CLOCK基因敲除HCT116细胞

The circadian clock generates 24 h rhythms in behavioural, cellular and molecular processes. Malfunctions of the clock are associated with enhanced susceptibility to cancer, worse treatment response and poor prognosis. Clock-controlled genes are involved in cellular processes associated with tumour development and progression including metabolism of drugs and the cell cycle. , a plant of the family, has been reported to have antiproliferative effects on breast cancer cells. Here, we used the human colorectal cancer (CRC) cell line HCT116 and its knockout variants for different core-clock genes (, , ), to investigate the treatment effect of lipophilic leaf extract under different clock scenarios. Our results show a direct effect of on the circadian phenotype of the cells, as indicated by alterations in the phase, amplitude, and period length of core-clock gene oscillations. Furthermore, our data indicate a role for the circadian clock in sensitivity to treatment. In particular, the treatment inhibited proliferation and induced cytotoxicity and apoptosis in a clock knockout-specific manner, in CRC cells. These results point to a potential effect of lipophilic leaf extracts as a modulator of the circadian clock, in addition to its anti-proliferative properties.

The circadian clock coordinates the timing of several cellular processes including transcription, the cell cycle, and metabolism. Disruptions in the clock machinery trigger the abnormal regulation of cancer hallmarks, impair cellular homeostasis, and stimulate tumourigenesis. Here we investigated the role of a disrupted clock by knocking out or knocking down the core-clock (CC) genes , or in cancer progression (e.g., cell proliferation and invasion) using colorectal cancer (CRC) cell lines HCT116, SW480 and SW620, from different progression stages with distinct clock phenotypes, and identified mechanistic links from the clock to altered cancer-promoting cellular properties. We identified (metastasis-associated in colon cancer 1), a known driver for metastasis and an EMT (epithelial-to-mesenchymal transition)-related gene, to be significantly differentially expressed in CC manipulated cells and analysed the effect of manipulation (knockout or overexpression) in terms of circadian clock phenotype as well as cancer progression. Our data points to a bi-directional -circadian clock interplay in CRC, via CC genes. In particular, knocking out reduced the period of oscillations, while its overexpression increased it. Interestingly, we found the MACC1 protein to be circadian expressed in HCT116 WT cells, which was disrupted after the knockout of CC genes, and identified a MACC1-NR1D1 protein-protein interaction. In addition, manipulation and CC knockout altered cell invasion properties of HCT116 cells, pointing to a regulation of clock and cancer progression in CRC, possibly via the interaction of with core-clock genes.

Emerging evidence points towards a regulatory role of the circadian clock in alternative splicing (AS). Whether alterations in core-clock components may contribute to differential AS events is largely unknown. To address this, we carried out a computational analysis on recently generated time-series RNA-seq datasets from three core-clock knockout (KO) genes (ARNTL, NR1D1, PER2) and WT of a colorectal cancer (CRC) cell line, and time-series RNA-seq datasets for additional CRC and Hodgkin's lymphoma (HL) cells, murine WT, Arntl KO, and Nr1d1/2 KO, and murine SCN WT tissue. The deletion of individual core-clock genes resulted in the loss of circadian expression in crucial spliceosome components such as SF3A1 (in ARNTL), SNW1 (in NR1D1), and HNRNPC (in PER2), which led to a differential pattern of KO-specific AS events. All HCT116 cells showed a rhythmicity loss of a crucial spliceosome gene U2AF1, which was also not rhythmic in higher progression stage CRC and HL cancer cells. AS analysis revealed an increase in alternative first exon events specific to PER2 and NR1D1 KO in HCT116 cells, and a KO-specific change in expression and rhythmicity pattern of AS transcripts related to cancer hallmarks genes including FGFR2 in HCT116_ARNTL, CD44 in HCT116_NR1D1, and MET in HCT116_PER2. KO-specific changes in rhythmic properties of known spliced variants of these genes (e.g. FGFR2 IIIb/FGFR2 IIIc) correlated with epithelial-mesenchymal-transition signalling. Altogether, our bioinformatic analysis highlights a role for the circadian clock in the regulation of AS, and reveals a potential impact of clock disruption in aberrant splicing in cancer hallmark genes.