MiR-125a-5p抑制3T3-L1前体脂肪细胞分化

MicroRNAs(miRNAs) 是一类在脂肪组织发育中发挥重要作用的小非编码RNA. 为探明miR-125a-5p在3T3-L1前体脂肪细胞中的作用,采用实时qPCR检测了miR-125a-5p在小鼠各组织及3T3-L1前体脂肪细胞分化过程中的表达|使用经化学修饰的miR-125a-5p模拟物agomir及抑制剂antagomir转染3T3-L1前体脂肪细胞,采用实时qPCR 和 Western印迹检测成脂标志基因Pparγ和aP2的表达,油红O染色观察脂肪细胞脂质积累. 结果显示,miR-125-5p在小鼠脂肪组织中高丰度表达,在3T3-L1前体脂肪细胞分化过程中表达下降.过表达miR-125a-5p,与对照组相比,成脂标志基因Pparγ和aP2在mRNA和蛋白质水平均明显下降|油红O染色及定量结果显示脂质积累减少. 抑制剂处理结果显示,Pparγ和aP2在mRNA和蛋白质水平均有不同程度上升,但油红O染色及定量结果差异不显著. 以上结果表明,miR-125a-5p在脂肪细胞分化中发挥负调控作用.     

Aged regulatory T cells protect from autoimmune inflammation despite reduced STAT3 activation and decreased constraint of IL-17 producing T cells

Regulatory T cells (Tregs) are specialized CD4⁺ T lymphocytes helping defend against autoimmunity and inflammation. Although age is associated with increased inflammation and autoimmunity, few reports address age effects of immune regulation or auto‐aggressive T cells. We show here that young and aged naïve CD4⁺ T cells are equivalently auto‐aggressive in vivo in T cell‐driven autoimmune colitis. Young and aged CD4⁺ Tregs equally suppressed age‐matched T cell proliferation in vitro and controlled clinical and pathologic T cell‐driven autoimmune colitis, suggesting equivalent regulatory function. However, whereas young and aged CD4⁺ Tregs suppressed interferon (IFN)‐γ⁺ T cells equivalently in this model, aged CD4⁺ Tregs unexpectedly failed to restrain interleukin (IL)‐17⁺ T cells. Nonetheless, young and aged CD4⁺ Tregs equally restrained IL‐17⁺ T cells in vivo during acute inflammation, suggesting a chronic inflammation‐related defect in aged CD4⁺ Tregs. In support, aged Tregs expressed reduced STAT3 activation, a defect associated with poor IL‐17‐producing T cell restraint. Aged naïve mice had markedly increased programmed death (PD)‐1⁺ T cells, but these exhibited no significant auto‐aggressive or regulatory functions in T cell‐driven colitis. Young CD8⁺ CD122^? T cells induce autoimmune bone marrow failure, but we show that aged CD8⁺ CD122^? T cells do not. These data demonstrate no apparent age‐related increase in auto‐aggressive T cell behavior, but disclose previously unrecognized functional defects in aged CD4⁺ Tregs during chronic inflammation. IL‐17 can be inflammatory and contributes to certain autoimmune disorders. Reduced aged Treg function during chronic inflammation and reduced IL‐17 restraint could contribute to age‐related inflammation or autoimmunity.     

MiR-17-5p and MKL-1 modulate stem cell characteristics of gastric cancer cells

Effectively targeting cancer stem cells to treat cancer has great therapeutic prospects. However, the effect of microRNA miR-17/MKL-1 on gastric cancer stem cells has not been studied yet. This study preliminarily explored the mechanism of miR-17/MKL-1 in gastric cancer stem cells. Many previous reports have indicated that microRNA and EMT regulated cancer stem cell characteristics, and miR-17 and MKL-1 were involved as a critical gene in migration and invasion in the EMT pathway. Through RT-PCR, Western Blot, flow cytometry, immunofluorescence, sphere formation xenograft tumor assays and drug resistance, the role of miR-17-5p and MKL-1 on promoting stem cell-like properties of gastric cancer were verified in vivo and vitro. Next, MKL-1 targets CD44, EpCAM, and miR -17-5p promoter verified by luciferase assay and ChIP. Besides, the TCGA database analysis found that both miR-17-5p and MKL-1 increased in gastric cancer, and the prognostic survival of the MKL-1 high expression group was reduced. It is found that MKL-1 promotes expression by targeting miR-17, CD44 and EpCAM promoters. Besides, the TCGA database analysis found that both miR-17-5p and MKL-1 increased in gastric cancer, and the prognostic survival of the MKL-1 high expression group was reduced. These findings reveal new regulatory signaling pathways for gastric cancer stem cells, thus it give new insights on potential early diagnosis and/or molecular therapy for gastric cancer.     

MiR-490-5p inhibits the stemness of hepatocellular carcinoma cells by targeting ECT2

To explore the targeting relationship between miR-490-5p and ECT2 in hepatocellular carcinoma (HCC) and the influences of miR-490-5p and ECT2 on the stemness of HCC cells. The expressions of miR-490-5p and ECT2 in HCC tissues and adjacent tissues were identified by quantitative real-time polymerase chain reaction (qRT-PCR). The relationships between the expression levels of miR-490-5p/ ECT2 and the overall/disease-free survival (OS/DFS) of patients with HCC were evaluated using correlative curves. In addition, the targeting relationship between miR-490-5p and ECT2 was predicted by TargetScan and verified by dual-luciferase reporter assay. Plasmid transfection was used for overexpression of ECT2 in HepG2 cells, and transfection efficiency was verified by qRT-PCR. Cell Counting Kit-8 assay and cell sphere-formation assay were conducted to detect the proliferation and sphere-formation ability of HCC cells, respectively. Cell populations with different cell transfections were sorted using flow cytometry. The expression levels of proteins in the stem cell signaling pathway were determined using Western blot analysis. MiR-490-5p was remarkably downregulated, yet ECT2 was upregulated in HCC tissues compared with adjacent tissues. MiR-490-5p expression was positively correlated with OS and DFS of patients with HCC, which were otherwise negatively correlated with ECT2 expression. ECT2 was validated to be the downstream target of miR-490-5p. Overexpression of miR-490-5p restrained the sphere formation ability, stemness, and proliferation of HCC cells. MiR-490-5p repressed the stemness of HCC cells through inhibiting the expression of ECT2. MiR-490-5p may be an underlying therapeutic target in HCC treatment.     

LncRNA-UCA1 modulates progression of colon cancer through regulating the miR-28-5p/HOXB3 axis

Emerging evidence has shown that the long noncoding RNA urothelial carcinoma–associated 1 (UCA1) plays a tumor-promoting role in colorectal cancer, while miR-28-5p shows tumor-inhibitory activity in several tumor types. However, the mechanisms both of these in colon cancer progression are still unknown. In this work, the detailed roles and mechanisms of UCA1 and its target genes in colon cancer were studied. The results showed that UCA1 was upregulated in colon cancer tissues when compared with the adjacent nonhumorous tissues, as well as in the various colon cancer cell lines, but the expression of miR-28-5p showed an opposite trend. Furthermore, a high UCA1 level in colon cancer tissues is positively associated with the tumor size and advanced tumor stages. Functional assays revealed that both UCA1 knockdown and miR-28-5p overexpression could inhibit colon cancer cell growth and migration. Further mechanistic studies indicated that UCA1 knockdown played tumor suppressive roles in SW480 and HT116 cells through binding with miR-28-5p. We also, for the first time, identified HOXB3 as the target gene of miR-28-5p and that HOXB3 overexpression could mediate the functions of UCA1 in cell proliferation and migration of colon cancer cells. In conclusion, our data provided evidence for the regulatory network of UCA1/miR-28-5p/HOXB3 in colon cancer, suggesting that UCA1, miR-28-5p, and HOXB3 are the potential targets for colon cancer therapy.     

LOC103691336/miR-138-5p/BMPR2 axis modulates Mg-mediated osteogenic differentiation in rat femoral fracture model and rat primary bone marrow stromal cells

Intramedullary stabilization is frequently used to treat long bone fractures. Since implant removal can become technically very challenging with the potential to cause further tissue damage, biodegradable materials are emerging as alternative options. Magnesium (Mg)-based biodegradable implants have a controllable degradation rate and good tissue compatibility, which makes them attractive for musculoskeletal research. Herein, the degradation of Mg and steel implants, the pathological characteristics and osteoblast differentiation in mice femora were examined. To investigate the molecular mechanism, we analyzed the differentially expressed long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) in Mg-implanted or stain-steel-implanted callus tissues. lncRNA LOC103691336 was upregulated in Mg-implanted tissues and most relevant to BMPR2, a kinase receptor of BMPs with an established role in osteogenesis. The knockdown of LOC103691336 attenuated Mg-mediated osteogenic differentiation. Furthermore, miR-138-5p, previously reported to inhibit osteogenic differentiation, could bind to LOC103691336 and BMPR2 in bone marrow stromal cells (BMSCs). LOC103691336 competed with BMPR2 for miR-138-5p binding in BMSCs to attenuate the inhibitory effect of miR-138-5p on BMPR2 expression. Finally, the effect of LOC103691336 knockdown on Mg-mediated osteogenic differentiation could be attenuated by miR-138-5p inhibition. In conclusion, we provided a novel mechanism of Mg implants mediating the osteogenesis differentiation and demonstrated that Mg implants may be promising for improving fracture healing.     

Depletion of CUL4B in macrophages ameliorates diabetic kidney disease via miR-194-5p/ITGA9 axis

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Chondrocyte mTORC1 activation stimulates miR-483-5p via HDAC4 in osteoarthritis progression

The hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) in chondrocytes has been shown to accelerate the severity of destabilization of the medial meniscus-induced and age-related osteoarthritis (OA) phenotypes with aberrant chondrocyte hypertrophy and angiogenesis. Meanwhile, we previously reported that miR-483-5p is essential for the initiation and development of OA by stimulating chondrocyte hypertrophy and angiogenesis. The connection between mTORC1 and miR-483-5p activation in OA progression, however, remains unclear. In this study, we elucidated their relationship and identified the underlying mechanisms. The expression of miR-483-5p in the articular cartilage of cartilage-specific TSC1 knockout mice was assessed compared with control mice using the Agilent Mouse miRNA (8*60K) V19.0 array and real-time polymerase chain reaction (RT-PCR). The functional effects of the stimulation of miR-483-5p via histone deacetylase 4 (HDAC4) by mTORC1 in OA development, subsequently modulating its downstream targets matrilin 3 and tissue inhibitor of metalloproteinase 2, were examined by immunostaining, western blotting, and real-time PCR. This study revealed that miR-483-5p was responsible for mTORC1 activation-stimulated OA. Mechanistically, mTORC1 controls the HDAC4-dependent expression of miR-483-5p to stimulate chondrocyte hypertrophy, extracellular matrix degradation, and subchondral bone angiogenesis, and it consequently initiates and accelerates the development of OA. Our findings revealed a novel mTORC1-HDAC4-miR-483-5p pathway that is critical for OA development.     

miR-17-5p promotes proliferation and epithelial-mesenchymal transition in human osteosarcoma cells by targeting SRC kinase signaling inhibitor 1

MicroRNA-17-5p (miR-17-5p) and epithelial-mesenchymal transition (EMT) have been reported to participate in the development and progression of multiple cancers. However, the relationship between the miR-17-5p and EMT in osteosarcoma (OS) is still poorly understood. This study was to investigate the effects of the miR-17-5p and its potential mechanism in regulating proliferation, apoptosis, and EMT of human OS. Quantitative real-time PCR was used to detect the miR-17-5p and SRC kinase signaling inhibitor 1 (SRCIN1) messenger RNA expression in OS specimens and cell lines. After transfection with miR-17-5p inhibitors, proliferation, apoptosis, migration, and invasion of OS cells were assessed by using the Cell Counting Kit-8, the annexin V-FITC apoptosis, wound-healing, and transwell assays. The SRCIN1 was validated as a target of the miR-17-5p through bioinformatics algorithms and luciferase reporter assay. Moreover, the expression of EMT markers, E-cadherin, N-cadherin, and Snail was identified by the Western blot analysis. MiR-17-5p was significantly upregulated in OS tumor samples and cell lines. It inhibited proliferation and EMT, and promoted apoptosis in OS. The SRCIN1 was identified as a direct target of the miR-17-5p. Silenced miR-17-5p could change the expression of EMT markers, such as upregulating the expression of E-cadherin, and downregulating the expression of N-cadherin and Snail through targeting the antioncogenic SRCIN1. These findings suggest that the miR-17-5p promotes cell proliferation, and EMT in human OS by directly targeting the SRCIN1, and reveal a branch of the miR-17-5p/SRCIN1/EMT signaling pathway involved in the progression of OS.     

DGCR5 attenuates neuropathic pain through sponging miR-330-3p and regulating PDCD4 in CCI rat models

Neuropathic pain caused by somatosensory nervous system dysfunction is a serious public health problem. Some long noncoding RNAs (lncRNAs) can participate in physiological processes involved in neuropathic pain. However, the effects of lncRNA DGCR5 in neuropathic pain have not been explored. Therefore, in our current study, we concentrated on the biological roles of DGCR5 in neuropathic pain. Here, it was observed that DGCR5 was significantly decreased in chronic sciatic nerve injury (CCI) rat models. DGCR5 overexpression was able to alleviate neuropathic pain development including mechanical and thermal hyperalgesia. In addition, the current understanding of miR-330-3p function in neuropathic pain remains largely incomplete. Here, we found that miR-330-3p was greatly increased in CCI rats and DGCR5 can modulate miR-330-3p expression negatively. Upregulation of DGCR5 repressed inflammation-correlated biomarkers including interleukin 6 (IL-6), tumor necrosis factor α, and IL-1β in CCI rats by sponging miR-330-3p. The negative correlation between DGCR5 and miR-330-3p was confirmed in our current study. Inhibition of miR-330-3p suppressed neuropathic pain progression by restraining neuroinflammation in vivo. In addition, PDCD4 was predicted as a downstream target of miR-330-3p. Furthermore, PDCD4 was significantly increased in CCI rats and DGCR5 regulated PDCD4 expression through sponging miR-330-3p in CCI rat models. Taken these together, it was implied that DGCR5/miR-330-3p/PDCD4 axis participated in neuropathic pain treatment.     

LncRNA PCED1B-AS1 activates the proliferation and restricts the apoptosis of glioma through cooperating with miR-194-5p/PCED1B axis

Glioma with poor prognosis is accepted as a lethal, malignant intracranial tumor among central nervous system diseases. It has been frequently exhibited that long noncoding RNAs (lncRNAs) exert indispensable functions in glioma through regulating gene expression through various molecular mechanisms. To unveil a novel lncRNA functioning in glioma, we browsed the cancer genome atlas dataset and chose the lncRNA PC-esterase domain containing 1B antisense RNA 1 (PCED1B-AS1) for further investigations. Loss-of-function experiments depicted that the proliferation ability was restrained and apoptosis ability was induced in glioma cells by PCED1B-AS1 silencing and this phenomenon was also observed when PCED1B was knocked down. In view of the position of PCED1B-AS1 in the cytoplasm, we produced the Venn diagram and discovered one shared microRNA of PCED1B-AS1 and PCED1B. The competing endogenous RNA network formed by PCED1B-AS1, miR-194-5p, and PCED1B was attested by mechanism assays. Rescue experiments reconfirmed that miR-194-5p suppression or PCED1B overexpression neutralized the obstructive impacts of PCED1B-AS1 silence on proliferation and the promoting effects of PCED1B-AS1 silence on apoptosis. The modulation mechanism of the PCED1B-AS1/miR-194-5p/PCED1B axis in glioma was investigated and affirmed, which supports researchers with a new insight into the therapy of patients with glioma.     

Silencing of circTASP1 inhibits proliferation and induces apoptosis of acute myeloid leukaemia cells through modulating miR-515-5p/HMGA2 axis

Acute myeloid leukaemia (AML) is a common hematopoietic disease that is harmful to the lives of children and adults. CircRNAs are aberrantly expressed in the haematologic malignancy cells. However, the expression of circTASP1 and its function in AML remain unclear. In this study, we showed that circTASP1 was significantly up-regulated in AML peripheral blood samples and cells. Knockdown of circTASP1 inhibited proliferation and promoted apoptosis of HL60 and THP-1 cells in vitro. Bioinformatics prediction and luciferase reporter assay proved that circTASP1 sponged miR-515-5p and negatively regulated miR-515-5p expression in HL60 and THP-1 cells. High mobility group A2 (HMGA2) was proved to be a downstream target of miR-515-5p. The rescue experiments confirmed that knockdown of circTASP1 inhibited proliferation and induced apoptosis by modulating miR-515-5p/HMGA2 pathway. Moreover, the in vivo experiment indicated that knockdown of circTASP1 suppressed tumour growth. In conclusion, circTASP1 acts as a sponge for miR-515-5p to regulate HMGA2, thereby promoting proliferation and inhibiting apoptosis during AML progression. Thus, circTASP1 has the potential to be explored as a therapeutic target for AML treatment.