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Epiblast stem cells (EpiSCs) derived from postimplantation epiblast are pluripotent stem cells, epigenetically distinct from embryonic stem cells (ESCs), which are widely used in reprogramming studies. Recent achieved haploid cell lines in mammalian species open a new era for high‐throughput genetic screening, due to their homozygous phenotypes. Here, we report the generation of mouse haploid EpiSCs (haEpiSCs) from postimplantation chimeric embryos at embryonic day 6.5 (E6.5). These cells maintain one set of chromosomes, express EpiSC‐specific genes, and have potentials to differentiate into three germ layers. We also develop a massive mutagenesis protocol with haEpiSCs, and subsequently perform reprogramming selection using this genome‐wide mutation library. Multiple modules related to various pathways are implicated. The validation experiments prove that knockout of Hst3st3b1 (one of the candidates) can promote reprogramming of EpiSCs to the ground state efficiently. Our results open the feasibility of utilizing haEpiSCs to elucidate fundamental biological processes including cell fate alternations., Mouse haploid epiblast stem cells (haEpiSCs) can be derived from E6.5 postimplantation chimeric epiblast, which are pluripotent with a single genome. Genome‐scale gene trapping in haEpiSCs provides a platform to uncover essential genes regulating cellular reprograming. Knocking out one of the candidate genes, Hs3st3b1, greatly improves the efficiency of reprogramming from EpiSCs to embryonic stem cells.

Reproduction is a key event in life guaranteeing the propagation and evolution of a species. Infertility caused by abnormal germ cell development is a topic of extensive concern. Herein, in vitro germline specification studies provide a modeling platform to investigate gametogenesis. The differentiation of pluripotent stem cells (PSCs) into germ cells has been studied for more than 30 years, and there have been many astonishing breakthroughs in the last decade. Fertile sperm and oocytes can be obtained from mouse embryonic stem cells (ESCs) through a primordial germ cell (PGC)-based method. Moreover, human PGC-like cells (PGCLCs) can be derived with a similar strategy as that used for mouse PGCLC derivation. In this review, we describe the reconstitution of PGCs and the subsequent meiosis, as well as the signaling pathways and factors involved in these processes.

Summary Haploid trophoblast stem cells (TSCs) are advanced in studying placental development for their placental precursor and homozygous features. Here, we describe how to generate haploid-induced TSCs (haiTSCs) from haploid embryonic stem cells with a Tet-on system. Our haiTSCs can maintain haploidy long-term and can produce genome-wide mutants combined with transposons. It is promising in high-throughput genetic screening of trophoblast-specific modulators. For complete details on the use and execution of this protocol, please refer to Peng et al. (2019)., Graphical abstract, Highlights • Protocol for inducing a Cdx2-OE Tet-on system into haploid ESCs • Protocol for conversion and purification of haploid induced TSCs from haploid ESCs • Protocol for construction of genome-wide mutated homozygous TSCs by piggyBac transposon, Haploid trophoblast stem cells (TSCs) are advanced in studying placental development for their placental precursor and homozygous features. Here, we describe how to generate haploid-induced TSCs (haiTSCs) from haploid embryonic stem cells with a Tet-on system. Our haiTSCs can maintain haploidy long-term and can produce genome-wide mutants combined with transposons. It is promising in high-throughput genetic screening of trophoblast-specific modulators.

Reactive oxygen species (ROS) are extensively assessed in physiological and pathological studies; however, the genes and mechanisms involved in antioxidant reactions are elusive. To address this knowledge gap, we used a forward genetic approach with mouse haploid embryonic stem cells (haESCs) to generate high-throughput mutant libraries, from which numerous oxidative stress-targeting genes were screened out. We performed proof-of-concept experiments to validate the potential inserted genes. Slc25a43 (one of the candidates) knockout (KO) ESCs presented reduced damage caused by ROS and higher cell viability when exposed to H2O2. Subsequently, ROS production and mitochondrial function analysis also confirmed that Slc25a43 was a main target gene of oxidative toxicity. In addition, we identified that KO of Slc25a43 activated mitochondria-related genes including Nlrx1 to protect ESCs from oxidative damage. Overall, our findings facilitated revealing target genes of oxidative stress and shed lights on the mechanism underlying oxidative death.

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with very poor prognosis. Therefore, it is important to fully understand the molecular mechanism underlying its occurrence and development. Pumilio RNA-binding family member 1 (PUM1) has been reported to function as an oncogene in ovarian cancer and nonsmall cell lung cancer. However, its role and mechanism in PDAC have not been fully illuminated. Here, we found that the PUM1 protein levels were higher in PDAC tissues than in adjacent tissues and that PUM1 levels were significantly associated with TNM stage and overall survival time, indicating a correlation between high PUM1 expression and poor prognosis in patients with PDAC. In vitro and in vivo assays showed that PUM1 knockdown inhibited cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT), and promoted apoptosis in MIA PaCa-2 and PANC-1 cells. Through cDNA microarrays and ingenuity pathway analysis, we found that the activation of the eIF2 signaling pathway significantly correlated with PUM1 knockdown. These results were further confirmed by the increased levels of key components of the eIF2 signaling pathway, p-PERK, p-EIF2A, and ATF4 in PUM1 knockdown cells. We also found that PUM1 levels have a significant negative correlation with p-PERK levels in PDAC tissues and that PERK overexpression inhibited cell proliferation, migration, invasion, and EMT, and promoted apoptosis in vitro. Moreover, a PERK inhibitor alleviated the effects of PUM1 knockdown on cell proliferation, apoptosis, migration, invasion, and EMT. Taken together, our results revealed that PUM1 knockdown suppressed cell growth, invasion, and metastasis, and promoted apoptosis by activating the PERK/eIF2/ATF4 signaling pathway in PDAC cells. PUM1 could be a potential target to develop pharmaceuticals and novel therapeutic strategies for the treatment of PDAC.

Aim: To investigate the function of Kdm2a in embryonic stem cells (ESCs). Materials & methods: Expression profile analysis after Kdm2a knockout. Analysis of Kdm2a, H3K4me3 and H3K27me3 ChIP-seq data in ESCs. qPCR analysis and ChIP-qPCR analysis of epigenetic changes after Kdm2a loss. Results: Kdm2a was dispensable for pluripotency maintenance in ESCs. Kdm2a genomic binding profile was positively correlated with that of H3K4me3, Zfx and Tet1. Kdm2a directly regulated germ cell genes in primordial germ cell-like cells. Kdm2a loss led to the reduced expression of endogenous retrovirus IAPEy and resulted in the gain of H3K36me2 and loss of H3K4me3 on IAPEy. Conclusion: Kdm2a regulates germ cell genes and endogenous retroviruses in ESCs possibly through demethylating H3K36me2 and influencing H3K4me3 deposition.

The appearance of trophectoderm (TE) is a hallmark event in preimplantation development during murine embryogenesis. However, little is known about the mechanisms underlying TE specification. We find that the depletion of Rif1 breaks down the barrier to the transition from embryonic stem cells (ESCs) to trophoblast stem cells (TSCs). Rif1-null-induced TSCs show typical TE properties and the potential to differentiate into terminal trophoblast lineages. Global transcriptome analysis reveal that Rif1 deletion activates 2-cell embryo (2C)-related genes and induces a totipotent-like state. Chimeric assays further confirm that Rif1-null ESCs contribute to the functional placenta in addition to the fetus on embryonic day 12.5. Furthermore, we show overexpression of Hmgn3, one of the key upregulated gene in Rif1-null ESCs, facilitates the induction of TSCs. Therefore, we report two key genes regulating the conversion of TSCs and provide insights for investigating TE specification.

Background & Aims p53 mutations occur frequently in human HCC. Activation of the mammalian target of rapamycin (mTOR) pathway is also associated with HCC. However, it is still unknown whether these changes together initiate HCC and can be targeted as a potential therapeutic strategy. Methods We generated mouse models in which mTOR was hyperactivated by loss of tuberous sclerosis complex 1 (Tsc1) with or without p53 haplodeficiency. Primary cells were isolated from mouse livers. Oncogenic signalling was assessed in vitro and in vivo, with or without targeted inhibition of a single molecule or multiple molecules. Transcriptional profiling was used to identify biomarkers predictive of HCC. Human HCC materials were used to corroborate the findings from mouse models. Results p53 haploinsufficiency facilitates mTOR signalling via the PTEN/PI3K/Akt axis, promoting HCC tumorigenesis and lung metastasis. Inhibition of PI3K/Akt reduced mTOR activity, which effectively enhanced the anticancer effort of an mTOR inhibitor. ATP-binding cassette subfamily C member 4 (Abcc4) was found to be responsible for p53 haploinsufficiency- and Tsc1 loss-driven HCC tumorigenesis. Moreover, in clinical HCC samples, Abcc4 was specifically identified an aggressive subtype. The mTOR inhibitor rapamycin significantly reduced hepatocarcinogenesis triggered by Tsc1 loss and p53 haploinsufficiency in vivo, as well as the biomarker Abcc4. Conclusions Our data advance the current understanding of the activation of the PTEN/PI3K/Akt/mTOR axis and its downstream target Abcc4 in hepatocarcinogenesis driven by p53 reduction and Tsc1 loss. Targeting mTOR, an unexpected vulnerability in p53 (haplo)deficiency HCC, can be exploited therapeutically to treat Abcc4-positive patients with HCC. Lay summary Tsc1 loss facilitates the p53 (haplo)insufficiency-mediated activation of the PTEN/Akt/mTOR axis, leading to the elevated expression of Abcc4 to drive HCC tumorigenesis and metastasis in mice. Inhibition of mTOR protects against p53 haploinsufficiency and Tsc1 loss-triggered tumour-promoting activity, providing a new approach for treating an aggressive subtype of HCC exhibiting high Abcc4 expression.

Lin28a is a pluripotent factor that promotes somatic cell reprogramming. Unlike other pluripotent factors, Lin28a expression is transient and accumulated in primed embryonic stem (ES) cells, but its exact function and mechanism in the conversion of ES cells from naïve to primed state remain unclear. Here, we present evidence for Dppa3, a protein originally known for its role in germ cell development, as a downstream target of Lin28a in naïve–primed conversion. Using rescue experiment, we demonstrate that Dppa3 functions predominantly downstream of Lin28a during naïve–primed state conversion. Higher level of Lin28a prevents let-7 maturation and results in Dnmt3a/b (target of let-7) upregulation, which in turn induces hypermethylation of the Dppa3 promoter. Dppa3 demarcates naïve versus primed pluripotency states. These results emphasize that Lin28a plays an important role during the naïve–primed state conversion of ES cells, which is partially mediated by a Lin28a–let-7–Dnmt3a/b–Dppa3 axis.

Background: Plant natural products have many different biological activities but the precise mechanisms underlying these activities remain largely unknown. Hypericum longistylum has long been recorded in Chinese medicine as a cure for depression and related disorders, but how it repairs neural lineages has not been addressed. Methods: We extracted compounds from Hypericum longistylum and determined their effect on neural differentiation of embryonic stem cells (ESCs) in vitro by using the Pax6-GFP reporter system. The amount of serotonin released during differentiation was measured by HPLC. The tail suspension test and forced swimming test was performed for determining the effect of compounds on depression-like behaviors in mice. Results: We found that one of the phloroglucinol derivatives not only facilitated differentiation of neural progenitor cells, but also increased the efficiency of differentiation into serotonergic neurons. This compound also improved the behaviors of mice placed in a stressful environment and reduced signs of depression. Conclusions: This is the first use of Chinese herb derived-natural products to promote neurogenesis of ESCs, including the generation of serotonergic neurons, and the first attempt to identify the active compound in Hypericum longistylum responsible for its beneficial effects on depressive diseases. Keywords: Cell differentiation, Chemical modification, Depression, Chinese medicine, Neural differentiation, Serotonergic neuron

Summary Phenotypes of haploid embryonic stem cells (haESCs) are dominant for recessive traits in mice. However, one major obstacle to their use is self-diploidization in daily culture. Although haESCs maintain haploidy well by deleting p53, whether they can sustain haploidy in differentiated status and the mechanism behind it remain unknown. To address this, we induced p53-deficient haESCs into multiple differentiated lineages maintain haploid status in vitro. Haploid cells also remained in chimeric embryos and teratomas arising from p53-null haESCs. Transcriptome analysis revealed that apoptosis genes were downregulated in p53-null haESCs compared with that in wild-type haESCs. Finally, we knocked out p73, another apoptosis-related gene, and observed stabilization of haploidy in haESCs. These results indicated that the main mechanism of diploidization was apoptosis-related gene-triggered cell death in haploid cell cultures. Thus, we can derive haploid somatic cells by manipulating the apoptosis gene, facilitating genetic screens of lineage-specific development., Graphical Abstract, Highlights • haEpiLCs and haNSCLCs differentiated from p53-null haESCs in vitro • p53-null haESCs contributed to chimeric embryos and teratoma • Downregulation of apoptosis genes resulted in haploidy stabilization • Deletion of p73 was also of benefit for haploidy sustenance, In this article, Shuai and colleagues showed that p53-null haESCs could sustain haploidy in differentiated status both in vitro and in vivo, and the main reason for diploidization was apoptosis gene-triggered cell death in haploid cell cultures. Through modulating apoptosis genes, desired haploid somatic cells could be derived easily, which facilitated genetic screening in lineage-specific development.

Parthenogenetic embryonic stem cells (pESCs) derived from bi-maternal genomes do not have competency of tetraploid complementation, due to lacking of paternal imprinting genes. To make pESCs possess fully development potentials and similar pluripotency to zygote-derived ESCs, we knocked out one allelic gene of the two essential maternal imprinting genes (H19 and IG) in their differentially methylated regions (DMR) via CRISPR/Cas9 system and obtained double knock out (DKO) pESCs. Maternal pESCs had similar morphology, expression levels of pluripotent makers and in vitro neural differentiation potentials to zygotes-derived ESCs. Besides that, DKO pESCs could contribute to full-term fetuses through tetraploid complementation, proving that they held fully development potentials. Derivation of DKO pESCs provided a type of major histocompatibility complex (MHC) matched pluripotent stem cells, which would benefit research in regenerative medicine.孤雌胚胎干细胞 (Parthenogenetic embryonic stem cells，pESCs) 的遗传物质全部来源于母源基因组，因缺失父源基因而不具备四倍体补偿的能力。为了使pESCs 也具备发育到个体的能力，呈现与受精卵来源ESCs 类似的多能性，文中借助CRISPR/Cas9 系统对孤雌来源的pESCs 中的2 个重要母源印迹基因的差异甲基化区域(Differentially methylated region，DMR) 进行单等位基因敲除 (H19-DMR，IG-DMR)，获得双基因敲除的 (DKO)pESCs。结果表明，pESCs 虽然来源于母源基因组，但是其形态特征、多能干性标记分子的表达水平、体外神经分化能力与受精卵来源的ESCs 基本一致。最后，通过基因修饰的DKO pESCs 可以通过四倍体补偿获得发育到期的胎儿，表明经过印迹基因修饰的pESCs 也具有发育到一个完整个体的多能性。从而为再生医学研究提供了一类具有主要组织相容性复合基因匹配且多能性良好的资源细胞。.

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

A previously undescribed polysaccharide, GMP70-1, was isolated from the rinds of Garcinia mangostana Lin. Physicochemical characterization analysis showed that GMP70-1 (absolute molecular weight: 2.01 × 104 g/mol) is a multi-branched acidic heteropolysaccharide with a compact coil chain conformation in sodium chloride solution. The repeated unit of GMP70-1 was mainly composed of (1 → 5)-linked α-L-Araf, (1 → 3, 5)-linked α-L-Araf, (1 → 2, 4)-linked α-L-Rhap, (1 → 4)-linked β-D-Galp, terminating with t-α-L-Araf, t-α-D-GalpA, and t-β-D-Galp. To explore the medicinal potential responsible for the bioactivity of G. mangostana, an immunomodulatory assay was performed. The in vitro cell test showed that GMP70-1 possessed a prominent immunoregulatory activity by enhancing the phagocytic uptake of neutral red and promoting the secretion of nitric oxide (NO), reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β of macrophages. Furthermore, an in vivo zebrafish evaluation revealed that the production of ROS and NO was significantly increased after treated with GMP70-1.

Haploid cells facilitate genetic screening of recessive mutations for a single set of chromosomes. Haploid embryonic stem cells (haESCs) have been achieved in several species and widely utilized in genetic screens. The fact that haESCs undergo substantial diploidization during differentiation has limited the screening to other haploid cell types. In this study, we report a method to establish haploid neural stem cells (haNSCs) by selection for a Pax6 reporter. We inserted a green fluorescence protein (GFP) marker gene by homologous recombination into the Pax6 locus of an haESC line. GFP-positive haploid cells could be sorted and further cultured in the NSC medium for more than 30 passages. The established haNSCs expressed neural lineage markers and could differentiate into neurons, oligodendroglia, and astrocytes. Our study shows the feasibility of deriving haploid proliferative somatic cell lines using a genetically encoded reporter that suggest a system for genetic screening of neural and retinal development.

Background Cancer stem cells (CSCs) are important in the tumorigenesis and progression of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play crucial roles regulating CD133+ and EpCAM+ CSCs in HCC, although it is unclear whether miRNAs regulate CD90+ CSCs in HCC. Methods The miRNA profiles of CD90+ and CD90- HCC cells were analyzed using a miRNA microarray and quantitative real-time PCR (qRT-PCR). CSC characteristics were examined by qRT-PCR and Western blot of pluripotency-associated genes, clone and sphere formation assay, transwell migration assay, and nude mice tumorigenicity assay. miR-589-5p mimic transfection was used to overexpress miR-589-5p in vitro. The CD90 and miR-589-5p expressions of HCC samples were detected by immunohistochemistry and qRT-PCR, respectively. Results miR-589-5p and miR-33b-5p were down-regulated in CD90+ cells. Overexpression of miR-589-5p suppressed CD90+ CSC characteristics such as Oct4, Sox2 and Nanog expression, a high likelihood of forming cell spheres, high invasiveness and high tumorigenicity. Luciferase reporter assays demonstrated that miR-589-5p directly binds to the 3ˈ-untranslated region of mitogen-activated protein kinase kinase kinase 8 (MAP3K8) mRNA, and exogenous miR-589-5p down-regulated MAP3K8 expression. In addition, siRNA inhibition of MAP3K8 also suppressed CD90+ CSC characteristics, even in the absence of miR-589-5p overexpression. In HCC tissues, miR-589-5p expression was inversely correlated with CD90 expression, and high CD90 expression and low miR-589-5p expression were positively correlated with vascular invasion and recurrence and significantly decreased disease-free and overall survival by clinical analysis. Conclusion In HCC, miR-589-5p down-regulates the stemness characteristics of CD90+ CSCs in part by silencing MAP3K8. CD90 and miR-589-5p expression predict HCC outcomes and might be novel molecular targets for HCC treatment. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0452-6) contains supplementary material, which is available to authorized users.

Haploid pluripotent stem cells, such as haploid embryonic stem cells (haESCs), facilitate the genetic study of recessive traits. In vitro, fish haESCs maintain haploidy in both undifferentiated and differentiated states, but whether mammalian haESCs can preserve pluripotency in the haploid state has not been tested. Here, we report that mouse haESCs can differentiate in vitro into haploid epiblast stem cells (haEpiSCs), which maintain an intact haploid genome, unlimited self-renewal potential, and durable pluripotency to differentiate into various tissues in vitro and in vivo. Mechanistically, the maintenance of self-renewal potential depends on the Activin/bFGF pathway. We further show that haEpiSCs can differentiate in vitro into haploid progenitor-like cells. When injected into the cytoplasm of an oocyte, androgenetic haEpiSC (ahaEpiSCs) can support embryonic development until midgestation (E12.5). Together, these results demonstrate durable pluripotency in mouse haESCs and haEpiSCs, as well as the valuable potential of using these haploid pluripotent stem cells in high-throughput genetic screening.

In this unit we introduce the derivation and genetic modification of mouse haploid embryonic stem (ES) cells. We detail how to produce haploid embryos and the subsequent ES derivation and cell culture. We further introduce readers to the intracytoplasmic injection processes of two types of haploid ES cells [androgenetic haploid ES (ahES) and parthenogenetic ES (phES)], both of which possess potential to produce fertile progenies by microinjection. This unit will be interesting to researchers who focus on recessive screens and transgenic animal model production with haploid stem cells.

Recently, reprogramming of somatic cells from a differentiated to pluripotent state by overexpression of specific external transcription factors has been accomplished. It has been widely speculated that an undifferentiated state may make donor cells more efficient for nuclear transfer. To test this hypothesis, we derived induced pluripotent stem cells (iPS cells) from several somatic cell lines: mouse embryonic fibroblast (MEF), adult tail tip fibroblast (TTF), and brain neural stem cells (NSCs). Three dimensional (3D)-fluorescent in situ hybridization (FISH) and quantitative-FISH (Q-FISH) were then used to evaluate constitutive (pericentric and telomeric) heterochromatin organization in these iPS cells and in their parental differentiated cells. Here, we show that important nuclear remodeling and telomeres rejuvenation occur in these iPS cells regardless of their parental origin. When we used these cells as donors for nuclear transfer, we produced live-born cloned mice at much higher rates with the iPS-induced cells than with the parental cell lines. Interestingly, we noticed that developmental potential after nuclear transfer could be correlated with telomere length of the donor cells. Altogether, our findings suggest that constitutive heterochromatin organization from differentiated somatic cells can be reprogrammed to the pluripotent state by induction of iPS cells, which in turn support nuclear transfer procedure quite efficiently.

Haploids and double haploids are important resources for studying recessive traits and have large impacts on crop breeding, but natural haploids are rare in animals. Mammalian haploids are restricted to germline cells and are occasionally found in tumours with massive chromosome loss. Recent success in establishing haploid embryonic stem (ES) cells in medaka fish and mice raised the possibility of using engineered mammalian haploid cells in genetic studies. However, the availability and functional characterization of mammalian haploid ES cells are still limited. Here we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into an enucleated oocyte. The ahES cells maintain haploidy and stable growth over 30 passages, express pluripotent markers, possess the ability to differentiate into all three germ layers in vitro and in vivo, and contribute to germlines of chimaeras when injected into blastocysts. Although epigenetically distinct from sperm cells, the ahES cells can produce viable and fertile progenies after intracytoplasmic injection into mature oocytes. The oocyte-injection procedure can also produce viable transgenic mice from genetically engineered ahES cells. Our findings show the developmental pluripotency of androgenentic haploids and provide a new tool to quickly produce genetic models for recessive traits. They may also shed new light on assisted reproduction.

Due to the lack of effective diagnostic tools, most patients with cholangiocarcinoma (CCA) have no chance of surgical resection. Ars2 is a protein that was reported to be important for microRNA (miR) biogenesis, and its depletion can reduce the levels of several miRs, including miR-21, which is overexpressed in CCAs. We hypothesized that Ars2 was also present in CCAs and could be an early diagnostic marker. In our experiments, Ars2, PTEN, PDCD4, and miR-21 were evaluated in 18 CCAs and paired normal tissues. ShArs2, miR-21 mimics, and Ars2 were transfected into CCA and bile duct epithelial cells either alone or together. Cell proliferation, tumorigenicity analysis and expression changes of Ars2, PTEN, PDCD4, and miR-21 were evaluated. We found that both Ars2 and miR-21 were overexpressed, with 95% sensitivity and 100% specificity, and an ROC of 0.995 in distinguishing between CCAs and paired normal tissues by qRT-PCR. PTEN and PDCD4 were reversed in immunohistochemistry, but no difference was observed using qRT-PCR. The knockdown of Ars2 in CCA cells decreased the level of miR-21, inhibited cell proliferation and prevented tumor formation in nude mice. Ars2 knockdown also led to an increase in both PTEN and PDCD4 protein levels. Both proteins decreased when the miR-21 mimic was con-transfected. However, the overexpression of Ars2 alone could not get the opposite results. Based on our data, we conclude that Ars2 is overexpressed in human CCA and may be a diagnostic marker. Ars2 depletion increases PTEN and PDCD4 protein levels via the reduction of miR-21.