Pax2-cre-mediated deletion of Lgl1 causes abnormal development of the midbrain.

Lgl1 protein plays a critical role in neurodevelopment, including hippocampus, olfactory bulb, and Purkinje cell. However, the specific mechanism of LGL1 function in the midbrain remains elusive. In this study, we generated Lgl1 conditional knockout mice using Pax2-Cre, which is expressed in the midbrain, and examined the functions of Lgl1 in the midbrain. Histological analysis exhibited abnormal midbrain development characterized by enlarged ventricular aqueduct and thinning tectum cortex. Lgl1 deletion caused excessive proliferation and heightened apoptosis of neural progenitor cells in the tectum of LP cko mice. BrdU labeling studies demonstrated abnormal neuronal migration. Immunofluorescence analysis of Nestin demonstrated an irregular and clustered distribution of glial cell fibers, with the adhesion junction marker N-cadherin employed for immunofluorescent labeling, unveiling abnormal epithelial connections within the tectum of LP cko mice. The current findings suggest that the deletion of Lgl1 leads to the disruption of the expression pattern of N-cadherin, resulting in abnormal development of the midbrain.

Role of LGL1 in cerebellar primordium of embryonic mice.

Lethal giant larvae 1 (LGL1) is originally recognized as a tumor suppressor, implicated in maintaining cell polarity in Drosophila and mammalian cells. Cell polarity plays a crucial role in tumorigenesis. We previously established Pax2-LGL1 -/- conditional knockout mice but did not focus on the tumorigenesis in cerebellar primordium. HE staining was used to detect the morphological structure of the cerebellar primordium during early embryonic development in Pax2-LGL1 -/- mice. Immunofluorescence assays were used to detect the expression of polar molecules. TUNEL staining assessed tissue apoptosis. Our findings reveal that deletion of LGL1 leads to the emergence of neuroblastoma-like tissues within the cerebellum primordium during early embryogenesis. This outcome can be attributed to alterations in expression patterns of polar molecules Cdc42 and ß-catenin following early deletion of LGL1, resulting in loss of cell polarity among neuroepithelial cells and subsequent formation of tumor-like tissues. However, further histological examination demonstrated that these tumor-like tissues disappear from embryonic day 15.5 onwards within the cerebellar primordium of Pax2-LGL1 -/- mice due to apoptosis-mediated cellular compensation. Our data emphasize the importance of LGL1 in maintaining neuroepithelial cell polarity and reveal a novel role for LGL1 in regulating tumorigenesis and ablation in the cerebellar primordium.

Loss of BAF (mSWI/SNF) chromatin-remodeling ATPase Brg1 causes multiple malformations of cortical development in mice.

Mutations in genes encoding subunits of the BAF (BRG1/BRM-associated factor) complex cause various neurodevelopmental diseases. However, the underlying pathophysiology remains largely unknown. Here, we analyzed the function of Brahma-related gene 1 (Brg1), a core ATPase of BAF complexes, in the developing cerebral cortex. Loss of Brg1 causes several morphological defects resembling human malformations of cortical developments (MCDs), including microcephaly, cortical dysplasia, cobblestone lissencephaly and periventricular heterotopia. We demonstrated that neural progenitor cell renewal, neuronal differentiation, neuronal migration, apoptotic cell death, pial basement membrane and apical junctional complexes, which are associated with MCD formation, were impaired after Brg1 deletion. Furthermore, transcriptome profiling indicated that a large number of genes were deregulated. The deregulated genes were closely related to MCD formation, and most of these genes were bound by Brg1. Cumulatively, our study indicates an essential role of Brg1 in cortical development and provides a new possible pathogenesis underlying Brg1-based BAF complex-related neurodevelopmental disorders.

Lgl1 deficiency disrupts hippocampal development and impairs cognitive performance in mice.

Cellular polarity is crucial for brain development and morphogenesis. Lethal giant larvae 1 (Lgl1) plays a crucial role in the establishment of cell polarity from Drosophila to mammalian cells. Previous studies have found the importance of Lgl1 in the development of cerebellar, olfactory bulb, and cerebral cortex. However, the role of Lgl1 in hippocampal development during the embryonic stage and function in adult mice is still unknown. In our study, we created Lgl1-deficient hippocampus mice by using Emx1-Cre mice. Histological analysis showed that the Emx1-Lgl1-/- mice exhibited reduced size of the hippocampus with severe malformations of hippocampal cytoarchitecture. These defects mainly originated from the disrupted hippocampal neuroepithelium, including increased cell proliferation, abnormal interkinetic nuclear migration, reduced differentiation, increased apoptosis, gradual disruption of adherens junctions, and abnormal neuronal migration. The radial glial scaffold was disorganized in the Lgl1-deficient hippocampus. Thus, Lgl1 plays a distinct role in hippocampal neurogenesis. In addition, the Emx1-Lgl1-/- mice displayed impaired behavioral performance in the Morris water maze and fear conditioning test.

Up-regulation of long intergenic noncoding RNA 01296 in ovarian cancer impacts invasion, apoptosis and cell cycle distribution via regulating EMT.

BACKGROUND: Recently, long intergenic non-coding RNA 01296 (LINC01296) has been demonstrated to regulate the initiation and progression of several cancers, but the functions of LINC01296 in ovarian cancer still remain unclear. The objective of our study was to determine the expression, biological roles, and clinical significance of LINC01296 in ovarian cancer. METHODS: LINC01296 expression was measured in ovarian cancer tissues or cell lines. Next, the relationships between LINC01296 levels and the clinical factors of ovarian cancer, such as progression-free survival and overall survival were analyzed. Additionally, cell proliferation, migration and invasion capacities, apoptosis, cell cycle distribution were investigated after silencing of LINC01296. To confirm whether LINC01296 mediates EMT initiation in ovarian cancer cells, the effect of LINC01296 silence on E-cadherin, N-cadherin and vimentin was assessed in SKOV3 and OVCAR3 cells. RESULTS: We found that LINC01296 was over-expressed in ovarian cancer tissues and cell lines, when comparing with adjacent normal tissue samples and normal cells. Higher LINC01296 expression was significantly correlated with shorter progression-free survival and overall survival. For the functional experiments, knockdown of LINC01296 suppressed cell proliferation, inhibited colony formation ability, abrogated cell migration and invasion potential, and enhanced cell apoptosis. Cell cycle analysis suggested that LINC01296 positively regulated cell cycle progression in ovarian cancer cells. Moreover, western blotting analysis displayed that knockdown of LINC01296 significantly increased E-cadherin, but reduced N-cadherin and vimentin expressions in SKOV3 and OVCAR3 cells, compared with no-transfection cells. CONCLUSIONS: LINC01296 plays an important role in promoting the progression of ovarian cancer. Over-expression of LINC01296 might function as an indicator for diagnosis and prognosis of ovarian cancer patients.

Loss of Lgl1 Disrupts the Radial Glial Fiber-guided Cortical Neuronal Migration and Causes Subcortical Band Heterotopia in Mice.

Radial glial cells (RGCs) are neuronal progenitors and function as scaffolds for neuronal radial migration in the developing cerebral cortex. These functions depend on a polarized radial glial scaffold, which is of fundamental importance for brain development. Lethal giant larvae 1 (Lgl1), a key regulator for cell polarity from Drosophila to mammals, plays a key role in tumorigenesis and brain development. To overcome neonatal lethality in Lgl1-null mice and clarify the role of Lgl1 in mouse cerebral cortex development and function, we created Lgl1 dorsal telencephalon-specific knockout mice mediated by Emx1-Cre. Lgl1Emx1 conditional knockout (CKO) mice had normal life spans and could be used for function research. Histology results revealed that the mutant mice displayed an ectopic cortical mass in the dorsolateral hemispheric region between the normotopic cortex and the subcortical white matter, resembling human subcortical band heterotopia (SBH). The Lgl1Emx1 CKO cortex showed disrupted adherens junctions (AJs), which were accompanied by ectopic RGCs and intermediate progenitors, and disorganization of the radial glial fiber system. The early- and late-born neurons failed to reach the destined position along the disrupted radial glial fiber scaffold and instead accumulated in ectopic positions and formed SBH. Additionally, the absence of Lgl1 led to severe abnormalities in RGCs, including hyperproliferation, impaired differentiation, and increased apoptosis. Lgl1Emx1 CKO mice also displayed deficiencies in anxiety-related behaviors. We concluded that Lgl1 is essential for RGC development and neural migration during cerebral cortex development.

Tprn is essential for the integrity of stereociliary rootlet in cochlear hair cells in mice.

Tprn encodes the taperin protein, which is concentrated in the tapered region of hair cell stereocilia in the inner ear. In humans, TPRN mutations cause autosomal recessive nonsyndromic deafness (DFNB79) by an unknown mechanism. To determine the role of Tprn in hearing, we generated Tprn-null mice by clustered regularly interspaced short palindromic repeat/Cas9 genome-editing technology from a CBA/CaJ background. We observed significant hearing loss and progressive degeneration of stereocilia in the outer hair cells of Tprn-null mice starting from postnatal day 30. Transmission electron microscopy images of stereociliary bundles in the mutant mice showed some stereociliary rootlets with curved shafts. The central cores of the stereociliary rootlets possessed hollow structures with surrounding loose peripheral dense rings. Radixin, a protein expressed at stereocilia tapering, was abnormally dispersed along the stereocilia shafts in Tprn-null mice. The expression levels of radixin and ß-actin significantly decreased.We propose that Tprn is critical to the retention of the integrity of the stereociliary rootlet. Loss of Tprn in Tprn-null mice caused the disruption of the stereociliary rootlet, which resulted in damage to stereociliary bundles and hearing impairments. The generated Tprn-null mice are ideal models of human hereditary deafness DFNB79.

Knockdown of Trio by CRISPR/Cas9 suppresses migration and invasion of cervical cancer cells.

Triple functional domain protein (Trio) is an evolutionarily conserved protein with guanine nucleotide exchange factors that regulate different physiological processes in some types of cancer. However, the expression and function of Trio in cervical cancer are still unknown. The purpose of this study was to detect the expression of Trio in cervical cancer tissues and to evaluate its clinical value. Furthermore, the effects of the Trio on the migration and invasion of cervical cancer cells and its mechanism were investigated in vitro. The results of the present study revealed that Trio expression levels were significantly higher in most of the clinical cervical cancer samples than in adjacent tissues. The clinicopathological significance of Trio expression was also analyzed, and the results revealed that high expression levels in cervical cancer were correlated with lymph node metastasis (P=0.005). The CRISPR/Cas9 system was used to knockdown the endogenous Trio. The inhibition of Trio significantly decreased the migration and invasion abilities of cervical cancer cells. Meanwhile, levels of RhoA/ROCK signaling factors (RhoA, Rock, and p-LIMK), which contributed to cell migration and invasion, were decreased along with the inhibition of Trio. Therefore, Trio may regulate the migration and invasion of cervical cancer through the RhoA/ROCK signaling pathway.

Hypermethylation of WIF1 and its inhibitory role in the tumor growth of endometrial adenocarcinoma.

Endometrial carcinoma is the most common malignancy of the female genital tract and is the fourth most common malignancy among women worldwide. Endometrial adenocarcinoma (EAC) accounts for ~80% of endometrial carcinoma cases. Numerous critical genetic events have been determined to serve an essential role in EAC progression; however, the precise molecular mechanisms underlying EAC progression remain unclear. Pyrosequencing and methylation­specific PCR were used to detect the methylation status of Wnt inhibitory factor 1 (WIF1). Immunohistochemistry and western blot were used to detect the expression of WIF1, Wnt family member 1 and other related pathways. The anticancer role of WIF1 in EAC was investigated in vitro and in vivo. Two of the three EAC cases exhibited significantly high methylation in five CpG sites, and the WIF1 methylation rate in EAC and endometrial tissues was 43.4 and 8%, respectively (P<0.05). The kappa consistency coefficient was ­0.369 between methylation and mRNA expression (P<0.05) and WIF1 expression levels were significantly downregulated in EAC tissues compared with non­tumorous tissues (P<0.01). The 5­year overall survival rates were significantly lower for patients with tumors that negatively expressed WIF1 when compared with the 77.9% exhibited by those with positive WIF1 expression. Furthermore, the proliferation rate of KLE cells was significantly reduced following 5­aza­20­deoxycytidine treatment or WIF1 overexpression in vitro and in vivo, which may be associated with downregulated c­Myc and phosphorylated­extracellular signal­regulated kinase expression. These results demonstrated the important role of WIF1 in EAC tumorigenesis, and suggested that WIF1 may be a potential drug target in EAC treatment.

miR-202 Suppresses Cell Proliferation by Targeting FOXR2 in Endometrial Adenocarcinoma.

BACKGROUND: MicroRNA-202 (miR-202) has been reported to be aberrantly regulated in several cancers. The aim of this study is to explore the functional role of miR-202 in EAC tumor growth. MATERIAL AND METHODS: miR-202 expression was detected by qRT-PCR. TargetScan and luciferase reporter assay were used to elucidate the candidate target gene of miR-202. The FOXR2 protein level was assessed by Western blot and immunohistochemistry. Survival analysis was explored for FOXR2 expression in EAC patients. RESULTS: miR-202 expression was significantly decreased in EAC tissues (P < 0.01) compared with that in control tissues. And the downregulate miR-202 was significantly associated with poor prognosis (P < 0.01). Re-expression of miR-202 dramatically suppressed cell proliferation in vitro and tumor growth in vivo. FOXR2 was identified as a direct target of miR-202. In EAC tissues, FOXR2 was upregulated and the increased FOXR2 was significantly associated with poor prognosis. In miR-202-transfected cells, the FOXR2 expression was inversely changed. The analysis of FOXR2 protein expression and miR-202 transcription in EAC tissues showed negative correlation (R = -0.429). CONCLUSION: miR-202 may function as a tumor suppressor in EAC tumor growth by targeting FOXR2 oncogene, which may provide new insights into the molecular mechanism and new targets for treatment of EAC.

Correction: Lgl1 Is Required for Olfaction and Development of Olfactory Bulb in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0162126.].

MicroRNA-326 suppresses the proliferation, migration and invasion of cervical cancer cells by targeting ELK1.

Although microRNAs (miRNAs or miRs) are able to function as oncogenes or tumor suppressors, the role of miR-326 in regulating human cervical cancer cells remains unclear. In the present study, the expression of miR-326 was identified to be downregulated in cervical cancer cell lines and primary tumor samples, and the overexpression of miR-326 decreased cell proliferation, migration and invasion in cervical cell lines. Bioinformatics prediction and experimental validation results revealed that the function of miR-326 was achieved by targeting and repressing ETS domain-containing protein Elk-1 (ELK1) expression. ELK1 was targeted directly by miR-326, which was downregulated in human cervical cancer tissues compared with that in adjacent normal tissues. The results of the present study suggest that miR-326, a potential tumor suppressor, may be used in the treatment of cervical cancer.

Lgl1 Is Required for Olfaction and Development of Olfactory Bulb in Mice.

Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and functioned as a key regulator of epithelial polarity and asymmetric cell division. In this study, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in olfactory bulb (OB). Next, we examined the effects of Lgl1 loss in the OB. First, we determined the expression patterns of Lgl1 in the neurogenic regions of the embryonic dorsal region of the LGE (dLGE) and postnatal OB. Furthermore, the Lgl1 conditional mutants exhibited abnormal morphological characteristics of the OB. Our behavioral analysis exhibited greatly impaired olfaction in Lgl1 mutant mice. To elucidate the possible mechanisms of impaired olfaction in Lgl1 mutant mice, we investigated the development of the OB. Interestingly, reduced thickness of the MCL and decreased density of mitral cells (MCs) were observed in Lgl1 mutant mice. Additionally, we observed a dramatic loss in SP8+ interneurons (e.g. calretinin and GABAergic/non-dopaminergic interneurons) in the GL of the OB. Our results demonstrate that Lgl1 is required for the development of the OB and the deletion of Lgl1 results in impaired olfaction in mice.

Loss of lysyl oxidase-like 3 causes cleft palate and spinal deformity in mice.

In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can result in embryonic malformation. The lysyl oxidase (LOX) family genes are closely related to human diseases. In this study, we investigated the essential role of lysyl oxidase-like 3 (LOXL3), a member of the LOX family, in embryonic development. Mice lacking LOXL3 exhibited perinatal lethality, and the deletion of the Loxl3 gene led to impaired development of the palate shelves, abnormalities in the cartilage primordia of the thoracic vertebrae and mild alveolar shrinkage. We found that the obvious decrease of collagen cross-links in palate and spine that was induced by the lack of LOXL3 resulted in cleft palate and spinal deformity. Thus, we provide critical in vivo evidence that LOXL3 is indispensable for mouse palatogenesis and vertebral column development. The Loxl3 gene may be a candidate disease gene resulting in cleft palate and spinal deformity.

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs.

PTEN regulation of the proliferation and differentiation of auditory progenitors through the PTEN/PI3K/Akt-signaling pathway in mice.

The organ of Corti, which is the sensory organ of hearing, consists of a single row of inner hair cells and three rows of outer hair cells in mice. The auditory hair cells develop from auditory progenitors. Hair cell development is related to several genes, including PTEN. Homozygous null mutant (PTEN(-/-)) mice die at around embryonic day 9, when hair cells are extremely immature. Moreover, in heterozygous PTEN knockout mice, it was found that PTEN regulates the proliferation of auditory progenitors. However, little is known about the molecular mechanism underlying this regulation. In the present study, we generated PTEN conditional knockout in the inner ear of mice and studied the aforementioned molecular mechanisms. Our results showed that PTEN knockout resulted in supernumerary hair cells, increased p-Akt level, and decreased p27(kip1) level. Furthermore, the presence of supernumerary hair cells could be explained by the delayed withdrawal of auditory progenitors from the cell cycle. The increased p-Akt level correlates with p27(kip1) downregulation in the cochlea in the Pax2-PTEN mice. The reduced p27(kip1) could not maintain the auditory progenitors in the nonproliferative state and some progenitors continued to divide. Consequently, additional progenitors differentiated into supernumerary hair cells. We suggest that PTEN regulates p27(kip1) through p-Akt, thereby regulating the proliferation and differentiation of auditory progenitors.

Induced pluripotent stem cells: origins, applications, and future perspectives.

Embryonic stem (ES) cells are widely used for different purposes, including gene targeting, cell therapy, tissue repair, organ regeneration, and so on. However, studies and applications of ES cells are hindered by ethical issues regarding cell sources. To circumvent ethical disputes, great efforts have been taken to generate ES cell-like cells, which are not derived from the inner cell mass of blastocyst-stage embryos. In 2006, Yamanaka et al. first reprogrammed mouse embryonic fibroblasts into ES cell-like cells called induced pluripotent stem (iPS) cells. About one year later, Yamanaka et al. and Thomson et al. independently reprogrammed human somatic cells into iPS cells. Since the first generation of iPS cells, they have now been derived from quite a few different kinds of cell types. In particular, the use of peripheral blood facilitates research on iPS cells because of safety, easy availability, and plenty of cell sources. Now iPS cells have been used for cell therapy, disease modeling, and drug discovery. In this review, we describe the generations, applications, potential issues, and future perspectives of iPS cells.