CircLRP6 Regulation of ZEB1 via miR-455 Is Involved in the Epithelial-Mesenchymal Transition During Arsenite-Induced Malignant Transformation of Human Keratinocytes
Junchao XueChao ChenFei LuoXueli PanHui XuPing YangQian SunXinlu LiuLu LuQianlei YangTian XiaoXiangyu DaiPeng LuoJiachun LüAihua ZhangQizhan Liu
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Circular RNAs (circRNAs), a class of noncoding RNAs generated from pre-mRNAs, participate in the regulation of tumorigenesis. The mechanism for regulation, however, is unclear. Here, to determine whether circRNAs are involved in arsenite-induced epithelial-mesenchymal transition (EMT) and malignant transformation in human keratinocyte (HaCaT) cells, the up-regulation of circLRP6 was confirmed in arsenite-transformed HaCaT (T-HaCaT) cells. In HaCaT cells, circLRP6 acted as an microRNA (miR)-455 sponge. For these cells, chronic exposure to arsenite caused an increase of circLRP6 and the transcription factor ZEB1, which induced the EMT. miR-455 suppressed the expression of ZEB1. Further, in T-HaCaT cells, knockdown of circLRP6 with siRNA inhibited ZEB1 expression, but cotransfection with circLRP6 siRNA and an miR-455 inhibitor reversed this inhibition. These results suggest that, in HaCaT cells, arsenite increases circLRP6 levels, which act as a sponge for miR-455 and up-regulate the miR-455 target, ZEB1, which subsequently induces the EMT, thus promoting malignant transformation. Thus, for HaCaT cells chronically exposed to arsenite, circLRP6 via miR-455 regulation of ZEB1 is involved in the EMT during malignant transformation. The results establish a previously unknown mechanism for arsenite-induced carcinogenesis.Keywords:
HaCaT
Malignant Transformation
Circular RNAs (circRNAs), a class of noncoding RNAs generated from pre-mRNAs, participate in the regulation of tumorigenesis. The mechanism for regulation, however, is unclear. Here, to determine whether circRNAs are involved in arsenite-induced epithelial-mesenchymal transition (EMT) and malignant transformation in human keratinocyte (HaCaT) cells, the up-regulation of circLRP6 was confirmed in arsenite-transformed HaCaT (T-HaCaT) cells. In HaCaT cells, circLRP6 acted as an microRNA (miR)-455 sponge. For these cells, chronic exposure to arsenite caused an increase of circLRP6 and the transcription factor ZEB1, which induced the EMT. miR-455 suppressed the expression of ZEB1. Further, in T-HaCaT cells, knockdown of circLRP6 with siRNA inhibited ZEB1 expression, but cotransfection with circLRP6 siRNA and an miR-455 inhibitor reversed this inhibition. These results suggest that, in HaCaT cells, arsenite increases circLRP6 levels, which act as a sponge for miR-455 and up-regulate the miR-455 target, ZEB1, which subsequently induces the EMT, thus promoting malignant transformation. Thus, for HaCaT cells chronically exposed to arsenite, circLRP6 via miR-455 regulation of ZEB1 is involved in the EMT during malignant transformation. The results establish a previously unknown mechanism for arsenite-induced carcinogenesis.
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Inorganic arsenic is a ubiquitous environmental contaminant associated with an increased risk of skin hyperkeratosis and cancer. Although several hypotheses that relate to arsenic-induced carcinogenesis have been suggested, the mechanism of action remains obscure. In the present study, molecular mechanisms underlying the inactivation of p53 function and the genomic instability in malignant transformation of the human keratinocyte cell line, HaCaT, induced by low levels of arsenic were investigated. Our results show that long-term exposure of HaCaT cells to sodium arsenite (1.0μM) increases their proliferation, causes DNA double-strand breaks, and induce anchorage-independent growth. In arsenite-exposed cells, the levels of phospho-p53, p21, and mdm2 increase at early times after exposure. The levels, however, decrease with longer times. Interaction of the promoter of mot-2 (a p53 inhibitor) with nuclear factor κB (NF-κB) was established by Southwestern and Western blot assays. Blockage of NF-κB prevents the increases of arsenite-induced mot-2 levels, and knockdown of mot-2 facilitates the nuclear translocation of p53, indicating that, in HaCaT cells exposed to arsenite, NF-κB inhibits p53 function by mot-2. Moreover, inactivation of NF-κB facilitated p53-mediated DNA repair and prevented arsenite-induced malignant transformation. Together, the results suggest that the repressive effect of NF-κB on p53 by mot-2 leads to genomic instability, which is involved in arsenite-induced malignant transformation of human keratinocytes.
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Human skin
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Trop2, an oncogenic cell surface protein under investigation as a therapeutic target, is commonly overexpressed in several epithelial tumor types yet its function in tumor biology remains relatively unexplored. To investigate the role of Trop2 in epithelial carcinogenesis, we generated Trop2(-/-) mice, which are viable and possess a normal lifespan. Contrary to expectations, Trop2 loss fails to suppress keratinocyte transformation. Instead, ras-transformed Trop2(-/-) keratinocytes preferentially pass through an epithelial to mesenchymal transition (EMT) and form tumors with spindle cell histology. Furthermore, Trop2 loss renders Arf-null mice susceptible to the formation of biphasic sarcomatoid carcinomas containing both squamous and spindle cell components upon carcinogen exposure in an otherwise skin cancer-resistant strain (C57BL/6). Immortalized keratinocytes derived from Trop2(-/-)Arf(-/-) mice exhibit enhanced proliferative and migratory capacity as well as increased activation of mitogen-activated protein kinase and Src prior to transformation. The clinical relevance of these findings was supported by studying the molecular epidemiology of Trop2 in primary head and neck squamous cell carcinomas. This analysis revealed that Trop2 mRNA levels are decreased in a subset of tumors with features of EMT, and total loss of Trop2 protein expression is observed in the spindle cell component of sarcomatoid carcinomas. Therefore, while previous studies have emphasized the potential importance of Trop2 gain of function, these results uncover a role for Trop2 loss in tumorigenesis and the mesenchymal transdifferentiation observed in a subset of squamous cell carcinomas.
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We have studied the gap junctional intercellular communication (GJIC) of immortalized and tumourigenic human keratinocyte cell lines and of a spontaneously immortalized non-tumourigenic and a highly differentiating keratinocyte cell line (HaCaT) as the control. In homologous cultures, the GJIC capacity of five squamous cell carcinoma-derived cell lines was 1-27% that of the HaCaT cells. Ha-ras-transfected HaCaT cells with tumourigenic potential and an SV40 DNA-immortalized cell line had markedly reduced GJIC capacities. Northern analysis and immunohistochemistry showed that connexin (Cx) 43 is the major gap junction protein expressed in the communicating cells. They do not express Cx 26 or 32. The low or absent communication observed in certain cell lines was due in some to a lack of Cx 43 gene expression, but in others to aberrant localization of the gap junction protein. GJIC of these cell lines, as well as that of primary normal human epidermal keratinocytes, was susceptible to 12-O-tetradecanoylphorbol-13-acetate-mediated inhibition. Moreover, GJIC of HaCaT cells and their tumourigenic derivatives is Ca(2+)-dependent. These results, when compared with those previously obtained for mouse keratinocyte cell lines, reveal that GJIC of human keratinocytes was correlated to the degree of differentiation and is controlled in a similar way to that of murine keratinocytes. Aberrant GJIC seems to be a common feature of human and murine skin carcinogenesis.
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One of the major complications in diabetes is impaired wound healing. Unfortunately, effective therapies are currently lacking. Epithelial to mesenchymal transition (EMT) is a critical process involved in cutaneous wound healing. In response to injury, EMT is required to activate and mobilize stationary keratinocytes in the skin toward the wound bed, which allows for re-epithelialization. This process is stalled in diabetic wounds. In this study, we investigate the role of long non-coding RNA (lncRNA), MALAT1, in transforming growth factor beta 1(TGF-β1)-induced EMT of human keratinocyte (HaCaT) cells. Initially, we detected MALAT1 and TGF-β1 expression in non-diabetic and diabetic wounds and found that these expression are significantly up-regulated in diabetic wounds. Then, HaCaT cells were cultured and exposed to TGF-β1. The EMT of HaCaT cells were confirmed by the increased expression of CDH2, KRT10, and ACTA2, in addition to the down-regulation of CDH1. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA). MALAT1 silencing attenuates TGFβ1-induced EMT. Mechanistically, MALAT1 is involved in TGF-β1 mediated EMT through significantly induced ZEB1 expression, a critical transcription factor for EMT. In summary, lncRNA MALAT1 is involved in TGFβ1-induced EMT of human HaCaT cells and provides new understanding for the pathogenesis of diabetic wounds.
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Arsenic is widely present in nature and is a common environmental poison that seriously damages human health. Chronic exposure to arsenic is a major environmental poisoning factor that promotes cell proliferation and leads to malignant transformation. However, its molecular mechanism remains unclear. In this study, we found that arsenite can promote the transformation of immortalized human keratinocyte cells (HaCaT) from the G0/G1 phase to S phase and demonstrated malignant phenotypes. This phenomenon is accompanied by obviously elevated levels of NRF2, NQO1, Cyclin E, and Cyclin-dependent kinase 2 (CDK2). Silencing the NRF2 expression with small interfering RNA (siRNA) in arsenite-transformed (T-HaCaT) cells was shown to reverse the malignant phenotype. Furthermore, the siRNA silencing of NQO1 significantly decreased the levels of the cyclin E-CDK2 complex, inhibiting the G0/G1 to S phase cell cycle progression and transformation to the T-HaCaT phenotypes. Thus, we hypothesized that the NRF2/NQO1 pathway played a key role in the arsenite-induced malignancy of HaCaT cells. By increasing the expression of Cyclin E-CDK2, the NRF2/NQO1 pathway can affect cell cycle progression and cell proliferation. A new common health effect mechanism of arsenic carcinogenesis has been identified; thus, it would contribute to the development of novel treatments to prevent and treat skin cancer caused by arsenic.
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Long-term exposure to arsenic has been linked to cancer in different organs and tissues, including skin. Here, non-malignant human keratinocytes (HaCaT) were exposed to arsenic and its effects on microRNAs (miRNAs; miR) expression were analyzed via miRCURY LNA array analyses. A total of 30 miRNAs were found differentially expressed in arsenic-treated cells, as compared to untreated controls. Among the up-regulated miRNAs, miR-21, miR-200a and miR-141, are well known to be involved in carcinogenesis. Additional findings confirmed that those three miRNAs were indeed up-regulated in arsenic-stimulated keratinocytes as demonstrated by quantitative PCR assay. Furthermore, bioinformatics analysis of both potential cancer-related pathways and targeted genes affected by miR-21, miR-200a and/or miR-141 was performed. Results revealed that miR-21, miR-200a and miR-141 are implicated in skin carcinogenesis related with melanoma development. Conclusively, our results indicate that arsenic-treated keratinocytes exhibited alteration in the miRNAs expression profile and that miR-21, miR-200a and miR-141 could be promising early biomarkers of the epithelial phenotype of cancer cells and they could be potential novel targets for melanoma therapeutic interventions.
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Alternative methods using various human or animal cell lines are under development to classify chemical ingredients to skin sensitizing or irritant chemicals. Keratinocyte cell line could be a good target cell, in that the cells are the first contact point for chemical absorption through skin, and immune reactions including inflammation are occurred through interactions among keratinocyte, Langerhans` cell, and T cells. This study introduced current research activities on development of alternative methods using keratinocyte cell lines for identifying skin sensitizers. In addition, a good laboratory practice was introduced to perform serial passage culture of HaCaT human keratinocyte cell line. Furthermore, optimal cell density and culture duration were demonstrated to determine both intracellular and extracellular levels of IL-1α, IL-6, IL-8, and IL-18 from HaCaT cells.
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