Characterization and regulation of cell cycle-independent noncanonical gene targeting.

Homology-dependent targeted DNA integration, generally referred to as gene targeting, provides a powerful tool for precise genome modification; however, its fundamental mechanisms remain poorly understood in human cells. Here we reveal a noncanonical gene targeting mechanism that does not rely on the homologous recombination (HR) protein Rad51. This mechanism is suppressed by Rad52 inhibition, suggesting the involvement of single-strand annealing (SSA). The SSA-mediated gene targeting becomes prominent when DSB repair by HR or end-joining pathways is defective and does not require isogenic DNA, permitting 5% sequence divergence. Intriguingly, loss of Msh2, loss of BLM, and induction of a target-site DNA break all significantly and synergistically enhance SSA-mediated targeted integration. Most notably, SSA-mediated integration is cell cycle-independent, occurring in the G1 phase as well. Our findings provide unequivocal evidence for Rad51-independent targeted integration and unveil multiple mechanisms to regulate SSA-mediated targeted as well as random integration.

Control of meiotic entry by dual inhibition of a key mitotic transcription factor.

The mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a functional homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role of SWI4LUTI in establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity.

Activation of the p53 signaling pathway by piRNA-MW557525 overexpression induces a G0/G1 phase arrest thus inhibiting neuroblastoma growth.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children. Due to drug resistance to radiotherapy and chemotherapy, mainly due to the existence of cancer stem cells (CSCs), some children still have a poor prognosis. Therefore, researchers have focused their attention on CSCs. Our research group successfully constructed cancer stem cell-like cells named Piwil2-iCSCs by reprogramming human preputial fibroblasts (FBs) with the PIWIL2 gene in the early stage, and Piwil2-iCSCs were confirmed to induce the formation of embryonic tumors. PiRNAs, noncoding small RNAs that interact with PIWI proteins, play important roles in a variety of tumors. Therefore, our study aimed to explore the role of differentially expressed (DE) piRNAs derived from sequencing of Piwil2-iCSCs in NB. METHODS: The DE piRNAs in Piwil2-iCSCs were screened using high-throughput sequencing and further verified in NB tissues and cells. An unknown piRNA, named piRNA-MW557525, showed obvious downregulation in NB. Thus we studied the effect of piRNA-MW557525 on the biological behavior of NB through in vitro and in vivo experiments. On this basis, we successfully constructed a stably transfected NB cell line overexpressing piRNA-MW557525 and performed transcriptome sequencing to further explore the mechanism of piRNA-MW557525 in NB. RESULTS: In vitro, piRNA-MW557525 inhibited NB cell proliferation, migration and invasion and induced apoptosis; in vivo, piRNA-MW557525 significantly reduced the volume and weight of tumors and inhibited their proliferation, migration and invasion. piRNA-MW557525 overexpression induced G0/G1 phase arrest in NB cells via activation of the P53-P21-CDK2-Cyclin E signaling pathway thus inhibiting NB growth. CONCLUSIONS: Our findings show that piRNA-MW557525 functions as a tumor suppressor gene in NB and may serve as an innovative biomarker and possible therapeutic target for NB.

A specific anti-cyclin D1 intrabody represses breast cancer cell proliferation by interrupting the cyclin D1-CDK4 interaction.

BACKGROUND: Cyclin D1 overexpression may contribute to development of various cancers, including breast cancer, and thus may serve as a key cancer diagnostic marker and therapeutic target. In our previous study, we generated a cyclin D1-specific single-chain variable fragment antibody (ADκ) from a human semi-synthetic single-chain variable fragment library. ADκ specifically interacted with recombinant and endogenous cyclin D1 proteins through an unknown molecular basis to inhibit HepG2 cell growth and proliferation. RESULTS: Here, using phage display and in silico protein structure modeling methods combined with cyclin D1 mutational analysis, key residues that bind to ADκ were identified. Notably, residue K112 within the cyclin box was required for cyclin D1-ADκ binding. In order to elucidate the molecular mechanism underlying ADκ anti-tumor effects, a cyclin D1-specific nuclear localization signal-containing intrabody (NLS-ADκ) was constructed. When expressed within cells, NLS-ADκ interacted specifically with cyclin D1 to significantly inhibit cell proliferation, induce G1-phase arrest, and trigger apoptosis of MCF-7 and MDA-MB-231 breast cancer cells. Moreover, the NLS-ADκ-cyclin D1 interaction blocked binding of cyclin D1 to CDK4 and inhibited RB protein phosphorylation, resulting in altered expression of downstream cell proliferation-related target genes. CONCLUSION: We identified amino acid residues in cyclin D1 that may play key roles in the ADκ-cyclin D1 interaction. A nuclear localization antibody against cyclin D1 (NLS-ADκ) was constructed and successfully expressed in breast cancer cells. NLS-ADκ exerted tumor suppressor effects via blocking the binding of CDK4 to cyclin D1 and inhibiting phosphorylation of RB. The results presented here demonstrate anti-tumor potential of intrabody-based cyclin D1-targeted breast cancer therapy.

IMP-1 inhibits renal cell carcinoma 786-O cell growth by targeting EphrinB2 signaling pathway

Abstract EphrinB2 plays a critical role in tumor growth. In this study, we studied the antitumor activity of imperatorin derivative IMP-1 in renal cell carcinoma (RCC) by regulating EphrinB2 pathway.. Results showed that IMP-1 inhibited the proliferation of 786-O cells in a dose- and time-dependent manner. More importantly, knockdown and transfection of EphrinB2 altered the inhibitory effect of IMP-1 on the activity of 786-O cells. IMP-1 arrested 786-O cell cycle at G0/G1 phase by decreasing the expression of cyclin D1 and cyclin E. Moreover, IMP-1 regulated Bcl-2 family proteins' expression, thus inducing apoptosis of 786-O cells. IMP-1 down-regulated the expression of EphrinB2, Syntenin1 and PICK1. Then, IMP-1 decreased the phosphorylation of Erk1/2 and AKT. In all, IMP-1 could regulate the EphrinB2 pathway in order to inhibit 786-O cell growth by arresting the cell cycle at G0/G1 phase and inducing cell apoptosis. Thus, IMP-1 may present as a potential strategy for RCC treatment.

The Ubiquitin Ligase RNF138 Cooperates with CtIP to Stimulate Resection of Complex DNA Double-Strand Breaks in Human G1-Phase Cells.

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138's participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells.

ZNF703 promotes triple-negative breast cancer cells through cell-cycle signaling and associated with poor prognosis.

BACKGROUND: The oncogenic drivers of triple-negative breast cancer (TNBC), which is characterized by worst prognosis compared with other subtypes, are poorly understood. Although next-generation sequencing technology has facilitated identifying potential targets, few of the findings have been translated into daily clinical practice. The present study is aimed to explore ZNF703 (Zinc finger 703) function and its underlying mechanism in TNBC. METHODS: ZNF703 expressions in tissue microarray were retrospectively examined by immunohistochemistry. The cell proliferation by SRB assay and colony formation assay, as well as cell cycle distribution by flow cytometry were assessed. The protein levels associated with possible underlying molecular mechanisms were evaluated by western blotting. Kaplan-Meier analysis was used to plot survival analysis. RESULTS: Our data suggest that ZNF703 expressed in 34.2% of triple-negative human breast tumors by immunohistochemistry. In vitro, ZNF703 knockdown had potent inhibitory effects on TNBC cell proliferation and cell cycle, with cyclin D1, CDK4, CDK6, and E2F1 downregulated, while Rb1 upregulated. Moreover, Kaplan-Meier analysis showed that high mRNA expression of ZNF703 was correlated to worse overall survival (HR for high expression was 3.04; 95% CI, 1.22 to 7.57, P = 0.017). CONCLUSIONS: Taken together, the results identified that targeting ZNF703 contributed to the anti-proliferative effects in TNBC cells, due to induced G1-phase arrest. This study is the first to identify ZNF703 as a potentially important protein that is involved in TNBC progression.

Suppression of CDCA3 inhibits prostate cancer progression via NF­κB/cyclin D1 signaling inactivation and p21 accumulation.

Dysregulation of the cell cycle contributes to tumor progression. Cell division cycle­associated 3 (CDCA3) is a known trigger of mitotic entry and has been demonstrated to be constitutively upregulated in tumors. It is therefore associated with carcinogenic properties reported in various cancers. However, the role of CDCA3 in prostate cancer is unclear. In the present study, western blotting and analysis of gene expression profiling datasets determined that CDCA3 expression was upregulated in prostate cancer and was associated with a poor prognosis. CDCA3 knockdown in DU145 and PC­3 cells led to decreased cell proliferation and increased apoptosis, with increased protein expression levels of cleaved­caspase3. Further experiments demonstrated that downregulated CDCA3 expression levels induced G0/G1 phase arrest, which was attributed to increased p21 protein expression levels and decreased cyclin D1 expression levels via the regulation of NF­κB signaling proteins (NFκB­p105/p50, IKKα/ß, and pho­NFκB­p65). In conclusion, these results indicated that CDCA3 may serve a crucial role in prostate cancer and consequently, CDCA3 knockdown may be used as a potential therapeutic target.

LncRNA SNHG5 promotes the glycolysis and proliferation of breast cancer cell through regulating BACH1 via targeting miR-299.

BACKGROUND: Breast cancer (BC) is one of the most common malignant tumors in women. Accumulating studies have been reported that long non-coding RNA (lncRNA) SNHG5 is highly expressed in BC. However, the specific molecular mechanism of SNHG5 in BC is unclear. METHODS: Gene and protein expressions in BC cell were detected by qRT-PCR and western blotting. The proliferation and cell cycle were measured using colony formation assay and flow cytometry analysis, separately. The glucose consumption and lactate production were determined by using the glucose assay kit and lactate assay kit. A dual-luciferase reporter assay was performed to measure the interaction between miR-299 and SNHG5 or BACH1. RESULTS: SNHG5 and BACH1 expressions were increased in BC cell while miR-299 level was decreased. SNHG5 increased BACH1 expression by directly targeting miR-299. SNHG5 silencing or miR-299 overexpression suppressed the proliferation of BC cell, arrested the cell cycle in the G1 cell phase, and decreased the glucose consumption and lactate production of BC cell. However, inhibition of miR-299 or overexpression of BACH1 could reverse the inhibitory effects of sh-SNHG5 on cell proliferation and glycolysis in BC. CONCLUSION: SNHG5 promoted the BC cell growth and glycolysis through up-regulating BACH1 expression via targeting miR-299. These findings may improve the diagnostic and therapeutic approaches to BC.

Elevated expression of NFE2L3 promotes the development of gastric cancer through epithelial-mesenchymal transformation.

Gastric cancer (GC) is a malignant tumor with high mortality, but research on its molecular mechanisms remain limited. This study is the first to explore the biological role of nuclear factor NFE2L3 (nuclear factor, erythroid 2 like 3) in GC. We used Western blot and RT-qPCR to detect gene expression at the protein or mRNA level. Short hairpin RNA (shRNA) transfection was used to inhibit NFE2L3 expression. CCK-8 and colony formation assays were used to detect cell proliferation. Cell migration, invasion, cell cycle and apoptosis were detected by Transwell assays and flow cytometry. The results showed that NFE2L3 was highly expressed in gastric cancer tissues and promoted gastric cancer cell proliferation and metastasis. Inhibiting NFE2L3 expression blocks the cell cycle and increases the proportion of apoptotic cells, whereas NFE2L3 expression promotes the epithelial-mesenchymal transformation (EMT) process. In summary, NFE2L3 is highly expressed in gastric cancer and promotes gastric cancer cell proliferation and metastasis and the EMT process.

LncRNA SNHG17 interacts with LRPPRC to stabilize c-Myc protein and promote G1/S transition and cell proliferation.

Oncogenic c-Myc is a master regulator of G1/S transition. Long non-coding RNAs (lncRNAs) emerge as new regulators of various cell activities. Here, we found that lncRNA SnoRNA Host Gene 17 (SNHG17) was elevated at the early G1-phase of cell cycle. Both gain- and loss-of function studies disclosed that SNHG17 increased c-Myc protein level, accelerated G1/S transition and cell proliferation, and consequently promoted tumor cell growth in vitro and in vivo. Mechanistically, the 1-150-nt of SNHG17 physically interacted with the 1035-1369-aa of leucine rich pentatricopeptide repeat containing (LRPPRC) protein, and disrupting this interaction abrogated the promoting role of SNHG17 in c-Myc expression, G1/S transition, and cell proliferation. The effect of SNHG17 in stimulating cell proliferation was attenuated by silencing c-Myc or LRPPRC. Furthermore, silencing SNHG17 or LRPPRC increased the level of ubiquitylated c-Myc and reduced the stability of c-Myc protein. Analysis of human hepatocellular carcinoma (HCC) tissues revealed that SNHG17, LRPPRC, and c-Myc were significantly upregulated in HCC, and they showed a positive correlation with each other. High level of SNHG17 or LRPPRC was associated with worse survival of HCC patients. These data suggest that SNHG17 may inhibit c-Myc ubiquitination and thus enhance c-Myc level and facilitate proliferation by interacting with LRPPRC. Our findings identify a novel SNHG17-LRPPRC-c-Myc regulatory axis and elucidate its roles in G1/S transition and tumor growth, which may provide potential targets for cancer therapy.

[Knockdown of lysine-specific demethylase 3A (KDM3A) inhibits the invasion and migration of MDA-MB-231 breast cancer cells and arrests the cell cycle in the G0/G1 phase].

Objective To investigate the effect of lysine-specific demethylase 3A (KDM3A) on the invasion and migration of MDA-MB-231 breast cancer cells. Methods The mRNA and the protein expressions of KDM3A in MDA-MB-231 breast cancer cells and MCF-10A normal breast cells were detected by real-time quantitative PCR and Western blotting, respectively; the KDM3A level of MDA-MB-231 cells was knocked down by lentivirus infection of KDM3A short hairpin RNA (shKDM3A). The change of invasion and migration ability of MDA-MB-231 cells was detected by TranswellTM assay, and the change in the cell cycle was detected by flow cytometry. Results The expression of KDM3A in MDA-MB-231 breast cancer cells was significantly increased compared with that in MCF-10A epithelial cells; after KDM3A knockdown, the invasion and migration abilities of MDA-MB-231 cells were significantly decreased, and the cell cycle was arrested in the G0/G1 phase. Conclusion Knockdown of KDM3A inhibits the invasion and migration of MDA-MB-231 breast cancer cells and arrests the cell cycle in G0/G1 phase.

Stromal interaction molecule 1 (STIM1) knock down attenuates invasion and proliferation and enhances the expression of thyroid-specific proteins in human follicular thyroid cancer cells.

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.

A novel role of ADGRF1 (GPR110) in promoting cellular quiescence and chemoresistance in human epidermal growth factor receptor 2-positive breast cancer.

While G protein-coupled receptors (GPCRs) are known to be excellent drug targets, the second largest family of adhesion-GPCRs is less explored for their role in health and disease. ADGRF1 (GPR110) is an adhesion-GPCR and has an important function in neurodevelopment and cancer. Despite serving as a poor predictor of survival, ADGRF1's coupling to G proteins and downstream pathways remain unknown in cancer. We evaluated the effects of ADGRF1 overexpression on tumorigenesis and signaling pathways using two human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC) cell-line models. We also interrogated publicly available clinical datasets to determine the expression of ADGRF1 in various BC subtypes and its impact on BC-specific survival (BCSS) and overall survival (OS) in patients. ADGRF1 overexpression in HER2+ BC cells increased secondary mammosphere formation, soft agar colony formation, and % of Aldefluor-positive tumorigenic population in vitro and promoted tumor growth in vivo. ADGRF1 co-immunoprecipitated with both Gαs and Gαq proteins and increased cAMP and IP1 when overexpressed. However, inhibition of only the Gαs pathway by SQ22536 reversed the pro-tumorigenic effects of ADGRF1 overexpression. RNA-sequencing and RPPA analysis revealed inhibition of cell cycle pathways with ADGRF1 overexpression, suggesting cellular quiescence, as also evidenced by cell cycle arrest at the G0/1 phase and resistance to chemotherapy in HER2+ BC. ADGRF1 was significantly overexpressed in the HER2-enriched BC compared to luminal A and B subtypes and predicted worse BCSS and OS in these patients. Therefore, ADGRF1 represents a novel drug target in HER2+ BC, warranting discovery of novel ADGRF1 antagonists.

H Long Terminal Repeat-Associating 2 (HHLA2) is a Biomarker of Advanced Stage Hepatocellular Carcinoma and Promotes Tumor Cell Development In Vitro.

BACKGROUND Several risk factors contribute to the inflammation promoting hepatocellular carcinoma (HCC) development, but the underlying mechanisms are unknown. Human endogenous retrovirus H long terminal repeat-associating 2 (HHLA2), a B7 family member, is highly expressed in various malignant tumor tissues and is related to tumor progression and metastasis. MATERIAL AND METHODS Bioinformatics analysis was used to analyze the gene expression chip GSE33006 of HCC tissue in the GEO database, draw a heat map of differentially expressed genes, and analyze the GO pathway of gene function annotation. Then, we compared HCC tissues with para-carcinoma liver tissues from 55 patients for expression patterns and associations with HHLA2. Effects of HHLA2 knockdown were examined in the human HCC cell line HepG2 to explore effects of HHLA2 on HepG2 cells. RESULTS A significantly higher expression of HHLA2 at the mRNA and protein levels was detected in HCC tissues than in para-carcinoma liver tissues, which was similar to HHLA2 expression in the GSE33006 data. A higher expression of HHLA2 protein was associated with advanced cancer stage, tumor differentiation, and invasion of adjacent structures. In vitro knockdown of HHLA2 expression significantly increased HepG2 cell adhesion, promoted cell apoptosis, induced cell cycle arrest in the G1/S phase, and inhibited cell proliferation, migration, and invasion. CONCLUSIONS Our data indicated there was a higher expression of HHLA2 in HCC tissues than in para-carcinoma liver tissues, and HHLA2 plays a major role in the development and progression of HCC. Owing to its higher expression, HHLA2 is a potential prognostic biomarker for HCC.

PRMT1 is a novel molecular therapeutic target for clear cell renal cell carcinoma.

Background and Objective: Epigenetic alterations are common events in clear cell renal cell carcinoma (ccRCC), and protein arginine methyltransferase 1 (PRMT1) is an important epigenetic regulator in cancers. However, its role in ccRCC remains unclear. Methods: We investigated PRMT1 expression level and its correlations to clinicopathological factors and prognosis in ccRCC patients based on ccRCC tissue microarrays (TMAs). Genetic knockdown and pharmacological inhibition using a novel PRMT1 inhibitor DCPT1061 were performed to investigate the functional role of PRMT1 in ccRCC proliferation. Besides, we confirmed the antitumor effect of PRMT1 inhibitor DCPT1061 in ccRCC cell-derived tumor xenograft (CDX) models as well as patient-derived tumor xenograft (PDX) models. Results: We found PRMT1 expression was remarkably upregulated in tumor tissues and associated with poor pathologic characters and outcomes of ccRCC patients. Furthermore, genetic knockdown and pharmacological inhibition of PRMT1 by a novel potent inhibitor DCPT1061 dramatically induced G1 cell cycle arrest and suppressed ccRCC cell growth. Mechanistically, RNA sequencing and further validation identified Lipocalin2 (LCN2), a secreted glycoprotein implicated in tumorigenesis, as a crucial regulator of ccRCC growth and functional downstream effector of PRMT1. Epigenetic silencing of LCN2 autocrine secretion by PRMT1 deficiency decreased downstream p-AKT, leading to reduced p-RB and cell growth arrest through the neutrophil gelatinase associated lipocalin receptor (NGALR). Moreover, PRMT1 inhibition by DCPT1061 not only inhibited tumor growth but also sensitized ccRCC to sunitinib treatment in vivo by attenuating sunitinib-induced upregulation of LCN2-AKT-RB signaling. Conclusion: Taken together, our study revealed a PRMT1-dependent epigenetic mechanism in the control of ccRCC tumor growth and drug resistance, indicating PRMT1 may serve as a promising target for therapeutic intervention in ccRCC patients.

FBXW8 regulates G1 and S phases of cell cycle progression by restricting ß-TrCP1 function.

Sequential alteration in the expression levels of cell cycle regulatory proteins is crucial for faithful cell cycle progression to maintain the cellular homeostasis. F-box protein ß-TrCP1 is known to control the expression levels of several important cell cycle regulatory proteins. However, how the function of ß-TrCP1 is regulated in spatiotemporal manner during cell cycle progression remains elusive. Here, we show that expression levels of ß-TrCP1 oscillate during cell cycle progression with a minimum level at the G1 and S phases of cell cycle. Using biochemical, flow cytometry, and immunofluorescence techniques, we found that oscillation of ß-TrCP1 expression is controlled by another F-box protein FBXW8. FBXW8 directs the proteasomal degradation of ß-TrCP1 in MAPK pathway-dependent manner. Interestingly, we found that the attenuation of ß-TrCP1 by FBXW8 is important for Cdc25A-mediated cell cycle transition from G1 phase to S phase as well as DNA damage-free progression of S phase. Overall, our study reveals the intriguing molecular mechanism and significance of maintenance of ß-TrCP1 levels during cell cycle progression by FBXW8-mediated proteasomal degradation.

FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner.

N6-Methyladenosine (m6A) modification is the most abundant chemical modification in mRNA, and it participates in various biological processes, such as cell differentiation and proliferation. However, little is known about the function of m6A demethylase fat mass and obesity-associated (FTO) in myoblast proliferation. Here, we demonstrated that knockdown of FTO can significantly inhibit myoblast proliferation and promote apoptosis. RNA sequencing analysis revealed that a lot of downregulated genes in FTO knockdown cells are associated with cell cycle and apoptosis. Furthermore, silencing FTO drastically decreased cyclin D1 (CCND1) expression through YTHDF2-mediated mRNA degradation, thereby delaying the progression of G1 phase, and leading to impaired myoblast proliferation. These findings unraveled that FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner, which highlights the critical roles of m6A modification in myoblast proliferation.

APOBEC3B is preferentially expressed at the G2/M phase of cell cycle.

APOBEC3B (A3B) is a cytosine deaminase that converts cytosine to uracil in single-stranded DNA. Cytosine-to-thymine and cytosine-to-guanine base substitution mutations in trinucleotide motifs (APOBEC mutational signatures) were found in various cancers including lymphoid hematological malignancies such as multiple myeloma and A3B has been shown to be an enzymatic source of mutations in those cancers. Although the importance of A3B is being increasingly recognized, it is unclear how A3B expression is regulated in cancer cells as well as normal cells. To answer these fundamental questions, we analyzed 1276 primary myeloma cells using single-cell RNA-sequencing (scRNA-seq) and found that A3B was preferentially expressed at the G2/M phase, in sharp contrast to the expression patterns of other APOBEC3 genes. Consistently, we demonstrated that A3B protein was preferentially expressed at the G2/M phase in myeloma cells by cell sorting. We also demonstrated that normal blood cells expressing A3B were also enriched in G2/M-phase cells by analyzing scRNA-seq data from 86,493 normal bone marrow mononuclear cells. Furthermore, we revealed that A3B was expressed mainly in plasma cells, CD10+ B cells and erythroid cells, but not in granulocyte-macrophage progenitors. A3B expression profiling in normal blood cells may contribute to understanding the defense mechanism of A3B against viruses, and partially explain the bias of APOBEC mutational signatures in lymphoid but not myeloid malignancies. This study identified the cells and cellular phase in which A3B is highly expressed, which may help reveal the mechanisms behind carcinogenesis and cancer heterogeneity, as well as the biological functions of A3B in normal blood cells.

Inhibition of Growth of Colon Tumors and Proliferation of HT-29 Cells by Warburgia ugandensis Extract through Mediating G0/G1 Cell Cycle Arrest, Cell Apoptosis, and Intracellular ROS Generation.

Warburgia ugandensis Sprague (W. ugandensis), widely distributed in Africa, is a traditional medicinal plant used for the treatment of various diseases including cancer. We intended to evaluate the anticolorectal cancer (CRC) activities of the crude extract from W. ugandensis (WUD) and reveal the underlying molecular mechanisms of its action. We found that WUD inhibited the proliferation of HT-29 and HCT116 cells in a time- and dose-dependent manner and induced intracellular ROS generation. The inhibitory effect of WUD on the proliferation of HT-29 and HCT116 cells could be attenuated by NAC (a ROS scavenger) in a dose-dependent manner. WUD induced G0/G1 phase arrest, down-regulated the protein expression of Cyclin D1 via ROS accumulation in HT-29 cells. In search of the molecular mechanism involved in WUD-induced Cyclin D1 down-regulation, it was found that WUD can suppress PI3K/Akt/GSK3ß signaling pathway in HT-29 cells. Next, it was found that WUD also activated apoptosis, poly-ADP ribose polymerase 1 (PARP1) cleavage and down-regulated pro-caspase 3 in HT-29 and HCT116 cells. Besides, WUD decreased the growth of colon tumors in vivo in the xenograft mouse model. We demonstrated for the first time that ROS and their modulation in the corresponding intracellular signaling could play a significant role in the potential activity of WUD against CRC cells.