Optimized rAAV8 targeting acinar KLF4 ameliorates fibrosis in chronic pancreatitis via exosomes-enriched let-7s suppressing pancreatic stellate cells activation.

Chronic pancreatitis (CP) is marked by progressive fibrosis and the activation of pancreatic stellate cells (PSCs), accompanied by the destruction of pancreatic parenchyma, leading to the loss of acinar cells (ACs). Few research studies have explored the mechanism by which damaged ACs (DACs) contribute to PSCs activation and pancreatic fibrosis. Currently, there are no effective drugs for curing CP or limiting the progression of pancreatic fibrosis. In this research, co-culture with intact acinar cells (IACs) suppressed PSC activation, while co-culture with DACs did the opposite. Krüppel-like factor 4 (KLF4) was significantly upregulated in DACs and was established as the key molecule that switches ACs from PSCs-suppressor to PSCs-activator. We revealed the exosomes of IACs contributed to the anti-activated function of IACs-CS on PSCs. MiRNome profiling showed that let-7 family is significantly enriched in IAC-derived exosomes (>30% miRNome), which partially mediates IACs' suppressive impacts on PSCs. Furthermore, it has been observed that the enrichment of let-7 in exosomes was influenced by the expression level of KLF4. Mechanistic studies demonstrated that KLF4 in ACs upregulated Lin28A, thereby decreasing let-7 levels in AC-derived exosomes, and thus promoting PSCs activation. We utilized an adeno-associated virus specifically targeting KLF4 in ACs (shKLF4-pAAV) to suppress PSCs activation in CP, resulting in reduced pancreatic fibrosis. IAC-derived exosomes hold potential as potent weapons against PSCs activation via let-7s, while activated KLF4/Lin28A signaling in DACs diminished such functions. ShKLF4-pAAV holds promise as a novel therapeutic approach for CP.

RNA-binding protein LIN28A upregulates transcription factor HIF1α by posttranscriptional regulation via direct binding to UGAU motifs.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.

RNA uridyl transferases TUT4/7 differentially regulate miRNA variants depending on the cancer cell type.

The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyze the additions of uridines at the 3' end of RNAs, including the precursors of the tumor suppressor miRNA let-7 upon recruitment by the oncoprotein LIN28A. As a consequence, let-7 family miRNAs are down-regulated. Disruption of this TUT4/7 activity inhibits tumorigenesis. Hence, targeting TUT4/7 could be a potential anticancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell-type-specific manner. Subsequently, BCL2, which is a well-established target of miR-200c is up-regulated. Therefore, TUT4/7 loss causes deregulation of miRNA-mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be an important aspect of targeting TUT4/7 for potential cancer therapies.

Exosomes derived from cancer-associated fibroblasts promote tumorigenesis, metastasis and chemoresistance of colorectal cancer by upregulating circ_0067557 to target Lin28.

BACKGROUND: Cancer associated fibroblasts (CAFs) can remodel tumor microenvironment by secreting exosomes. This study aimed to investigate the role of exosomes derived from cancer-associated fibroblasts in colorectal cancer (CRC) progression. METHODS: Circular RNA (circRNA) array was used to identify differentially expressed circRNAs in exosomes from normal fibroblasts (NFs) and CAFs, and confirmed one differentially expressed circRNA circ_0067557 by real-time PCR. The effect of circ_0067557 on proliferation, metastasis, chemoresistance and apoptosis was verified by wound heal, tranwell, CCK8, sphere-forming and flow cytometry assay. RESULTS: Circ_0067557 expression in exosomes from CAFs was higher than those from NFs. CAF-derived exosomes promoted the proliferation, migration, invasion and chemoresistance of CRC cells while suppressed apoptosis. Silencing of circ_0067557 inhibited malignant phenotypes of CRC cells by targeting Lin28A and Lin28B. Moreover, CAF-derived exosomes enhanced the growth of CRC xenograft tumors. CONCLUSION: Circ_0067557/Lin28A and Lin28B signal axis may be a potential therapy target for CRC.

The human FOXM1 homolog promotes basal progenitor cell proliferation and cortical folding in mouse.

Cortical expansion and folding are key processes in human brain development and evolution and are considered to be principal elements of intellectual ability. How cortical folding has evolved and is induced during embryo development is not well understood. Here, we show that the expression of human FOXM1 promotes basal progenitor cell proliferation and induces cortical thickening and folding in mice. Human-specific protein sequences further promote the generation of basal progenitor cells. Human FOXM1 increases the proliferation of neural progenitors by binding to the Lin28a promoter and increasing Lin28a expression. Furthermore, overexpression of LIN28A rescues the proliferation of human FOXM1 knockout neural progenitor cells. Together, our findings demonstrate that a human gene can increase the number of basal progenitor cells in mice, leading to brain size increase and gyrification, and may thus contribute to evolutionary brain development and cortical expansion.

Progress on the role of LIN28A/B in tumor development and progression.

LIN28A and its homolog LIN28B are highly conserved RNA-binding proteins that play important roles in early embryonic development, somatic cell reprogramming, metabolism and tumorigenesis. LIN28A/B are highly expressed in a variety of malignant tumors such as breast cancer. They play important roles in the initiation, maintenance, and metastasis of tumors and are associated with poor prognosis. Previous studies have shown that the main regulatory mechanisms of LIN28A/B include let-7s dependent ways and let-7s independent ways, such as directly targeting mRNA. In this review, we summarize the function and molecular regulatory mechanisms of LIN28A/B in malignant tumors such as liver cancer, breast cancer and colorectal cancer, in order to provide references for further exploring the function and mechanism of LIN28A/B and their possible roles in clinical applications.

LIN28 expression and function in medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current treatment modalities are not completely effective and can lead to severe neurological and cognitive adverse effects. In addition to urgently needing better treatment approaches, new diagnostic and prognostic biomarkers are required to improve the therapy outcomes of MB patients. The RNA-binding proteins, LIN28A and LIN28B, are known to regulate invasive phenotypes in many different cancer types. However, the expression and function of these proteins in MB had not been studied to date. This study identified the expression of LIN28A and LIN28B in MB patient samples and cell lines and assessed the effect of LIN28 inhibition on MB cell growth, metabolism and stemness. LIN28B expression was significantly upregulated in MB tissues compared to normal brain tissues. This upregulation, which was not observed in other brain tumors, was specific for the aggressive MB subgroups and correlated with patient survival and metastasis rates. Functionally, pharmacological inhibition of LIN28 activity concentration-dependently reduced LIN28B expression, as well as the growth of D283 MB cells. While LIN28 inhibition did not affect the levels of intracellular ATP, it reduced the expression of the stemness marker CD133 in D283 cells and the sphere formation of CHLA-01R cells. LIN28B, which is highly expressed in the human cerebellum during the first few months after birth, subsequently decreased with age. The results of this study highlight the potential of LIN28B as a diagnostic and prognostic marker for MB and open the possibility to utilize LIN28 as a pharmacological target to suppress MB cell growth and stemness.

BMP4 activates the Wnt-Lin28A-Blimp1-Wnt pathway to promote primordial germ cell formation via altering H3K4me2.

The unique developmental characteristics of chicken primordial germ cells (PGCs) enable them to be used in recovery of endangered bird species, gene editing and the generation of transgenic birds, but the limited number of PGCs greatly limits their application. Studies have shown that the formation of mammalian PGCs is induced by BMP4 signal, but the mechanism underlying chicken PGC formation has not been determined. Here, we confirmed that Wnt signaling activated via BMP4 activates transcription of Lin28A by inducing ß-catenin to compete with LSD1 for binding to TCF7L2, causing LSD1 to dissociate from the Lin28A promoter and enhancing H3K4me2 methylation in this region. Lin28A promotes PGC formation by inhibiting gga-let7a-3p maturation to initiate Blimp1 expression. Interestingly, expression of Blimp1 helped sustain Wnt5A expression by preventing LSD1 binding to the Wnt5A promoter. We thus elucidated a positive feedback pathway involving Wnt-Lin28A-Blimp1-Wnt that ensures PGC formation. In summary, our data provide new insight into the development of PGCs in chickens.

Embryonal tumor with multilayered rosettes arising from the internal auditory canal of an adult: Illustrative case with molecular investigations.

Embryonal tumors with multilayered rosettes (ETMRs) are aggressive central nervous system (CNS) tumors that usually occur in young children. Here, we describe the first incidence of ETMR in an adult patient that also originated in the novel location of the internal auditory canal (IAC). The 36-year-old patient initially presented with unsteadiness, diplopia, and tinnitus. The tumor in the IAC was discovered on brain magnetic resonance imaging, and gross total resection was performed followed by pathological and molecular diagnosis. The patient received whole brain and spinal cord radiotherapy after an intracranial recurrence and adjuvant chemotherapy consisting of four cycles of ifosfamide, cisplatin, and etoposide. Progression was rapid; however, the patient survived for 22 months after diagnosis before succumbing to the disease. Molecular investigation revealed a DICER1 mutation at exon 25, and methylation classification categorized the tumor as ETMR, non-C19MC-altered. This case underscores the diverse possible presentations of ETMR, DICER1-mutated and the importance of molecular techniques to characterize and promptly treat atypical ETMR.

LncRNA DDX11-AS1 Promotes Chemoresistance through LIN28A-Mediated ATG12 mRNA Stabilization in Breast Cancer.

INTRODUCTION: During breast cancer chemotherapy, the chemoresistance that frequently accompanies the treatment has become a big challenge. Long noncoding RNAs (LncRNAs) have been related to the development of chemoresistance in multiple cancer types. LncRNA DDX11-AS1 has shown a carcinogenic role in lung and colorectal cancer and was reported to enhance oxaliplatin resistance in gastric cancer and Taxol insensitivity in esophageal cancer. But its role in breast cancer chemotherapy drug resistance remains unknown. This study aimed to investigate the function and mechanism of lncRNA DDX11-AS1 in breast cancer chemoresistance. METHODS: The relationship between DDX11-AS1 and adriamycin (ADR) resistance was confirmed by qPCR, cell viability tests, and survival analysis. Then, RNA immunoprecipitation was conducted to evaluate the interaction between DDX11-AS1 and RNA-binding protein LIN28A. The regulation effect of LIN28A on autophagy-related genes ATG7 or ATG12 was detected by RNA stability assay and Western blot. Their correlation analysis was evaluated in GEO datasets and further validated by immunohistochemical results. The clinical significance of DDX11-AS1, ATG7, or ATG12 was evaluated by Kaplan-Meier Plotter analysis. RESULTS: Here, we reported DDX11-AS1 was significantly upregulated in chemoresistant breast cancer cells and overexpression of DDX11-AS1 promoted ADR resistance in breast cancer. LIN28A could interact with DDX11-AS1 and was involved in DDX11-AS1-mediated ADR resistance. Interfering with LIN28A reversed DDX11-AS1-induced ADR resistance. LIN28A could increase the protein level of ATG7 and ATG12 by increasing their mRNA stability. Survival analysis showed that ATG12 expression level was negatively correlated with the prognosis of breast cancer patients. CONCLUSION: This study clarifies the role of DDX11-AS1 in breast cancer chemoresistance and revealed a new mechanism, that is, interacting with LIN28A to stabilize ATG7 and ATG12 and jointly promote chemorefractory. These findings warrant further in vivo investigations to study DDX11-AS1 as a potential target to overcome chemoresistance.

miR-188-5p silencing improves cerebral ischemia/reperfusion injury by targeting Lin28a.

This report aimed to explore whether miR-188-5p regulated the pathological regulatory network of cerebral ischemia/reperfusion (I/R) injury. We simulated the cerebral I/R injury model with MACO/R and OGD/R treatments. Neuronal viability and apoptosis were assessed. The contents of miR-188-5p and Lin 28a were evaluated. The abundances of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) and pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) were measured. The interaction of miR-188-5p and Lin28a was confirmed. Lin28a silencing was supplemented to determine the delicate regulation of miR-188-5p. We revealed that miR-188-5p was upregulated and Lin28a was downregulated in I/R rats and OGD/R-induced cells. miR-188-5p silencing remarkably reduced the cerebral infarction volume, neurobehavioral score, brain edema, and Evans blue leakage. miR-188-5p silencing enhanced neuronal viability and alleviated apoptosis. The abundance of Bax and cleaved caspase-3 was reduced by miR-188-5p silencing, while Bcl-2 was augmented. miR-188-5p silencing impeded the contents of TNF-α, IL-1ß, and IL-6. miR-188-5p interacted with Lin28a and negatively regulated its expression. Interestingly, extra Lin28a silencing reversed apoptosis and the content of inflammatory cytokines. Our studies confirmed that miR-188-5p silencing alleviated neuronal apoptosis and inflammation by mediating the expression of Lin28a. The crosstalk of miR-188-5p and Lin28a offered a different direction for ischemic stroke therapy.

LIN28A binds to meiotic gene transcripts and modulates their translation in male germ cells.

The RNA-binding protein LIN28A is required for maintaining tissue homeostasis, including in the reproductive system, but the underlying mechanisms on how LIN28A regulates germline progenitors remain unclear. Here, we dissected LIN28A-binding targets using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse testes. LIN28A preferentially binds to mRNA coding sequence (CDS) or 3'UTR regions at sites enriched with GGAG(A) sequences. Further investigation of Lin28a-null mouse testes indicated that meiosis-associated mRNAs bound by LIN28A were differentially expressed. Next, ribosome profiling revealed that the mRNA levels of these targets were significantly reduced in the polysome fractions, and their protein expression levels decreased, in Lin28a-null mouse testes, even when meiotic arrest in the null mouse testes was not apparent. Collectively, these findings provide a set of LIN28A-regulated target mRNAs, and show that LIN28A binding might be a mechanism through which LIN28A acts to regulate undifferentiated spermatogonia fates and male fertility in mammals.

Lin 28A/Occludin axis: An aberrantly activated pathway in intestinal epithelial cells leading to impaired barrier function under total parenteral nutrition.

Disassembly of tight junctions is a major cause of intestinal barrier dysfunction under total parenteral nutrition (TPN), but the precise mechanisms have not been fully understood. Normally, RNA binding protein Lin 28A is highly restricted to embryonic stem cells and dramatically decreases as differentiation progresses; however, in our preliminary study it was found aberrantly increased in the intestinal epithelial cells of TPN rats, and thus its mechanism of action needs to be addressed. Herein, we report a pivotal role of Lin 28A in the regulation of tight junctions, which induces a sustained translational repression of Occludin, leading to disruption of intestinal barrier function under TPN. Using a rat model of TPN, we found time-dependent upregulation of Lin 28A, negatively correlated with Occludin. Using mouse intestinal organoids and human gut-derived Caco-2 cells as in vitro models, we found that expression of Occludin could be significantly suppressed by ectopic overexpression of Lin 28A. The underlying mechanisms may be partially attributed to translational repression, as the abundance of Occludin transcripts in polysomes was dramatically reduced by Lin 28A (polysomal profiling). Furthermore, Lin 28A was found to directly bind to Occludin mRNA 3' untranslated coding region (UTR), thereby repressing the translation of Occludin transcripts through decapping enzyme 1A (DCP1a). Taken together, our findings revealed that Lin 28A/Occludin axis may be a novel mechanism accounting for the development of barrier dysfunction under TPN.

LIN28 Family in Testis: Control of Cell Renewal, Maturation, Fertility and Aging.

Male reproductive development starts early in the embryogenesis with somatic and germ cell differentiation in the testis. The LIN28 family of RNA-binding proteins promoting pluripotency has two members-LIN28A and LIN28B. Their function in the testis has been investigated but many questions about their exact role based on the expression patterns remain unclear. LIN28 expression is detected in the gonocytes and the migrating, mitotically active germ cells of the fetal testis. Postnatal expression of LIN28 A and B showed differential expression, with LIN28A expressed in the undifferentiated spermatogonia and LIN28B in the elongating spermatids and Leydig cells. LIN28 interferes with many signaling pathways, leading to cell proliferation, and it is involved in important testicular physiological processes, such as cell renewal, maturation, fertility, and aging. In addition, aberrant LIN28 expression is associated with testicular cancer and testicular disorders, such as hypogonadotropic hypogonadism and Klinefelter's syndrome. This comprehensive review encompasses current knowledge of the function of LIN28 paralogs in testis and other tissues and cells because many studies suggest LIN28AB as a promising target for developing novel therapeutic agents.

Persistent TLR4 Activation Promotes Hepatocellular Carcinoma Growth through Positive Feedback Regulation by LIN28A/Let-7g miRNA.

Chronic inflammation caused by liver damage or infection plays an important role in the development and progression of hepatocellular carcinoma (HCC). The activation of Toll-like receptors 4 (TLR4) is involved in HCC tumorigenesis. Moreover, high TLR4 expression in HCC has been linked to poor prognosis. Although the expression of TLR4 in HCC is relatively low compared to hematopoietic cells, it is important to explore the molecular mechanism leading to the elevation of TLR4 in HCC. In this study, we aimed to investigate the positive regulating loop for TLR4 expression in HCC in response to chronic inflammation. Our results confirm that the mRNA expression of TLR4 and proinflammatory cytokines, including interleukin 6 (IL6) and C-C motif chemokine ligand 2 (CCL2), positively correlate in human HCC samples. High TLR4 expression in HCC is more susceptible to lipopolysaccharide (LPS); TLR4 activation in HCC provides growth and survival advantages and thus promotes tumorigenesis. It has been shown that the LIN28/let-7 microRNA (miRNA) axis is a downstream effector of the TLR4 signal pathway, and let-7 miRNA is a potential post-transcriptional regulator for TLR4. Thus, we investigated the correlation between TLR4 and LIN28A mRNA and let-7g miRNA in HCC clinical samples and found that the expression of TLR4 was positively correlated with LIN28A and negatively correlated with let-7g miRNA. Moreover, by culturing PLC/PRF5 (PLC5) HCC cells in low-dose LPS-containing medium to mimic chronic inflammation for persistent TLR4 activation, the mRNA and protein levels of TLR4 and LIN28A were elevated, and let-7g miRNA was decreased. Furthermore, the 3' untranslated region (3'UTR) of TLR4 mRNA was shown to be the target of let-7g miRNA, suggesting that inhibition of let-7g miRNA is able to increase TLR4 mRNA. While parental PLC5 cells have a low susceptibility to LPS-induced cell growth, long-term LPS exposure for PLC5 cells leads to increased proliferation, cytokine expression and stemness properties. In conclusion, our studies demonstrate positive feedback regulation for chronic TLR4 activation in the modulation of TLR4 expression level through the LIN28A/let-7g pathway in HCC and suggest a connection between chronic inflammation and TLR4 expression level in HCC for promoting tumorigenesis.

MicroRNA-126 suppresses the invasion of trophoblast-model JEG-3 cells by targeting LIN28A.

Inadequate trophoblast invasion and impaired trophoblast-induced vascular remodeling are features of preeclampsia. In this context, an angiogenesis-related microRNA, miR-126, is abnormally expressed in preeclampsia placentas, but its role in trophoblast development remains unclear. The purpose of this study was to investigate the roles of miR-126 in the proliferation, migration, and invasion processes of trophoblast cells using the human choriocarcinoma-derived JEG-3 cell line as a model. The mRNA expression profiling of JEG-3 cells with and without miR-126 overexpression, in combination with bioinformatics analysis, identified LIN28A as a putative target of miR-126. The results of real-time RT-PCR and luciferase assay were consistent with this idea. Overexpression of miR-126 in JEG-3 cells decreased the invasive ability of the cells without affecting proliferation or migration. The invasiveness of JEG-3 cells was significantly reduced to a similar extent by knockdown of LIN28A with siRNA and by miR-126-overexpression-induced downregulation of LIN28A, although the level of LIN28A protein was much lower in the siLIN28A-transfected cells. These results indicate that miR-126 suppresses JEG-3 cell invasion by targeting LIN28A, and suggest that miR-126-mediated downregulation of LIN28A might contribute to the onset/deterioration of preeclampsia.

Association Between LIN28A Gene Polymorphisms and Glioma Susceptibility in Chinese Children.

Gliomas are the most prevalent brain tumors among children and adolescents. The occurrence and development of various malignant tumors is closely related with LIN28A gene, but its relationship with glioma susceptibility has not been widely discovered. In this case-control study, we conducted four single nucleotide polymorphisms (SNPs) (rs3811464 G>A, rs3811463 T>C, rs34787247 G>A, and rs11247957 G>A) of LIN28A gene to investigate whether they increase the risk of glioma. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate their relationship. There was no significant correlation between four SNPs and glioma risk in single polymorphism and conjoint analysis. However, in stratification analysis, we found that rs3811463 TC/CC may add to the risk of glioma with clinical stage III (adjusted OR = 3.16, 95% CI = 1.15-8.70, P = .026) or stage III+IV patients (adjusted OR = 2.05, 95% CI = 1.02-4.13, P = .044). Our research suggested that four SNPs of LIN28A gene have a weak relationship with the risk of glioma in Chinese children. LIN28A rs3811463 TC/CC may increase the possibility of glioma in clinical stage III or stage III+IV patients which need larger samples and further confirmation.

Targeting LIN28: a new hope in prostate cancer theranostics.

The mortality and morbidity rates for prostate cancer have recently increased to alarming levels, rising higher than lung cancer. Due to a lack of drug targets and molecular probes, existing theranostic techniques are limited. Human LIN28A and its paralog LIN28B overexpression are associated with a number of tumors resulting in a remarkable increase in cancer aggression and poor prognoses. The current review aims to highlight recent work identifying the key roles of LIN28A and LIN28B in prostate cancer, and to instigate further preclinical and clinical research in this important area.

Regulation of human trophoblast surrogate Jeg-3 spheroids implantation potential by Wnt/ß-catenin pathway and lin28a/let-7a axis.

Successful implantation happens only when the development of a competent blastocyst synchronized with the differentiation of a receptive uterus. The exact mechanism affecting embryo implantation competency is still unclear. Previous data from our laboratory showed that several members of the let-7 family were up-regulated in the implanting dormant blastocysts and prohibited embryo activation through down-regulation integrin-ß3. However, how the mir-let-7 family is regulated is still a question. In this study, the in vitro co-culture model was applied to imitate implantation. Human embryo surrogate Jeg-3 spheroids and endometrium epithelial cells Ishikawa were used. The following views were demonstrated. Firstly，Wnt/ß-catenin signaling is essential for Jeg-3 spheroids implantation. Secondly, mir-let-7a is repressed by Wnt signaling, and low let-7a is beneficial for spheroids attachment and outgrowth. Third, in contrast with let-7a, lin28a is up-regulated by Wnt and promotes attachment and outgrowth. Lastly, the function of Wnt in embryo surrogate spheroids in implantation is mediated through lin28a/let-7a axis. In summary, our findings suggest Wnt/ß-catenin signaling strength human embryo surrogate spheroids implanting potential through regulation lin28a/let-7a axis.

C19MC amplification and expression of Lin28A and Olig2 in the classification of embryonal tumors of the central nervous system: A 14-year retrospective study from a tertiary care center.

INTRODUCTION: CNS embryonal tumors (CET) other than medulloblastomas (MB) and atypical teratoid/rhabdoid tumors (AT/RTs), previously designated as 'central nervous system primitive neuroectodermal tumors' ('CNS PNETs'), are a heterogenous subset of tumors with poorly defined diagnostic criteria. Other than the subset of embryonal tumor with multilayered rosettes (ETMR) defined by C19MC amplification, most CETs are diagnosed by exclusion of other molecularly defined entities and histological mimics including MB, AT/RTs, and high-grade gliomas, and termed as CET, not otherwise specified (NOS) in the 2016 WHO classification. AIM: To reclassify 'CNS PNETs' as per WHO 2016, and estimate the true proportion of CET, NOS in a tertiary healthcare setting, and to evaluate the diagnostic utility of C19MC amplification, Lin28A and Olig2 expression in the subclassification of CETs. METHODS: Previously diagnosed cases of 'CNS PNETs' (2002-2016) were first evaluated by immunohistochemistry (IHC) for MIC2, RelaA, L1CAM, IDH1R132H, H3K27M, H3G34R, H3G34V, INI1, and BRG1 proteins and by fluorescence in-situ hybridization (FISH) for EWSR1 translocation to exclude histological mimics. The selected CETs (case cohort) and 79 histological mimics (comparison cohort) comprising of MB, AT/RT, pineal parenchymal tumors, Ewing sarcoma, esthesioneuroblastoma, intraocular medulloepithelioma, and H3G34R mutant high-grade glioma were subject to IHC for Olig2 and Lin28A, and FISH for C19MC amplification. RESULTS: Twenty-two cases of 'CNS PNETs' were retrieved, all of which were negative for the first panel of markers and showed retained INI-1/BRG1 expression. Three of them (3/22, 13.6%) showed C19MC amplification (ETMR, C19MC-altered) with ETMR histology, Lin28A positivity, and Olig2 negativity. Among the remaining 19 CETs, one showed medulloepithelioma histology (Medulloepithelioma, NOS) and remaining were non-descript small round cell tumors (CET, NOS), all negative for Lin28A. Olig2 was positive in only 3 CETs (13.6%), all being CET, NOS. All tumors in the comparison cohort were negative for C19MC amplification, Lin28A and Olig2 except for 27% of ATRTs that were Lin28A positive. CONCLUSION: ETMR, C19MC-altered constitute less than 14% of CETs, with majority remaining uncharacterized as CET, NOS. Lin28A is 100% sensitive for the detection of C19MC amplification; however, its specificity is limited by its expression in ATRTs. Olig2 expression is seen only in a small subset of CET, NOS and is of limited diagnostic utility.