally observed long half-lives may constrain possible dynamics. Here, we profile miRNA expression throughout C. elegans postembryonic development at high temporal resolution, which identifies dynamically expressed miRNAs. We use mathematical models to explore the underlying mechanisms. For let-7, we can explain, and experimentally confirm, a striking stepwise accumulation pattern through a combination of rhythmic transcription and stage-specific regulation of precursor processing by the RNA-binding protein LIN-28. By contrast, the dynamics of several other miRNAs cannot be explained by regulation of production rates alone. Specifically, we show that a combination of oscillatory transcription and rhythmic decay drive rhythmic accumulation of miR-235, orthologous to miR-92 in other animals. We demonstrate that decay of miR-235 and additional miRNAs depends on EBAX-1, previously implicated in target-directed miRNA degradation (TDMD). Taken together, our results provide insight into dynamic miRNA decay and establish a resource to studying both the developmental functions of, and the regulatory mechanisms acting on, miRNAs.

N-Biphenyl Pyrrolinones and Dibenzofurans as RNA-Binding Protein LIN28 Inhibitors Disrupting the LIN28-Let-7 Interaction.

The RNA-binding protein LIN28 is a regulator of miRNA let-7 biogenesis. Inhibitors of LIN28 are highly sought after given the central role that LIN28 plays in tumorigenesis and development of cancer stem cells as well as LIN28's association with poor clinical prognosis. Although LIN28 inhibitors of different scaffolds have been reported, the potential of most LIN28 inhibiting small molecules was not fully explored since very limited structure-activity relationship (SAR) studies have been performed. We previously identified trisubstituted pyrrolinones as a new class of LIN28 inhibitors disrupting the LIN28-let-7 interaction. Here, we performed extensive SAR by evaluating 95 small molecules and identified new trisubstituted pyrrolinones featuring either an N-biphenyl or N-dibenzofuran substituent, overthrowing the existing conclusion that a salicylic acid moiety is indispensable for activity. Exchange of the negatively charged salicylic acid moiety in LIN28 inhibitors with a heterocyclic substituent is beneficial for membrane permeability, leading to increased activity in a cellular assay, and will potentially reduce toxicity.

The LIN28B-let-7-PBK pathway is essential for group 3 medulloblastoma tumor growth and survival.

Children with Group 3 medulloblastoma (G3 MB) have a very poor prognosis, and many do not survive beyond 5 years after diagnosis. A factor that may contribute to this is the lack of available targeted therapy. Expression of protein lin-28 homolog B (LIN28B), a regulator of developmental timing, is upregulated in several cancers, including G3 MB, and is associated with worse survival in this disease. Here, we investigate the role of the LIN28B pathway in G3 MB and demonstrate that the LIN28B-lethal-7 (let-7; a microRNA that is a tumor suppressor)-lymphokine-activated killer T-cell-originated protein kinase (PBK; also known as PDZ-binding kinase) axis promotes G3 MB proliferation. LIN28B knockdown in G3-MB-patient-derived cell lines leads to a significant reduction in cell viability and proliferation in vitro and in prolonged survival of mice with orthotopic tumors. The LIN28 inhibitor N-methyl-N-[3-(3-methyl-1,2,4-triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide (1632) significantly reduces G3 MB cell growth and demonstrates efficacy in reducing tumor growth in mouse xenograft models. Inhibiting PBK using HI-TOPK-032 also results in a significant reduction in G3 MB cell viability and proliferation. Together, these results highlight a critical role for the LIN28B-let-7-PBK pathway in G3 MB and provide preliminary preclinical results for drugs targeting this pathway.

Spirocyclic Chromenopyrazole Inhibitors Disrupting the Interaction between the RNA-binding protein LIN28 and Let-7.

Small-molecule inhibitors of the RNA-binding and regulating protein LIN28 have the potential to be developed as chemical probes for biological perturbation and as therapeutic candidates. Reported small molecules disrupting the interaction between LIN28 and let-7 miRNA suffer from moderate to weak inhibitory activity and flat structure-activity relationship, which hindered the development of next-generation LIN28 inhibitors that warrant further evaluations. We report herein the identification of new LIN28 inhibitors utilizing a spirocyclization strategy based on a chromenopyrazole scaffold. Representative compounds 2-5 showed potent in vitro inhibitory activity against LIN28-let-7 interaction and single-digit micromolar potency in inhibiting the proliferation of LIN28-expressed JAR cancer cells. The spirocyclic compound 5 incorporated a position that is amenable for functional group appendage and further structural modifications. The binding mode of compound 5 with the LIN28 cold shock domain was rationalized via a molecular docking analysis.

MEL regulates miR-21 and let-7b through the STAT3 cascade in the follicular granulosa cells of Tibetan sheep

Under physiological and pathological conditions, melatonin (MEL) can regulate microRNA (miRNA) expression. However, the mechanisms underlying the regulatory effects of MEL on miRNAs in ovaries are not understood. Firstly, by using fluorescence in situ hybridisation, we found that in ovaries and follicular granulosa cells (FGCs), MT1 co-located with miR-21 and let-7b. Additionally, immunofluorescence revealed that MT1, STAT3, c-MYC and LIN28 proteins co-located. The mRNA and protein levels of STAT3, c-MYC and LIN28 increased under treatment with 10−7 M MEL. MEL induced an increase in miR-21 and a decrease in let-7b.The LIN28/let-7b and STAT3/miR-21 axes are related to cell differentiation, apoptosis and proliferation. We explored whether the STAT3/c-MYC/LIN28 pathway was involved in miRNA regulation by MEL to explore the putative mechanism of the above relationship. AG490, an inhibitor of the STAT3 pathway, was added before MEL treatment. AG490 inhibited the MEL-induced increases in STAT3, c-MYC, LIN28 and MT1 and changes in miRNA. Through live-cell detection, we discovered that MEL enhanced the proliferation of FGCs. However, the ki67 protein levels decreased when AG490 was added in advance. Furthermore, the dual-luciferase reporter assay verified that STAT3, LIN28 and MT1 were target genes of let-7b. Furthermore, STAT3 and SMAD7 were target genes of miR-21. In addition, the protein levels of the STAT3, c-MYC, LIN28 and MEL receptors decreased when let-7b was overexpressed in FGCs. Overall, MEL might regulate miRNA expression through the STAT3 pathway. In addition, a negative feedback loop between the STAT3 and miR-21 formed; MEL and let-7b antagonized each other in FGCs. These findings may provide a theoretical basis for improving the reproductive performance of Tibetan sheep through MEL and miRNAs.

Lin28 promoting the protective effect of PMSCs on hepatic ischaemia-reperfusion injury by regulating glucose metabolism.

Human placental mesenchymal stem cells (PMSCs) can prevent liver ischaemia-reperfusion injury (LIRI). However, their therapeutic effects are limited. Therefore, additional research is required to elucidate the mechanisms of PMSC-mediated LIRI prevention and enhance the related therapeutic effects. This study aimed to examine the role of the Lin28 protein in the regulation of glucose metabolism in PMSCs. Further, it explored whether Lin28 could enhance the protective effects of PMSCs against LIRI and investigated the underlying mechanisms. Western blotting was performed to examine Lin28 expression in PMSCs under hypoxic conditions. A Lin28 overexpression construct was introduced into PMSCs, and the effect on glucose metabolism was examined using a glucose metabolism kit. Further, the expression of some proteins involved in glucose metabolism and the PI3K-AKT pathway and the levels of microRNA Let-7a-g were examined using western blots and real-time quantitative PCR, respectively. To examine the relationship between Lin28 and the PI3K-Akt pathway, the effects of AKT inhibitor treatment on the changes induced by Lin28 overexpression were examined. Subsequently, AML12 cells were co-cultured with PMSCs to elucidate the mechanisms via which PMSCs prevent hypoxic injury in liver cells in vitro. Finally, C57BL/6J mice were used to establish a partial warm ischaemia-reperfusion model. The mice received intravenous injections containing PMSCs (control and Lin28-overexpressing PMSCs). Finally, their serum transaminase levels and degree of liver injury were assessed using biochemical and histopathological methods, respectively. Lin28 was upregulated under hypoxic conditions in PMSCs. Lin28 exerted protective effects against hypoxia-induced cell proliferation. Moreover, it increased the glycolytic capacity of PMSCs, allowing PMSCs to produce more energy under hypoxic conditions. Lin28 also activated the PI3K-Akt signalling pathway under hypoxic conditions, and its effects were attenuated by AKT inhibition. Lin28 overexpression could protect cells against LIRI-induced liver damage, inflammation and apoptosis and could also attenuate hypoxia-induced hepatocyte injury. Lin28 enhances glucose metabolism under hypoxic conditions in PMSCs, thereby exerting protective effects against LIRI by activating the PI3K-Akt signalling pathway. Our study is the first to report the potential of genetically modified PMSCs for LIRI treatment.

Ontogenetic Changes in the Expression of the Lin28 Protein in the Rat Hypothalamic Tuberal Nuclei.

The hypothalamus is a primary regulator of homeostasis, biological rhythms and adaptation to different environment factors. It also participates in the aging regulation. The expression of neurons containing Lin28 was studied by immunohistochemistry in male rats aged 2, 6, 12, and 24 months in the tuberal region of the rat hypothalamus. We have shown for the first time the presence of Lin28-immunoreactive (IR) neurons in the ventromedial nucleus (VMH) and their absence in the dorsomedial and arcuate nuclei in all studied animals. With aging, the percentage of Lin28-IR neurons increases from 37 ± 4.7 in 2-month-old rat until 76 ± 4.6 in 6-month-old and further decreases to 41 ± 7.3 in 12-month-old rat and 28 ± 5.5 in 24-month-old rats. Many VMH Lin28-IR neurons colocalized components of insulin signaling including mTOR, Raptor, PI3K and Akt. The percentage of Lin28/Akt-IR neurons was maximal in 6-month-old and 1-year-old rats compared to 2-month-old and 2-year-old animals. The proportion of Lin28/PI3K-IR neurons significantly increased from 77 ± 1.2 in 2-month-old rat until 99 ± 0.3 in 24-month-old rats and 96-99% of Lin28-IR neurons colocalized mTOR and mTORC1 component Raptor without statistically significant differences in all studied age groups. Thus, Lin28 expresses only in the VMH neurons of the tuberal nuclei of the hypothalamus and the Lin 28 expression changes during the development together with the components of PI3K-Akt-mTOR signaling.

Lin-28 Homolog B-Activated Protein Disulfide Isomerase A4 Regulates Cell Proliferation, Migration and Invasion of Glioma

The goal of this study is to elucidate the role of protein disulfide isomerase A4 (PDIA4) in glioma, as well as its regulatory mechanism. Cell transfection was performed to adjust the expression level of PDIA4 and RNA-binding protein lin-28 homolog B (LIN28B). The expression of PDIA4 in human astrocytes and glioma cell lines was determined by quantitative real-time PCR and western blot. CCK-8, colony formation, Transwell and wound-healing assays were applied to determine the capabilities of cells to proliferate, invade and migrate. The connection between PDIA4 and LIN28B was demonstrated by RNA immunoprecipitation (RIP) and RNA pull down assays. As a result, PDIA4 was elevated in glioma. PDIA4 depletion hugely suppressed cell proliferative ability, which was characterized by the reduced cell viability and colony formation, and declined contents of PCNA and Ki67. Meanwhile, PDIA4 knockdown repressed the cell capabilities to migrate and invade, accompanied with downregulated MMP2 and MMP9. LIN28N was also found to be upregulated in glioma cells, and was verified to bind with PDIA4 and positively regulate PDIA4 expression. Additionally, LIN28B overexpression partly hindered the suppressive impacts of PDIA4 knockdown on cell abilities to proliferate, migrate and invade. In conclusion, this study delineates that LIN28B-mediated PDIA4 plays a critical role in the progression of glioma.

RNA-binding protein complex LIN28/MSI2 enhances cancer stem cell-like properties by modulating Hippo-YAP1 signaling and independently of Let-7

The RNA binding protein LIN28 directly modulates the stability and translation of target mRNAs independently of Let-7; however, the key downstream targets of LIN28 in this process are largely unknown. Here, we revealed that Hippo signaling effector YAP1 functioned as a key downstream regulator of LIN28 to modulate the cancer stem cell (CSC)-like properties and tumor progressions in triple negative breast cancer (TNBC). LIN28 was overexpressed in BC tissues and cell lines, and significantly correlated with poorer overall survivals in patients. Ectopic LIN28 expression enhanced, while knockdown of LIN28A inhibited the CSC-like properties, cell growth and invasive phenotypes of TNBC cells in vitro and in vivo. Transcriptome analysis demonstrated LIN28 overexpression significantly induced the expressions of YAP1 downstream genes, while reduced the transcripts of YAP1 upstream kinases, such as MST1/2 and LATS1/2, and knockdown of LIN28A exhibited the opposite effects. Furthermore, constitutive activation of YAP1 in LIN28 knockdown TNBC cells could rescue the cell growth and invasive phenotypes in vitro and in vivo. Mechanistically, instead of the dependence of Let-7, LIN28 recruited RNA binding protein MSI2 in a manner dependent on the LIN28 CSD domain and MSI2 RRM domain, to directly induce the mRNA decay of YAP1 upstream kinases, leading to the inhibition of Hippo pathway and activation of YAP1, which eventually gave rise to increased CSC populations, enhanced tumor cell growth and invasive phenotypes. Accordingly, co-upregulations of LIN28 and MSI2 in TNBC tissues were strongly associated with YAP1 protein level and tumor malignance. Taken together, our findings unravel a novel LIN28/MSI2-YAP1 regulatory axis to induce the CSC-like properties, tumor growth and metastasis, independently of Let-7, which may serve as a potential therapeutic strategy for the treatment of a subset of TNBC with LIN28 overexpression.

Mechanism of RNA-binding protein Lin28 in neuronal ferroptosis after intracerebral haemorrhage.

Intracerebral haemorrhage (ICH) is a highly risky cerebrovascular disease with poor prognosis. Lin-28 homolog A (Lin28) has been identified as a crucial regulator in ICH. This study aims to analyse the mechanism of Lin28 in neuronal ferroptosis after ICH and provide theoretical basis for ICH treatment. An ICH mouse model was established via injection of collagenase VII, followed by neurological impairment assessment, and haematoxylin-eosin staining. An in vitro ICH model was established using hemin treatment. Next, cell viability and ferroptosis parameters were detected via cell counting kit-8, assay kits, enzyme-linked immunosorbent assay and western blot. Lin28 expression and tripartite motif-containing 37 (Trim37) mRNA level were detected via western blot and quantitative real-time polymerase chain reaction (qRT-PCR). The binding relationship of Lin28 and Trim37 was verified. ICH mice exhibited neuronal ferroptosis and upregulation of Lin28. Lin28 inhibition alleviated neurological impairment, manifested by decreased hematoma, oedema, neuronal necrosis, glial cell swelling, intracellular vacuoles and inflammatory cell infiltration, reduced Fe2+ concentration and reactive oxygen species content, and increased glutathione and glutathione peroxidase 4 activity. In the hemin-induced HT-22 cells, Lin28 inhibition promoted cell viability and alleviated neuronal ferroptosis. Lin28 bound to Trim37 mRNA to stabilize the mRNA level of Trim37. Overexpression of Trim37 reversed the alleviating role of silencing Lin28 in neuronal ferroptosis after ICH. Overall, Lin28 stabilized the mRNA level of Trim37 to aggravate neuronal ferroptosis after ICH.

Small molecules with tetrahydroquinoline-containing Povarov scaffolds as inhibitors disrupting the Protein–RNA interaction of LIN28–let-7

Inhibition of the RNA-binding protein LIN28 and disruption of the protein–RNA interaction of LIN28–let-7 with small molecules holds great potential to develop new anticancer therapeutics. Herein, we report the LIN28 inhibitory activities of a series of 30 small molecules with a tricyclic tetrahydroquinoline (THQ)-containing scaffold obtained from a Povarov reaction. The THQ molecules were structurally optimized by varying the 2-benzoic acid substituent, the fused ring at 3- and 4-positions, and the substituents at the phenyl moiety of the tetrahydroquinoline core. Among the tested compounds, GG-43 showed dose-dependent inhibition in an EMSA validation assay and low micromolar inhibitory activity in a fluorescence polarization-based assay measuring disruption of LIN28–let-7 interaction. Binding mode between GG-43 and the cold shock domain of LIN28 was proposed via a molecular docking analysis. The study provides one of the first systematic analyses on structural features that are required for LIN28 inhibition, and indicates the necessity to develop small molecules with new scaffolds as LIN28-targeting probes and therapeutic candidates. In parallel, this study demonstrates the polypharmacological nature of tricyclic THQ-containing scaffolds accessible through Povarov reactions.

LINC01094 promotes pancreatic cancer progression by sponging miR-577 to regulate LIN28B expression and the PI3K/AKT pathway

The leading cause of death in pancreatic cancer (PC) patients is the progression of cancer metastasis. Recently, long non-coding RNAs (lncRNAs) have been shown to play an important role in regulating cancer cell proliferation and metastasis; however, its molecular basis in PC remains to be explored. In this study, we observed that LINC01094 was markedly overexpressed in PC tissues and was associated with poor patient prognosis. Downregulation of LINC01094 decreased the proliferation and metastasis of PC cells and inhibited tumorigenesis and metastasis in mouse xenografts. Mechanically, LINC01094 acted as an endogenous miR-577 sponge to increase the expression of its target gene, the RNA-binding protein lin-28 homolog B (LIN28B), by decoying the miR-577, thereby activating the PI3K/AKT pathway. Our findings suggest that LINC01094 plays critical roles in proliferation and metastasis of PC, implying that LINC01094 can be regarded as a new biomarker or therapeutic target for the treatment of PC.

NSUN2-mediated RNA 5-methylcytosine promotes esophageal squamous cell carcinoma progression via LIN28B-dependent GRB2 mRNA stabilization

5-Methylcytosine (m5C) is a posttranscriptional RNA modification participating in many critical bioprocesses, but its functions in human cancer remain unclear. Here, by detecting the transcriptome-wide m5C profiling in esophageal squamous cell carcinoma (ESCC), we showed increased m5C methylation in ESCC tumors due to the overexpressed m5C methyltransferase NSUN2. Aberrant expression of NSUN2 was positively regulated by E2F Transcription Factor 1 (E2F1). High NSUN2 levels predicted poor survival of ESCC patients. Moreover, silencing NSUN2 suppressed ESCC tumorigenesis and progression in Nsun2 knockout mouse models. Mechanistically, NSUN2 induced m5C modification of growth factor receptor-bound protein 2 (GRB2) and stabilized its mRNA, which was mediated by a novel m5C mediator, protein lin-28 homolog B (LIN28B). Elevated GRB2 levels increased the activation of PI3K/AKT and ERK/MAPK signalling. These results demonstrate that NSUN2 enhances the initiation and progression of ESCC via m5C-LIN28B dependent stabilization of GRB2 transcript, providing a promising epitranscriptomic-targeted therapeutic strategy for ESCC.

Non-microRNA binding competitively inhibits LIN28 regulation.

SUMMARYRNA binding protein (RBP) expression is finite. For RBPs that are vastly outnumbered by their potential target sites, a simple competition for binding can set the magnitude of post-transcriptional control. Here, we show that LIN28, best known for its direct regulation of let-7 miRNA biogenesis, is also indirectly regulated by its widespread binding of non-miRNA transcripts. Approximately 99% of LIN28 binding sites are found on non-miRNA transcripts, like protein coding and ribosomal RNAs. These sites are bound specifically and strongly, but they do not appear to mediate direct post-transcriptional regulation. Instead, non-miRNA sites act to sequester LIN28 protein and effectively change its functional availability, thus impeding the regulation of let-7 in cells. Together, these data show that the binding properties of the transcriptome broadly influence the ability of an RBP to mediate changes in RNA metabolism and gene expression.

LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28’s role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved β-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.