Role and mechanism of miR-130a-3p in the chemosensitivity of retinoblastoma cells to vincristine

Purpose Chemoresistance remains the primary obstacle threatening the prognosis of retinoblastoma (RB). microRNAs (miRNAs) are acknowledged as critical regulator of drug resistance. This study explored the molecular mechanism of miR-130a-3p affecting the chemosensitivity of RB to vincristine (VCR). Methods miR-130a-3p expression of VCR-sensitive and VCR-resistant RB tissues was detected using RT-qPCR. VCR-resistant RB cell line Y79/VCR was induced. miR-130a-3p expression of Y79/VCR cell line and its corresponding parental cell line was detected. Y79/VCR cells were subjected to miR-130a-3p overexpression treatment. The cell proliferation was measured using MTT assay, and the IC50 value and drug resistance index were examined using CCK-8 assay. The targeting relationship between miR-130a-3p and PAX6 was predicted through bioinformatics analysis and verified using dual-luciferase assay. Functional rescue experiments were conducted to confirm the role of PAX6 in chemosensitivity of RB cells. The effect of miR-130a-3p on tumorigenesis and VCR sensitivity was observed in vivo. Results miR-130a-3p was downregulated in VCR-resistant RB tissues and cells. Overexpression of miR-130a-3p repressed the proliferation of VCR-resistant RB cells and enhanced chemosensitivity. miR-130a-3p targeted PAX6 expression. Overexpression of PAX6 reversed the effect of miR-130a-3p on chemosensitivity of RB. Overexpression of miR-130a-3p suppressed tumor growth and reduced VCR resistance in vivo. Conclusion miR-130a-3p enhanced the chemosensitivity of RB cells to VCR by targeting PAX6 expression.

Chromatin interaction maps identify Wnt responsive cis-regulatory elements coordinating Paupar-Pax6 expression in neuronal cells

Central nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map the cis-regulatory interaction landscape of the key neuro-developmental Paupar-Pax6 lncRNA-mRNA locus. The results defined chromatin architecture changes associated with high Paupar-Pax6 expression in neurons and identified both promoter selective as well as shared cis-regulatory interactions with the Paupar and Pax6 promoters involved in regulating Paupar-Pax6 co-expression in neuronal cells. The TCF7L2 transcription factor, a major regulator of chromatin architecture and effector of the Wnt signalling pathway, binds to a subset of these candidate cis-regulatory elements to coordinate Paupar and Pax6 co-expression. We identify a functional TCF7L2 bound cis-regulatory element within the Paupar gene, suggesting that the Paupar DNA locus itself regulates Pax6 expression in addition to its previously described transcript-dependent modes of action. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain.

Pax6 regulates the morphological and electrophysiological development of mouse prethalamic neurons

SummaryPax6 is a well-known regulator of early neuroepithelial progenitor development. We discovered that it can also influence the later morphological and functional development of neurons in the prethalamus, which are unusual in retaining Pax6 expression as they mature. We used conditional mutagenesis to induce Pax6 loss-of-function mutations in prethalamic neurons and found that they showed developmental abnormalities when grown in vitro. Their rates of neurite elongation were disturbed and their axon initial segments were longer and extended further away from the soma than their control counterparts. They exhibited altered electrophysiological properties, including abnormal action potential (AP) waveforms and enhanced excitability, with abnormally low levels of current stimulus able to initiate APs. Our results significantly broaden the known functions of Pax6, suggesting that it has direct cell autonomous effects on the structural and functional development of some neurons. They provide new insights into the genetic regulation of the later stages of neurodevelopment.Graphical abstract

Dynamic Enhancement Pattern on CT for Predicting Pancreatic Neuroendocrine Neoplasms with Low PAX6 Expression: A Retrospective Observational Study

Paired box 6 (PAX6) is a transcription factor that plays a critical role in tumor suppression, implying that the downregulation of PAX6 promotes tumor growth and invasiveness. This study aimed to examine dynamic computed tomography (CT) features for predicting pancreatic neuroendocrine neoplasms (Pan-NENs) with low PAX6 expression. We retrospectively evaluated 51 patients with Pan-NENs without synchronous liver metastasis to assess the pathological expression of PAX6. Two radiologists analyzed preoperative dynamic CT images to determine morphological features and enhancement patterns. We compared the CT findings between low and high PAX6 expression groups. Pathological analysis identified 11 and 40 patients with low and high PAX6 expression, respectively. Iso- or hypoenhancement types in the arterial and portal phases were significantly associated with low PAX6 expression (p = 0.009; p = 0.001, respectively). Low PAX6 Pan-NENs showed a lower portal enhancement ratio than high PAX6 Pan-NENs (p = 0.044). The combination based on enhancement types (iso- or hypoenhancement during arterial and portal phases) and portal enhancement ratio (≤1.22) had 54.5% sensitivity, 92.5% specificity, and 84.3% accuracy in identifying low PAX6 Pan-NENs. Dynamic CT features, including iso- or hypoenhancement types in the arterial and portal phases and lower portal enhancement ratio may help predict Pan-NENs with low PAX6 expression.

Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

Different subtypes of interneurons, destined for the olfactory bulb, are continuously generated by neural stem cells located in the ventricular and subventricular zones along the lateral forebrain ventricles of mice. Neuronal identity in the olfactory bulb depends on the existence of defined microdomains of pre-determined neural stem cells along the ventricle walls. The molecular mechanisms underlying positional identity of these neural stem cells are poorly understood. Here, we show that the transcription factor Vax1 controls the production of two specific neuronal subtypes. First, it is directly necessary to generate Calbindin expressing interneurons from ventro-lateral progenitors. Second, it represses the generation of dopaminergic neurons by dorsolateral progenitors through inhibition of Pax6 expression. We present data indicating that this repression occurs, at least in part, via activation of microRNA miR-7.

PDTM-28. AN OTX2-PAX3 SIGNALLING AXIS REGULATES GROUP 3 MEDULLOBLASTOMA CELL FATE

The majority of Group 3 medulloblastomas (MB) exhibit amplification or increased expression of OTX2. OTX2 is primarily known as an oncogenic driver of tumor growth and cell cycle progression in Group 3 MB; however, its role as a repressor of differentiation is poorly characterized. Therefore, we utilized extensive patient data and mapped Group 3 MB chromatin dynamics in stem cell-enriched cultures to evaluate the divergent role of OTX2 in cell fate decisions in Group 3 MB pathogenesis. Several PAX genes were identified as novel OTX2 targets in Group 3 MB. Examination of patient data revealed that PAX3 and PAX6 expression is significantly reduced in Group 3 MB patients and is associated with significantly reduced survival. Functional evaluation of PAX3 and PAX6 expression showed that PAX3 expression significantly reduced self-renewal capacity of Group 3 MB tumorspheres in vitro and significantly prolonged survival and reduced tumor size in orthotopic xenograft models in vivo. RNA-sequencing of PAX3 and PAX6 gain of function (GOF) tumorspheres revealed mTORC1 signalling was specifically downregulated in PAX3 GOF, indicating this pathway may be critical for the survival and self-renewal differences observed between PAX3/PAX6 GOF models. Treatment of Group 3 MB with mTOR inhibitors reduced self-renewal in vitro and significantly prolonged survival and reduced tumor size in vivo. To further evaluate the role for this signalling axis in the Group 3 MB neural lineage hierarchy, we carried out scRNA-sequencing in tumorspheres from 4 Group 3 MB cell lines. Interestingly, a broad range of OTX2 expression was observed across single cell clusters, suggesting distinct OTX2 regulatory hierarchies are present in Group 3 MB. Collectively, our work demonstrates the multifaceted role of OTX2 as a regulator of cell fate decisions in Group 3 MB and identifies a novel role for mTORC1 signalling in Group 3 MB self-renewal and differentiation.

Effect of Ionizing Radiation from Computed Tomography on Differentiation of Human Embryonic Stem Cells into Neural Precursors

We studied the effect of radiation from computed tomography (CT) scans on differentiation of human embryonic stem cells (hESCs) into neuronal lineage. hESCs were divided into three radiation exposure groups: 0-dose, low-dose, or high-dose exposure. Low dose was accomplished with a single 15 mGy CT dose index (CTDI) CT scan that approximated the dose for abdominal/pelvic CT examinations in adults while the high dose was achieved with several consecutive CT scans yielding a cumulative dose of 500 mGy CTDI. The neural induction was characterized by immunocytochemistry. Quantitative polymerase chain reaction (qPCR) and Western blots were used to measure expression of the neuronal markers PAX6 and NES and pluripotency marker OCT4. We did not find any visible morphological differences between neural precursors from irradiated and non-irradiated cells. However, quantitative analyses of neuronal markers showed that PAX6 expression was reduced following exposure to the high dose compared to 0-dose controls, while no such decrease in PAX6 expression was observed following exposure to the low dose. Similarly, a statistically significant reduction in expression of NES was observed following high-dose exposure, while after low-dose exposure, a modest but statistically significant reduction in NES expression was only observed on Day 8 of differentiation. Further studies are warranted to elucidate how lower or delayed expression of PAX6 and NES can impact human fetal brain development.