KRT5 Expression Research Articles

3, 3′, 5-Triiodo-L-thyronine affects polarity proteins of bovine Sertoli cells via WT1/non-canonical Wnt signaling pathway

To determine the role of 3, 3′, 5-triiodo-L thyroxine (T3) in the differentiation of Sertoli cells (SCs) and the factors influencing maturity via the Wilms’ tumor 1 (WT1)/non-canonical Wnt signaling pathway, high purity SCs were isolated from newborn calves’ testes and cultured in vitro. The SCs were stimulated with T3, and co-treated with short interference (si) RNA to knockdown endogenous WT1 and non-canonical Wnt signalling inhibitor Wnt-c59. Our results suggested that the addition of different concentrations (0, 25, 50, and 100 nM) of T3 in the culture medium changed the expression of KRT-18 (SCs immature marker) and accelerated the differentiation of SCs. T3 (100 nM) treatment induced up-regulated expression of WT1 over time (p < 0.05), while the expression of polarity proteins (Par3, Par6b, and E-cadherin) and Wnt4 were affected to varying degrees (p < 0.05). SCs were treated simultaneously with T3 + Wnt-c59 and T3 + WT1 siRNA, and the results showed that T3 could affect the expression of polarity proteins via WT1/non-canonical Wnt signaling pathway. These data put together indicate that T3 plays a dependent role in the induction of bovine SCs differentiation via WT1/non-canonical Wnt signaling pathway in vitro. This study proposes for the first time that WT1 is a major target for T3.

Dimethylarsinic acid (DMA) enhanced lung carcinogenesis via histone H3K9 modification in a transplacental mouse model.

Pregnant CD-1 mice received 200ppm dimethylarsinic acid (DMA) in the drinking water from gestation day 8-18, and tumor formation was assessed in offspring at the age of 84weeks. DMA elevated the incidence of lung adenocarcinoma (10.0%) and total tumors (33.3%) in male offspring compared to male control offspring (1.9 and 15.1%, respectively). DMA also elevated the incidence of hepatocellular carcinoma (10.0%) in male offspring compared to male control offspring (0.0%). DMA and its metabolites were detected in the lungs of transplacental DMA-treated neonatal mice. Transplacental DMA exposure increased cell proliferation in the epithelium in the lungs of both neonatal and 6-week-old male mice. Microarray and real-time PCR analyses detected high expression of keratin 8 (Krt8) in the lungs of both neonatal and 6-week-old DMA-treated mice. Western blot analysis indicated that DMA elevated methylation of histone H3K9, but not H3K27, in the lungs of male mice. Importantly, chromatin immunoprecipitation sequencing (ChIP-seq) analysis using an H3K9me3 antibody found differences in heterochromatin formation between mice exposed to DMA and the controls. Notably, ChIP-seq analysis also found regions of lower heterochromatin formation in DMA-treated mice, and one of these regions contained the Krt8 gene, agreeing with the results obtained by microarray analysis. High expression of Krt8 was also detected in adenoma and adenocarcinoma of the lung in male offspring. Overall, these data indicate that transplacental DMA treatment enhanced lung and liver carcinogenesis in male mice. In the lung, DMA caused aberrant methylation of histone H3K9, increased Krt8 expression, and enhanced cell proliferation.

Activation of extracellular signal-regulated kinase is associated with hepatocellular carcinoma with aggressive phenotypes.

Sorafenib inhibits multiple kinase signaling pathways, including the rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and is a promising therapy for hepatocellular carcinoma (HCC). However, the role of ERK activation in HCC remains unclear. This study was designed to investigate the potential link between ERK activation and aggressive HCC phenotypes. We evaluated nuclear ERK expression by immunohistochemistry in 154 resected HCC nodules from 136 patients. We then investigated the associations of ERK expression with the clinicopathological characteristics of HCC, c-MET expression, and the molecular subclass biomarkers Ki-67, keratin19 (KRT19, CK19, or K19), and sal-like protein4. Multivariate Cox regression analysis was carried out to determine independent prognostic factors for overall survival and recurrence-free survival. The effects of ERK activation by hepatocyte growth factor (HGF) on eight HCC cell lines were further examined. High-level nuclear expression of ERK was observed in 20 (13%) of 154 nodules and was significantly associated with higher serum alpha-fetoprotein levels (P = 0.034), poorer differentiation (P = 0.003), a higher Ki-67 index (P < 0.001), high-level expression of c-MET (P = 0.008), KRT19 (P = 0.002), or sal-like protein4 (P < 0.001), and shorter overall survival (multivariate hazard ratio 3.448; P = 0.028) and recurrence-free survival (multivariate hazard ratio 2.755; P = 0.004). HCC cells treated with hepatocyte growth factor showed enhanced cell proliferation together with ERK activation and upregulated KRT19 expression, both of which were inhibited by sorafenib. High-level ERK activation is associated with a KRT19-positive highly proliferative subtype of HCC with a dismal prognosis. These findings support the key role of the hepatocyte growth factor/c-MET/ERK axis in HCC progression.

RNA Sequencing of Collecting Duct Renal Cell Carcinoma Suggests an Interaction between miRNA and Target Genes and a Predominance of Deregulated Solute Carrier Genes.

Collecting duct carcinoma (CDC) is a rare renal cell carcinoma subtype with a very poor prognosis. There have been only a few studies on gene expression analysis in CDCs. We compared the gene expression profiles of two CDC cases with those of eight normal tissues of renal cell carcinoma patients. At a threshold of |log2fold-change| ≥1, 3349 genes were upregulated and 1947 genes were downregulated in CDCs compared to the normal samples. Pathway analysis of the deregulated genes revealed that cancer pathways and cell cycle pathways were most prominent in CDCs. The most upregulated gene was keratin 17, and the most downregulated gene was cubilin. Among the most downregulated genes were four solute carrier genes (SLC3A1, SLC9A3, SLC26A7, and SLC47A1). The strongest negative correlations between miRNAs and mRNAs were found between the downregulated miR-374b-5p and its upregulated target genes HIST1H3B, HK2, and SLC7A11 and between upregulated miR-26b-5p and its downregulated target genes PPARGC1A, ALDH6A1, and MARC2. An upregulation of HK2 and a downregulation of PPARGC1A, ALDH6A1, and MARC2 were observed at the protein level. Survival analysis of the cancer genome atlas (TCGA) dataset showed for the first time that low gene expression of MARC2, cubilin, and SLC47A1 and high gene expression of KRT17 are associated with poor overall survival in clear cell renal cell carcinoma patients. Altogether, we identified dysregulated protein-coding genes, potential miRNA-target interactions, and prognostic markers that could be associated with CDC.

NANOG is required to form the epiblast and maintain pluripotency in the bovine embryo.

During preimplantation development, the embryo undergoes two consecutive lineages specifications. The first cell fate decision determines which cells give rise to the trophectoderm (TE) and the inner cell mass (ICM). Subsequently, the ICM differentiates into hypoblast and epiblast, the latter giving rise to the embryo proper. The transcription factors that govern these cell fate decisions have been extensively studied in the mouse, but are still poorly understood in other mammalian species. In the present study, the role of NANOG in the formation of the epiblast and maintenance of pluripotency in the bovine embryo was investigated. Using a CRISPR-Cas9 approach, guide RNAs were designed to target exon 2, resulting in a functional deletion of bovine NANOG at the zygote stage. Disruption of NANOG resulted in the embryos that form a blastocoel and an ICM composed of hypoblast cells. Furthermore, NANOG-null embryos showed lower expression of epiblast cell markers SOX2 and HA2AFZ, and hypoblast marker GATA6; without affecting the expression of TE markers CDX2 and KRT8. Results indicate that NANOG, has no apparent role in segregation or maintenance of the TE, but it is required to derive and maintain the pluripotent epiblast and during the second lineage commitment in the bovine embryo.

174 Use of mesenchymal stem cell treatment to improve oocyte yield and invitro embryo production in cattle

Cumulative tissue damage and chronic inflammation associated with frequent ovum pickup (OPU) may lead to a progressive reduction in the number and quality of the oocytes recovered, particularly in donors with a high antral follicle count. The aim of this study was to evaluate the effect of an intraovarian treatment with mesenchymal stem cells (MSC) on oocyte yield, quality, and development potential during invitro embryo production (IVEP) in cattle donors undergoing repeated OPU. Mesenchymal stem cells were previously isolated from adipose tissue, cultured in Dulbecco's modified Eagle medium until reaching 80% confluence, isolated with trypsin, and frozen in liquid N2 until use. Characterisation of MSC was carried out according to the guidelines of the International Society for Cellular Therapy. Nelore (Bos indicus) cows (n=5) were used in this study, with the ovaries as replicates. The cows underwent eight OPU sessions at 15-day intervals, and the oocytes recovered were graded and used for IVEP with the semen of a single sire and batch under similar invitro culture conditions. To ensure a high inflammatory response, immediately after the fourth OPU session all ovaries received 30 additional punctures, performed with a 16-gauge Jelco needle. Six hours later, the left ovary of each cow was injected with 500µL of Dulbecco's modified phosphate buffered saline (control ovary) and the right ovary received 500µL of Dulbecco's modified phosphate buffered saline with 2.5×106 allogenic MSC (treated ovary). Oocyte yield and embryo production before and after treatment were recorded for each ovary and donor. Grade I blastocysts produced from control and treated ovaries were used for gene expression evaluation. Data was analysed using the repeated-measures procedure of SAS (SAS Institute Inc.) to account for the effects of treatment, time, and interactions. There was no difference (P&amp;gt;0.05) in any endpoint before treatment (sessions 1-4) between the right and left ovaries. Thus, differences between ovaries observed in OPU sessions 5-8 were assumed to be due to the treatment. After the injection of MSC, more total and viable oocytes were collected from the right ovaries compared with the left ovaries (15.3±2.2 vs. 8.7±1.2 (P&amp;lt;0.02) and 13.6±2.1 vs. 7.1±1.0 (P&amp;lt;0.01), respectively), resulting in more embryos produced invitro (7.6±1.2 vs. 3.6±0.6, respectively; P&amp;lt;0.01) as well as more initial and expanded blastocysts (1.4±0.3 vs. 0.4±0.1 and 4.4±0.9 vs. 2.1±0.4, respectively; P&amp;lt;0.04). The proportion of viable oocytes recovered from the right ovary after treatment was greater than that from the left ovary (89.1% vs. 81.5%; P&amp;lt;0.05). However, blastocyst rates did not differ between ovaries before or after treatment (50.4% vs. 55.5%: P&amp;gt;0.05). In the blastocysts produced from treated ovaries, SLC2A3 was overexpressed (P&amp;lt;0.04), whereas there was no difference for the expression of KRT8, PLAC8, SLC2A1, CASP3, PRDX3, or SOD2 (P&amp;gt;0.05), suggesting potential differences in glucose uptake and metabolism. In conclusion, intraovarian treatment with MSC improved oocyte yield and quality and may be an alternative to increase IVEP from donors under intensive OPU schedules. This research was supported by CNPq, CAPES, and Fazenda Grupo Esplanada.

Knockdown of cytokeratin 8 overcomes chemoresistance of chordoma cells by aggravating endoplasmic reticulum stress through PERK/eIF2\u03b1 arm of unfolded protein response and blocking autophagy

Chordoma is a malignant primary osseous spinal tumor with pronounced chemoresistance. However, the mechanisms of how chordoma cells develop chemoresistance are still not fully understood. Cytokeratin 8 (KRT8) is a molecular marker of notochordal cells, from which chordoma cells were believed to be originated. In this study, we showed that either doxorubicin or irinotecan promoted KRT8 expression in both CM319 and UCH1 cell lines, accompanied by an increased unfolded protein response and autophagy activity. Then, siRNA-mediated knockdown of KRT8 chemosensitized chordoma cells in vitro. Mechanistic studies showed that knockdown of KRT8 followed by chemotherapy aggravated endoplasmic reticulum stress through PERK/eIF2α arm of unfolded protein response and blocked late-stage autophagy. Moreover, suppression of the PERK/eIF2α arm of unfolded protein response using PERK inhibitor GSK2606414 partially rescued the apoptotic chordoma cells but did not reverse the blockage of the autophagy flux. Finally, tumor xenograft model further confirmed the chemosensitizing effects of siKRT8. This study represents the first systematic investigation into the role of KRT8 in chemoresistance of chordoma and our results highlight a possible strategy of targeting KRT8 to overcome chordoma chemoresistance.

P11 KRT17 PROMOTES PROLIFERATION AND METASTASIS OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA LEADING TO POOR PROGNOSIS

Abstract Background Keratin17(KRT17), as a multifunction cytoskeletal protein, associated with a multitudinous of biological processes, including cell proliferation, migration, and invasion. Previously, we have found overexpression of KRT17 genes in ESCC tissues. Currently, published kinds of researches claimed KRT17 is engaged in the tumorigenesis and progression of multiple cancers. However, the prognostic significance of KRT17 in ESCC patients and its effects in ESCC progression remain indistinct. Methods We verified the expression of KRT17 in ESCC tissues by Western blotting and q-PCR. And then the immunochemistry was performed to investigate the KRT17 expression in 64 ESCC tissue specimens. KRT17 upregulated, and knockdown EC9706 and Eca109 cells were constructed by Lentivirus overexpression and CRISPR-Cas9. The function of KRT17 in ESCC proliferation and metastasis were studied by CCK-8 assays, plate colony formation assays, trans-well and wound healing in vitro. Results overexpression of KRT17 was verified in 12 pairs of fresh ESCC tissues. We found that the expression of KRT17 was remarkably positively correlated with the depth of invasion (T) (P&lt;0.05), lymph node metastasis (N) (P&lt;0.05), tumor node metastasis (TNM) stage (P&lt;0.0001). High expression of KRT17 predicted a lower 5-year survival rate(P&lt;0.001) of ESCC patients. We verified the KRT17 malignant behavior of ESCC in vitro. We conformed proliferation and migration capacity of ESCC were significantly enhanced by overexpression of KRT17 but compromised when KRT17 was knocked down. Collectively, it supported that the increased expression of KRT17 promoted proliferation and migratory phenotype of ESCC. Conclusion The breakthrough indicated that the upregulation of KRT17 in ESCC is closely related to malignant progression, our findings revealed that KRT17 plays a vital role in facilitating proliferation and metastasis, and could be used as a novel predictive biomarker, thus providing a novel diagnosis and therapeutic target for ESCC patients. Keywords: Molecular target ESCC Keratin17 Therapeutic target

P8 KERATIN 17 PROMOTES CELLS PROLIFERATION AND METASTASIS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Keratin17(KRT17), as a multifunction cytoskeletal protein, associated with a multitudinous of biological processes, including cell proliferation, migration, and invasion. Previously, we have found up-expression of KRT17 genes in ESCC tissues. Currently, published kinds of researches claimed KRT17 is engaged in the tumorigenesis and progression of multiple cancers. However, the prognostic significance of KRT17 in ESCC patients and its effects in ESCC progression remains indistinct. Methods We verified the expression level of KRT17 in ESCC tissues by Western blotting and q-PCR and constructed KRT17 upregulated and knockdown EC9706 and Eca109 cells by Lentivirus overexpression and CAS9. The function of KRT17 in ESCC proliferation and metastasis were studied in vitro. We performed CCK-8 assays and plate colony formation assays and the plate colony formation assays to explore the effect of KRT17 on the proliferation, Moreover, the trans-well migration chamber experiment and wound healing test was taken to reveal the impact of KRT17 on migration capabilities. Results We verified that KRT17 is overexpressed in ESCC tissues and the role of KRT17 in the malignant behavior of ESCC in vitro and vivo . In CCK-8 assays and plate colony formation assays. We conformed proliferation capacity was notably enhanced by overexpression of KRT17 but compromised when KRT17 was knocked out. On the other hand, we used the plate colony formation assays to explore the effect of KRT17 on the proliferation of ESCC cells. KRT17 up-regulated significantly strengthened the proliferation while the KRT17 knockdown was weakened. To reveal the impact of KRT17 on migration, the trans-well migration chamber experiment was used to compare the role of down-regulation and up-regulation of KRT17 in migration; the results demonstrated that reducing expression of KRT17 significantly blocked the movement of ESCC cells as compared with the empty vector control. Otherwise, it has been confirmed that the cell migration ability employs a wound healing/scratch test because wound closure is generally a measure of cell motility. The results showed KRT17 overexpression notably reinforced the mobility of ESCC cells compared with the empty groups, whereas KRT17 knockdown cells prolonged the time to close the injury compared to blank groups. Collectively, these discoveries support that the increased expression of KRT17 in ESCC cells promoted a more proliferation and migratory phenotype in vitro. Conclusion The breakthrough indicated that the up-regulation of KRT17 in ESCC is closely related to malignant progression. Our data confirmed that KRT17 plays an essential role in reinforcing proliferation and metastasis in ESCC. Thence, KRT17 may serve as a significant molecular target for the diagnosis and therapy of ESCC.

Long non-coding RNA MIR205HG regulates KRT17 and tumor processes in cervical cancer via interaction with SRSF1

Abnormal expression of long non-coding RNAs (lncRNAs) has been demonstrated to be a vital regulatory factor in a large number of malignancies. The investigation in cervical cancer and the associated modulation mechanisms are yet to be probed. The aim of this study is to specifically investigate the expression pattern and modulatory mechanism of MIR205HG in cervical cancer. Our paper firstly revealed the up-regulation of KRT17 in cervical cancer. Function assays further displayed that KRT17 silencing impaired the proliferation and migration, and activated the apoptosis of cervical cancer cells. Based on the finding that MIR205HG could regulate KRT17 expression, we further probed the detailed mechanism between MIR205HG and KRT17. It was observed from mechanism experiments that MIR205HG depleted SRSF1 to increase KRT17 expression. The whole mechanism of MIR205HG/SRSF1/KRT17 axis affecting cell proliferation, apoptosis and migration in cervical cancer was validated using rescue assays. In conclusion, MIR205HG modulated the biological activities of cervical cancer cells via targeting SRSF1 and regulating KRT17, which better understood the pathogenesis of cervical carcinoma and excavated a novel therapeutic target.

KRT18 is correlated with the malignant status and acts as an oncogene in colorectal cancer.

Keratin 18 (KRT18) has been suggested to be overexpressed in most types of human tumor, but the expression pattern of KRT18 in colorectal cancer (CRC) remained unknown. In our research, KRT18 protein expression was markedly increased in CRC cancer tissues and cell lines compared with adjacent normal colorectal tissues and normal colonic epithelial cell line, respectively. Meanwhile, we observed high KRT18 expression was associated with advanced clinical stage, deep tumor invasion, lymph node metastasis, distant metastasis, poor differentiation and unfavorable prognosis in CRC patients. Multivariate Cox regression analysis showed high expression of KRT18 was an unfavorable independent predictor for overall survival in CRC patients. The in vitro studies indicated down-regulation of KRT18 expression depressed CRC cell viability, migration and invasion. In conclusion, KRT18 serves as an oncogenic role in CRC progression and may be a therapeutic target for promoting CRC patients’ prognosis.

Characterization of a BAC transgenic mouse expressing Krt19-driven iCre recombinase in its digestive organs.

Cytokeratin 19 (KRT19) protein is highly expressed in the epithelium of the gastrointestinal (GI) tract, hepatobiliary tissues, and pancreas of humans and mice. In the present study, we used an improved Cre (iCre) gene to enhance the efficiency of Cre expression in mammalian cells. We established a new transgenic Krt19-iCre bacterial artificial chromosome (BAC) mouse model using the BAC recombineering strategy. Site-specific iCre expression pattern was examined in embryos, adults, and elderly Krt19-iCre mice crossed with Tomato or LacZ reporter mice. Both iCre and reporter protein expressions in adult Krt19-iCre;Tomatoflox/+ (Krt19-iCre Tomato reporter) mice were observed mainly in the epithelial cells of the GI tract, hepatobiliary tissues, and pancreas. However, the expression in the intrahepatic and small pancreatic duct were lower than those in the common bile and large pancreatic duct. In the Krt19-iCre; LacZ reporter embryos, β-galactosidase for the LacZ reporter was expressed in the glandular epithelial cells of the GI tract in 9.5-day embryos, 12-day embryos, and newborn mice. The reporter protein expression in Krt19-iCre-Tomato reporter mice was consistent with the KRT19 expression in human GI tissues. In conclusion, Krt19-iCre BAC transgenic mice can be used to investigate developmental and pathological conditions using the iCre-loxP system.

189 Comparative transcriptomic analysis between Total RNA and mRNA isolated from feces of neonatal dairy calves

Abstract The fecal RNA method can be used to evaluate biological adaptations of the gastrointestinal tract of dairy calves through gene expression analysis. The process of RNA isolation from fecal samples presents several challenges, including the potential enrichment of prokaryotic (bacterial), RNA which can dilute the targeted eukaryotic RNA and consequently affect the sensitivity of the fecal RNA method to low expressed genes. Therefore, our objective in this study was to determine the differential eukaryotic RNA enrichment in total RNA vs mRNA from feces of healthy neonatal dairy calves. To test this, a comparative transcriptomic profiling of genes specific for epithelial cells, including cytokeratin 8 (KRT8) and aquaporin (AQP3), as well as inflammatory-related genes (TLR4 and IL1B) was performed in fecal samples collected from 6 pre-weaned Holstein calves. The total RNA was isolated from 200 mg of feces, using a Trizol based method along with the RNeasy Plus Mini Kit (Qiagen). Then, 45 μg of fecal total RNA was used to isolate mRNA through magnetic selection using Dynabeads® Oligo (dT)25 (Invitrogen). The standard curve was composite from all samples including cDNA from total RNA and mRNA. The internal control genes used in this experiment were B2M, ACTB, GAPDH, RPS9, and PPIA. Normalized gene expression data were log-transformed prior to statistical analysis using the Proc Mixed of SAS (SAS 9.4). The expression of KRT8 was greater (P = 0.03) in fecal mRNA than in fecal total RNA. A trend (P = 0.09) was observed for greater expression of TLR4 in fecal total RNA than in fecal mRNA. The expression of AQP3 and IL1B was not different. These preliminary data further confirms that fecal RNA method has potential to be used as a tool to evaluate gastrointestinal tract health in dairy calves, but further adjustments are needed to improve accuracy and robustness.

Long-term culture and characterization of patient-derived primary hepatocytes using conditional reprogramming.

Cultivation of primary human hepatocytes (PHHs) often faces obstacles including failure of long-term in vitro culture, weak proliferation ability, rapid loss of liver-specific function and morphology, and tendency of fibrosis. Previous research focused on immortalization methods, such as telomerase and viral, to culture immortalized primary human hepatocytes, which may lose some of the normal properties. However, non-immortalized PHHs often fail to maintain long-term viability and functionality. These highlight the urgent need for developing new culture strategy for PHHs. In the present study, we isolated PHHs from fresh human liver tissues representing different liver diseases and age groups. We used conditional reprogramming, without permanent immortalization, for long-term in vitro primary human hepatocytes cultivation and characterization. For functional characterization, we assessed CYP3A4, 1A1 and 2C9 activities and measured the mRNA expression of albumin, s100a4, krt8, krt18, cyp1a1, cyp3a4, cyp2b6, cyp2c8, cyp2c9, and cyp2d6. Additionally, we compared the DNA fingerprint of the cells against their original liver tissues using short tandem repeat (STR) analysis. We found that PHHs-derived from young patients can survive for more than three months, while the lifespan of primary human hepatocytes derived from adult patients ranges from two to three months, which is longer than most commercial primary hepatocytes. Importantly, the cells at early passages retain strong CYP3A4, 1A1 and 2C9 activities and the DNA fingerprints are identical with their original tissues. Through conditional programming, we achieved, for the first time, a high level of success rate in the long-term in vitro cultivation of primary human hepatocytes-derived patients representing diverse liver disease. Moreover, the conditional programming cell culture technology reported in this paper requires neither co-culture with additive cells, nor complex and expensive components, such as collagen sandwich or spheroid culture. We thus believe that the patient-derived PHHs cultivation using conditional programming may provide a viable and valuable cell model to study liver disease-related mechanisms. Impact statement Commercially available primary human hepatocytes rapidly lose their proliferative ability and liver-specific functions over a few cultivation days. The demand for pharmaceutical toxicity screening and liver disease research requires the development of long-term primary hepatocyte culture methods. This manuscript addresses this challenge by introducing for the first time successful long-term in vitro cultivation of primary human hepatocytes from a range of liver transplantation patients using conditional reprogramming technique. The beauty of this technique is that it is not a permanent immortalization and does not require co-culture with additive cells. The primary human hepatocytes retain proliferative capacity, genetic stability, and hepatocyte-specific functions at early passages. In view of these, we believe that scientists and researchers will benefit from using these highly valuable cell models to study diverse liver diseases.

Netrin-1 plays a critical role in regulating capacities of epidermal stem cells upon ultraviolet-B (UV-B) irradiation

Loss of the capacities of epidermal stem cells (ESCs) induced by ultraviolet-B (UV-B) irradiation has been widely associated with various skin diseases. Netrin-1, a member of the axonal guidance protein family, has displayed diverse biological functions in different types of cells and tissues, mediated by its specific receptor UNC-5 homolog B (UNC5b). In this study, we examined the physiological functions of netrin-1 and UNC5b in ESCs upon UV-B exposure. Our results indicate that UNC5b is expressed in ESCs, and its expression is upregulated in response to UV-B radiation. We found that treatment with netrin-1 prevented UV-B radiation-induced oxidative stress by reducing the generation of reactive oxygen species (ROS) and expression of NADPH oxidase 4 (NOX-4). Additionally, treatment with netrin-1 improved UV-B radiation-induced mitochondrial dysfunction by increasing mitochondrial membrane potential (MMP) levels and adenosine triphosphate (ATP) production. The presence of netrin-1 attenuated UV-B radiation-induced lactic dehydrogenase (LDH) release. UV-B exposure resulted in the loss of the capacities of ESCs by reducing the expressions of integrin β1 and Krt19, the two major ESC markers. Importantly, this process was prevented by netrin-1. Silencing of UNC5b abolished the effects of netrin-1 on the expression of integrin β1 and Krt19, suggesting that the effects of netrin-1 in maintaining the capacities of ESCs are dependent on UNC5b. Mechanistically, we found that the Wnt/β-catenin signalling may be involved. Our findings suggest that netrin-1 may serve as a therapeutic agent for the treatment of skin diseases.

KRT17 confers paclitaxel-induced resistance and migration to cervical cancer cells

AimTo understand potential pro-oncological effects of lower dose paclitaxel treatment in cervical cancer cells, we investigated the potential roles of KRT17 on migration and proliferation of cervical cancer cells which might respond to cytoskeletal-based drugs treatments. Materials and methodsWe extracted the clinic data of cervical cancer patients from TCGA database to investigate mRNA expression of different keratins. HPV genotypes were identified by reverse transcription PCR. krt17 mRNA and EMT markers were quantified by real-time PCR. krt17 and EMT markers protein were immunoblotted by western blot. Cell viability was detected by CCK8. Cell migration was performed by transwell migration assay. Key findingsOur results showed that HPV16 infection correlated with the expression of KRT17 in cervical cancer cell lines. KRT17 knockdown would decrease Snail2 and elevate E-Cadherin to inhibit migration of Caski cells and SiHa cells. Lower dose of paclitaxel promoted SiHa proliferation, it also significantly promoted the migration of Caski cells. Otherwise, colchicine and higher dose of paclitaxel dose-dependently suppressed the proliferation and migration of Caski cells and SiHa cells. Moreover, KRT17 knockdown significantly facilitated cytoskeletal-based drugs to inhibit migration and induce cytotoxicity in cervical cancer cells. SignificanceKRT17 played pivotal oncogenic roles in cell survival, migration and paclitaxel-induced resistance of cervical cancer cells. Thus, KRT17 would serve as a promising target for compromising paclitaxel-induced resistance and metastasis.

Screening of acetaminophen-induced alterations in epithelial-to-mesenchymal transition-related expression of microRNAs in a model of stem-like triple-negative breast cancer cells: The possible functional impacts

Current protocols for therapy inefficiently targets triple negative breast cancer and barely eradicate cancer stem cells. Elucidation of the pleiotropic effect of clinically proven therapeutics on cancer cells shed light on novel application of old friends. The pleiotropic effect of acetaminophen (APAP) on breast cancer was previously reported. In a cell model of triple negative breast cancer with stem-like CD44high/CD24low phenotype, we screened the impacts of APAP (1 mM, 72 h) on the Epithelial to mesenchymal transition (EMT)-related expression of miRs. APAP significantly overexpressed hsa-miR-130a-3p, 192-5p, 214-3p, 101-3p, 30d-5p, 10a-5p, 99a-5p, 200c-3p, 143-3p, 30b-5p and let-7f-5p showed significant overexpression, but suppressed the expression of hsa-miR-7-5p, 149-3p, 215, 150-5p, 205-5p, 206, 10b-5p, 20b-5p, 145-5p, 26b-5p, 223-3p, 17-5p, 186-5p, 146a-5p and let-7c. It also altered on the expression of selected EMT-related genes, significantly upregulated the expression of KRT19, AKT2, CD24, and TIMP1; but downregulated the expression of MMP2, ALDH1, MMP9, TWIST, NOTCH1, and AKT1. Such shifts in expression profiles increased the population of the cells with CD44high/CD24high, and CD44low/CD24high phenotypes, significantly reduced the Twist protein and shifted the balance of E-cadherin and Vimentin proteins in favor of differentiation. Treated cells showed a significant reduction of in vitro migration and were significantly chemosensitized to Camptothecin. In conclusion, APAP, at a safe clinical dose, induced a set of targeted alterations in the EMT-related miRs which implicate, even in part, significant mitigation in chemoresistance and in vitro migration. Further studies should also be piloted to elucidate the most crucial miRs and to evaluate its clinical effectiveness.

P16Ink4a deletion in cells of the intervertebral disc affects their matrix homeostasis and senescence associated secretory phenotype without altering onset of senescence

Intervertebral disc degeneration is an important contributor to chronic low back and neck pain. Although many environmental and genetic factors are known to contribute to disc degeneration, age is still the most significant risk factor. Recent studies have shown that senescence may play a role in age-related disc degeneration and matrix catabolism in humans and mouse models. Clearance of p16Ink4a-positive senescent cells reduces the degenerative phenotype in many age-associated diseases. Whether p16Ink4a plays a functional role in intervertebral disc degeneration and senescence is unknown. We first characterized the senescence status of discs in young and old mice. Quantitative histology, gene expression and a novel p16tdTom reporter mice showed an increase in p16Ink4a, p21 and IL-6, with a decrease in Ki67 with aging. Accordingly, we studied the spinal-phenotype of 18-month-old mice with conditional deletion of p16Ink4a in the disc driven by Acan-CreERT2 (cKO). The analyses of discs of cKO and age-matched control mice showed little change in cell morphology and tissue architecture. The cKO mice exhibited changes in functional attributes of aggrecan as well as in collagen composition of the intervertebral disc. While cKO discs exhibited a small decrease in TUNEL positive cells, lineage tracing experiments using ZsGreen reporter indicated that the overall changes in cell fate or numbers were minimal. The cKO mice maintained expression of NP-cell phenotypic markers CA3, Krt19 and GLUT-1. Moreover, in cKO discs, levels of p19Arf and RB were higher without alterations in Ki67, γH2AX, CDK4 and Lipofuscin deposition. Interestingly, the cKO discs showed lower levels of SASP markers, IL-1β, IL-6, MCP1 and TGF-β1. These results show that while, p16Ink4a is dispensable for induction and maintenance of senescence, conditional loss of p16Ink4a reduces apoptosis, limits the SASP phenotype and alters matrix homeostasis of disc cells.

Dual role of KRT17: development of papillary renal cell tumor and progression of conventional renal cell carcinoma.

Expression of KRT17 has been described in multi-layered epithelia as well as in tumors derived from these cells. In cancers arising from KRT17 negative single layered epithelia neo-expression of KRT17 has been associated with tumor progression. To obtain more insight into the biology of kidney cancers we have investigated KRT17 expression by immunohistochemistry in normal kidney, in papillary preneoplastic lesions and in 151 papillary and 692 conventional renal cell carcinomas placed on tissue microarray. We found a positive staining in ureteric bud and collecting duct cells in foetal kidney, in all papillary preneoplastic lesions and also in 77% of the 151 papillary renal cell tumors indicating a continuos KRT17 expression during tumor development. The neo-expression of KRT17 in conventional renal cell carcinomas, which derives from KRT17 negative proximal tubules showed a significant correlation with postoperative tumor relapse (RR=2.50; 95% CI=1.59-3.94; p<0.001). In conclusion, the continuous expression of KRT17 from emerging fetal kidney tubules and microscopic pre-neoplastic lesions towards papillary renal cell tumors and its neo-expression in aggressive growing conventional renal cell carcinomas reflects the multiple function of KRT17 in kidney cancers with distinct natural history. This should be taken into account in clinical managements and therapy.

Potential of Human Nucleus Pulposus-Like Cells Derived From Umbilical Cord to Treat Degenerative Disc Disease.

BACKGROUNDDegenerative disc disease (DDD) is a common spinal disorder that manifests with neck and lower back pain caused by the degeneration of intervertebral discs (IVDs). Currently, there is no treatment to cure this debilitating ailment.OBJECTIVETo investigate the potential of nucleus pulposus (NP)-like cells (NPCs) derived from human umbilical cord mesenchymal stem cells (MSCs) to restore degenerated IVDs using a rabbit DDD model.METHODSNPCs differentiated from MSCs were characterized using quantitative real-time reverse transcription polymerase chain reaction and immunocytochemical analysis. MSCs and NPCs were labeled with fluorescent dye, PKH26, and transplanted into degenerated IVDs of a rabbit model of DDD (n = 9 each). Magnetic resonance imaging of the IVDs was performed before and after IVD degeneration, and following cell transplantation. IVDs were extracted 8 wk post-transplantation and analyzed by various biochemical, immunohistological, and molecular techniques.RESULTSNPC derivatives of MSCs expressed known NP-specific genes, SOX9, ACAN, COL2, FOXF1, and KRT19. Transplanted cells survived, dispersed, and integrated into the degenerated IVDs. IVDs augmented with NPCs showed significant improvement in the histology, cellularity, sulfated glycosaminoglycan and water contents of the NP. In addition, expression of human genes, SOX9, ACAN, COL2, FOXF1, KRT19, PAX6, CA12, and COMP, as well as proteins, SOX9, ACAN, COL2, and FOXF1, suggest NP biosynthesis due to transplantation of NPCs. Based on these results, a molecular mechanism for NP regeneration was proposed.CONCLUSIONThe findings of this study demonstrating feasibility and efficacy of NPCs to regenerate NP should spur interest for clinical studies to treat DDD using cell therapy.