Sanhong Liu

The histone H3 lysine-27 demethylase Jmjd3 plays a critical role in specific regulation of Th17 cell differentiation

Interleukin (IL) 17-producing T helper (Th17) cells play critical roles in the clearance of extracellular bacteria and fungi as well as the pathogenesis of various autoimmune diseases, such as multiple sclerosis, psoriasis, and ulcerative colitis. Although a global transcriptional regulatory network of Th17 cell differentiation has been mapped recently, the participation of epigenetic modifications in the differentiation process has yet to be elucidated. We demonstrated here that histone H3 lysine-27 (H3K27) demethylation, predominantly mediated by the H3K27 demethylase Jmjd3, crucially regulated Th17 cell differentiation. Activation of naïve CD4(+) T cells immediately induced high expression of Jmjd3. Genetic depletion of Jmjd3 in CD4(+) T cells specifically impaired Th17 cell differentiation both in vitro and in vivo. Ectopic expression of Jmjd3 largely rescued the impaired differentiation of Th17 cells in vitro in Jmjd3-deficient CD4(+) T cells. Importantly, Jmjd3-deficient mice were resistant to the induction of experimental autoimmune encephalomyelitis (EAE). Furthermore, inhibition of the H3K27 demethylase activity with the specific inhibitor GSK-J4 dramatically suppressed Th17 cell differentiation in vitro. At the molecular level, Jmjd3 directly bound to and reduced the level of H3K27 trimethylation (me3) at the genomic sites of Rorc, which encodes the master Th17 transcription factor Rorγt, and Th17 cytokine genes such as Il17, Il17f, and Il22. Therefore, our studies established a critical role of Jmjd3-mediated H3K27 demethylation in Th17 cell differentiation and suggest that Jmjd3 can be a novel therapeutic target for suppressing autoimmune responses.

The IκB family member Bcl-3 stabilizes c-Myc in colorectal cancer

Dear Editor, The proto-oncogene c-myc has been thought to play a critical role during the tumor-initiating process in multiple human cancers. Among others, colorectal cancer (CRC) is particularly associated with deregulated expression of c-Myc (Meyer and Penn, 2008; Wilkins and Sansom, 2008). Physiologically, Myc mRNA and protein levels are tightly regulated, and the Myc protein is highly unstable. The high levels of Myc protein in human CRC could be attributed to the altered Myc turnover and aberrant transcriptional activation of the myc genes (Ikegaki et al., 1986; Welcker and Clurman, 2008). The atypical member of the IkB family Bcl-3 can bind to p50 and p52 homodimers on DNA, thereby positively or negatively regulating the expression of NF-kB target genes, depending on the context (Fujita et al., 1993; Wang et al., 2012). Recently, high levels of Bcl-3 have been noted in a variety of solid cancers including CRC (Puvvada et al., 2010; Maldonado and Melendez-Zajgla, 2011). However, the function of Bcl-3 in colorectal tumorigenesis remains to be elucidated. We found that human CRC tissues exhibited increased levels of Bcl-3 compared with colorectal normal tissues (Figure 1A and B). In order to explore the role of Bcl-3 in human colorectal tumorigenesis, we transduced the human CRC cell line HCT116 with a teton lentiviral vector containing the shRNA against bcl-3 gene to establish the doxycycline (DOX)-inducible Bcl-3 knockdown cell line (HCT116/shBcl-3) (Supplementary Figure S1A). The in vitro cell growth was significantly slower upon Bcl-3 knockdown in HCT116 cells (Figure 1C and Supplementary Figure S2A). Bcl-3 knockdown also significantly suppressed the ability of HCT116 cells to form colonies in plate and soft agar (Supplementary Figure S2B and C). The inhibition was associated with a block in the G1/S transition of cell cycle (Supplementary Figure S2D). The inhibitory effects of Bcl-3 knockdown on in vitro CRC cell growth were also observed in mouse CRC cell line CT26 WT (Supplementary Figures S1B, S3A–C), excluding that the growth inhibition was due to the off-target effects of Bcl-3 knockdown. These results indicate that Bcl-3 knockdown suppresses colorectal tumor cell growth in vitro. To elucidate the mechanism by which Bcl-3 regulated the cell cycle, we compared the expressions of cell cycle-related genes between wild-type and Bcl-3 knockdown cells. We found that the level of c-Myc protein, but not the mRNA, was markedly decreased upon Bcl-3 knockdown. Accordingly, p21, which is negatively regulated by c-Myc, was increased. Bcl-3 knockdown, however, did not significantly affect the expression of other genes such as cyclin D1, cyclin E1, Skp2, p27, and IL-6 (Figure 1D and Supplementary Figure S4A). This suggests that reduced c-Myc protein level is associated with cell growth inhibition by Bcl-3 knockdown. We over-expressed c-Myc in Bcl-3 knockdown HCT116 cells, and found that the inhibited cell growth by Bcl-3 knockdown could be partially rescued by the over-expression of c-Myc (Figure 1E), indicating that reduced c-Myc protein contributes to reduced cell proliferation upon Bcl-3 knockdown. To verify that Bcl-3 regulates c-Myc protein level, we determined the half-life of c-Myc protein after applying the protein synthesis inhibitor cyclohemimide (CHX). c-Myc levels decreased faster in Bcl-3 knockdown cells than in control cells, while cyclin D1 levels decreased similarly in both cells (Figure 1F). Intriguingly, the inhibition of proteasomal function by MG-132 restored the decreased c-Myc protein in Bcl-3 knockdown cells to levels seen in control cells (Figure 1G). Moreover, Bcl-3 over-expression in a Bcl-3-deficient cell line significantly extended the half-life of c-Myc protein and reduced the levels of ubiquitinated c-Myc (Supplementary Figure S4B and C). The results above suggest that Bcl-3 regulates ubiquitination-mediated degradation of c-Myc. c-Myc protein stability can be differentially regulated by phosphorylation at threonine 58 (Thr58) and at serine 62 (Ser62). The phosphorylation at Thr58 by GSK-3 leads to degradation of c-Myc, while the phosphorylation at Ser62 likely mediated by ERK1/2 is required for RAS-induced stabilization of the c-Myc protein (Sears et al., 1999, 2000). In this study, we did not observe any notable difference on AKT phosphorylation or the expression of its targets such as p27 and cyclin E1 upon the absence of Bcl-3 in HCT116 cells. By contrast, we consistently found reduced levels of c-Myc, p-c-Myc at Ser62 and p-ERK1/2 upon Bcl-3 knockdown (Figure 1H and Supplementary Figure S5). Collectively, our data indicate that Bcl-3 may stabilize c-Myc protein by enhancing the ERK1/2-mediated phosphorylation of c-Myc at Ser62. Cell growth inhibition in vitro by Bcl-3 knockdown prompted us to investigate the effect of Bcl-3 on tumor cell growth in vivo. Bcl-3 knockdown induced by Dox significantly inhibited tumor cell growth in vivo when compared with tumor growth in control mice without Dox treatment (Figure 1I–K). There were significantly fewer Ki67 positive cells in xenograft tumors of Bcl-3 knockdown cells compared with controls (Supplementary Figure S6). We also noted decreased levels of c-Myc protein and ERK phosphorylation, but not c-myc mRNA, in xenograft tumor tissue grown under Bcl-3 knockdown conditions (Figure 1L and Supplementary Figure S7). Similar inhibitory effects of Bcl-3 280 | Journal of Molecular Cell Biology (2013), 5, 280–282 doi:10.1093/jmcb/mjt020 Published online June 20, 2013

The Role of PIWIL4, an Argonaute Family Protein, in Breast Cancer

P-element-induced wimpy testis (PIWI) proteins bind to PIWI-interacting RNAs and play key roles in the biogenesis and functions of PIWI-interacting RNAs. It has been reported that PIWI proteins are essential for stem cell self-renewal and germline development in diverse organisms and that they are ectopically expressed in multiple forms of cancer. However, the role of PIWI in cancer remains elusive. Here we report that one of the four PIWI proteins in humans, PIWIL4, is highly expressed in both breast cancer tissues and the cytoplasm of MDA-MB-231 cells derived from breast cancer. Reducing PIWIL4 expression drastically impairs the migration ability of MDA-MB-231 cells, significantly increases their apoptosis, and mildly affects their proliferation. Our transcriptome and proteome analysis reveal that these functions are at least partially achieved via the PIWIL4 regulation of TGF-β and FGF signaling pathways and MHC class II proteins. These findings suggest that PIWIL4 may serve as a potential therapeutic target for breast cancer.

miR-449a inhibits colorectal cancer progression by targeting SATB2

miR-449a has been reported to act as a tumor suppressor in several cancers, however, it is controversial whether it inhibits tumor growth in colorectal cancer. The mechanisms underlying its expression and functions in colorectal cancers are still largely unknown. SATB2 is a sensitive and specific marker for CRC diagnosis. However, the mechanisms by which the expression and functions of SATB2 are regulated still remain to be clarified. We investigated the expression and functional significance of miR-449a and SATB2 and the mechanisms of their dysregulation in human CRC cells. miR-449a overexpression or SATB2 depletion inhibited tumor growth and promoted apoptosis in colorectal tumor cells in vitro and in xenograft mouse model, partially by downregulating SATB2. Expression of miR-449a was increased epigenetically via knocking down their targets, particularly SATB2. miR-449a was downregulated and STAB2 expression was upregulated in human CRCs. Their expressions were significantly associated with overall survival of CRC patients. Our findings demonstrate the existence of a miR-449a-SATB2 negative feedback loop that maintains low levels of miR-449a as well as high level of SATB2, thereby promoting CRC development.

Primary investigation on GISH-NOR in cotton

Six loci of nucleolar organizer region (NOR) were detected in genomicin situ hybridization (GISH) of cotton (Gossypium). NOR was the characteristic of 45S rDNA but could be generated by genomic DNA (gDNA) extracted fromGossypium species as probe. With twice FISH to the same mitotic cell ofG. herbaceum orG. hirsutum, number, position and size for NORs generated from 45S rDNA and gDNA were identified largely similar or even the same. The NORs with gDNA as probe were therefore permanently defined as GISH-NORs. GISH-NORs fromG. hirsutum andG. raimondii mitotic images were all terminal types. Four and two GISH-NORs fromG. herbaceum (var.africanum) were terminal and centromere types, respectively. Six GISH-NORs inG. hirsutum were chromosome mapped with two in A- and four in D-subgenomes. There were also GISH-NORs in mitotic image ofG. raimondii with its own gDNA as probe. From mitotic image ofG. herbaceum with its own gDNA as probe, GISH-NOR could not be observed but non-whole-recovery of hybridized signals was distinguished. These non-whole-recovery of hybridized signals were detected on long arm terminals of most chromosomes and especially existed in nearly half long arm of a pair of chromosomes inG. herbaceum gDNA probed itself GISH image, which may be possibly induced by low copy genes within the regions rather than inter-subgenomic segment translocations. GISH-NORs in G.hirsutum mitotic images were dominantly observed when gDNAs from D and A genome species were used as probes and block, respectively, but not when the reverse probe and block gDNA from the two diploid progenitor genomes were designed. There may be two speculations to this special phenomenon: rDNA concerted evolution; content of rDNA in genome D more than genome A.

Downregulated miR-29a/b/c during Contact Inhibition Stage Promote 3T3-L1 Adipogenesis by Targeting DNMT3A.

Differentiation of 3T3-L1 cells into adipocytes involves a highly-orchestrated series of events including contact inhibition (CI), clonal expansion, growth arrest, and terminal differentiation. Recent study demonstrated that 3T3-L1 preadipocytes will not be differentiated into mature adipocytes without CI stage, which indicated that CI stage plays an important role during 3T3-L1 adipogenesis. However, the molecular mechanism is not yet fully understood. In the present study, we found that the expression level of miR-29a/b/c was decreased and the expression of DNMT3A was up-regulated during CI stage, respectively. Furthermore, overexpression of miR-29a/b/c during CI stage inhibits adipogenesis significantly but not at other stages. In addition, miR-29a/b/c repressed DNMT3A expression by directly targeting its 3’ untranslated region (3’ UTR). Our data reveal a novel mechanism of miR-29a/b/c in the regulation of adipogenesis.

Bcl-3 regulates TGFβ signaling by stabilizing Smad3 during breast cancer pulmonary metastasis.

Transforming growth factor beta (TGFβ) signaling in breast cancer is selectively associated with pulmonary metastasis. However, the underlying mechanisms remain unclear. Here we show that Bcl-3, a member of the IκB family, serves as a critical regulator in TGFβ signaling to modulate breast cancer pulmonary metastasis. Bcl-3 expression was significantly associated with metastasis-free survival in breast cancer patients. Bcl-3 deletion inhibited the migration and invasion of breast cancer cells in vitro, as well as breast cancer lung metastasis in vivo. Bcl-3 was required for the expression of downstream TGFβ signaling genes that are involved in breast cancer lung metastasis. Bcl-3 knockdown enhanced the degradation of Smad3 but not Smad2 following TGFβ treatment. Bcl-3 could bind to Smad3 and prevent the ubiquitination and degradation of Smad3 protein. These results indicate that Bcl-3 serves as a promising target to prevent breast tumor lung metastasis.

Cesarean section and risks of overweight and obesity in school-aged children: a population-based study

Background Obesity puts a great health burden in the world. Previous studies suggest that caesarean section (CS) may increase the risk of obesity in children, but it is still uncertain whether this association is causal or due to residual confounding by medical indication. Aim To assess the association between CS, CS without medical indications in particular and the risk of overweight and obesity in school-aged children. Design Cross-sectional survey. Methods The 2014 Shanghai Child Health, Education and Lifestyle Evaluation was a large population-based survey with cluster random probability sampling in 26 primary schools in Shanghai, China, in 2014. The mode of delivery was reported by parents. The height, weight and waist circumference of the children were measured. Logistic regression models with SURVEYLOGISTIC procedure were used to estimate the risk of childhood obesity. Pupils delivered vaginally were served as the reference group. Results A total of 17 571 pupils completed this survey, and 13 724 of them who were singleton, born term and between 5 and 13 years old were included in our analysis. CS was associated with increased risks of overweight and obesity (BMI: adjusted OR = 1.28 [95%CI 1.13-1.45] and 1.44 [1.26-1.66], respectively; weight for height ratio [WHtR] >0.46: 1.33 [1.20-1.48]). Similar results were found in CS without medical indication (BMI: overweight = 1.24 [1.05-1.47], obesity = 1.43 [1.19-1.72]; WHtR > 0.46: 1.30 [1.13-1.50]). Conclusions CS overall and CS without medical indications were associated with increased risks of overweight and obesity in primary school children.

Alternative NF-κB signaling promotes colorectal tumorigenesis through transcriptionally upregulating Bcl-3.

Multiple studies have shown that chronic inflammation is closely related to the occurrence and development of colorectal cancer (CRC). Classical NF-κB signaling, the key factor in controlling inflammation, has been found to be of great importance to CRC development. However, the role of alternative NF-κB signaling in CRC is still elusive. Here, we found aberrant constitutive activation of alternative NF-κB signaling both in CRC tissue and CRC cells. Knockdown of RelB downregulates c-Myc and upregulates p27Kip1 protein level, which inhibits CRC cell proliferation and retards CRC xenograft growth. Conversely, overexpression of RelB increases proliferation of CRC cells. In addition, we revealed a significant correlation between Bcl-3 and RelB in CRC tissues. The expression of RelB was consistent with the expression of Bcl-3 and the phosphorylation of Bcl-3 downstream proteins p-Akt (S473) and p-GSK3β (S9). Bcl-3 overexpression can restore the phenotype changes caused by RelB knockdown. Importantly, we demonstrated that alternative NF-κB transcriptional factor (p52:RelB) can directly bind to the promoter region of Bcl-3 gene and upregulate its transcription. Moreover, the expression of RelB, NF-κB2 p52, and Bcl-3 was associated with poor survival of CRC patients. Taken together, these results represent that alternative NF-κB signaling may function as an oncogenic driver in CRC, and also provide new ideas and research directions for the pathogenesis, prevention, and treatment of other inflammatory-related diseases.

miR-221/222 activate the Wnt/β-catenin signaling to promote triple-negative breast cancer

Triple-negative breast cancer (TNBC), characterized by the lack of expression of the estrogen receptor, the progesterone receptor, and the human epidermal growth factor receptor 2, is an aggressive form of cancer that conveys unpredictable and poor prognosis due to limited treatment options and lack of effective targeted therapies. Wnt/β-catenin signaling is hyperactivated in TNBC, which promotes the progression of TNBC. However, the molecular mechanism of Wnt/β-catenin activation in TNBC remains unknown. Here, we report the drastic overexpression of miR-221/222 in all of four TNBC cell lines and TNBC primary tumor samples from patients. Furthermore, we demonstrate by both ex vivo and xenograft experiments that inhibiting miR-221/222 expression in a TNBC cell line (MDA-MB-231) suppresses its proliferation, viability, epithelial-to-mesenchymal transition, and migration; whereas expressing miR-221/222 in a non-TNBC line (MCF7) promotes all of the above cancer properties. miR-221/222 achieve so by directly repressing multiple negative regulators of the Wnt/β-catenin signaling pathway, including WIF1, SFRP2, DKK2, and AXIN2, to activate the pathway. Notably, the level of miR-221/222 expression is inversely correlated whereas that of WIF1, DKK2, SFRP2, and AXIN2 expression is positively correlated with the patient survival. Last, we show that anti-miR-221/222 significantly increases apoptotic cells with tamoxifen/Wnt3a treatment but not with cyclophosphamide/Wnt3a treatment. These results demonstrate that miR-221/222 activate the Wnt/β-catenin signaling to promote the aggressiveness and TNBC properties of breast cancers, and thus reveal a new prospect for TNBC treatment.