ABSTRACT
Chitin deacetylases (CDAs, including CDA1 and CDA2) are considered key enzymes for body cuticle formation and tracheal morphogenesis in various insect species. However, their functions in the formation of the cuticular intima of the foregut and hindgut are unclear. Here, we investigated the roles of their respective genes LmCDA1 and LmCDA2 in this process, in the hemimetabolous insect Locusta migratoria. Transcripts of LmCDA1 and LmCDA2 were highly expressed both before and after molting in the foregut. In the hindgut, their expression was high only before molting. In both the foregut and hindgut, LmCDA1 protein was localized in the basal half of the chitin matrix (procuticle), whereas LmCDA2 was detected in the upper half of the procuticle. Knockdown of LmCDA1 by RNA interference (RNAi) in 5th-instar nymphs caused no visible defects of the hindgut cuticle. By contrast, the chitinous lamellae of the cuticular intima in the foregut of knockdown animals were less compact than in control animals. RNAi against LmCDA2 led to thickening of both the foregut and hindgut cuticles, with a greater number of thinner laminae than in the respective control cuticles. Taken together, our results show that LmCDA1 and LmCDA2 have distinct, but overlapping, functions in chitin organization in the foregut cuticle. However, in the hindgut, this process seems independent of LmCDA1 activity but requires LmCDA2 function. Thus, the CDAs reflect tissue-specific differences in cuticular organization and function, which need further detailed molecular and histological analyses for full comprehension.
Subject(s)
Chitin , Gastrointestinal Tract/metabolism , Insect Proteins , Locusta migratoria , Animal Shells , Animals , Chitin/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Locusta migratoria/genetics , Locusta migratoria/metabolism , Molting , Nymph/genetics , Nymph/metabolism , RNA InterferenceABSTRACT
Neuroblastoma is the second most common extracranial malignant solid tumor that occurs in childhood, and metastasis is one of the major causes of death in neuroblastoma patients. The epithelial-mesenchymal transition (EMT) is an important mechanism for both the initiation of tumor invasion and subsequent metastasis. Therefore, this study investigated the mechanism by which transforming growth factor (TGF)-ß1 induces EMT in human neuroblastoma cells. Using quantitative RT-qPCR and western blot analyses, we found that the mRNA and protein expression levels of E-cadherin were significantly decreased, whereas that of α-SMA was significantly increased after neuroblastoma cells were treated with different concentrations of TGF-ß1. A scratch test and Transwell migration assay revealed that cell migration significantly and directly correlated with the concentration of TGF-ß1 indicating that TGF-ß1 induced EMT in neuroblastoma cells and led to their migration. Inhibiting Smad2/3 expression did not affect the expression of the key molecules involved in EMT. Further investigation found that the expression of the glioblastoma transcription factor (Gli) significantly increased in TGF-ß1-stimulated neuroblastoma cells undergoing EMT, accordingly, interfering with Gli1/2 expression inhibited TGF-ß1-induced EMT in neuroblastoma cells. GANT61, which is a targeted inhibitor of Gli1 and Gli2, decreased cell viability and promoted cell apoptosis. Thus, TGF-ß1 induced EMT in neuroblastoma cells to increase their migration. Specifically, EMT induced by TGF-ß1 in neuroblastoma cells did not depend on the Smad signaling pathway, and the transcription factor Gli participated in TGF-ß1-induced EMT independent of Smad signaling.
