PTBP1-activated co-transcriptional splicing controls epigenetic status of pluripotent stem cells.

Many spliceosomal introns are excised from nascent transcripts emerging from RNA polymerase II (RNA Pol II). The extent of cell-type-specific regulation and possible functions of such co-transcriptional events remain poorly understood. We examined the role of the RNA-binding protein PTBP1 in this process using an acute depletion approach followed by the analysis of chromatin- and RNA Pol II-associated transcripts. We show that PTBP1 activates the co-transcriptional excision of hundreds of introns, a surprising effect given that this protein is known to promote intron retention. Importantly, some co-transcriptionally activated introns fail to complete their splicing without PTBP1. In a striking example, retention of a PTBP1-dependent intron triggers nonsense-mediated decay of transcripts encoding DNA methyltransferase DNMT3B. We provide evidence that this regulation facilitates the natural decline in DNMT3B levels in developing neurons and protects differentiation-specific genes from ectopic methylation. Thus, PTBP1-activated co-transcriptional splicing is a widespread phenomenon mediating epigenetic control of cellular identity.

The inactive Dnmt3b3 isoform preferentially enhances Dnmt3b-mediated DNA methylation.

The de novo DNA methyltransferases Dnmt3a and Dnmt3b play crucial roles in developmental and cellular processes. Their enzymatic activities are stimulated by a regulatory protein Dnmt3L (Dnmt3-like) in vitro. However, genetic evidence indicates that Dnmt3L functions predominantly as a regulator of Dnmt3a in germ cells. How Dnmt3a and Dnmt3b activities are regulated during embryonic development and in somatic cells remains largely unknown. Here we show that Dnmt3b3, a catalytically inactive Dnmt3b isoform expressed in differentiated cells, positively regulates de novo methylation by Dnmt3a and Dnmt3b with a preference for Dnmt3b. Dnmt3b3 is equally potent as Dnmt3L in stimulating the activities of Dnmt3a2 and Dnmt3b2 in vitro. Like Dnmt3L, Dnmt3b3 forms a complex with Dnmt3a2 with a stoichiometry of 2:2. However, rescue experiments in Dnmt3a/3b/3l triple-knockout (TKO) mouse embryonic stem cells (mESCs) reveal that Dnmt3b3 prefers Dnmt3b2 over Dnmt3a2 in remethylating genomic sequences. Dnmt3a2, an active isoform that lacks the N-terminal uncharacterized region of Dnmt3a1 including a nuclear localization signal, has very low activity in TKO mESCs, indicating that an accessory protein is absolutely required for its function. Our results suggest that Dnmt3b3 and perhaps similar Dnmt3b isoforms facilitate de novo DNA methylation during embryonic development and in somatic cells.

The structure of DNA methyltransferase DNMT3C reveals an activity-tuning mechanism for DNA methylation.

DNA methylation is one of the major epigenetic mechanisms crucial for gene regulation and genome stability. De novo DNA methyltransferase DNMT3C is required for silencing evolutionarily young transposons during mice spermatogenesis. Mutation of DNMT3C led to a sterility phenotype that cannot be rescued by its homologs DNMT3A and DNMT3B. However, the structural basis of DNMT3C-mediated DNA methylation remains unknown. Here, we report the structure and mechanism of DNMT3C-mediated DNA methylation. The DNMT3C methyltransferase domain recognizes CpG-containing DNA in a manner similar to that of DNMT3A and DNMT3B, in line with their high sequence similarity. However, two evolutionary covariation sites, C543 and E590, diversify the substrate interaction among DNMT3C, DNMT3A, and DNMT3B, resulting in distinct DNA methylation activity and specificity between DNMT3C, DNMT3A, and DNMT3B in vitro. In addition, our combined structural and biochemical analysis reveals that the disease-causing rahu mutation of DNMT3C compromises its oligomerization and DNA-binding activities, explaining the loss of DNA methylation activity caused by this mutation. This study provides a mechanistic insight into DNMT3C-mediated DNA methylation that complements DNMT3A- and DNMT3B-mediated DNA methylation in mice, unraveling a regulatory mechanism by which evolutionary conservation and diversification fine-tune the activity of de novo DNA methyltransferases.

Liver-specific deletion of de novo DNA methyltransferases protects against glucose intolerance in high-fat diet-fed male mice.

Alterations to gene transcription and DNA methylation are a feature of many liver diseases including fatty liver disease and liver cancer. However, it is unclear whether the DNA methylation changes are a cause or a consequence of the transcriptional changes. It is even possible that the methylation changes are not required for the transcriptional changes. If DNA methylation is just a minor player in, or a consequence of liver transcriptional change, then future studies in this area should focus on other systems such as histone tail modifications. To interrogate the importance of de novo DNA methylation, we generated mice that are homozygous mutants for both Dnmt3a and Dnmt3b in post-natal liver. These mice are viable and fertile with normal sized livers. Males, but not females, showed increased adipose depots, yet paradoxically, improved glucose tolerance on both control diet and high-fat diets (HFD). Comparison of the transcriptome and methylome with RNA sequencing and whole-genome bisulfite sequencing in adult hepatocytes revealed that widespread loss of methylation in CpG-rich regions in the mutant did not induce loss of homeostatic transcriptional regulation. Similarly, extensive transcriptional changes induced by HFD did not require de novo DNA methylation. The improved metabolic phenotype of the Dnmt3a/3b mutant mice may be mediated through the dysregulation of a subset of glucose and fat metabolism genes which increase both glucose uptake and lipid export by the liver. However, further work is needed to confirm this.

Isorhapontigenin inhibition of basal muscle-invasive bladder cancer attributed to its downregulation of SNHG1 and DNMT3b.

BACKGROUND: Bladder cancer (BC) is among the most prevalent malignant urothelial tumors globally, yet the prognosis for patients with muscle-invasive bladder cancer (MIBC) remains dismal, with a very poor 5-year survival rate. Consequently, identifying more effective and less toxic chemotherapeutic alternatives is critical for enhancing clinical outcomes for BC patients. Isorhapontigenin (ISO), a novel stilbene isolated from a Gnetum found in certain provinces of China, has shown potential as an anticancer agent due to its diverse anticancer activities. Despite its promising profile, the specific anticancer effects of ISO on BC and the underlying mechanisms are still largely unexplored. METHODS: The anchorage-independent growth, migration and invasion of BC cells were assessed by soft agar and transwell invasion assays, respectively. The RNA levels of SOX2, miR-129 and SNHG1 were quantified by qRT-PCR, while the protein expression levels were validated through Western blotting. Furthermore, methylation-specific PCR was employed to assess the methylation status of the miR-129 promoter. Functional assays utilized siRNA knockdown, plasmid-mediated overexpression, and chemical inhibition approaches. RESULTS: Our study demonstrated that ISO treatment significantly reduced SNHG1 expression in a dose- and time-dependent manner in BC cells, leading to the inhibition of anchorage-independent growth and invasion in human basal MIBC cells. This effect was accompanied by the downregulation of MMP-2 and MMP-9 and the upregulation of the tumor suppressor PTEN. Further mechanistic investigations revealed that SOX2, a key upstream regulator of SNHG1, played a crucial role in mediating the ISO-induced transcriptional suppression of SNHG1. Additionally, we found that ISO treatment led to a decrease in DNMT3b protein levels, which in turn mediated the hypomethylation of the miR-129 promoter and the subsequent suppression of SOX2 mRNA 3'-UTR activity, highlighting a novel pathway through which ISO exerts its anticancer effects. CONCLUSIONS: Collectively, our study highlights the critical role of SNHG1 downregulation as well as its upstream DNMT3b/miR-129/SOX2 axis in mediating ISO anticancer activity. These findings not only elucidate the mechanism of action of ISO but also suggest novel targets for BC therapy.

DNMT3B activates FGFR3-mediated endoplasmic reticulum stress by regulating PTPN2 promoter methylation to promote the development of atherosclerosis.

Endoplasmic reticulum (ER) stress is closely associated with atherosclerosis (AS). Nevertheless, the regulatory mechanism of ER stress in endothelial cells during AS progression is unclear. Here, the role and regulatory mechanism of DNA (cytosine-5-)- methyltransferase 3 beta (DNMT3B) in ER stress during AS progression were investigated. ApoE-/- mice were fed with high fat diet to construct AS model in vivo. HE and Masson staining were performed to analyze histopathological changes and collagen deposition. HUVECs stimulated by ox-LDL were used as AS cellular model. Cell apoptosis was examined using flow cytometry. DCFH-DA staining was performed to examine ROS level. The levels of pro-inflammatory cytokines were assessed using ELISA. In addition, MSP was employed to detect PTPN2 promoter methylation level. Our results revealed that DNMT3B and FGFR3 were significantly upregulated in AS patient tissues, whereas PTPN2 was downregulated. PTPN2 overexpression attenuate ox-LDL-induced ER stress, inflammation and apoptosis in HUVECs and ameliorated AS symptoms in vivo. PTPN2 could suppress FGFR3 expression in ox-LDL-treated HUVECs, and FGFR3 knockdown inhibited ER stress to attenuate ox-LDL-induced endothelial cell apoptosis. DNMT3B could negatively regulate PTPN2 expression and positively FGFR2 expression in ox-LDL-treated HUVECs; DNMT3B activated FGFR2 expression by increasing PTPN2 promoter methylation level. DNMT3B downregulation repressed ox-LDL-induced ER stress, inflammation and cell apoptosis in endothelial cells, which was reversed by PTPN2 silencing. DNMT3B activated FGFR3-mediated ER stress by increasing PTPN2 promoter methylation level and suppressed its expression, thereby boosting ER stress to facilitate AS progression.

Methyltransferase DNMT3B promotes colorectal cancer cell proliferation by inhibiting PLCG2.

Aberrant DNA methylation patterns in the promoter region of PLCG2 are associated with dysregulated signaling pathways and cellular functions. Its role in colorectal cancer cells is still unknown. In this study, qRT-PCR is used to measure DNMT3B expression in colorectal cancer. Western blot analysis and immunohistochemistry are used to analyze DNMT3B and PLCG2 protein levels in colorectal tissues and cell lines. Cell Counting Kit-8 (CCK-8) and colony formation assays are used to assess the proliferation of colorectal cancer cells. Methylation-specific PCR (MSP) and bisulfite-sequencing PCR (BSP) are used to measure DNA methylation level. Our results show that DNMT3B is overexpressed in colorectal cells in the TCGA datasets according to Kaplan-Meier plots. DNMT3B is significantly overexpressed in tumor tissues compared to that in adjacent nontumor tissues. Western blot analysis results demonstrate high expression of DNMT3B in tumor tissues. Compared to normal colonic epithelial cells, colorectal cancer cell lines exhibit elevated level of PLCG2 methylation. Overexpression of PLCG2 effectively prevents the growth of colorectal cancer xenograft tumors in vivo. PLCG2 is identified as a key downstream regulatory protein of DNMT3B in colorectal cancer. DNMT3B inhibits PLCG2 transcription through methylation of the PLCG2 promoter region. DNMT3B controls colorectal cancer cell proliferation through PLCG2, which is useful for developing therapeutic approaches that target PLCG2 expression for the treatment of colorectal cancer.

Dnmt3b ablation affects fracture repair process by regulating apoptosis.

PURPOSE: Previous studies have shown that DNA methyltransferase 3b (Dnmt3b) is the only Dnmt responsive to fracture repair and Dnmt3b ablation in Prx1-positive stem cells and chondrocyte cells both delayed fracture repair. Our study aims to explore the influence of Dnmt3b ablation in Gli1-positive stem cells in fracture healing mice and the underlying mechanism. METHODS: We generated Gli1-CreERT2; Dnmt3bflox/flox (Dnmt3bGli1ER) mice to operated tibia fracture. Fracture callus tissues of Dnmt3bGli1ER mice and control mice were collected and analyzed by X-ray, micro-CT, biomechanical testing, histopathology and TUNEL assay. RESULTS: The cartilaginous callus significantly decrease in ablation of Dnmt3b in Gli1-positive stem cells during fracture repair. The chondrogenic and osteogenic indicators (Sox9 and Runx2) in the fracture healing tissues in Dnmt3bGli1ER mice much less than control mice. Dnmt3bGli1ER mice led to delayed bone callus remodeling and decreased biomechanical properties of the newly formed bone during fracture repair. Both the expressions of Caspase-3 and Caspase-8 were upregulated in Dnmt3bGli1ER mice as well as the expressions of BCL-2. CONCLUSIONS: Our study provides an evidence that Dnmt3b ablation Gli1-positive stem cells can affect fracture healing and lead to poor fracture healing by regulating apoptosis to decrease chondrocyte hypertrophic maturation.

FOXC1 Promotes Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem Cells via the Dnmt3b/CXCL12 Axis.

Bone defects have remained a clinical problem in current orthopedics. Bone marrow mesenchymal stem cells (BM-MSCs) with multi-directional differentiation ability have become a research hotspot for repairing bone defects. In vitro and in vivo models were constructed, respectively. Alkaline phosphatase (ALP) staining and alizarin red staining were performed to detect osteogenic differentiation ability. Western blotting (WB) was used to detect the expression of osteogenic differentiation-related proteins. Serum inflammatory cytokine levels were detected by ELISA. Fracture recovery was evaluated by HE staining. The binding relationship between FOXC1 and Dnmt3b was verified by dual-luciferase reporter assay. The relationship between Dnmt3b and CXCL12 was explored by MSP and ChIP assays. FOXC1 overexpression promoted calcium nodule formation, upregulated osteogenic differentiation-related protein expression, promoted osteogenic differentiation, and decreased inflammatory factor levels in BM-MSCs, and promoted callus formation, upregulated osteogenic differentiation-related protein expression, and downregulated CXCL12 expression in the mouse model. Furthermore, FOXC1 targeted Dnmt3b, with Dnmt3b knockdown decreasing calcium nodule formation and downregulating osteogenic differentiation-related protein expression. Additionally, inhibiting Dnmt3b expression upregulated CXCL12 protein expression and inhibited CXCL12 methylation. Dnmt3b could be binded to CXCL12. CXCL12 overexpression attenuated the effects of FOXC1 overexpression and inhibited BM-MSCs osteogenic differentiation. This study confirmed that the FOXC1-mediated regulation of the Dnmt3b/CXCL12 axis had positive effects on the osteogenic differentiation of BM-MSCs.

A novel mutation in DNMT3B gene causing ICF1 syndrome in an infant with refractory thrombocytopenia.

BACKGROUND: ICF syndrome is a rare autosomal recessive condition characterized by immunodeficiency, centromeric instability, and facial abnormalities. It is a clinical condition that depends on the mutation of a few particular genes and is caused by methylation disruption in chromosomes 1, 9, and 16 to varying degrees. CASE PRESENTATION: The 9-months old, female patient was admitted to our clinic for treatment-resistant thrombocytopenia, chronic diarrhea and sepsis. Immunological investigations revealed agammaglobulinemia. In the genetic analysis by NGS of the patient, who had dysmorphic facial findings as well as a history of parental consanguinity, it was determined that she had a novel mutation in the DNMT3B gene, which is one of the responsible genes of ICF, as homozygous. The patient, who was started on regular immunoglobulin replacement therapy and antibiotic therapy, was referred to a center with a stem cell transplant unit to continue her follow-up. CONCLUSIONS: Although autoimmunity has not been commonly reported in previous studies in ICF syndrome, which has a varied clinical presentation, a homozygous mutation in the DNMT3B gene was discovered in a 9-month-old patient with refractory thrombocytopenia and agammaglobulinemia. Examining the literature reveals that this mutation is a novel mutation.

Genetic aetiology of Down syndrome birth: novel variants of maternal DNMT3B and RFC1 genes increase risk of meiosis II nondisjunction in the oocyte.

The aim of the present work was to explore the intriguing association of maternal folate regulator gene polymorphisms and mutations with the incidence of chromosome 21 nondisjunction and Down syndrome birth. We tested polymorphisms/mutations of DNMT3B and RFC1 genes for their association with meiotic errors in oocyte among the 1215 Down syndrome child-bearing women and 900 controls. We observed that 23 out of 31 variants of DNMT3B and RFC1 exhibited an association with meiosis II nondisjunction in maternal age-independent manner. Additionally, we have reported 17 novel mutations and 1 novel polymorphic variant that are unique to the Indian Bengali speaking cohort and increased odds in favour of meiosis II nondisjunction. We hypothesize that the risk variants and mutations of DNMT3B and RFC1 genes may cause reduction in two or more recombination events and also cause peri-centromeric single exchange that increases the risk of nondisjunction at any age of women. In silico analyses predicted the probable damages of the transcripts or proteins from the respective genes owing to the said polymorphisms. These findings from the largest population sample tested ever revealed that mutations/polymorphisms of the genes DNMT3B and RFC1 impair recombination that leads to chromosome 21 nondisjunction in the oocyte at meiosis II stage and bring us a significant step closer towards understanding the aetiology of chromosome 21 nondisjunction and birth of a child with Down syndrome to women at any age.

Non-coding RNAs/DNMT3B axis in human cancers: from pathogenesis to clinical significance.

Cancer is a complex disease with many contributing factors, and researchers have gained extensive knowledge that has helped them understand the diverse and varied nature of cancer. The altered patterns of DNA methylation found in numerous types of cancer imply that they may play a part in the disease's progression. The human cancer condition involves dysregulation of the DNA methyltransferase 3 beta (DNMT3B) gene, a prominent de novo DNA methyltransferase, and its abnormal behavior serves as an indicator for tumor prognosis and staging. The expression of non-coding RNAs (ncRNAs), which include microRNAs (miRNA), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), is critical in controlling targeted gene expression and protein translation and their dysregulation correlates with the onset of tumors. NcRNAs dysregulation of is a critical factor that influences the modulation of several cellular characteristics in cancerous cells. These characteristics include but are not limited to, drug responsiveness, angiogenesis, metastasis, apoptosis, proliferation, and properties of tumor stem cell. The reciprocal regulation of ncRNAs and DNMT3B can act in synergy to influence the destiny of tumor cells. Thus, a critical avenue for advancing cancer prevention and treatment is an inquiry into the interplay between DNMT3B and ncRNAs. In this review, we present a comprehensive overview of the ncRNAs/DNMT3B axis in cancer pathogenesis. This brings about valuable insights into the intricate mechanisms of tumorigenesis and provides a foundation for developing effective therapeutic interventions.

Induced PSIG expression by Herbacetin contributes to suppressing the proliferation, migration, and invasion of melanoma cells.

Melanoma is a very common malignant tumor with poor prognosis. Herbacetin is a flavonol compound with outstanding anti-tumor effects. Our work investigated the biological effects and mechanism of Herbacetin in melanoma. In our study, the mRNA and protein expressions were assessed using qRT-PCR, Western blot and IHC. MSP was performed to evaluated PGIS promoter methylation level. Cell viability, migration and invasion were examined by MTT assay, transwell migration and invasion assay, respectively. Our results revealed that DNMT3B was markedly upregulated in melanoma, while PGIS was lowly expressed. Herbacetin treatment could not only inhibit the proliferation, migration, invasion of melanoma cells and inhibit the growth of melanoma in vivo. Herbacetin could also restore the abnormal expressions of DNMT3B and PGIS in melanoma cells and tumor tissues. PGIS silencing neutralized the inhibitory effects of Herbacetin on the malignant behaviors of melanoma cells. Besides, DNMT3B knockdown promoted PGIS expression via reducing PGIS promoter methylation level in melanoma cells, thereby inhibiting malignant behaviors of melanoma cells. And as expected, the inhibitory effects of Herbacetin on malignant behaviors of melanoma cells were all abolished by DNMT3B overexpression. Collectively, Herbacetin reduced DNMT3B expression to upregulate PGIS in melanoma cells and participated in suppressing the proliferation, migration, and invasion of melanoma cells.

Uterine deficiency of Dnmt3b impairs decidualization and causes consequent embryo implantation defects.

Uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects. Recent advances in molecular technologies have allowed the unprecedented mapping of epigenetic modifications during embryo implantation. DNA methyltransferase 3a (DNMT3A) and DNMT3B are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. It was reported that conditional knockout of Dnmt3a in the uterus does not markedly affect endometrial function during embryo implantation, but the tissue-specific functions of Dnmt3b in the endometrium during embryo implantation remain poorly understood to investigate the role of Dnmt3b during peri-implantation period. Here, we generated Dnmt3b conditional knockout (Dnmt3bd/d) female mice using progesterone receptor-Cre mice and examined the role of Dnmt3b during embryo implantation. Dnmt3bd/d female mice exhibited compromised fertility, which was associated with defective decidualization, but not endometrial receptivity. Furthermore, results showed loss of Dnmt3b did not lead to altered genomic methylation patterns of the decidual endometrium during early pregnancy. Transcriptome sequencing analysis of uteri from day 6 pregnant mice identified phosphoglycerate kinase 1 (Pgk1) as one of the most variable genes in Dnmt3bd/d decidual endometrium. Potential roles of PGK1 in the decidualization process during early pregnancy were confirmed. Lastly, the compromised decidualization upon the downregulation of Dnmt3b could be reversed by overexpression of Pgk1. Collectively, our findings indicate that uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects.

The relationship between DNA methyltransferase 3B (DNMT3B) and miR 124-3pa expressions in bladder cancer tissues.

BACKGROUND: Cancer bladder is the most common malignant tumor affecting the urinary tract. Genetic alterations are tightly associated with the development of cancer bladder. MicroRNAs (miRNA) are small, noncoding single-stranded RNA molecules that have been linked to bladder cancer. miR-124-3pa exhibits altered expression in various types of human malignancies. DNA methyltransferase 3B (DNMT3B) is responsible for de novo DNA methylation which is a fundamental epigenetic process in carcinogenesis. This work was performed to study the expression of DNMT3B and miR 124-3pa in bladder cancer tissues, and investigate their significance in the diagnosis and prognosis of the disease. SUBJECTS & METHODS: This case-control study included one hundred and six tissue samples of patients with primary urothelial bladder cancer. The tissues were separated into two parts. The first part was immediately frozen and kept at - 80 °C for total RNA extraction with subsequent detection of miR 124-3pa and DNMT3B expressions. The other part was preserved in formalin solution for histopathological examination. RESULTS: There was a highly statistically significant difference between the cancerous and the normal tissues as regarding miRNA-124-3pa and DNMT3B expression (P < 0.001) for each. Also, there was a highly statistically significant strong negative correlation between miRNA-124-3pa and DNMT3B expression (r=-0.750, P < 0.001). The combined performance of miR-124-3pa and DNMT3B revealed that the cutoff point of ≥ 3.3 can be used as a predictor of the presence of cancer bladder with sensitivity of 98.1% and specificity of 80%. CONCLUSION: miR-124-3pa and DNMT3B can be used as predictors of the presence of cancer bladder.

RAMP2-AS1 inhibits CXCL11 expression to suppress malignant phenotype of breast cancer by recruiting DNMT1 and DNMT3B.

BACKGROUND: Breast cancer is the most common malignancy in women populations. METHODS: RAMP2-AS1 and CXCL11 expression in breast cancer tissues and cells were determined using RT-qPCR or Western blot. RIP analysis confirmed the interaction between DNMT1, DNMT3B and RAMP2-AS1. ChIP assay verified that RAMP2-AS1 recruited DNMT1 and DNMT3B to the promoter region of CXCL11. FISH detected the sub-localization of RAMP2-AS1 in breast cancer cells. Bisulfite sequencing PCR (BSP) tested the methylation level of CXCL11. The cell viability, proliferation, migration and apoptosis were assessed by CCK-8, colony formation, transwell and flow cytometry assays, respectively. IHC was performed to evaluate the expression of Ki67, CXCL11, MMP2 in tumor tissues. RESULTS: The level of RAMP2-AS1 was decreased in breast cancer tissues and cells, whereas CXCL11 was highly expressed. Patients with decreased RAMP2-AS1 had a poor prognosis. RAMP2-AS1 inhibited breast cancer cell malignant phenotype. Besides, RAMP2-AS1 regulated the methylation of CXCL11 by recruiting DNMT1 and DNMT3B to the promoter region of CXCL11. RAMP2-AS1 overexpression suppressed the malignant phenotype through CXCL11 and inhibited tumor growth in vivo. CONCLUSION: RAMP2-AS1 suppresses breast cancer malignant phenotype via DNMT1 and DNMT3B mediated inhibition of CXCL11.

Reduced expression of microRNA-432-5p by DNA methyltransferase 3B leads to development of colorectal cancer through upregulation of CCND2.

BACKGROUND: The tumor suppressive function of microRNA-432-5p (miR-432-5p) has been reported in several human malignances. This study aimed to probe the expression profile and role of miR-432-5p in colorectal cancer (CRC) and the molecular mechanism. METHODS: Differentially expressed miRNAs between CRC and healthy samples were screened using a miRNA expression dataset GSE136020. The related molecules were identified by integrated bioinformatic analyses. A murine model of primary CRC was established and xenograft tumors were induced in mice. Altered expression of DNMT3B, miR-432-5p and cyclin D2 (CCND2) were introduced in CRC cells to determine their roles in the development of CRC. RESULTS: miR-432-5p was downregulated in CRC according to the GSE136020 dataset. CCND2 mRNA was confirmed as a target of miR-432-5p. miR-432-5p was downregulated, whereas CCND2 was abundantly expressed in CRC tissues and cells. DNA methyltransferase 3B (DNMT3B) induced DNA methylation at the CpG island of miR-432-5p to inhibit its expression. miR-432-5p mimic significantly suppressed tumorigenesis of primary CRC in mice. Downregulation of DNMT3B weakened viability, invasiveness, blocked the cell cycle progression of CRC cells in vitro, and inhibited xenograft tumor growth and metastasis in nude mice. However, additional downregulation of miR-432-5p or upregulation of CCND2 restored the malignant behaviors of CRC cells. CONCLUSION: This study showed that DNMT3B induced DNA methylation and downregulation of miR-432-5p to promote development of CRC by upregulating CCND2.

PCDH17 is regulated by methylation of DNMT3B and affects the malignant biological behavior of HCC through EMT.

Hepatocellular carcinoma (HCC) is one of the malignant tumors most frequently encountered in the clinic. Studies have shown that the abnormal expression of various genes leads to the malignant progression of tumors, and the modification in DNA methylation can cause a change in gene expression. Increasing evidence has shown that abnormal expression of PCDH17 is found in many human cancers. However, its functional role in HCC remains unexplored. Herein, we found that PCDH17 was expressed at low levels in HCC tissues and cell lines. There is a significant correlation between low expression of PCDH17 and poor prognosis of HCC patients. Increased expression of PCDH17 significantly suppressed cell proliferation, migration and invasion in HCC. The low expression of PCDH17 was due to its high DNA methylation level, and changing the expression of DNMT3B significantly affect the DNA methylation level of PCDH17 and increase its protein expression. Furthermore, methylation of PCDH17 regulated by DNMT3B affects the malignant biological behavior of HCC through EMT. In conclusion, PCDH17 participates in malignant biological behavior of HCC and that DNMT3B plays an important role in the regulation of PCDH17 methylation, which affects the malignant progression of HCC.

microRNA-150 targets major epigenetic repressors and inhibits cell proliferation.

The Polycomb Repressive Complex (PRC) proteins, EZH2 and EZH1 regulate many biological processes by generating the repressive H3K27me3 modifications in the chromatin. However, the factors that regulate the EZH1/EZH2 functions are poorly studied. We identify that the 3'UTRs of EZH2 and EZH1 mRNAs contain the binding sites for the miRNA, miR-150. MicroRNA-150 (miR-150) controls numerous biological processes including cell proliferation, differentiation and pathogenesis of a variety of diseases including cancer. We find that miR-150 regulates the levels of EZH1 and EZH2 through various experimental investigations. Since EZH2 is known to form a repressive complex with other epigenetic repressors especially DNMT3A and DNMT3B, we investigated whether miR-150 also regulates the DNMT3A and DNMT3B levels. We report that miR-150 regulates DNMT3A and DNMT3B levels through direct and indirect mechanisms respectively. Since these epigenetic repressors promote cell proliferation, we investigated the effect of miR-150 perturbation on HEK293 cell proliferation. We found that miR-150 inhibits cell proliferation and induces S-phase arrest by increasing the levels of tumor suppressors and decreasing the cell cycle regulators. Collectively, our study shows that miR-150 act as a tumor suppressor by down-regulating the oncogenic major epigenetic repressors and controls cell proliferation.

Investigation of the association between goat DNMT3B gene polymorphism and growth traits.

The DNA methyltransferase 3 beta (DNMT3B) gene is key for DNA methylation and has been well recognized in regulating growth and development. A previous observation indicated that an 11-bp indel of DNMT3B affected the reproductive traits in goats, yet the effect of this polymorphism on body measurement traits in goats has not been reported. This study aims to investigate the associations between DNMT3B gene polymorphism and goat growth traits. We investigated this 11-bp indel in 2184 goats and three genotypes have been found in Shaanbei white cashmere goat (SBWC): insertion/insertion (II), deletion/deletion (DD) and insertion/deletion (ID). Only ID and DD genotypes were detected in Nubian goats and Guizhou heima goat (GZHM). The allele frequencies analyzed revealed that the 'D' allele frequencies were higher in all three goat breeds. Further association analysis demonstrated that this indel is markedly associated with the cannon circumference (CC) and cannon circumference index (CCI) of SBWC and cannon circumference (CC) of Nubian goats (p < .05). The CC and CCI are essential indicators to measure the growth status of goats. In summary, our study sheds some light on the potential impact of the 11-bp indel polymorphism of the DNMT3B gene on improving the growth traits in goats.