Grainyhead-like 2 Expression Research Articles

Prognostic value of blood GRHL2 in patients with non-small-cell lung cancer after radiotherapy and chemotherapy.

Aim: We aimed to investigate the predictive value of the Grainyhead-like 2 (GRHL2) expression from circulating blood for recurrence, metastasis and overall death on patients with non-small-cell lung cancer (NSCLC).Materials & Methods: We collected blood samples from 122 patients who were admitted to our hospital for NSCLC.Results: Multivariable Cox proportional-hazards analysis in adjusted Model II showed that compared with GRHL2-negative expression, positive expression in patients with NSCLC was associated with increased death risk (HR=7.0, 95% CI: 2.1-20.9, p=0.03) and risk for composite end point (HR=8.2, 95% CI: 4.0-27.1, p <0.01).Conclusion: This study supported that elevated circulating GRHL2 expression might be considered as a candidate prognostic biomarker for poor prognosis among these NSCLC patients.

Grainyhead-like 2 Deficiency and Kidney Cyst Growth in a Mouse Model.

The transcription factor Grainyhead-like 2 (GRHL2) plays a crucial role in maintaining the epithelial barrier properties of the renal collecting duct and is essential for osmoregulation. We noticed a reduction in GRHL2 expression in cysts derived from the collecting ducts in kidneys affected by Autosomal Dominant Polycystic Kidney Disease (ADPKD). However, the specific role of GRHL2 in cystic kidney disease remains unknown. The functional role of the transcription factor Grhl2 in the context of cystic kidney disease was examined through analysis of its expression pattern in patient samples with ADPKD and generating a transgenic cystic kidney disease (TCKD) mouse model by overexpressing the human proto-oncogene c-MYC in kidney collecting ducts. Next, TCKD mice bred with collecting duct-specific Grhl2 knockout mice (Grhl2KO). The resulting TCKD-Grhl2KO mice and their littermates were examined by various types of histological and biochemical assays and gene profiling analysis via RNA-seq. A comprehensive examination of kidney samples from patients with ADPKD revealed GRHL2 downregulation in collecting duct-derived cyst epithelia. Comparative analysis of TCKD and TCKD-Grhl2KO mice exhibited that the collecting duct-specific deletion of Grhl2 resulted in markedly aggravated cyst growth, worsened kidney dysfunction, and shortened life span. Furthermore, transcriptomic analyses indicated sequential downregulation of kidney epithelial cyst development regulators (Frem2, Muc1, Cdkn2c, Pkd2, and Tsc1) during cyst progression in kidneys of TCKD-Grhl2KO mice which included presumed direct Grhl2 target genes. These results suggest GRHL2 as a potential progression modifier, especially for cysts originating from collecting ducts.

Silencing of GRHL2 induces epithelial‑to‑mesenchymal transition in lung cancer cell lines with different effects on proliferation and clonogenic growth.

Grainyhead-like 2 (GRHL2) is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT). It has been previously shown that GRHL2 can confer both oncogenic and tumor-suppressive roles in human cancers, including breast, pancreatic and colorectal cancers. However, its role in lung cancer remains elusive. In the present study, a meta-analysis of multiple gene expression datasets with clinical data revealed that GRHL2 expression was increased in lung cancer compared with that in the normal tissues. Copy number analysis of GRHL2, performed using datasets of whole exome sequencing involving 151 lung cancer cell lines, revealed frequent amplifications, suggesting that the increased GRHL2 expression may have resulted from gene amplification. A survival meta-analysis of GRHL2 using The Cancer Genome Atlas (TCGA) dataset showed no association of GRHL2 expression with overall survival. GRHL2 expression was found to be associated with EMT status in lung cancer in TCGA dataset and lung cancer cell lines. GRHL2 knockdown induced partial EMT in the hTERT/Cdk4-immortalized normal lung epithelial cell line HBEC4KT without affecting proliferation measured by CCK-8 assays. In addition, GRHL2 silencing caused three lung cancer cell lines, H1975, H2009 and H441, to undergo partial EMT. However, the proliferative effects differed significantly. GRHL2 silencing promoted proliferation but not colony formation in H1975 cells whilst suppressing colony formation without affecting proliferation in H2009 cells, but it did not affect proliferation in H441 cells. These results suggest cell type-dependent effects of GRHL2 knockdown. Downstream, GRHL2 silencing enhanced the phosphorylation of AKT and ERK, assessed by western blotting with phospho-specific antibodies, in HBEC4KT, H1975 and H2009 cell lines but not in the H441 cell line. By contrast, transient GRHL2 overexpression did not affect A549 cell proliferation, which lack detectable endogenous expression of the GRHL2 protein. However, GRHL2 overexpression did suppress E-cadherin expression in A549 cells. These results suggested that GRHL2 does not only function as a tumor suppressor of EMT but can also behave as an oncogene depending on the lung cancer cell-type context.

Abstract P3-11-06: Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance

Abstract Current data using gene expression and DNA modification profiles yield limited information for evaluating the potential resistance to therapies. Recent studies show that the chromatin landscape is dynamic and defines cell identity and disease status. Thus, changes in open chromatin sites could provide important information on disease progression and identify potential therapeutic targets. The majority of breast cancers express estrogen receptor (ER) and initially respond to endocrine therapies blocking ER activity. However, endocrine therapy resistance (ETR) occurs de novo or follows an initial response. Recent studies have identified new drivers of ETR, such as mTOR and CDK4/6. Therapy with these inhibitors in combination with endocrine therapy have improved patient survival, but the molecular mechanism of ETR is not fully understood and additional therapeutic options for ETR are needed. To investigate the molecular mechanisms of ETR, we established in vitro Long-Term Estrogen Deprivation (LTED) model using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression upon LTED with ESR1 upregulation in some cell lines and downregulation in others. LTEDs with ESR1 upregulation were sensitive to fulvestrant, while the others were resistant. We analyzed time-course of chromatin landscape transitions during acquisition of ETR in ESR1 up- and downregulated LTEDs cells by Assay for Transposase Accessible Chromatin (ATAC-seq) combined with RNA-seq. Because transcription factors play a primary key role in gene expression and in cell-fate changes including response to therapies, we performed bioinformatic analysis to uncover differential open chromatin sites, select enhancer signatures and identify digital footprints of transcription factors. Unlike ChIP-seq, which identifies binding sites for a known transcription factor, ATAC-seq permits analysis of all sites potentially accessible to the transcriptional machinery and predict occupancy by ~700 different transcription factors at once. With these algorithms, we found that Grainyhead-like 2 (GRHL2) is associated with development of both ESR1 overexpressed and downregulated types of LTEDs. Furthermore, RNA-seq analysis showed that GRHL2 target genes are upregulated in LTEDs. Furthermore, in the METABRIC database, high GRHL2 expression is significantly associated with poor outcome in ER+ breast cancer patients treated with endocrine therapy. Taken together, these data suggest that GRHL2 may be a novel important transcription factor in endocrine therapy resistant breast cancer regardless of fulvestrant sensitivity. Our studies provide evidence that chromatin landscape analysis, coupled with the transcription factor network algorithm, is capable of identifying novel biomarkers or therapeutic targets, This approach will expand the range of translational research as it is applicable to many cancers and diseases. Citation Format: Saori Fujiwara, Songjoon Baek, Kaustubh Wagh, Diana Stavreva, Lyuba Varticovski, Gordon Hager. Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-06.

GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer.

The transcription factor Grainyhead-like 2 (GRHL2) is a critical transcription factor for epithelial tissues that has been reported to promote cancer growth in some and suppress aspects of cancer progression in other studies. We investigated its role in different breast cancer subtypes. In breast cancer patients, GRHL2 expression was increased in all subtypes and inversely correlated with overall survival in basal-like breast cancer patients. In a large cell line panel, GRHL2 was expressed in luminal and basal A cells, but low or absent in basal B cells. The intersection of ChIP-Seq analysis in 3 luminal and 3 basal A cell lines identified conserved GRHL2 binding sites for both subtypes. A pathway analysis of ChIP-seq data revealed cell-cell junction regulation and epithelial migration as well as epithelial proliferation, as candidate GRHL2-regulated processes and further analysis of hub genes in these pathways showed similar regulatory networks in both subtypes. However, GRHL2 deletion in a luminal cell line caused cell cycle arrest while this was less prominent in a basal A cell line. Conversely, GRHL2 loss triggered enhanced migration in the basal A cells but failed to do so in the luminal cell line. ChIP-Seq and ChIP-qPCR demonstrated GRHL2 binding to CLDN4 and OVOL2 in both subtypes but not to other GRHL2 targets controlling cell-cell adhesion that were previously identified in other cell types, including CDH1 and ZEB1. Nevertheless, E-cadherin protein expression was decreased upon GRHL2 deletion especially in the luminal line and, in agreement with its selectively enhanced migration, only the basal A cell line showed concomitant induction of vimentin and N-cadherin. To address how the balance between growth reduction and aspects of EMT upon loss of GRHL2 affected in vivo behavior, we used a mouse basal A orthotopic transplantation model in which the GRHL2 gene was silenced. This resulted in reduced primary tumor growth and a reduction in number and size of lung colonies, indicating that growth suppression was the predominant consequence of GRHL2 loss. Altogether, these findings point to largely common but also distinct roles for GRHL2 in luminal and basal breast cancers with respect to growth and motility and indicate that, in agreement with its negative association with patient survival, growth suppression is the dominant response to GRHL2 loss.

Circ_0111277 suppresses trophoblast cell proliferation, angiogenesis, migration, invasion and EMT via regulating miR-188-3p/GRHL2 axis.

Preeclampsia (PE) is the main factor threatening the life of primipara. Defective migration and invasion of trophoblast cells was one of the causes of PE. Circ_0111277 had been reported to be related to the development of PE, but the mechanism of its effect on trophoblast cells needed further study. The expression of circ_0111277, microRNA-188-3p (miR-188-3p) and grainyhead-like 2 (GRHL2) mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8) assay, 5-Ethynyl-20-deoxyuridine (EdU) and colony formation assay were used to examine cell proliferation ability. Tube formation and transwell assay were performed to assess the angiogenesis and metastasis ability of cells. Western blot was applied to measure the levels of epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin and Vimentin) and GRHL2 protein. The relationship between miR-188-3p and circ_0111277 or GRHL2 was verified by the dual luciferase reporter experiment. Circ_0111277 and GRHL2 were elevated, and miR-188-3p was declined in PE patients. Overexpression of circ_0111277 could inhibit the proliferation, angiogenesis, migration, invasion and EMT of trophoblast cells (HTR-8/Svneo). Circ_0111277 was the molecular sponge of miR-188-3p. MiR-188-3p up-regulation could reduce the inhibition of HTR-8/Svneo cell growth caused by overexpression of circ_0111277. GRHL2 was a target gene of miR-188-3p, and GRHL2 silencing relieved the adverse effects of miR-188-3p inhibitors on HTR-8/Svneo. In general, circ_0111277 up-regulated GRHL2 expression through sponge miR-188-3p. Highly expressed circ_0111277 up-regulated the expression of GRHL2 through sponge miR-188-3p, thereby inhibiting trophoblast cells function, which suggested a new molecular mechanism for the pathogenesis of PE.

Abnormal GRHL2 Methylation Confers Malignant Progression to Acute Leukemia.

Purpose Abnormal methylation of Grainyhead-like 2 (GRHL2) is associated with a substantial role in the malignant phenotype of tumor patients. Our present research is aimed at studying the abnormal expression of GRHL2 and the association of methylation in patients with acute leukemia and its relationship with prognosis. Materials and Methods We used quantitative real-time polymerase chain reaction (qRT-PCR) for detecting the aberrant expression level of GRHL2 in 60 patients with acute leukemia and 60 normal controls. We analyzed the significant correlation between the expression level of GRHL2 with clinicopathological features and patients' prognosis in acute leukemia using the corresponding statistical methods. Secondly, we employed qRT-PCR and Western blotting to detect the mRNA and protein levels of GRHL2 in leukemia cell lines. Next, we used methylation-specific polymerase chain reaction (MSP) technology for detecting the methylation of GRHL2 in clinical samples with acute leukemia and cell lines. Then we investigated the demethylating effect of arsenic trioxide and 5-azacitidine on the mRNA and protein expression levels of GRHL2 in cell lines of acute leukemia. Finally, we studied the effects of arsenide trioxide and 5-azacitidine on the proliferation of leukemia cells and the TGF-β signaling pathway. Results We found a lower level of GRHL2 expression not only in acute leukemia patients but also in cell lines when compared with normal controls. At the same time, the expression level of GRHL2 in patients with acute leukemia was significantly correlated with leukocyte count, platelet count, and cytogenetic risk grouping. In addition, the lower GRHL2 expression group showed a significantly lower overall survival rate in acute leukemia patients than that of patients with a higher GRHL2 expression group. Univariate and multivariate analyses revealed that the expression of GRHL2 is an independent risk factor in acute leukemia patients. The methylation level of the GRHL2 promoter region in acute leukemia patients and cell lines was significantly higher than the normal control group, and we found the elevated mRNA and protein levels of GRHL2 in acute leukemia cell lines after the use of the demethylation drug arsenic trioxide and 5-azacitidine. At the same time, arsenide trioxide and 5-azacitidine are associated with the inhibition of cellular proliferation of acute leukemia cells and also promote the elevated expression of TGF-β signaling pathway-linked proteins, including TGF-β, Smad2, Smad3, and Smad4. Conclusion Increased expression and methylation level of GRHL2 are closely associated with the prognosis and malignant phenotype of acute leukemia patients and play an irreplaceable role in the occurrence and development of patients with acute leukemia.

LncRNA XIST sponges microRNA-448 to promote malignant behaviors of colorectal cancer cells via regulating GRHL2.

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are essential regulators in human cancers, while the role of lncRNA X-inactive-specific transcript (XIST) in colorectal cancer (CRC) via regulating miR-448 remains largely unknown. Herein, we aimed to elucidate the effect of the XIST/miR-448/grainyhead-like 2 (GRHL2) axis on CRC development. XIST, miR-448, and GRHL2 expression in CRC tissues from patients and in human CRC cell lines was assessed. Loss- and gain-function assays were implemented to unveil the roles of XIST, miR-448, and GRHL2 in screened CRC cells. The tumor growth in vivo was observed in nude mice. Binding relations among XIST, miR-448, and GRHL2 were evaluated. XIST and GRHL2 expressed highly whereas miR-448 expressed lowly in CRC tissues and cells. XIST or GRHL2 downregulation, or miR-448 elevation suppressed the malignant behaviors of CRC cells in vitro, and downregulated XIST or upregulated miR-448 also inhibited the tumor growth in vivo. miR-448 upregulation reversed the role of XIST elevation in CRC cells. XIST particularly bound to miR-448, and miR-448 targeted GRHL2. Knockdown of XIST upregulates miR-448 to impede malignant behaviors of CRC cells via inhibiting GRHL2. This study may provide novel biomarkers for CRC diagnosis and treatment.

Soy isoflavones alleviate periodontal destruction in ovariectomized rats.

The purpose of this study was to investigate whether soy isoflavone supplementation is effective in preventing periodontal destruction exacerbated by estrogen deficiency (ED) and its potential mechanism. The progression of periodontitis is affected by host factors, such as smoking, diabetes mellitus, and steroid use. Bone loss in periodontitis can be aggravated by ED. A rat model of experimental periodontitis (EP) with ED was established by silk ligature and inoculation with Porphyromonas gingivalis, and some EP rats were subjected to bilateral ovariectomy (OVX). The treatment groups received an intravenous injection of 17-β-estradiol (E2 B) or soy isoflavones (SI) by gavage. The rats were euthanized, and the maxillary jaws, gingiva, and serum were harvested. Tight junction protein and interleukin (IL)-17 expression, reactive oxygen species (ROS) level, and periodontal destruction were assessed. In addition, we determined whether grainyhead-like 2 (GRHL2) is required for enhancing the epithelial barrier by SI in an in vitro P.gingivalis infection model. Estrogen deficiency impaired the expression of genes encoding tight junction proteins in the gingiva, increased IL-17 level, and accelerated alveolar bone resorption. SI treatment alleviated tight junction protein expression, decreased IL-17 and ROS levels, and prevented the absorption of alveolar bone. Furthermore, GRHL2 expression was significantly induced by SI in human oral keratinocytes-1 (HOK-1) cells; GRHL2 knockdown impaired the expression of OCLN and ZO-1 induced by SI treatment. Soy isoflavones alleviates periodontitis in OVX rats, as observed by the increased expression of tight junction proteins, and reduced IL-17 level and alveolar bone loss. The in vitro studies suggested that the enhancement of oral epithelial barrier by SI treatment was partially dependent on GRHL2.

GRHL2 Expression Functions in Breast Cancer Aggressiveness and Could Serve as Prognostic and Diagnostic Biomarker for Breast Cancer.

Background:Breast cancer (BC) is the most frequent malignancy in women worldwide and the leading cause of female cancer–associated death in the world. Grainyhead-like 2 (GRHL2) is an important gene involved in human cancer progression. However, the role of GRHL2 in BC is unknown.Methods:In this study, we used in vitro experiments to verify the role of GRHL2 expression in BC progression. We used 14 databases to analyse the expression level of GRHL2 in BC and its prognostic and diagnostic value. In addition, the correlation between GRHL2 expression and immune cell infiltration and DNA methylation was also analysed.Results:At the cellular level, overexpression of GRHL2 induced E-cadherin expression in BC cells with a mesenchymal phenotype and resulted in a hybrid epithelial/mesenchymal (E/M) phenotype, which is more strongly correlated with tumour aggressiveness than a pure mesenchymal phenotype. Through analysis of various databases, we found that tumour tissue had a higher expression level of GRHL2. High expression of GRHL2 was associated with worse prognosis of BC patients and indicated that GRHL2 had significant diagnostic value. Grainyhead-like 2 is also related to immune infiltration and regulated by DNA methylation. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses showed that GRHL2-related signalling pathways in BC were related to tumour cell proliferation, invasion, and angiogenesis.Conclusions:In summary, evidence indicates that GRHL2 can be used as a prognostic and diagnostic biomarker for BC.

Whole blood GRHL2 expression as a prognostic biomarker in metastatic hormone-sensitive and castration-resistant prostate cancer.

BackgroundAs potent systemic therapies transition earlier in the prostate cancer disease course, molecular biomarkers are needed to guide optimal treatment selection for metastatic hormone-sensitive prostate cancer (mHSPC). The value of whole blood RNA to detect candidate biomarkers in mHSPC remains largely undefined.MethodsIn this cohort study, we used a previously optimised whole blood reverse transcription polymerase chain reaction assay to assess the prognostic utility [measured by seven-month undetectable prostate-specific antigen (PSA) and time to castration-resistance (TTCR)] of eight prostate cancer-associated gene transcripts in 43 mHSPC patients. Transcripts with statistically significant associations (P<0.05) were further investigated in a metastatic castration-resistant prostate cancer (mCRPC) cohort (n=119) receiving contemporary systemic therapy, exploring associations with PSA >50% response (PSA50), progression-free survival (PFS) and overall survival (OS). Clinical outcomes were prospectively collected in a protected digital database. Kaplan-Meier estimates and multivariable Cox proportional-hazards models assessed associations between gene transcripts and clinical outcomes (mHSPC covariates: disease volume, docetaxel use and haemoglobin level; mCRPC covariates: prior exposure to chemotherapy or ARPIs, haemoglobin, performance status and presence of visceral disease). Follow-up was performed monthly during ARPI treatment, three-weekly during taxane chemotherapy, and three-monthly during androgen deprivation therapy (ADT) monotherapy. Serial PSA measurements were performed before each follow-up visit and repeat imaging was at the discretion of the investigator.ResultsDetection of circulating Grainyhead-like 2 (GRHL2) transcript was associated with poor outcomes in mHSPC and mCRPC patients. Detectable GRHL2 expression in mHSPC was associated with a lower rate of seven-month undetectable PSA levels (25% vs. 65%, P=0.059), and independently associated with shorter TTCR (HR 7.3, 95% CI: 1.5–36, P=0.01). In the mCRPC cohort, GRHL2 expression predicted significantly lower PSA50 response rates (46% vs. 69%, P=0.01), and was independently associated with shorter PFS (HR 3.1, 95% CI: 1.8–5.2, P<0.001) and OS (HR 2.9, 95% CI: 1.6–5.1, P<0.001). Associations were most apparent in patients receiving ARPIs.ConclusionsDetectable circulating GRHL2 was a negative prognostic biomarker in our mHSPC and mCRPC cohorts. These data support further investigation of GRHL2 as a candidate prognostic biomarker in metastatic prostate cancer, in addition to expanding efforts to better understand a putative role in therapeutic resistance to AR targeted therapies.

CircZDHHC20 represses the proliferation, migration and invasion in trophoblast cells by miR-144/GRHL2 axis

BackgroundPreeclampsia (PE) is a prevalent pregnancy disorder that has been one of the leading causes of maternal and perinatal mortality worldwide. Circular RNAs (circRNAs) have recently considered as important regulators in PE pathogenesis. In the current study, we aimed to explore the impact and mechanisms of circRNA zinc finger DHHC-type palmitoyltransferase 20 (circZDHHC20) in PE pathogenesis.MethodsRNase R assay and reverse transcription with Oligo(dT)18 primers were performed to confirm that circZDHHC20 was indeed circular transcript. The expression of circZDHHC20, grainyhead-like 2 (GRHL2) and miR-144 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Subcellular localization assay was used to determine whether circZDHHC20 was predominantly present in the cytoplasm. The target correlations between miR-144 and circZDHHC20 or GRHL2 were confirmed using dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Cell proliferation, migration, and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetr-azolium (MTS), wound healing and transwell assays, respectively. Western blot was used for the quantification of GRHL2 protein level.ResultsOur data indicated that circZDHHC20 was up-regulated and miR-144 was down-regulated in PE placenta. CircZDHHC20 sequestered miR-144 by acting as a miR-144 sponge. CircZDHHC20 overexpression repressed trophoblast cell proliferation, migration, and invasion, while its knockdown exerted opposite effects. Moreover, miR-144 mediated the regulation of circZDHHC20 on trophoblast cell behaviors. GRHL2 was directly targeted and inhibited by miR-144. MiR-144 exerted regulatory effects on trophoblast cell proliferation, migration and invasion by GRHL2. Furthermore, circZDHHC20 modulated GRHL2 expression through sponging miR-144.ConclusionOur study suggested that a high level of circZDHHC20 inhibited the proliferation, migration, and invasion in trophoblast cells at least partially through sponging miR-144 and up-regulating GRHL2, providing a novel mechanism of PE pathogenesis.

Porphyromonas gingivalis Impairs Oral Epithelial Barrier through Targeting GRHL2

Oral mucosa provides the first line of defense against a diverse array of environmental and microbial irritants by forming the barrier of epithelial cells interconnected by multiprotein tight junctions (TJ), adherens junctions, desmosomes, and gap junction complexes. Grainyhead-like 2 (GRHL2), an epithelial-specific transcription factor, may play a role in the formation of the mucosal epithelial barrier, as it regulates the expression of the junction proteins. The current study investigated the role of GRHL2 in the Porphyromonas gingivalis (Pg)–induced impairment of epithelial barrier functions. Exposure of human oral keratinocytes (HOK-16B and OKF6 cells) to Pg or Pg-derived lipopolysaccharides (Pg LPSs) led to rapid loss of endogenous GRHL2 and the junction proteins (e.g., zonula occludens, E-cadherin, claudins, and occludin). GRHL2 directly regulated the expression levels of the junction proteins and the epithelial permeability for small molecules (e.g., dextrans and Pg bacteria). To explore the functional role of GRHL2 in oral mucosal barrier, we used a Grhl2 conditional knockout (KO) mouse model, which allows for epithelial tissue-specific Grhl2 KO in an inducible manner. Grhl2 KO impaired the expression of the junction proteins at the junctional epithelium and increased the alveolar bone loss in the ligature-induced periodontitis model. Fluorescence in situ hybridization revealed increased epithelial penetration of oral bacteria in Grhl2 KO mice compared with the wild-type mice. Also, blood loadings of oral bacteria (e.g., Bacteroides, Bacillus, Firmicutes, β-proteobacteria, and Spirochetes) were significantly elevated in Grhl2 KO mice compared to the wild-type littermates. These data indicate that Pg bacteria may enhance paracellular penetration through oral mucosa in part by targeting the expression of GRHL2 in the oral epithelial cells, which then impairs the epithelial barrier by inhibition of junction protein expression, resulting in increased alveolar tissue destruction and systemic bacteremia.

GRHL2 inhibits colorectal cancer progression and metastasis via oppressing epithelial-mesenchymal transition

ABSTRACTOur previous study has demonstrated that knockdown of Grainyhead-like 2(GRHL2) in colorectal cancer (CRC) cells inhibited cell proliferation by targeting ZEB1. This study aimed at researching whether knockdown of GRHL2 promoted CRC progression and metastasis via inducing epithelial-mesenchymal transition (EMT). GRHL2-upregulated SW-620/GRHL2+ and GRHL2-knockdown HCT116/GRHL2-KD, HT29/GRHL2-KD cells and their control cells were generated. The morphological changes after overexpression and knockdown GRHL2 were observed. qRT-PCR, Western blotting, and Immunofluorescence were used to detect EMT markers: E-cadherin, Vimentin, p-catein, ZO-1 and ZEB1 expression. Then, sh-ZEB1 was transfected to GRHL2 knockdown cells to research the relationship between GRHL2 and ZEB1. Transwell and wound healing assays were further performed to detect the impact of GRHL2 on invasion and migration in vitro. CRC cells were injected into mice tail vein to verify the impact of GRHL2 on CRC metastasis. Morphological change of mesenchymal-epithelial transition (MET) could be observed in SW620/GRHL2+ cell. The expression of epithelial markers: E-cadherin, β-catenin, ZO-1 were up-regulated, while mesenchymal markers: Vimentin was decreased. Meanwhile, opposite EMT morphological change could be observed in HCT116/GRHL2-KD cell, accompanied by reverse change of E-cadherin, β-catenin, ZO-1, and Vimentin. The expression level of GRHL2 and ZEB1 was found negative in both SW620/GRHL2+ and HCT116/GRHL2-KD cells. Knockdown of ZEB1 by siRNA in HCT116/GRHL2-KD and HT29/GRHL2-KD could upregulate expression of E-cadherin and GRHL2. GRHL2 knockdown also promoted migration, invasion in vitro and CRC metastasis in mice model. In conclusion, GRHL2/ZEB1 axis inhibits CRC progression and metastasis via oppressing EMT.

Knockdown of GRHL2 inhibited proliferation and induced apoptosis of colorectal cancer by suppressing the PI3K/Akt pathway

Grainyhead-like 2 (GRHL2) transcription factor is implicated in many types of cancers. However, the role of GRHL2 in colorectal cancer (CRC) has not been fully understood. The present study aimed to evaluate the expression and functional roles of GRHL2 in CRC. The expression of GRHL2 in normal human intestinal epithelial cells and colorectal cancer cells was measured by qRT-PCR and western blot. For knockdown of GRHL2, two small interfere RNAs (siRNAs) targeting GRHL2 or control siRNA was transfected into CRC cell lines (HCT116 and HT29). For GRHL2 overexpression, the GRHL2-overexpressing vector or empty lentiviral vector was infected into HCT116 and HT29 cells. Cell proliferation was measured by MTT assay. Cell apoptosis rate was analyzed by flow cytometry. The expression of proliferating cell nuclear antigen (PCNA), Bax, and Bcl-2 was detected by western blot. We found that GRHL2 was upregulated in CRC cells compared to normal human intestinal epithelial cells. Knockdown of GRHL2 inactivated the PI3K/Akt pathway in HCT116 and HT29 cells. Knockdown of GRHL2 inhibited cell viability, elevated the apoptosis rates, suppressed the expression of PCNA and Bcl-2, and induced the expression of Bax in HCT116 and HT29 cells, and these effects were reversed by activation of the PI3K/Akt pathway. Inhibition of PI3K/Akt pathway blocked the effects of GRHL2 overexpression on cell proliferation and apoptosis. In conclusion, GRHL2 acted as an oncoprotein through regulating cell proliferation and apoptosis in CRC cells. The PI3K/Akt pathway was closely involved in the effects of GRHL2. Therefore, GRHL2 might be a therapeutic target for the CRC treatment.

GRHL2-Dependent Enhancer Switching Maintains a Pluripotent Stem Cell Transcriptional Subnetwork after Exit from Naive Pluripotency

The enhancer landscape of pluripotent stem cells undergoes extensive reorganization during early mammalian development. The functions and mechanisms behind such reorganization, however, are unclear. Here, we show that the transcription factor GRHL2 is necessary and sufficient to activate an epithelial subset of enhancers as naive embryonic stem cells (ESCs) transition into formative epiblast-like cells (EpiLCs). Surprisingly, many GRHL2 target genes do not change in expression during the ESC-EpiLC transition. Instead, enhancers regulating these genes in ESCs diminish in activity in EpiLCs while GRHL2-dependent alternative enhancers become activated to maintain transcription. GRHL2 therefore assumes control over a subset of the naive network via enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency. These data evoke a model where the naive pluripotency network becomes partitioned into smaller, independent networks regulated by EpiLC-specific transcription factors, thereby priming cells for lineage specification.

Expression and significance of GRHL2 in esophageal cancer.

Esophageal cancer (EC) is one of the most common causes of cancer-related mortality in the world. Although much effort has been made to improve the 5-year survival rate of patients with EC, it still remains low due to diagnosis at an advanced stage, aggressive local invasion, early metastasis, and resistance to chemotherapy. Although grainyhead-like 2 (GRHL2) has attracted interest since it has been recently identified as a novel suppressor of the epithelial–mesenchymal transition, clinical values of GRHL2 and its relationship with the metastasis-related factors, such as hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF), remain unclear. In order to investigate the expression of GRHL2, HIF-1α, and VEGF, and their correlation with angiogenesis in EC, 63 patients with EC were examined. The expression of GRHL2, HIF-1α, and VEGF in tumor tissues was higher than that in adjacent tissues and was associated with tumor differentiation. GRHL2 expression was significantly correlated with lymph node metastasis and invasion depth, whereas VEGF expression was associated with tumor (TNM) stage. A significant correlation was found between the expression of GRHL2 and HIF-1α. The patients expressing low GRHL2 and high HIF-1α showed significant reduction in both overall survival rate and disease-free survival rate. The results demonstrated that abnormal expression of GRHL2 is common in EC, and low expression of GRHL2 accompanied by a high expression of HIF-1α indicates poor prognosis.

MicroRNA-194 regulates keratinocyte proliferation and differentiation by targeting Grainyhead-like 2 in psoriasis

MicroRNAs (miRNAs) are currently emerged as important regulators in psoriasis. Psoriasis is characterized by hyperproliferation and impaired differentiation of keratinocytes in skin lesions. miR-194 is a well-known regulator of cell proliferation and differentiation. However, the role of miR-194 in psoriasis pathogenesis remains unclear. In this study we aimed to investigate the role of miR-194 in keratinocyte hyperproliferation and differentiation. We found that miR-194 was significantly downregulated in psoriasis lesional skin. Overexpression of miR-194 inhibited the proliferation and promoted the differentiation of primary human keratinocytes, whereas miR-194 suppression promoted the proliferation and inhibited their differentiation. Bioinformatic analysis predicted that the Grainyhead-like 2 (GRHL2) was a target gene of miR-194, which we further validated with a dual-luciferase reporter assay, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot analysis. The effect of miR-194 on cell proliferation and differentiation was significantly reversed by overexpression of GRHL2. Moreover, the expression of miR-194 and GRHL2 was inversely correlated in psoriasis lesional skin. Taken together, our results suggest that miR-194 inhibits the proliferation and promotes the differentiation of keratinocytes through targeting GRHL2. The downregulation of miR-194 expression may contribute to the pathogenesis of psoriasis and targeting miR-194 may represent a novel and potential therapeutic strategy for psoriasis.

Identification of a novel progenitor cell marker, grainyhead-like 2 in the developing pituitary.

Pituitary stem/progenitor cells give rise to all of the endocrine cell types within the pituitary gland and are necessary for both development and gland homeostasis. Recent studies have identified several key factors that characterize the progenitor cell population. However, little is known about the factors that regulate progenitor cell differentiation and maintenance. Therefore, it is crucial to identify novel factors that help elucidate mechanisms of progenitor cell function in the developing pituitary. Our studies are the first to characterize the expression of Grainyhead-like 2 (GRHL2), a transcription factor known to regulate progenitor cell plasticity, in the developing pituitary. Our studies show GRHL2 expression is highest in the embryonic and early postnatal pituitary and is localized in pituitary progenitor cells. We demonstrate GRHL2 expression is changed in Notch2 cKO and Prop1df/df mice, mouse models that display progenitor cell number defects. In addition, our studies indicate a potential relationship between Notch signaling and GRHL2 expression in the developing pituitary. Taken together, our results indicate GRHL2 as a novel progenitor cell maker in the developing pituitary that may contribute to progenitor cell function and maintenance. Developmental Dynamics 245:1097-1106, 2016. © 2016 Wiley Periodicals, Inc.

Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells.

Grainyhead-like 2 (GRHL2) is one of the three mammalian homologues of Drosophila Grainyhead involved in epithelial morphogenesis. We recently showed that GRHL2 also controls normal epithelial cell proliferation and differentiation. In this study, we investigated the role of GRHL2 in oral carcinogenesis and the underlying mechanism. GRHL2 expression was elevated in cells and tissues of oral squamous cell carcinomas (OSCCs) compared with normal counterparts. Knockdown of GRHL2 resulted in the loss of in vivo tumorigenicity, cancer stemness and epithelial phenotype of oral cancer cells. GRHL2 loss also inhibited oral cancer cell proliferation and colony formation. GRHL2 regulated the expression of miR-200 family and Octamer-binding transcription factor 4 (Oct-4) genes through direct promoter DNA binding. Overexpression of miR-200 genes in the oral cancer cells depleted of GRHL2 partially restored the epithelial phenotype, proliferative rate and cancer stemness, indicating that miR-200 genes in part mediate the functional effects of GRHL2. Taken together, this study demonstrates a novel connection between GRHL2 and miR-200, and supports protumorigenic effect of GRHL2 on OSCCs.