Ethynyl Deoxyuridine Research Articles

CircPTK2 promotes cell viability, cell cycle process, and glycolysis and inhibits cell apoptosis in acute myeloid leukemia by regulating miR-582-3p/ALG3 axis

ABSTRACT Background Circular RNA (circRNA) regulates the pathogenesis of acute myeloid leukemia (AML). However, the mechanism of circRNA protein tyrosine kinase 2 (circPTK2) in AML remains unclear. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) assay was adopted for circPTK2, miR-582-3p and alpha-1,3-mannosyltransferase (ALG3) mRNA levels. 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and 5’-ethynyl-2’-deoxyuridine (EdU) assay were conducted for cell proliferation. Flow cytometry analysis was employed for cell apoptosis and cell cycle process. The glycolysis level was estimated by specific commercial kits. Western blot assay was utilized for protein levels. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the interaction between miR-582-3p and circPTK2 or ALG3. Results CircPTK2 level was enhanced in AML peripheral blood samples and cells. CircPTK2 knockdown restrained AML cell proliferation and glycolysis and promoted cell apoptosis and cell cycle arrest. Mechanically, circPTK2 functioned as the sponge for miR-582-3p to positively ALG3 expression in AML cells. Moreover, miR-582-3p inhibition ameliorated the impacts of circPTK2 knockdown on AML cell processes. MiR-582-3p overexpression regulated cell phenotypes by targeting ALG3. Conclusion CircPTK2 contributed to AML cell malignant behaviors by modulation of miR-582-3p/ALG3 axis, which might provide a potential target for AML therapy.

Circ_0000011 promotes cerebral ischemia/reperfusion injury via miR-27a-3p-dependent regulation of NRIP1.

Cerebral ischemia/reperfusion (I/R) can result in brain function impairments. Circular RNAs (circRNAs) have emerged as vital regulators in cerebral I/R injury. However, the functions of mmu_circ_0000011 in cerebral I/R injury are still unclear. Thus, in this study, we aimed to explore the effect of mmu_circ_0000011 on cerebral I/R injury. Oxygen-glucose deprivation and reperfusion (OGD/R)-induced HT-22 cells were used to mimic the condition of cerebral I/R injury in vitro. Cell Counting Kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, 5'-ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry analysis were utilized to assess cell viability, LDH release, proliferation and apoptosis, respectively. qRT-PCR and western blot were performed to determined the levels of circ_0000011, miR-27a-3p and NRIP1. Dual-luciferase reporter assay and RNA pull-down assay were utilized to analyze the targeting relation of circ_0000011, miR-27a-3p and NRIP1. OGD/R treatment inhibited HT-22 cell viability and promoted LDH release, cell apoptosis and inflammation. Circ_0000011 level was increased in OGD/R-induced HT-22 cells. Silencing of circ_0000011 promoted cell proliferation and inhibited LDH release, apoptosis and inflammation in OGD/R-treated HT-22 cells. For mechanism analysis, circ_0000011 was demonstrated to sponge miR-27a-3p, which directly targeted NRIP1. MiR-27a-3p inhibition or NRIP1 overexpression ameliorated the impacts of circ_0000011 silencing on cell proliferation, LDH release, apoptosis and inflammation in OGD/R-treated HT-22 cells. Circ_0000011 promotes OGD/R-induced HT-22 cell impairments by elevating NRIP1 through sponging miR-27a-3p.

Nuclear morphology is a deep learning biomarker of cellular senescence

Cellular senescence is an important factor in aging and many age-related diseases, but understanding its role in health is challenging due to the lack of exclusive or universal markers. Using neural networks, we predict senescence from the nuclear morphology of human fibroblasts with up to 95% accuracy, and investigate murine astrocytes, murine neurons, and fibroblasts with premature aging in culture. After generalizing our approach, the predictor recognizes higher rates of senescence in p21-positive and ethynyl-2’-deoxyuridine (EdU)-negative nuclei in tissues and shows an increasing rate of senescent cells with age in H&E-stained murine liver tissue and human dermal biopsies. Evaluating medical records reveals that higher rates of senescent cells correspond to decreased rates of malignant neoplasms and increased rates of osteoporosis, osteoarthritis, hypertension and cerebral infarction. In sum, we show that morphological alterations of the nucleus can serve as a deep learning predictor of senescence that is applicable across tissues and species and is associated with health outcomes in humans.

Circ_0018168 inhibits the proliferation and osteogenic differentiation of fibroblasts in ankylosing spondylitis via regulating miR-330-3p/DKK1 axis.

BackgroundCircular RNAs (circRNAs) play a crucial regulatory role in human diseases. However, the roles of circRNAs in ankylosing spondylitis (AS) are barely known. In this study, the functions of circ_0018168 in AS were investigated.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and western blot assay were used for circ_0018168, microRNA-330-3p (miR-330-3p), dickkopf-1 (DKK1), alkaline phosphatase (ALP), osteocalcin (OCN), Runt-related transcription factor 2 (Runx2) levels. Cell Counting Kit-8 (CCK-8) assay and 5′-ethynyl-2′-deoxyuridine (EdU) assay were conducted to analyze cell proliferation ability. Flow cytometry analysis was manipulated for cell cycle process. ALP activity was examined with a commercial kit. RNA immunoprecipitation (RIP) assay, RNA pull-down assay and dual-luciferase reporter assay were used to analyze the relationships of circ_0018168, miR-330-3p and DKK1.ResultsCirc_0018168 and DKK1 levels were lowly expressed in AS hip capsule specimens. Circ_0018168 overexpression repressed cell proliferation, cell cycle process as well as reduced ALP activity and ALP, OCN and Runx2 protein levels in AS fibroblasts. DKK1 silencing ameliorated the impact of circ_0018168 on AS progression. In addition, circ_0018168 served as the sponge for miR-330-3p, which could target DKK1. MiR-330-3p inhibition suppressed the proliferation, cell cycle and osteogenic differentiation in AS fibroblasts, but DKK1 silencing reversed the impacts. Besides, the effect of circ_0018168 on AS development was abolished by miR-330-3p upregulation.ConclusionCirc_0018168 overexpression restrained fibroblast proliferation and osteogenic differentiation in AS by elevating DKK1 through adsorbing miR-330-3p.

Circ_FURIN promotes trophoblast cell proliferation, migration and invasion in preeclampsia by regulating miR-34a-5p and TFAP2A.

Emerging evidence has shown that circular RNAs (circRNAs) play vital roles in the progression of diverse human diseases. However, the functions of circRNAs in preeclampsia (PE) are largely unknown. In this study, we aimed to explore the functions of the circRNA furin, paired basic amino acid cleaving enzyme (circ_FURIN) in PE development. qRT-PCR and western blot analyses were conducted to determine the levels of circ_FURIN, miR-34a-5p and transcription factor AP-2 alpha (TFAP2A). A Cell Counting Kit-8 (CCK-8) assay and a 5'-ethynyl-2'-deoxyuridine (EdU) incorporation assay were utilized to evaluate the cell proliferation ability. Transwell assays were adopted to estimate cell migration and invasion. A dual-luciferase reporter assay and an RNA pulldown assay were utilized to analyze the relationships among circ_FURIN, miR-34a-5p and TFAP2A. It was found that circ_FURIN was downregulated in PE placental tissues and hypoxia-treated placental trophoblast cells. Overexpression of circ_FURIN promoted trophoblast cell proliferation, migration and invasion under hypoxic conditions. Circ_FURIN functioned as the sponge for miR-34a-5p. MiR-34a-5p overexpression abrogated the effects of circ_FURIN on the proliferation, migration and invasion of trophoblast cells under hypoxic conditions. In addition, TFAP2A was demonstrated to be the target gene of miR-34a-5p. TFAP2A silencing ameliorated the promotive effects of miR-34a-5p inhibition on trophoblast cell proliferation, migration and invasion under hypoxic conditions. In conclusion, circ_FURIN enhanced trophoblast cell proliferation, migration and invasion under hypoxic conditions by elevating TFAP2A expression through sponging miR-34a-5p.

CircPCNX Promotes PDGF-BB-Induced Proliferation and Migration of Human Aortic Vascular Smooth Muscle Cells Through Regulating miR-1278/DNMT1 Axis.

Human aortic vascular smooth muscle cells (HA-VSMCs) play vital roles in the pathogenesis of vascular diseases. Circular RNAs (circRNAs) have been reported to regulate the biological functions of HA-VSMCs. In this study, the functions of circRNA pecanex homolog (circPCNX) in platelet-derived growth factor-BB (PDGF-BB)-induced HA-VSMCs were investigated. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine the expression of circPCNX, DNA methyltransferase 1 (DNMT1), and microRNA-1278 (miR-1278). 5'-Ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry analysis, wound healing assay, and transwell assay were used to examine cell proliferation, cell cycle, and migration. Western blot assay was utilized to measure protein levels. RNA immunoprecipitation (RIP) assay, RNA pull down assay, and dual-luciferase reporter assay were adopted to analyze the relationships among circPCNX, miR-1278, and DNMT1. CircPCNX was upregulated in PDGF-BB-treated HA-VSMCs in a dose- or time-dependent manner. CircPCNX knockdown alleviated PDGF-BB-induced cell proliferation, cell cycle progression, and migration in HA-VSMCs. CircPCNX knockdown could reverse PDGF-BB-induced HA-VSMC progression by regulating DNMT1. Moreover, circPCNX was identified to regulate DNMT1 expression by sponging miR-1278. Inhibition of miR-1278 reversed circPCNX knockdown-mediated effects on cell proliferation and migration in PDGF-BB-induced HA-VSMCs. MiR-1278 overexpression suppressed PDGF-BB-stimulated HA-VSMC proliferation and migration by targeting DNMT1. CircPCNX promoted PDGF-BB-induced HA-VSMC proliferation and migration by elevating DNMT1 expression through sponging miR-1278.

Exercise Attenuates Hindpaw Hypersensitivity, Depression-like Behaviors, and Morphological Changes in the Hippocampus of Mice Exposed to Early Life Stress

Early life stress (ELS) is associated with the development of chronic pain and mood disorders. Exposure to ELS has been correlated with suppressed hippocampal neurogenesis across the lifespan. Conversely, exercise promotes hippocampal neurogenesis, as well as prevents pain-like behaviors in ELS mice. Here, we are using an established mouse model of ELS, neonatal maternal separation (NMS), to test the hypothesis that increasing physical activity can attenuate ELS-induced pain- and depression-like behaviors and increase hippocampal neurogenesis. NMS was performed by separating whole litters from the dam for 3h/day from postnatal day 1 (P1) to P21. Mice were weaned and pair-housed with same-sex littermates on P22 and remained in sedentary caging (-Sed) or were given running wheels (-Ex) at 4 weeks of age. Mice underwent monthly hindpaw von Frey and Hargreaves testing for 4 months and were subsequently tested for sucrose preference and nest building. Ethynyldeoxyuridine (EdU) was used to quantify hippocampal neurogenesis at 5 months of age. A significant impact of NMS and exercise was observed on mechanical sensitivity in female mice, such that NMS-Ex mice had higher thresholds compared to NMS-Sed and naive-Ex mice. Male NMS-Sed mice had significantly lower withdrawal thresholds compared to naive-Sed mice, only at the end of the study. A significant impact of exercise and an NMS/Ex interaction was observed on female thermal latency with naive-Ex mice having lower withdrawal latencies. Male NMS-Ex mice had significantly lower sucrose preference compared to NMS-Sed and naive-Ex mice. Exercise significantly increased nest construction quality in female mice, with NMS-Sed mice having lower scores than naive-Sed or NMS-Ex mice. Finally, exercise increased the number of EdU-immuno-positive cells in the hippocampus of NMS mice. Taken together, our results suggest that increased voluntary physical activity can prevent the development of ELS-associated outcomes, including hypersensitivity, depression-like behaviors, and decreased hippocampal neurogenesis. This work was supported by RO1DK099611 (JAC), RO1DK103872 (JAC), T32HD057850 (BML), COBRE grant P20GM104936, IDeA grant P20GM103418, core support from IDDRC grant P30HD002528.

CircHIPK3 alleviates inflammatory response and neuronal apoptosis via regulating miR-382-5p/DUSP1 axis in spinal cord injury

BackgroundSpinal cord injury (SCI) is one of the serious neurological diseases with high morbidity which may be treated with hematopoietic stem cell (HSC) transplants. Circular RNAs (circRNAs) play vital roles in SCI. The study aimed to reveal the function and mechanism of circRNA homeodomain interacting protein kinase 3 (HIPK3) in SCI. MethodsSCI model in vitro was established by treating neuronal cells AGE1.HN with oxygen-glucose deprivation (OGD) and CoCl2. The levels of circHIPK3, miR-382-5p and dual specificity phosphatase 1 (DUSP1) were examined using quantitative real-time PCR (qRT-PCR) or western blot assay. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors (IL-6 and TNF-α). Cell proliferation and apoptosis were evaluated by 5′-ethynyl-2′-deoxyuridine (EdU) assay and flow cytometry. Caspase-3 Colorimetric Assay Kit was used to detect aaspase-3 activity. The interactions among circHIPK3, miR-382-5p and DUSP1 were confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. ResultsCircHIPK3 and DUSP1 were down-regulated, while miR-382-5p was up-regulated in OGD-induced AGE1.HN cells. Overexpression of circHIPK3 suppressed inflammatory response and cell apoptosis and promoted proliferation in OGD-induced AGE1.HN cells by sponging miR-382-5p. CircHIPK3 regulated DUSP1 expression by targeting miR-382-5p. MiR-382-5p inhibition hindered inflammatory response of IL-6 and TNF-α and neuronal apoptosis and promoted apoptosis via targeting DUSP1. ConclusionCircHIPK3 overexpression alleviated OGD-induced AGE1.HN cell inflammatory response and neuronal apoptosis via regulating miR-382-5p/DUSP1 axis, indicating that circHIPK3 might be a promising therapeutic target for SCI.

CircATIC inhibits esophageal carcinoma progression and promotes radiosensitivity by elevating RHCG through sponging miR-10-3p.

BackgroundCircular RNAs (circRNAs) are implicated in the progression and radiosensitivity of human cancers, including esophageal carcinoma (ESCA). In this study, we aimed to explore the functions of circRNA 5‐aminoimidazole‐4‐carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (circATIC) in ESCA progression.MethodsCircATIC expression, miR‐10b‐3p and Rh family C glycoprotein (RHCG) were examined via quantitative real‐time polymerase chain reaction (qRT‐PCR), western blot assay or immunohistochemistry (IHC) assay. 5′‐ethynyl‐2′‐deoxyuridine (EdU), wound‐healing, transwell, and cell colony formation assays and flow cytometry analysis were conducted to evaluate cell proliferation, migration, invasion, radiosensitivity and apoptosis, respectively. Dual‐luciferase reporter assay and RNA pulldown assay were conducted to analyze the relationships among circATIC, miR‐10b‐3p and RHCG. A murine xenograft model assay was performed to explore the functions of circATIC in tumor formation and radiosensitivity in vivo.ResultsCircATIC was decreased in ESCA. CircATIC overexpression suppressed cell proliferation, migration and invasion and promoted radiosensitivity and apoptosis in ESCA cells in vitro and repressed tumor formation and radioresistance in vivo. Functionally, circATIC served as the sponge for miR‐10b‐3p, which directly targeted RHCG. MiR‐10b‐3p elevation reversed circATIC‐mediated effect on ESCA cell progression. Moreover, miR‐10b‐3p inhibition suppressed cell growth and metastasis and enhanced radiosensitivity in ESCA cells by targeting RHCG.ConclusionsOverexpression of circATIC hampered ESCA progression and promoted radiosensitivity depending on the regulation of miR‐10b‐3p and RHCG.

Circ_0020123 promotes cell proliferation and migration in lung adenocarcinoma via PDZD8.

High circ_0020123 expression is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC) as previously reported. Whether circ_0020123 also plays an oncogenic role in lung adenocarcinoma (LUAD) is still unknown. Additionally, circ_0020123 is derived from part of exon (1312–3851) from its host gene PDZ domain-containing protein 8 (PDZD8). We hypothesized that circ_0020123 might affect malignant behaviors of LUAD cells by regulating PDZD8. Reverse transcription quantitative polymerase chain reaction revealed that PDZD8 was highly expressed in LUAD tissues and cells. PDZD8 knockdown suppressed LUAD cell proliferation and migration as shown by colony formation assays, Ethynyl deoxyuridine incorporation assays, Transwell assays, and wound healing assays. circ_0020123 was also found to be upregulated in LUAD tissues and cells. Moreover, circ_0020123 positively regulated PDZD8 expression in LUAD cells but exerted no significant effect on the transcriptional level of PDZD8. Mechanistically, circ_0020123 act as a competing endogenous RNA (ceRNA) to interact with miR-1283, thereby releasing the repression on PDZD8. Moreover, PDZD8 overexpression rescued the suppressive effect of circ_0020123 knockdown on LUAD cell proliferation and migration. In conclusion, circ_0020123 interacts with miR-1283 as a ceRNA to regulate PDZD8 expression, thus promoting the proliferation and migration of LUAD cells. The study might provide new biomarkers for future LUAD investigation.

LncRNA NCK1-AS1 Promotes Malignant Cellular Phenotypes of Laryngeal Squamous Cell Carcinoma via miR-137/NCK1 Axis.

Increasing evidence demonstrates that many long noncoding RNAs (lncRNAs) are implicated with the development of laryngeal squamous cell carcinoma (LSCC). As shown by bioinformatics analysis, lncRNA non-catalytic region of tyrosine kinase adaptor protein 1-antisense 1 (NCK1-AS1) is upregulated in tissues of head and neck squamous cell carcinoma. The study aimed to explore the role and mechanism of NCK1-AS1 in LSCC. NCK1-AS1 expression in LSCC cells was evaluated by reverse transcription qPCR. The viability, proliferation, invasion, migration, and apoptosis of LSCC cells with indicated transfection were evaluated by CCK-8 assays, Ethynyl deoxyuridine incorporation assays, Transwell assays, wound healing assays, and TUNEL assays, respectively. Subcellular fractionation assays were performed to evaluate the cellular distribution of NCK1-AS1 and NCK1. NCK1 protein level in LSCC cells with indicated transfection was quantified by western blotting. The binding relation between miR-137 and NCK1-AS1 (or NCK1) were determined using RNA immunoprecipitation assays and luciferase reporter assays. NCK1-AS1 was highly expressed in LSCC cell lines. NCK1-AS1 depletion suppressed LSCC cell viability, proliferation, invasion, and migration while enhancing cell apoptosis. NCK1, an adjacent gene of NCK1-AS1, is also highly expressed in LSCC cells and was positively regulated by NCK1-AS1. Moreover, NCK1-AS1 interact with miR-137 to upregulate NCK1 expression. NCK1 was the downstream target of miR-137 and was negatively correlated to miR-137. In addition, overexpressed NCK1 reversed the suppressive impact of NCK1-AS1 depletion on malignant behaviors of LSCC cells. NCK1-AS1 contributes to LSCC cellular behaviors by upregulating NCK1 via interaction with miR-137.

Circ_0000647 promotes cell injury by modulating miR-126-5p/TRAF3 axis in oxygen-glucose deprivation and reperfusion-induced SK-N-SH cell model

BackgroundEmerging evidence has shown that circular RNAs (circRNAs) are involved in the pathogenesis of ischemic stroke (IS). Nonetheless, the function of circ_0000647 was not reported. MethodsOxygen-glucose deprivation and reperfusion (OGD/R)-treated SK-N-SH cells were used to mimic cerebral ischemia/reperfusion (I/R) conditions. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure the levels of circ_0000647, microRNA-126-5p (miR-126-5p) and TNF receptor associated factor 3 (TRAF3). Cell Counting Kit-8 (CCK-8) assay, 5′-ethynyl-2′-deoxyuridine (EDU) assay and flow cytometry analysis were employed to assess cell proliferation and apoptosis. Enzyme-linked immunosorbent assay (ELISA) was conducted for the concentrations of IL-6 and TNF-α. Oxidative stress was assessed by determining malondialdehyde (MDA) level and superoxide dismutase (SOD) activity. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were adopted to estimate the relationships of circ_0000647, miR-126-5p and TRAF3. The morphology and size of exosomes were observed via transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) analysis. ResultsCirc_0000647 was elevated in OGD/R-treated SK-N-SH cells. OGD/R treatment suppressed the proliferation and promoted the apoptosis, inflammation and oxidative stress in SK-N-SH cells, while circ_0000647 knockdown reversed the effects. Circ_0000647 could sponge miR-126-5p, which directly targeted TRAF3. MiR-126-5p overexpression alleviated OGD/R-induced SK-N-SH cell damage and miR-126-5p inhibition reversed the effect of circ_0000647 knockdown on OGD/R-induced SK-N-SH cell damage. Moreover, TRAF3 elevation abated miR-126-5p-mediated effect on SK-N-SH cell injury. In addition, exosomal circ_0000647 level was increased in OGD/R-stimulated SK-N-SH cells. ConclusionCirc_0000647 interference relieved OGD/R-induced SK-N-SH cell damage by altering miR-126-5p/TRAF3 axis.

SNHG1 functions as an oncogenic lncRNA and promotes osteosarcoma progression by up-regulating S100A6 via miR-493-5p.

The mechanism behind the aberrant expression of S100A6 in osteosarcoma is seldom reported so far. This study sought to explore the regulatory axis targeting S100A6 involved in osteosarcoma progression. Clinical samples collected from osteosarcoma patients were used to detect the expressions of SNHG1, miR-493-5p, and S100A6 by western bolt analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The effects of S100A6 on proliferation and osteogenic differentiation were investigated by the CCK-8 assay, colony formation assay, Ethynyl deoxyuridine staining, matrix mineralization assay, and alkaline phosphatase assay. The potential of lncRNAs/miRNAs targeting S100A6 was identified by the bioinformatics approach, and the results were verified by the dual luciferase assay and RNA immunoprecipitation assay. Both and rescue experiments were performed to investigate the regulatory relationship between the identified lncRNAs and S100A6. The results showed that S100A6 is highly expressed in osteosarcoma. S100A6 overexpression not only increases the proliferation but also reduces the osteogenic differentiation of osteosarcoma cells, while S1006A silence exerts the opposite effects. Then, SNHG1 is identified to directly interact with miR-493-5p to attenuate miR-493-5p binding to the 3'-untranslated region of S100A6. Notably, S100A6 silence partially rescues the effect of SNHG1 overexpression on proliferation and osteogenic differentiation of osteosarcoma cells. Furthermore, the suppressive role of SNHG1 silence in the growth of osteosarcoma xenograft tumors is countered by S100A6 overexpression. Collectively, this study reveals that S100A6 plays an important role in osteosarcoma progression, and SNHG1 promotes S100A6 expression by competitively sponging miR-493-5p.

A ceRNA network mediated by LINC00475 in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most frequent histological type of differentiated thyroid carcinoma. Long noncoding RNAs (lncRNAs) have been widely reported to play a key role in human malignancies, and PTC is included. This study aimed to find out the functions and mechanism of lncRNA LINC00475 in PTC. LINC00475 was upregulated in PTC cells and was mainly located in the cytoplasm according to reverse-transcription polymerase chain reaction analyses and subcellular fractionation assays. As shown by cell counting kit-8 assays, ethynyl deoxyuridine incorporation assays, wound healing assays, and transwell assays, LINC00475 knockdown suppressed cell viability, proliferation, migration, and invasion. Mechanistically, LINC00475 upregulated the expression of messenger RNA zinc finger CCHC-type containing 12 (ZCCHC12) by binding to miR-376c-3p. ZCCHC12 was a direct target gene of miR-376c-3p in PTC cells. The relationship between miR-376c-3p and LINC00475 (or ZCCHC12) in PTC cells was probed by luciferase reporter assays, RNA pulldown assays, and RNA immunoprecipitation assays. In addition, both mRNA and protein levels of ZCCHC12 were downregulated due to miR-376c-3p overexpression or LINC00475 silencing. ZCCHC12 overexpression partially reversed the suppressive effect of LINC00475 knockdown on malignant behaviors of PTC cells. In conclusion, LINC00475 promotes PTC cell proliferation, migration, and invasion by upregulating ZCCHC12 via the interaction with miR-376c-3p.

DEVELOPMENT OF A METHOD TO STUDY SEQUENTIAL PROLIFERATION WITHIN HUMAN OVARIAN FOLLICLES UTILIZING THYMIDINE ANALOGUE INCORPORATION

To date, methods for quantifying cellular proliferation within the human ovary have relied primarily on static measurements at the time of tissue recovery, such as labeling for ki67 and phosphohistone-H3. Here, we describe a method based on consecutive injection of two thymidine analogues, ethynyl deoxyuridine (EdU) and 5-chloro-2′-deoxyuridine (CldU), in a human ovarian xenograft model, allowing resolution of cellular division within discrete temporal windows of follicle development and observation of a previously undescribed influence of lead follicles on the proliferative activity of surrounding stroma and preantral follicles.

CircWHSC1 serves as an oncogene to promote hepatocellular carcinoma progression.

Circular RNAs (circRNAs) function as vital regulators in multifarious cancers, including hepatocellular carcinoma (HCC). However, the roles of circRNA Wolf-Hirschhorn syndrome candidate gene-1 (circWHSC1) in HCC are barely known. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted for the levels of circWHSC1, miR-142-3p, miR-421, miR-665 and homeobox A1 (HOXA1) mRNA. Cell Counting Kit-8 (CCK-8) assay, colony formation assay and 5'-ethynyl-2'-deoxyuridine (EdU) assay were used to evaluate cell proliferation ability. Transwell assay was adopted for cell migration and invasion. Western blot assay was employed for protein levels. RNA pull-down assay, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were executed to verify the interaction between miR-142-3p and circWHSC1 or HOXA1. Murine xenograft model assay was conducted for the role of circWHSC1 in vivo. The morphology of exosomes was observed by transmission electron microscopy (TEM). CircWHSC1 was elevated in HCC tissues and cells, and high level of circWHSC1 was associated with worse overall survival of HCC patients. Knockdown of circWHSC1 suppressed HCC cell proliferation and metastasis in vitro and restrained tumorigenesis in vivo. CircWHSC1 functioned as the sponge for miR-142-3p, which directly targeted HOXA1. Inhibition of miR-142-3p ameliorated the effects of circWHSC1 knockdown on HCC cell proliferation and metastasis. Moreover, miR-142-3p overexpression restrained the growth and motility of HCC cells, with HOXA1 elevation reversing the impacts. Additionally, circWHSC1 was increased in HCC patients' serum and might be a diagnostic indicator for HCC. CircWHSC1 played a tumour-promoting role in HCC by elevating HOXA1 through sponging miR-142-3p.

New bitongling (NBTL) ameliorates rheumatoid arthritis in rats through inhibiting JAK2/STAT3 signaling pathway.

Rheumatoid arthritis (RA) is featured by a variety of physical symptoms and fibroblast-like synoviocytes (FLSs) abnormal proliferation. Increasing evidence has demonstrated that traditional Chinese medicine exerts an important role in RA treatment. New bitongling (NBTL) as one of the traditional Chinese medicine has been reported to be involved in the progression of RA, but the exact mechanism is unclear. In our study, we intended to investigate the effect of NBTL on RA to identify the mechanisms related to JAK2/STAT3 signaling pathway. Extracts of Tripterygium wilfordii (TW), a traditional Chinese herbal medicine, have been widely used for treating RA in China for several decades, so, TW was used as a positive control drug for TBNL. RA rats were constructed by immunization with collagen type II to evaluate the action of NBTL in vivo. Body weight and arthritic index were evaluated. Hematoxylin & Eosin staining was performed to analysis the morphological changes of ankle joints tissue. TUNEL and flow cytometry were performed to examine cell apoptosis, while CCK8 and Ethynyl-2′-deoxyuridine (EdU) were performed to examine cell proliferation. In addition, the markers of inflammation were detected by Western blot, ELISA, and RT-qPCR. Firstly, we find that rats treated with NBTL or TW not only reduced swelling degree and bone destruction, but also repressed IL-1 β and IL-6 levels. In addition, NBTL and TW could increase the weight of rats, and promote the level of IL-10 and IL-4 in vivo. Furthermore, NBTL inhibited inflammation of FLS, induced cell apoptosis and hindered cell proliferation, which was reversed by dipeptidyl peptidase (DPP), a JAK2/STAT3 pathway activator. Taken together, NBTL potentially retarded RA via JAK2/STAT3 pathway, highlighting novel mechanisms associated with RA

Long Non-coding RNA LINC01969 Promotes Ovarian Cancer by Regulating the miR-144-5p/LARP1 Axis as a Competing Endogenous RNA.

Accumulating evidence has shown that long non-coding RNAs (lncRNAs) can be used as biological markers and treatment targets in cancer and play various roles in cancer-related biological processes. However, the lncRNA expression profiles and their roles and action mechanisms in ovarian cancer (OC) are largely unknown. Here, we assessed the lncRNA expression profiles in OC tissues from The Cancer Genome Atlas (TCGA) database, and one upregulated lncRNA, LINC01969, was selected for further study. LINC01969 expression levels in 41 patients were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The in vitro effects of LINC01969 on OC cell migration, invasion, and proliferation were determined by the CCK-8, ethynyl-2-deoxyuridine (EdU), wound healing, and Transwell assays. Epithelial–mesenchymal transition (EMT) was evaluated using qRT-PCR and Western blotting. The molecular mechanisms of LINC01969 in OC were assessed through bioinformatics analysis, RNA-binding protein immunoprecipitation (RIP), dual luciferase reporter gene assays, and a rescue experiment. Finally, in vivo experiments were conducted to evaluate the functions of LINC01969. The results of the current study showed that LINC01969 was dramatically upregulated in OC, and patients with lower LINC01969 expression levels tended to have better overall survival. Further experiments demonstrated that LINC01969 promoted the migration, invasion, and proliferation of OC cells in vitro and sped up tumor growth in vivo. Additionally, LINC01969, which primarily exists in the cytoplasm, boosted LARP1 expression by sponging miR-144-5p and promoted the malignant phenotypes of OC cells. In conclusion, the LINC01969/miR-144-5p/LARP1 axis is a newly identified regulatory signaling pathway involved in OC progression.

Circ-ABCB10 promotes growth and metastasis of nasopharyngeal carcinoma by upregulating ROCK1.

Nasopharyngeal carcinoma (NPC) is one of the most ordinary head and neck tumors. This study aims to uncover the role of circ-ABCB10 functions in the progression of NPC. The circ-ABCB10 expression was detected in paired NPC patients' tissue samples and cell lines. The role of circ-ABCB10 in NPC proliferation was identified through proliferation assay, Ethynyl deoxyuridine (EdU) assay and colony formation assay. Next, the role of circ-ABCB10 in NPC metastasis was measured through wound healing assay and transwell assay. Finally, the underlying mechanism was further uncovered through Western blot assay and real-time quantitative polymerase chain reaction (RT-qPCR). It was found that circ-ABCB10 expression was significantly higher in NPC tissues than that in adjacent samples. In addition, cell proliferation, migration and invasion of NPC were promoted through overexpression of circ-ABCB10 in vitro. Results of further experiments revealed that ROCK1 was upregulated via overexpression of circ-ABCB10 in NPC. This study indicated that circ-ABCB10 promoted NPC cell proliferation and metastasis via upregulating ROCK1 in vitro.

Quantifying Cell Proliferation During Regeneration of Aquatic Worms.

Many species of aquatic worms, including members of the phyla Nemertea, Annelida, Platyhelminthes, and Xenacoelomorpha, can regenerate large parts of their body after amputation. In most species, cell proliferation plays key roles in the reconstruction of lost tissues. For example, in annelids and flatworms, inhibition of cell proliferation by irradiation or chemicals prevents regeneration. Cell proliferation also plays crucial roles in growth, body patterning (e.g., segmentation) and asexual reproduction in many groups of aquatic worms. Cell proliferation dynamics in these organisms can be studied using immunohistochemical detection of proteins expressed during proliferation-associated processes or by incorporation and labeling of thymidine analogues during DNA replication. In this chapter, we present protocols for labeling and quantifying cell proliferation by (a) antibody-based detection of either phosphorylated histone H3 during mitosis or proliferating cell nuclear antigen (PCNA) during S-phase, and (b) incorporation of two thymidine analogues, 5'-bromo-2'-deoxyuridine (BrdU) and 5'-ethynyl-2'-deoxyuridine (EdU), detected by immunohistochemistry or inorganic "click" chemistry, respectively. Although these protocols have been developed for whole mounts of small (<2cm) marine and freshwater worms, they can also be adapted for use in larger specimens or tissue sections.