Differently expressed genes (DEGs) were identified with a fold change 2 and a false discovery rate (FDR) 0

Differently expressed genes (DEGs) were identified with a fold change 2 and a false discovery rate (FDR) 0.05 in a comparison as significant DEGs, DEGs were then subjected to enrichment analysis of GO functions and KEGG pathways. CELF6 induces FCGR3A G1 phase arrest. The effect of CELF6 on cell proliferation is usually p53 and/or p21 dependent. Collectively, these data demonstrate that CELF6 might be a potential tumor suppressor, CELF6 regulates cell proliferation and cell cycle progression via modulating p21 stability. mice exhibit a partial autism spectrum disorder-like phenotype, polymorphisms in the CELF6 gene may contribute to autism risk in human31. expression in hypothalamic nuclei may impact a variety of behaviors downstream of neuropeptide activity32. In this statement, we aimed to study the function of CELF6 in malignancy cell proliferation. We show that the expression of CELF6 is usually cell cycle regulated. The cell cycle-dependent expression of CELF6 is usually mediated through the ubiquitin-proteasome pathway, the E3 ubiquitin ligase SCF (SKP1-CUL1-F-box)–TrCP is responsible for CELF6 degradation. Gene expression profiling and KEGG pathway enrichment analysis reveal that this p53 signaling is usually enriched in knockout cells. Depletion or overexpression of CELF6 results in dramatic switch of p21 expression. CELF6 binds to p21 mRNA and regulates its stability. CELF6 modulates cell cycle progression and cell proliferation in p53 and/or p21-dependent manner. Thus, we propose that CELF6 is usually a potential tumor suppressor, CELF6 regulates malignancy cell proliferation and cell cycle progression via modulating p21 stability. Results The expression of CELF6 is usually cell cycle regulated To examine whether the expression of CELF6 Manidipine 2HCl is usually cell cycle regulated, the HCT116 colorectal malignancy cells were synchronized at the G1/S boundary by a double-thymidine (DT) block, cells were released and harvested at different time points to perform circulation cytometry and immunoblotting analysis. Immunoblotting revealed that CELF6 protein was relatively higher at G1/S and early S phases, then decreased sharply 4? h post DT release and managed a relatively low level until most of the cells joined G2/M phase, following an increase in the amount of CELF6 at 10C12?h post DT release (G1 phase) (Fig. 1a, b). However, quantitative RT-PCR (qPCR) exhibited that the expression patterns of CELF6 protein and mRNA are different, mRNA levels increased dramatically 4?h post DT release, indicating that posttranscriptional modifications may regulate the fluctuation of CELF6 protein during the cell cycle (Fig. ?(Fig.1c).1c). Then, we used a selective CDK1 inhibitor RO-3306 to arrest cells at the G2/M phase border (Fig. ?(Fig.1d).1d). The G2/M phase marker cyclin B1 was used Manidipine 2HCl as an indicator for immunoblotting of synchronized cell extracts. CELF6 mRNA and protein maintained at relatively constant levels during G2/M and early G1 phases, followed by accumulation of CELF6 protein in late G1 (Fig. 1e, f). We also analyzed CELF6 expression in HCT116 cells, Manidipine 2HCl the protein level of CELF6 is still cell cycle regulated in cells (Supplementary Fig. 1). Open in a separate window Fig. 1 The expression of CELF6 is cell cycle regulated.a HCT116 cells were Manidipine 2HCl synchronized at the G1/S boundary by using double-thymidine (DT) block, cells were released from thymidine treatment at the indicated time points, fixed and stained with Propidium iodide (PI) for flow cytometry. b Cell extracts were collected at different time pointes after DT release and analyzed by immunoblotting, cyclin E1 was used as a G1/S phase protein marker. c Relative mRNA levels were determined by quantitative RT-PCR. d HCT116 cells were synchronized at the G2/M transition by CDK1 inhibitor RO-3306 treatment, cells were released from RO-3306 treatment at the indicated time points and cell cycle distribution was analyzed by flow cytometry. e Cell extracts were collected at different time pointes after RO-3306 release and analyzed by immunoblotting or f quantitative RT-PCR, cyclin B1 was used as a G2/M phase protein marker CELF6 is degraded by the ubiquitin-proteasome pathway Manidipine 2HCl Both autophagy-lysosomal pathway and the ubiquitin-proteasome system control degradation of the majority of eukaryotic proteins33. To investigate which pathway contributes to CELF6 degradation, HCT116 cells were treated with the lysosomal inhibitor bafilomycin A1 (BAF) or hydroxychloroquine (HCQ), or the proteasome inhibitor MG132 before harvesting cells for immunoblotting. Both BAF and HCQ did not affect CELF6 expression, whereas the proteasomal inhibitor MG132 stabilized CELF6, indicating that CELF6 is degraded by proteasome pathway (Fig. ?(Fig.2a).2a). The majority.