MiR-21-5p knockdown inhibits epithelial to mesenchymal transition in A549 lung adenocarcinoma cells by upregulating RhoB.

BACKGROUND: MiR-21-5p is a highly expressed microRNA that plays an important role in various cancer-promoting processes, including anchorage-independent growth, invasion, migration metastasis, and drug resistance in lung cancer. Studies indicate that miR-21-5p may contribute to these processes by promoting epithelial-mesenchymal transition (EMT). Ras homolog gene family member B (RhoB), a gene downregulated by miR-21-5p, has also been linked to EMT in lung cancer. However, the role of the miR-21-5p/RhoB axis in EMT regulation in lung adenocarcinoma remains unclear. In this study, we aimed to investigate the regulatory role of the miR-21-5p/RhoB axis in EMT and related in vitro functional characteristics such as migration, invasion, cisplatin resistance, and the formation of tumor spheroids. METHODS AND RESULTS: A549 cells were transfected with the miR-21-5p inhibitor, RhoB siRNA, and their corresponding negative controls. Wound healing, transwell invasion, Methyl thiazole tetrazolium (MTT), and sphere formation assays were also performed to evaluate the migration, invasion, cisplatin resistance, and anchorage-independent growth of A549 cells. RT-qPCR was used to determine the mRNA expression levels of EMT markers. MiR-21-5p knockdown inhibited migration, invasion, cisplatin resistance, and sphere formation while upregulating E-cadherin and downregulating Slug. Furthermore, RhoB silencing restored EMT and related in vitro functional characteristics in A549 cells. CONCLUSIONS: Knockdown of miR-21-5p inhibits EMT and related in vitro functional characteristics by upregulating RhoB, suggesting that miR-21-5p may promote EMT through downregulation of RhoB.

HMGA1 promotes the progression of esophageal squamous cell carcinoma by elevating TKT-mediated upregulation of pentose phosphate pathway.

Esophageal squamous cell carcinoma (ESCC) possesses a poor prognosis and treatment outcome. Dysregulated metabolism contributes to unrestricted growth of multiple cancers. However, abnormal metabolism, such as highly activated pentose phosphate pathway (PPP) in the progression of ESCC remains largely unknown. Herein, we report that high-mobility group AT-hook 1 (HMGA1), a structural transcriptional factor involved in chromatin remodeling, promoted the development of ESCC by upregulating the PPP. We found that HMGA1 was highly expressed in ESCC. Elevated HMGA1 promoted the malignant phenotype of ESCC cells. Conditional knockout of HMGA1 markedly reduced 4-nitroquinoline-1-oxide (4NQO)-induced esophageal tumorigenesis in mice. Through the metabolomic analysis and the validation assay, we found that HMGA1 upregulated the non-oxidative PPP. With the transcriptome sequencing, we identified that HMGA1 upregulated the expression of transketolase (TKT), which catalyzes the reversible reaction in non-oxidative PPP to exchange metabolites with glycolytic pathway. HMGA1 knockdown suppressed the PPP by downregulating TKT, resulting in the reduction of nucleotides in ESCC cells. Overexpression of HMGA1 upregulated PPP and promoted the survival of ESCC cells by activating TKT. We further characterized that HMGA1 promoted the transcription of TKT by interacting with and enhancing the binding of transcription factor SP1 to the promoter of TKT. Therapeutics targeting TKT with an inhibitor, oxythiamine, reduced HMGA1-induced ESCC cell proliferation and tumor growth. Together, in this study, we identified a new role of HMGA1 in ESCCs by upregulating TKT-mediated activation of PPP. Our results provided a new insight into the role of HMGA1/TKT/PPP in ESCC tumorigenesis and targeted therapy.

Uncovering the Interaction between TRAF1 and MAVS in the RIG-I Pathway to Enhance the Upregulation of IRF1/ISG15 during Classical Swine Fever Virus Infection.

Classical swine fever (CSF) is caused by the classical swine fever virus (CSFV), which poses a threat to swine production. The activation of host innate immunity through linker proteins such as tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) is crucial for the induction of the NF-κB pathway. Recent research has revealed the involvement of mitochondrial antiviral-signaling protein (MAVS) in the interaction with TRAF2, 3, 5, and 6 to activate both the NF-κB and IRF3 pathways. This study revealed that CSFV infection led to the upregulation of TRAF1 mRNA and protein levels; moreover, TRAF1 overexpression inhibited CSFV replication, while TRAF1 knockdown promoted replication, highlighting its importance in the host response to CSFV infection. Additionally, the expression of RIG-I, MAVS, TRAF1, IRF1, and ISG15 were detected in PK-15 cells infected with CSFV, revealing that TRAF1 plays a role in regulating IRF1 and ISG15 within the RIG-I pathway. Furthermore, Co-IP, GST pull-down, and IFA analyses demonstrated that TRAF1 interacted with MAVS and co-localized in the cytoplasm during CSFV infection. Ultimately, TRAF1 acted as a novel member of the TRAF family, bound to MAVS as a linker molecule, and functioned as a mediator downstream of MAVS in the RIG-I/MAVS pathway against CSFV replication.

Up-regulation of miR-126 via DNA methylation in hypoxia-preconditioned endothelial cells may contribute to hypoxic tolerance of neuronal cells.

BACKGROUND: Endothelial cells (ECs) can confer neuroprotection by secreting molecules. This study aimed to investigate whether DNA methylation contributes to the neuroprotective gene expression induced by hypoxia preconditioning (HPC) in ECs and to clarify that the secretion of molecules from HPC ECs may be one of the molecular mechanisms of neuroprotection. METHODS: Human microvascular endothelial cell-1 (HMEC-1) was cultured under normal conditions (C), hypoxia(H), and hypoxia preconditioning (HPC), followed by the isolation of culture medium (CM). SY5Y cell incubated with the isolated CM from HMEC-1 was exposed to oxygen-glucose deprivation (OGD). The DNA methyltransferases (DNMTs), global methylation level, miR-126 and its promotor DNA methylation level in HMEC-1 were measured. The cell viability and cell injury in SY5Y were detected. RESULTS: HPC decreased DNMTs level and global methylation level as well as increased miR-126 expression in HMEC-1. CM from HPC treated HMEC-1 also relieved SY5Y cell damage, while CM from HMEC-1 which over-expression of miR-126 can reduce injury in SY5Y under OGD condition. CONCLUSIONS: These findings indicate EC may secrete molecules, such as miR-126, to execute neuroprotection induced by HPC through regulating the expression of DNMTs.

APETALA2-like Floral Homeotic Protein Up-Regulating FaesAP1_2 Gene Involved in Floral Development in Long-Homostyle Common Buckwheat.

In the rosid species Arabidopsis thaliana, the AP2-type AP2 transcription factor (TF) is required for specifying the sepals and petals identities and confers a major A-function to antagonize the C-function in the outer floral whorls. In the asterid species Petunia, the AP2-type ROB TFs are required for perianth and pistil development, as well as repressing the B-function together with TOE-type TF BEN. In Long-homostyle (LH) Fagopyrum esculentum, VIGS-silencing showed that FaesAP2 is mainly involved in controlling filament and style length, but FaesTOE is mainly involved in regulating filament length and pollen grain development. Both FaesAP2 (AP2-type) and FaesTOE (TOE-type) are redundantly involved in style and/or filament length determination instead of perianth development. However, neither FaesAP2 nor FaesTOE could directly repress the B and/or C class genes in common buckwheat. Moreover, the FaesAP1_2 silenced flower showed tepal numbers, and filament length decreased obviously. Interestingly, yeast one-hybrid (Y1H) and dual-luciferase reporter (DR) further suggested that FaesTOE directly up-regulates FaesAP1_2 to be involved in filament length determination in LH common buckwheat. Moreover, the knockdown of FaesTOE expression could result in expression down-regulation of the directly target FaesAP1_2 in the FaesTOE-silenced LH plants. Our findings uncover a stamen development pathway in common buckwheat and offer deeper insight into the functional evolution of AP2 orthologs in the early-diverging core eudicots.

MCM8 promotes lung cancer progression through upregulating DNAJC10.

MCM8 is a helicase, which participates in DNA replication and tumorigenesis and is upregulated in many human cancers, including lung cancer (LC); however, the function of MCM8 in LC tumour progression is unclear. In this study, we found that MCM8 was expressed at high levels in LC cells and tissues. Further, MCM8 upregulation was associated with advanced tumour grade and lymph node metastasis, and indicated poor prognosis. Silencing of MCM8 suppressed cell growth and migration in vitro and in vivo, while ectopic MCM8 expression promoted cell cycle progression, as well as cell migration, proliferation, and apoptosis. Mechanistically, DNAJC10 was identified as a downstream target of MCM8, using gene array and CO-IP assays. DNAJC10 overexpression combatted the inhibitory activity of MCM8 knockdown on LC progression, while silencing DNAJC10 alleviated the oncogenic function of MCM8 overexpression. MCM8 expression was positively correlated with that of DNAJC10 in LC samples from The Cancer Genome Atlas database, and DNAJC10 upregulation was also associated with poor overall survival of patients with LC. This study indicated that MCM8/DNAJC10 axis plays an important role in in LC development, and maybe as a new potential therapeutic target or a diagnostic biomarker for treating patients with LC.

WTAP/IGF2BP3-mediated GBE1 expression accelerates the proliferation and enhances stemness in pancreatic cancer cells via upregulating c-Myc.

BACKGROUND: Pancreatic cancer (PC) is one of the most malignant cancers with highly aggressiveness and poor prognosis. N6-methyladenosine (m6A) have been indicated to be involved in PC development. Glucan Branching Enzyme 1 (GBE1) is mainly involved in cell glycogen metabolism. However, the function of GBE1 and Whether GBE1 occurs m6A modification in PC progression remains to be illustrated. METHODS: The clinical prognosis of GBE1 was analyzed through online platform. The expression of GBE1 was obtained from online platform and then verified in normal and PC cell lines. Lentivirus was used to generated GBE1 stable-overexpression or knockdown PC cells. Cell Counting Kit (CCK-8), colony formation assay, sphere formation assay and flow cytometry assay were conducted to analyze cell proliferation and stemness ability in vitro. Subcutaneous and orthotopic mouse models were used to verify the function of GBE1 in vivo. RNA immunoprecipitation (RIP) assay, RNA stability experiment and western blots were conducted to explore the molecular regulation of GBE1 in PC. RESULTS: GBE1 was significantly upregulated in PC and associated with poor prognosis of PC patients. Functionally, GBE1 overexpression facilitated PC cell proliferation and stemness-like properties, while knockdown of GBE1 attenuated the malignancy of PC cells. Importantly, we found the m6A modification of GBE1 RNA, and WTAP and IGF2BP3 was revealed as the m6A regulators to increase GBE1 mRNA stability and expression. Furthermore, c-Myc was discovered as a downstream gene of GBE1 and functional rescue experiments showed that overexpression of c-Myc could rescue GBE1 knockdown-induced PC cell growth inhibition. CONCLUSIONS: Our study uncovered the oncogenic role of GBE1/c-Myc axis in PC progression and revealed WTAP/IGF2BP3-mediated m6A modification of GBE1, which highlight the potential application of GBE1 in the targeted therapy of PC.

Upregulation of circ-IGF1R increased therapeutic effect of hypoxia-pretreated ADSC-derived extracellular vesicle by regulating miR-503-5p/HK2/VEGFA axis.

Diabetes mellitus is a major cause of blindness and chronic ulcers in the working-age population worldwide. Wound healing is deeply dependent on neovascularization to restore blood flow. Former research has found that differentially expressed circular RNAs (circRNAs) are associated with hyperglycaemia-induced endothelial cell damage, and hypoxia-pretreated adipose-derived stem cells (ADSCs)-extracellular vesicle (HEV) transplants have a more therapeutic effect to enhance wound healing in diabetic mice by delivery circRNA. The current investigation employed high-throughput sequencing to identify circRNAs that are abnormally expressed between EV and HEV. The regulatory mechanism and predicted targets of one differentially expressed circRNA, circ-IGF1R, were investigated utilizing bioinformatics analyses, luciferase reporter assays, angiogenic differentiation assays, flow cytometric apoptosis analysis and RT-qPCR. Circ-IGF1R expression increased in HEV, and downregulation of circ-IGF1R suppressed and reversed the promotion effect of HEV on angiogenesis in ulcerated tissue. Bioinformatics analyses and luciferase reporter assays confirmed that miR-503-5p was the downstream target of circ-IGF1R, and inhibiting miR-503-5p restored the promotion effect of HEV on angiogenesis after circ-IGF1R silence. The study also found that miR-503-5p can interact with 3'-UTR of both HK2 and VEGFA. Overexpression of HK2 or VEGFA restored the promotion effect of HExo on angiogenesis after circ-IGF1R silence. Overexpression miR-503-5p or silence HK2/VEGFA reversed the protective effect of circ-IGF1R to MLMECs angiogenic differentiation. Overexpression of circ-IGF1R increased the protective effect of HEV on the promotion of wound healing in mice with diabetes. Circ-IGF1R promotes HIF-1α expression through miR-503-5p sponging. Our data demonstrate that circ-IGF1R overexpression EVs from ADSCs suppress high glucose-induced endothelial cell damage by regulating miR-503-5p/HK2/VEGFA axis.

Increased level of GATA3-AS1 long non-coding RNA is correlated with the upregulation of GATA3 and IL-4 genes in multiple sclerosis patients.

BACKGROUND: Long non-coding RNAs (lncRNAs) play various roles in gene regulation. GATA3 antisense RNA 1 (GATA3-AS1) is an lncRNA gene neighboring GATA binding protein 3 (GATA3). The current study aims to quantitatively compare the levels of the expression of GATA3-AS1, GATA3, and Interleukin-4 (IL-4) in peripheral blood mononuclear cells (PBMC) samples of MS patients and healthy individuals under the hypothesis of regulation of GATA3 and IL-4 expression orchestrated by GATA3-AS1. METHODS AND RESULTS: In this case-control study, the GATA3-AS1, GATA3 and IL-4 expression profiles were assessed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Also, we assessed the IL-4 levels in the serum. The median fold changes in MS patients vs. controls were (4.39 ± 0.38 vs. 2.44 ± 0.20) for GATA3-AS1, (5.22 ± 0.51 vs. 2.86 ± 0.30) for GATA3, and (6.16 ± 0.52 vs. 3.57 ± 0.38) for IL-4, (P < 0.001). Furthermore, the mean serum levels of IL-4 were 30.85 ± 1.53 pg/ml in MS patients and 11.15 ± 4.23 pg/ml in healthy controls (P < 0.001). ROC curve analysis showed that the level of GATA3-AS1 might serve as a biomarker for diagnosing MS patients with the area under the curve (AUC = 0.918, P < 0.0001). Based on our results, this GATA3-AS1/GATA3/IL-4 pathway may increase IL-4 expression in MS patients. CONCLUSIONS: Our results indicate a probably regulatory function for GATA3-AS1and the levels of GATA3-AS1 in blood could be important biomarkers for MS diagnosis. To confirm and be more certain of these results, it is necessary to study neuromyelitis optica (NMO) and asthma patients in future studies.

Upregulation of Siglec-6 induces mitochondrial dysfunction by promoting GPR20 expression in early-onset preeclampsia.

BACKGROUND: Preeclampsia, especially early-onset preeclampsia (EO-PE), is a pregnancy complication that has serious consequences for the health of both the mother and the fetus. Although abnormal placentation due to mitochondrial dysfunction is speculated to contribute to the development of EO-PE, the underlying mechanisms have yet to be fully elucidated. METHODS: The expression and localization of Siglec-6 in the placenta from normal pregnancies, preterm birth and EO-PE patients were examined by RT-qPCR, Western blot and IHC. Transwell assays were performed to evaluate the effect of Siglec-6 on trophoblast cell migration and invasion. Seahorse experiments were conducted to assess the impact of disrupting Siglec-6 expression on mitochondrial function. Co-IP assay was used to examine the interaction of Siglec-6 with SHP1/SHP2. RNA-seq was employed to investigate the mechanism by which Siglec-6 inhibits mitochondrial function in trophoblast cells. RESULTS: The expression of Siglec-6 in extravillous trophoblasts is increased in placental tissues from EO-PE patients. Siglec-6 inhibits trophoblast cell migration and invasion and impairs mitochondrial function. Mechanismly, Siglec-6 inhibits the activation of NF-κB by recruiting SHP1/SHP2, leading to increased expression of GPR20. Notably, the importance of GPR20 function downstream of Siglec-6 in trophoblasts is supported by the observation that GPR20 downregulation rescues defects caused by Siglec-6 overexpression. Finally, overexpression of Siglec-6 in the placenta induces a preeclampsia-like phenotype in a pregnant mouse model. CONCLUSIONS: This study indicates that the regulatory pathway Siglec-6/GPR20 has a crucial role in regulating trophoblast mitochondrial function, and we suggest that Siglec-6 and GPR20 could serve as potential markers and targets for the clinical diagnosis and therapy of EO-PE.

Hypomethylation-associated Sox11 upregulation promotes oncogenesis via the PI3K/AKT pathway in OLP-associated OSCC.

Oral lichen planus (OLP) is a particularly prevalent oral disorder with the potential to progress to oral squamous cell carcinoma (OSCC). SRY-box transcription factor 11 (Sox11) has been reported to serve as a prognostic marker for various cancers. However, the role and mechanism of Sox11 in OLP-related OSCC are unknown. Our results indicated that Sox11 was highly expressed, and that Sox11 promoter methylation was significantly reduced in OLP-associated OSCC tissues. High Sox11 expression and Sox11 promoter hypomethylation indicate a poor patient prognosis. According to in vivo and in vitro experiments, the knockdown of Sox11 inhibited proliferation, invasion, and migration while driving its apoptotic death in OSSC cells; Sox11 overexpression exerted the opposite effect as Sox11 knockdown. Mechanistically, knockdown of Sox11 inhibited PI3K/AKT and glycolysis pathway, and overexpression of Sox11 enhanced the PI3K/AKT and glycolysis pathways in OSCC cells. In addition, we demonstrated that Sox11 overexpression accelerated the progression of OSCC, at least in part by promoting PI3K/AKT pathway activation. In conclusion, our data indicated that the DNA hypomethylation-associated upregulation of Sox11 could promote oncogenic transformation via the PI3K/AKT pathway in OLP-associated OSCC. Therefore, Sox11 might be a reliable biomarker for predicting the progression of precancerous oral tissues.

Epigenetically associated IGF2BP3 upregulation promotes cell proliferation by regulating E2F1 expression in hepatocellular carcinoma.

RNA-binding proteins (RBPs) are a class of proteins that primarily function by interacting with different types of RNAs and play a critical role in regulating the transcription and translation of cancer-related genes. However, their role in the progression of hepatocellular carcinoma (HCC) remains unclear. In this study, we analyzed RNA sequencing data and the corresponding clinical information of patients with HCC to screen for prognostic RBPs. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was identified as an independent prognostic factor for liver cancer. It is upregulated in HCC and is associated with a poor prognosis. Elevated IGF2BP3 expression was validated via immunohistochemical analysis using a tissue microarray of patients with HCC. IGF2BP3 knockdown inhibited the proliferation of Hep3B and HepG2 cells, whereas IGF2BP3 overexpression promoted the expansion of HuH-7 and MHCC97H cells. Mechanistically, IGF2BP3 modulates cell proliferation by regulating E2F1 expression. DNA hypomethylation of the IGF2BP3 gene may increase the expression of IGF2BP3, thereby enhancing cell proliferation in HCC. Therefore, IGF2BP3 may act as a novel prognostic biomarker and a potential therapeutic target for HCC.

TMB Signature-Related RCAN2 Promotes Apoptosis by Upregulating EHF/DR5 Pathway in Hepatocellular Carcinoma.

BACKGROUND: The tumour mutation burden (TMB) is a valuable indicator of the accumulation of somatic mutations, and is thought to be associated with the biological behaviour and prognosis of tumours. However, the related genetic mechanism for these association is still unclear. The aim of the present study was to identify the key gene(s) associated with TMB in hepatocellular carcinoma (HCC) and to investigate its biological functions, downstream transcription factors, and mechanism of action. METHODS: Patients in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database were classified according to TMB signature-related genes. Key genes related to the TMB signature and tumour prognosis were identified. Immunohistochemistry and Quantitative Real-Time Polymerase Chain Reaction (qPCR) were then used to assess gene expression in clinical HCC tissues and HCC cells. Cells with altered gene expression were evaluated for the effect on cell proliferation and apoptosis, both in vitro and in vivo. Three independent databases and cell sequencing data were used to identify the mechanisms involved and the downstream transcription factors. The mechanism was also studied by altering the expression of downstream transcription factors in vitro. RESULT: The integrated cluster (IC) 2 group, characterized by 99 TMB signature-related genes, showed a significant different TMB score compared to the IC1 group (p < 0.001), as well as more favourable tumour prognosis (p = 0.031). We identified five key prognostic genes that were differentially expressed between IC2 and IC1 and were associated with overall survival. The expression of one of these key prognostic genes, RCAN2, was negatively correlated with TMB in 18 out of 33 tumour types examined. A high level of RCAN2 was correlated with better overall survival in HCC (p = 0.0009). Overexpression of RCAN2 enhanced apoptosis in vitro and in vivo, whereas knockdown of RCAN2 attenuated apoptosis. The mechanism by which RCAN2 promotes apoptosis may involve upregulation of the expression of ETS homologous factor (EHF) and of death receptor 5 (DR5). CONCLUSIONS: Downregulation of RCAN2 expression was found to correlate with elevated TMB in multiple cancer types. RCAN2 was also found to be a biomarker of HCC prognosis, and to promote the apoptosis of HCC cells through the EHF/DR5 pathway. These findings provide a new perspective on systemic treatment for advanced HCC with a high TMB.

CTSD upregulation as a key driver of spinal ligament abnormalities in spinal stenosis.

Spinal stenosis (SS) is frequently caused by spinal ligament abnormalities, such as ossification and hypertrophy, which narrow the spinal canal and compress the spinal cord or nerve roots, leading to myelopathy or sciatic symptoms; however, the underlying pathological mechanism is poorly understood, hampering the development of effective nonsurgical treatments. Our study aims to investigate the role of co-expression hub genes in patients with spinal ligament ossification and hypertrophy. To achieve this, we conducted an integrated analysis by combining RNA-seq data of ossification of the posterior longitudinal ligament (OPLL) and microarray profiles of hypertrophy of the ligamentum flavum (HLF), consistently pinpointing CTSD as an upregulated hub gene in both OPLL and HLF. Subsequent RT-qPCR and IHC assessments confirmed the heightened expression of CTSD in human OPLL, ossification of the ligamentum flavum (OLF), and HLF samples. We observed an increase in CTSD expression in human PLL and LF primary cells during osteogenic differentiation, as indicated by western blotting (WB). To assess CTSD's impact on osteogenic differentiation, we manipulated its expression levels in human PLL and LF primary cells using siRNAs and lentivirus, as demonstrated by WB, ALP staining, and ARS. Our findings showed that suppressing CTSD hindered the osteogenic differentiation potential of PLL and LF cells, while overexpressing CTSD activated osteogenic differentiation. These findings identify CTSD as a potential therapeutic target for treating spinal stenosis associated with spinal ligament abnormalities.

TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.

SETDB1 deletion causes DNA demethylation and upregulation of multiple zinc-finger genes.

BACKGROUND: SETDB1 (SET domain bifurcated-1) is a histone H3-lysine 9 (H3K9)-specific methyltransferase that mediates heterochromatin formation and repression of target genes. Despite the assumed functional link between DNA methylation and SETDB1-mediated H3K9 trimethylations, several studies have shown that SETDB1 operates autonomously of DNA methylation in a region- and cell-specific manner. This study analyzes SETDB1-null HAP1 cells through a linked methylome and transcriptome analysis, intending to explore genes controlled by SETDB1-involved DNA methylation. METHODS AND RESULTS: We investigated SETDB1-mediated regulation of DNA methylation and gene transcription in human HAP1 cells using reduced-representation bisulfite sequencing (RRBS) and RNA sequencing. While two-thirds of differentially methylated CpGs (DMCs) in genic regions were hypomethylated in SETDB1-null cells, we detected a plethora of C2H2-type zinc-finger protein genes (C2H2-ZFP, 223 of 749) among the DMC-associated genes. Most C2H2-ZFPs with DMCs in their promoters were found hypomethylated in SETDB1-KO cells, while other non-ZFP genes with promoter DMCs were not. These C2H2-ZFPs with DMCs in their promoters were significantly upregulated in SETDB1-KO cells. Similarly, C2H2-ZFP genes were upregulated in SETDB1-null 293T cells, suggesting that SETDB1's function in ZFP gene repression is widespread. There are several C2H2-ZFP gene clusters on chromosome 19, which were selectively hypomethylated in SETDB1-KO cells. CONCLUSIONS: SETDB1 collectively and specifically represses a substantial fraction of the C2H2-ZFP gene family. Through the en-bloc silencing of a set of ZFP genes, SETDB1 may help establish a panel of ZFP proteins that are expressed cell-type specifically and thereby can serve as signature proteins for cellular identity.

Circ_0012152 Accelerates Acute Myeloid Leukemia Progression through the miR-652-3p/SOX4 Axis.

OBJECTIVE: Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by abnormal myeloid blast expansion. Recent studies have demonstrated that circular RNAs play a role in AML pathogenesis. In this study, we aimed to investigate the clinical significance of circ_0012152 in AML and elucidate its underlying molecular mechanism in the pathogenesis of this condition. METHODS: Circ_0012152 expression was detected by quantitative real-time polymerase chain reaction in samples obtained from 247 patients with AML and 40 healthy controls. A systematic analysis of clinical characteristics and prognostic factors was also conducted. Cell growth was assessed using the Cell Counting Kit-8 (CCK-8) assay, and apoptosis and cell cycle progression were evaluated by flow cytometry. Moreover, RNA pull-down was performed to identify target microRNAs, and transcriptome RNA sequencing and bioinformatics analyses were utilized to identify downstream mRNA targets. RESULTS: Circ_0012152 was significantly upregulated in samples from patients with AML and served as an independent adverse prognostic factor for overall survival (OS) (hazard ratio: 2.357; 95% confidence interval 1.258-4.415). The circ_0012152 knockdown reduced cell growth, increased apoptosis, and inhibited cell cycle progression in AML cell lines. RNA pull-down and sequencing identified miR-652-3p as a target microRNA of circ_0012152. Cell growth inhibition by circ_0012152 knockdown was significantly relieved by miR-652-3p inhibitors. We suggested that miR-652-3p targeted SOX4, as the decrease in SOX4 expression resulting from circ_0012152 knockdown was upregulated by miR-652-3p inhibitors in AML cells. CONCLUSION: Circ_0012152 is an independent poor prognostic factor for OS in AML, and it promotes AML cell growth by upregulating SOX4 through miR-652-3p.

Pseudogene OCT4-pg5 upregulates OCT4B expression to promote bladder cancer progression by competing with miR-145-5p.

Bladder cancer (BC) is one of the most common malignant neoplasms worldwide. Competing endogenous RNA (ceRNA) networks may identify potential biomarkers associated with the progression and prognosis of BC. The OCT4-pg5/miR-145-5p/OCT4B ceRNA network was found to be related to the progression and prognosis of BC. OCT4-pg5 expression was significantly higher in BC cell lines than in normal bladder cells, with OCT4-pg5 expression correlating with OCT4B expression and advanced tumor grade. Overexpression of OCT4-pg5 and OCT4B promoted the proliferation and invasion of BC cells, whereas miR-145-5p suppressed these activities. The 3' untranslated region (3'UTR) of OCT4-pg5 competed for miR-145-5p, thereby increasing OCT4B expression. In addition, OCT4-pg5 promoted epithelial-mesenchymal transition (EMT) by activating the Wnt/ß-catenin pathway and upregulating the expression of matrix metalloproteinases (MMPs) 2 and 9 as well as the transcription factors zinc finger E-box binding homeobox (ZEB) 1 and 2. Elevated expression of OCT4-pg5 and OCT4B reduced the sensitivity of BC cells to cisplatin by reducing apoptosis and increasing the proportion of cells in G1. The OCT4-pg5/miR-145-5p/OCT4B axis promotes the progression of BC by inducing EMT via the Wnt/ß-catenin pathway and enhances cisplatin resistance. This axis may represent a therapeutic target in patients with BC.

Circ_0050444 represses esophageal squamous cell carcinoma progression through sponging miR-486-3p to upregulate C10orf91.

Esophageal squamous cell carcinoma (ESCC) ranks as the fourth leading cause of tumor-related deaths in China. Circ_0050444 has been revealed to be downregulated in ESCC tissues, however, its function and molecular mechanism underlying ESCC progression is unknown. Therefore, we attempted to clarify the functional role and molecular mechanism of circ_0050444 underlying ESCC progression. RT-qPCR and RNase R digestion assays were used to evaluate circ_0050444 expression and stability characteristics in ESCC cells. Gain-of-function assays were conducted to clarify circ_0050444 role in ESCC cell malignant behaviors. Bioinformatics and mechanism experiments were performed to assess the relationship between circ_0050444 or C10orf91 and miR-486-3p in ESCC cells. Rescue assays were conducted to evaluate the regulatory function of the circ_0050444-miR-486-3p-C10orf91 axis in ESCC cellular processes. Circ_0050444 expression was found to be downregulated both in ESCC patient tissues and cell lines. Functionally, circ_0050444 overexpression repressed ESCC cell proliferative, migratory, and invasive capabilities in cultured cells. Mechanistically, circ_0050444 was found to be competitively bound with miR-486-3p to upregulate C10orf91 in ESCC cells. Moreover, the impact of circ_0050444 elevation on ESCC cell proliferation, migration, and invasion was countervailed by C10orf91 silencing. Circ_0050444 presents downregulation and functions as a tumor suppressor in ESCC progression. Circ_0050444 suppresses ESCC proliferation, migration, and invasion through sponging miR-486-3p to upregulate C10orf91, providing a potential new direction for seeking therapeutic plans for ESCC.

KCNK1 promotes proliferation and metastasis of breast cancer cells by activating lactate dehydrogenase A (LDHA) and up-regulating H3K18 lactylation.

Breast cancer is the most prevalent malignancy and the most significant contributor to mortality in female oncology patients. Potassium Two Pore Domain Channel Subfamily K Member 1 (KCNK1) is differentially expressed in a variety of tumors, but the mechanism of its function in breast cancer is unknown. In this study, we found for the first time that KCNK1 was significantly up-regulated in human breast cancer and was correlated with poor prognosis in breast cancer patients. KCNK1 promoted breast cancer proliferation, invasion, and metastasis in vitro and vivo. Further studies unexpectedly revealed that KCNK1 increased the glycolysis and lactate production in breast cancer cells by binding to and activating lactate dehydrogenase A (LDHA), which promoted histones lysine lactylation to induce the expression of a series of downstream genes and LDHA itself. Notably, increased expression of LDHA served as a vicious positive feedback to reduce tumor cell stiffness and adhesion, which eventually resulted in the proliferation, invasion, and metastasis of breast cancer. In conclusion, our results suggest that KCNK1 may serve as a potential breast cancer biomarker, and deeper insight into the cancer-promoting mechanism of KCNK1 may uncover a novel therapeutic target for breast cancer treatment.