Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells / 生物医学与环境科学（英文）

Objective@#To investigate the function of primary cilia in regulating the cellular response to temozolomide (TMZ) and ionizing radiation (IR) in glioblastoma (GBM).@*Methods@#GBM cells were treated with TMZ or X-ray/carbon ion. The primary cilia were examined by immunostaining with Arl13b and γ-tubulin, and the cellular resistance ability was measured by cell viability assay or survival fraction assay. Combining with cilia ablation by IFT88 depletion or chloral hydrate and induction by lithium chloride, the autophagy was measured by acridine orange staining assay. The DNA damage repair ability was estimated by the kinetic curve of γH2AX foci, and the DNA-dependent protein kinase (DNA-PK) activation was detected by immunostaining assay.@*Results@#Primary cilia were frequently preserved in GBM, and the induction of ciliogenesis decreased cell proliferation. TMZ and IR promoted ciliogenesis in dose- and time-dependent manners, and the suppression of ciliogenesis significantly enhanced the cellular sensitivity to TMZ and IR. The inhibition of ciliogenesis elevated the lethal effects of TMZ and IR via the impairment of autophagy and DNA damage repair. The interference of ciliogenesis reduced DNA-PK activation, and the knockdown of DNA-PK led to cilium formation and elongation.@*Conclusion@#Primary cilia play a vital role in regulating the cellular sensitivity to TMZ and IR in GBM cells through mediating autophagy and DNA damage repair.

Mechanism of highly expressed primary cilia in tibial growth plate chondrocyte accelerating chondrocyte differentiation in young rats with chronic renal failure / 中华肾脏病杂志

Objective:To explore the mechanism of highly expressed primary cilia in tibial growth plate chondrocytes accelerating chondrocytes differentiation in young rats with chronic renal failure (CRF).Methods:Forty male 4-week-old SD rats weighing (98±3) g were randomly divided into control group (intragastric administration with distilled water, n=20) and CRF group (intragastric administration with adenine suspension 150 mg·kg -1·d -1, n=20). All the young rats were sacrificed after continuous gavage for 6 weeks. The length of the growth plate was measured with histological sections. Immunofluorescence (IF) was used to detect the expression rate of primary cilia and the level of β-catenin, the key protein of Wnt/β-catenin signaling pathway in tibial growth plate chondrocytes. Chondrocytes isolated from growth plate in two groups were cultured in vitro to P3 generation, and the expression rate of primary cilia in chondrocytes, the levels of Indian hedgehog (IHH) and glycogen synthase kinase 3β (GSK3β) were detected by IF. Co-immunoprecipitation was used to detect the relationship between IHH and GSK3β. Results:Compared with the control group, the relative length of the growth plate was shorter in histological sections [(0.51±0.11) vs (1.00±0.08), t=16.11, P<0.001], the expression rate of primary cilia was higher [(26.3±5.5)% vs (7.6±1.9)%, t=14.37, P<0.001], and the level of β-catenin increased [(7.1±2.0) scores vs (3.6±1.0) scores, t=7.10, P<0.001] in CRF group. In vitro, the expression rate of primary cilia was higher in CRF group chondrocytes [(31.4±8.2)% vs (12.5±3.1)%, t=9.64, P<0.001] than that in control group. The level of IHH in CRF group increased than that in control group [(1 360±270) vs (310±84), t=16.61, P<0.001]. There was no significant difference in GSK3β level of chondrocytes between the two groups [(850±195) vs (780±140), t=1.30, P=0.200]. There was a direct interaction between IHH and GSK3β in CRF group chondrocytes. Conclusions:The expression levels of primary cilia and related protein IHH increase in tibial growth plate chondrocytes of CRF young rats. The IHH protein plays a direct interaction with GSK3β protein, Wnt/β-catenin signaling pathway antagonist, which leads to the activation of Wnt/β-catenin signaling pathway and final accelerated differentiation of chondrocytes. The rapid differentiation of chondrocytes causes the closing trend of growth plate.

Primary cilia in hard tissue development and diseases / 医学前沿

Bone and teeth are hard tissues. Hard tissue diseases have a serious effect on human survival and quality of life. Primary cilia are protrusions on the surfaces of cells. As antennas, they are distributed on the membrane surfaces of almost all mammalian cell types and participate in the development of organs and the maintenance of homeostasis. Mutations in cilium-related genes result in a variety of developmental and even lethal diseases. Patients with multiple ciliary gene mutations present overt changes in the skeletal system, suggesting that primary cilia are involved in hard tissue development and reconstruction. Furthermore, primary cilia act as sensors of external stimuli and regulate bone homeostasis. Specifically, substances are trafficked through primary cilia by intraflagellar transport, which affects key signaling pathways during hard tissue development. In this review, we summarize the roles of primary cilia in long bone development and remodeling from two perspectives: primary cilia signaling and sensory mechanisms. In addition, the cilium-related diseases of hard tissue and the manifestations of mutant cilia in the skeleton and teeth are described. We believe that all the findings will help with the intervention and treatment of related hard tissue genetic diseases.

Primary cilium and its role in tumorigenesis / 浙江大学学报·医学版

The primary cilium, a sensory organelle that protrudes from the surface of most eukaryotic cells, receives and transduces various critical signals that are essential for normal development and homeostasis. Structural or functional disruption of primary cilia causes a number of human diseases, including cancer. Primary cilia has cross talks with cell cycle and it may act as a cell cycle checkpoint to suppress cancer development. Moreover, primary cilia has cross-regulation with autophagy, which may affect tumor progression. We then discuss the association of the primary cilia with several oncogenic signaling pathways, including Shh, Wnt, Notch and platelet-derived growth factor receptor (PDGFR). Since these signaling pathways are often over-activated in many types of human cancers, primary cilia are likely to play a role in the tumorigenesis by modulating these pathways. Finally, we summarize current progress on the role of cilia during tumorigenesis and the challenges that the cilia-cancer field faces.

Freeing the brake: Proliferation needs primary cilium to disassemble

Primary cilia are non-motile, microtubule-based, antennae-like organelle that protrude out from the cell surfaceand perform sensory function or transduce physiological signals in majority of the vertebrate cells. Cilia areassembled on basal bodies that are transformed centrioles. The assembly-disassembly of primary cilia maypose an additional measure on regulating cell cycle in vertebrate cells. While primary cilia are commonly foundin differentiated or quiescent cells that are not cycling, disassembly of primary cilia may promote re-entry ofthese cells into the mitotic cycle, and support proliferation. Many cancer tissues or cancer-derived cells exhibitloss of primary cilia. However, primary cilia may also promote tumorigenesis in some contexts throughgrowth-promoting signalling. This review will shed light on recent advancements of temporal coordination ofciliary disassembly and cell cycle progression, with a focus on how cilia loss may support tumorigenesis invarious epithelial cancers

Biomechanical Effects of Primary Cilia in Articular Cartilage / 医用生物力学

Biomechanical factors play a crucial role in the steady-state maintenance of articular cartilage. The primary cilium (PC) is a kind of organelle which can sense mechanical and chemical signals at the same time. It is also distributed on the surface of chondrocyte membrane. It is involved in multiple signal transduction pathways as well as in the process of chondrocyte phenotype maintenance and material metabolism. Abnormalities in PC are also associated with a variety of human bone and joint diseases. This paper mainly discusses the mechanism of PC in mechanical microenvironment of chondrocytes and the interaction with other signaling pathways, and explores its relationship with bone and joint diseases, so as to provide some scientific basis for clinical and basic research in orthopedics.

Research progress on the distribution of primary cilia and related signaling pathways involved in odontogenesis / 口腔疾病防治

@#Primary cilia are organelles present on most mammalian cells that sense environmental changes and transduce signaling, and they are the key coordinators of various signaling pathways during tissue development. This article reviews the progress of research on the distribution of primary cilia in tooth development and the related signaling pathways. A literature review shows that in odontogenesis, primary cilia play an important role in the mutual induction of the epithelium and mesenchyme; during the continuous proliferation and differentiation of cells, the distribution of primary cilia is temporally and spatially dependent. Although the reason for this distribution is still unclear, some experimental evidence indicates that this phenomenon is compatible with the function of cells and tissues in which primary cilia are distributed. Primary cilia are involved in the regulation of two important signaling pathways, Hedgehog and Wnt, in odontogenesis. Genes encoding cilia (such as Kif3a, Evc/Evc2 and Ift) can affect the development of teeth by regulating these two signaling pathways, and there is an interaction between the two signaling pathways. Deletion of related genes (such as Ofd1 and Bbs) can damage the transmission of upstream and downstream signals by damaging the structure or function of cilia, thereby causing various types of dental dysplasia, including small teeth, enamel hypoplasia, missing teeth, or craniofacial deformities.

Primary cilia mediate Klf2-dependant Notch activation in regenerating heart

Unlike adult mammalian heart, zebrafish heart has a remarkable capacity to regenerate after injury. Previous study has shown Notch signaling activation in the endocardium is essential for regeneration of the myocardium and this activation is mediated by hemodynamic alteration after injury, however, the molecular mechanism has not been fully explored. In this study we demonstrated that blood flow change could be perceived and transmitted in a primary cilia dependent manner to control the hemodynamic responsive klf2 gene expression and subsequent activation of Notch signaling in the endocardium. First we showed that both homologues of human gene KLF2 in zebrafish, klf2a and klf2b, could respond to hemodynamic alteration and both were required for Notch signaling activation and heart regeneration. Further experiments indicated that the upregulation of klf2 gene expression was mediated by endocardial primary cilia. Overall, our findings reveal a novel aspect of mechanical shear stress signal in activating Notch pathway and regulating cardiac regeneration.

Primary cilia mediate Klf2-dependant Notch activation in regenerating heart

Unlike adult mammalian heart, zebrafish heart has a remarkable capacity to regenerate after injury. Previous study has shown Notch signaling activation in the endocardium is essential for regeneration of the myocardium and this activation is mediated by hemodynamic alteration after injury, however, the molecular mechanism has not been fully explored. In this study we demonstrated that blood flow change could be perceived and transmitted in a primary cilia dependent manner to control the hemodynamic responsive klf2 gene expression and subsequent activation of Notch signaling in the endocardium. First we showed that both homologues of human gene KLF2 in zebrafish, klf2a and klf2b, could respond to hemodynamic alteration and both were required for Notch signaling activation and heart regeneration. Further experiments indicated that the upregulation of klf2 gene expression was mediated by endocardial primary cilia. Overall, our findings reveal a novel aspect of mechanical shear stress signal in activating Notch pathway and regulating cardiac regeneration.

Effects of Fluid Shear Stress on PCP Signaling Pathway and Ciliogenesis in Vascular Endothelial Cells / 医用生物力学

Objective To investigate the effect of different fluid shear stress (FSS) on the regulation of planar cell polarity (PCP) signaling, and further to explore the relationship among FSS, PCP signaling pathway and ciliogenesis. Methods The hydrodynamic cell model of adjustable FSS was established. qPCR and immunofluorescence were used to detect the mRNA expression of PCP signaling pathway core protein Dvl2 and cilia assembly protein IFT88, cell targeting and co-localization under different FSS. Western blot (WB) was used to detect the protein expression of Dvl2 at 18 h under different FSS. Results The qPCR result showed that compared with 1.5 Pa FSS, under 0.1 Pa FSS, the mRNA expression of Dvl2 was higher at 6 h and 18 h (P＜0.05), significantly higher at 12 h (P＜0.01); the mRNA expression of IFT88 was significantly higher at 18 h (P＜0.01). The WB result showed that compared with 0 h, under 0.1 Pa FSS, the protein expression of Dvl2 was higher at 18 h (P＜0.05), significantly lower under 1.5 Pa FSS (P＜0.01); compared with 1.5 Pa FSS, the protein expression of Dvl2 was higher at 18 h under 0.1 Pa FSS (P＜0.05). The immunofluorescence result showed that the positive expression of Dvl2 increased with the loading time on FSS increasing, and gradually aggregated at a point around the nucleus; the positive expression of IFT88 was gradually transferred from the nucleus to the cytoplasm and aggregated at a point under 0.1 Pa FSS, and gradually decreased and depolymerized under 1.5 Pa FSS. Protein Dvl2 and IFT88 were located in the same position in cells under 0.1 Pa FSS and before 18 h under 1.5 Pa FSS, and colocalization of proteins Dvl2 and IFT88 was not observed after 18 h under 1.5 Pa FSS due to IFT88 depolymerization. Conclusions Laminar FSS played an inhibition on the transduction of PCP signaling pathway and a hindrance on the process of ciliogenesis, while low FSS played a promotion on the transduction. PCP signaling pathway might regulate FSS-induced ciliogenesis by Dvl2.

Role of primary cilia in common skin tumors / 中华皮肤科杂志

Primary cilium has attracted increasing attention in the biomedical field in recent years.It exists in the surface of various cells and is micro-sized,but its complex structures have not been clear.The primary cilium has an important role in sensory perception.Cells can receive extracellular mechanical and chemical signals through primary cilia,and primary cilia can assist in transferring signals into cells,followed by cellular responses.Recent studies have shown that the primary cilium also plays an important role in embryonic development and malignant transformation of cells.The investigation into primary cilia will facilitate the understanding of malignant transformation of cells and development of tumors.According to the related literature in recent years,this review summarizes the relationship between the primary cilium and common skin tumors,as well as potential targets for these tumors,so as to provide references and new sights for further researches.

Repeated restraint stress promotes hippocampal neuronal cell ciliogenesis and proliferation in mice / 한국실험동물학회지

Stress severely disturbs physiological and mental homeostasis which includes adult neurogenesis in hippocampus. Neurogenesis in hippocampus is a key feature to adapt to environmental changes and highly regulated by multiple cellular signaling pathways. The primary cilium is a cellular organelle, which acts as a signaling center during development and neurogenesis in adult mice. However, it is not clear how the primary cilia are involved in the process of restraint (RST) stress response. Using a mouse model, we examined the role of primary cilia in repeated and acute RST stress response. Interestingly, RST stress increased the number of ciliated cells in the adult hippocampal dentate gyrus (DG). In our RST model, cell proliferation in the DG also increased in a time-dependent manner. Moreover, the analysis of ciliated cells in the hippocampal DG with cell type markers indicated that cells that were ciliated in response to acute RST stress are neurons. Taken together, these findings suggest that RST stress response is closely associated with an increase in the number of ciliated neurons and leads to an increase in cell proliferation.

Cilia and congenital heart disease / 国际儿科学杂志

Cilia are membrane-bound organelles that play an important role in motility and sensation. The abnormal structure and function of cilia can cause a broad range of disorders and diseases called ciliopathies. The incidence of congenital heart disease(CHD)was significantly higher in patients with ciliopathies than that in general population.The process of cardiac development involves multiple regulation mechanisms,and the abnor-mal cardiac development can cause congenital heart disease.Recent studies have indicated that different types of cilia,including nodal cilia and primary cilia,play different roles in the process of cardiac development. And structure defects or abnormal function in cilia can cause congenital heart disease.Some pathogenic genes of con-genital heart disease are related to cilia and cilia-transduced cell signaling pathways.Abnormalities of these genes not only lead to the pathogenesis of congenital heart disease,but also influence cilia development or signaling pathways,which can cause other ciliopathies and affect the prognosis of CHD patients.In this review,we present the relationship between cilia and congenital heart disease.

Parathyroid Hormone Promotes the Proliferation and Invasion of Chondrosarcoma Cells by Regulating the Expression of Primary Cilia / 华中科技大学学报(医学版)

Objective To investigate the effect of parathyroid hormone(PTH)on the proliferation and invasion of chondrosarcoma cells,and the relationship between PTH and the regulation of primary cilia expression.Methods After stimulation of the chondrosarcoma cell line SW1353 with different concentrations of PTH,induction of the expression of cilia with hypoxia and destruction of cilia structure with chloral hydrate,the cell viability was detected by CCK8 assay,the proliferation and invasion of SW1353 by Western blotting and Transwell method,the primary cilia expression by immunofluorescence assay and the GLI1,PTCH1 and IFT88 expression levels by real-time PCR.Results PTH could promote the proliferation of chondrosarcoma cells in a concentration-dependent manner and this effect was correlated with the structural integrity of the primary cilia.PTH could up-regulate the invasive ability of SW1353 cells and increase the expression levels of MMP9,which was suppressed when the primary cilia structure was damaged.Additionally,it was found that PTH could down-regulate the number of primary cilia,which was related to the structural integrity of the primary cilia.It could also regulate the expression levels of GLI1 and PTCH1,the target genes in Hedgehog pathway,and the expression levels of IFT88,the gene associated with the cilia function.Conclusion PTH can promote the proliferation and invasion of chondrosarcoma cells,down-regulate the expression of primary cilia and the downstream target genes.PTH may regulate the malignant biological features of chondrosarcoma by regulating the primary cilia expression.

Autophagy Regulates Formation of Primary Cilia in Mefloquine-Treated Cells

Primary cilia have critical roles in coordinating multiple cellular signaling pathways. Dysregulation of primary cilia is implicated in various ciliopathies. To identify specific regulators of autophagy, we screened chemical libraries and identified mefloquine, an anti-malaria medicine, as a potent regulator of primary cilia in human retinal pigmented epithelial (RPE) cells. Not only ciliated cells but also primary cilium length was increased in mefloquine-treated RPE cells. Treatment with mefloquine strongly induced the elongation of primary cilia by blocking disassembly of primary cilium. In addition, we found that autophagy was increased in mefloquine-treated cells by enhancing autophagic flux. Both chemical and genetic inhibition of autophagy suppressed ciliogenesis in mefloquine-treated RPE cells. Taken together, these results suggest that autophagy induced by mefloquine positively regulates the elongation of primary cilia in RPE cells.

The effects of IFT80 proteins on tumors / 中国肿瘤临床

Objective:To investigate Intraflagellar Transport 80 (IFT80) protein expression in bone, lung, pancreatic, stomach, in-testinal, prostate, breast, and ovarian cancers to explore its mechanism in cancer cell proliferation and to diagnose and identify new tar-gets in cancer treatment. Methods:Immunohistochemistry was used to investigate the expression of IFT80 in gastric cancer tissue of different stages and in eight other kinds of human cancer tissues. We studied the relationship between cancer cell proliferation and inhi-bition of IFT80. Immunofluorescence method and cell culture were used to study the cilia and IFT80. Results:Results showed the fol-lowing:a) the expression of IFT80 was high in gastric and lung carcinoma tissues, moderate in breast and colorectal cancers, low in bone and ovarian cancers, and nearly absent in prostate and pancreatic cancers;b) inhibition of IFT80 in the A549 cancer cell line accel-erated cell proliferation and resulted in shorter, lower quality cilia;and c) IFT80 was abundantly expressed in cancer tissues of well-dif-ferentiated stage-IIA gastric cancer and normal gastric tissues, but was hardly expressed in late-stage, poorly differentiated gastric can-cer. IFT80 could have various degrees of expression in gastric carcinoma of other stages and differentiation. Conclusions:Different can-cer organs showed variation in IFT80 expression. IFT80 can be distributed in the organs with mechanical motion function, such as lungs and stomach. IFT80 is distributed on the cell cilia and can adjust the number and length of the cilia by reducing IFT80 protein ex-pression. Through a variety of ways, IFT80 directly or indirectly participates in the proliferation of cancer cells. Thus, the lowest or nearly zero expression of IFT80 can be seen in cancer tissues of high-grade malignancy, such as advanced cancers with poor differentia-tion.

Effect of primary cilia-mediated shh signaling on neuronal-like cell differentiation of rat bone marrow stromal cells / 第二军医大学学报

To study the effect of primary cilia-mediated sonic hedgehog (Shh) signaling on differentiation of rat bone marrow stromal cells (MSCs) into neuron-like cells. Methods Rat MSCs were isolated and cultured. The study was divided into normal resveratrol-cultured group, resveratrol-induced group, SAG (Smoothened [Smo] agonist) group, and cyclopamine (Smo inhibitor) group. Cell morphology was observed under inverted microscope; the expressions of Ac-Tu, Ptc, Smo, and Glil were examined by immunofluorescence method. Western blotting analysis was used to detect the protein expressions of Smo and Gli, and RT-PCR was applied to detect mRNA expressions of Smo and Glil. Results No expression of primary cilia was found in the normally cultured MSCs. After pre-induction or 24 h starvation, MSCs expressed primary cilia, Ptc, Smo and Glil proteins, with Ptc protein in the primary cilia and Smo, Glil in the cytoplasm. In normal cultivation, resveratrol did not promote the translocation of Smo or induce differentiation of MSCs into neuronal-like cells. When MSCs expressed primary cilia, resveratrol and SAG led to translocation of Smo from the cytoplasm into primary cilia, accompanied by MSCs differentiation into neuronal-like cells and significantly increased expression of Smo and Glil mRNA and protein (P< 0. 05). When cyclopamine was added, Smo remained expressed in the cytoplasm, the expression of Smo and Glil mRNA and protein was significantly decreased (P < 0. 05), and MSCs differentiation into neuronal-like cells was inhibited. Conclusion MSCs express primary cilia and have Shh signaling. Primary cilia-mediated Shh signaling participates in MSCs differentiation into neuronal-like cells.