Dual Luciferase Activity Assay Research Articles

Circular RNA CircCOL5A1 Sponges the MiR-7-5p/Epac1 Axis to Promote the Progression of Keloids Through Regulating PI3K/Akt Signaling Pathway.

Keloids, as a result of abnormal wound healing in susceptible individuals, are characterized by the hyper-proliferation of fibroblasts and exaggerated deposition of extracellular matrix. Current surgical and therapeutic modalities provide limited satisfactory results. Growing evidence has highlighted the roles of circRNAs in acting as miRNA sponges. However, up to date, the regulatory mechanism of circRNAs in the pathological process of keloids has rarely been reported. In this study, cell proliferation, cell migration, flow cytometry, western blotting, fluorescence in situ hybridization, dual-luciferase activity, and immunohistochemistry assays were applied to explore the roles and mechanisms of the circCOL5A1/miR-7-5p/Epac1 axis in the keloid. The therapeutic potential of circCOL5A1 was investigated by establishing keloid implantation models. The RT-qPCR result revealed that circCOL5A1 expression was obviously higher in keloid tissues and keloid fibroblasts. Subsequent cellular experiments demonstrated that circCOL5A1 knockdown repressed the proliferation, migration, extracellular matrix (ECM) deposition, whereas promoted cell apoptosis, through the PI3K/Akt signaling pathway. Furthermore, RNA-fluorescence in situ hybridization (RNA-FISH) illustrated that both circCOL5A1 and miR-7-5p were located in the cytoplasm. The luciferase reporter gene assay confirmed that exact binding sites were present between circCOL5A1 and miR-7-5p, as well as between miR-7-5p and Epac1. Collectively, the present study revealed that circCOL5A1 functioned as competing endogenous RNA (ceRNA) by adsorbing miR-7-5p to release Epac1, which contributed to pathological hyperplasia of keloids through activating the PI3K/Akt signaling pathway. Our data indicated that circCOL5A1 might serve as a novel promising therapeutic target and represent a new avenue to understand underlying pathogenesis for keloids.

LncRNA MAFG-AS1 regulates miR-125b-5p/SphK1 axis to promote the proliferation, migration, and invasion of bladder cancer cells

MAFG-AS1 is an oncogenic lncRNA in multiple types of cancer. However, its role in bladder cancer (BC) remains unclear. The present study aimed to investigate the function of MAFG-AS1 in BC. BC and paired non-tumor tissues were collected. Two BC cell lines HT01197 and HT-1376 were used. Dual luciferase activity assay, RT-qPCR, western blot, CCK-8, transwell invasion assay, and wound healing assay were performed. We found that MAFG-AS1 was significantly up-regulated in BC tissues and predicted a poor survival rate. MAFG-AS1 interacted with miR-125b-5p. However, the expression levels of MAFG‑AS1 and miR-125b-5p were not obviously correlated in BC tissues, and MAFG‑AS1 and miR-125b-5p did not regulate the expression of each other. Interestingly, we found that SphK1, a downstream target of miR-125b-5p, was negatively correlated with miR-125b-5p, while it was positively correlated with MAFG-AS1 across BC tissues. In addition, overexpression of MAFG‑AS1 upregulated the expression of SphK1 in BC cells, and attenuated the inhibitory effects of miR-125b-5p on the expression of SphK1. Functional assays showed that overexpression of MAFG‑AS1 promoted BC cell proliferation, migration, and invasion, while its effects were attenuated by overexpression of miR-125b-5p. Moreover, overexpression of miR-125b-5p inhibited BC cell proliferation, migration, and invasion, while its effects were alleviated by overexpression of SphK1. Taken together, our findings demonstrated that MAFG-AS1 has an oncogenic role in BC by regulating the miR-125b-5p/SphK1 axis. MAFG-AS1 might serve as a good diagnostic marker and a potential therapeutic target of BC.

Circular RNA Circ_0000677 promotes cell proliferation by regulating microRNA-106b-5p/CCND1 in non-small cell lung cancer

ABSTRACT Recently, circular RNAs (circRNAs) have become an intense focus of research and large numbers of circRNAs have been identified, awaiting functional elucidation. Thus, the present study aims to examine the regulation of circRNAs and its molecular mechanism in lung cancer growth. Here, we show that circular RNA circ_0000677 was overexpressed and correlated with poor prognosis in non‐small cell lung cancer (NSCLC) patients. Functionally, circ_0000677 knockdown markedly inhibited proliferation of NSCLC cells by observing of immunofluorescence staining of Ki67, clone formation assay, and xenograft experiments. In mechanism, circ_0000677 acted as a sponge of microRNA-106b and further regulated CCDND1 gene expression in NSCLC cells by dual luciferase activity assay and their expression examination. Taken together, these findings suggest a role for circ_0000677/miR-106b/CCND1 regulation axis in promoting NSCLC growth and progression.

Exosomal miR-10b-5p mediates cell communication of gastric cancer cells and fibroblasts and facilitates cell proliferation.

Tumor microenvironment interacts with gastric cancer (GC) cells and affects tumor development. The communication between GC cells and fibroblasts has not been clearly studied and understood. MiR-10b-5p was found highly expressed in tissue and serum samples of patients with advanced stages (stage III+IV) than that in early stage patients (stage I+II). The expression determination of serum exosomal microRNA was also shown with high expression of miR-10b-5p in GC patients with advanced stages. Dual-luciferase activity assays indicated that miR-10b-5p targeted PTEN in GC cells and KLF11 in fibroblasts. The silence of miR-10b-5p up-regulated the expression of PTEN and repressed PI3K/Akt/mTORC1 signaling in GC cells. Clonogenic assay and MTT assay demonstrated that miR-10b-5p inhibitor could significantly reduce the colony formation and cell viability of GC cells. And the incubation of exosomal miR-10b-5p could increase the proliferation of GC cells. Immunohistochemistry staining revealed that high expression of α-SMA was detected in GC tissues with advanced stages. The overexpression of miR-10b-5p down-regulated KLF11 expression and elevated TGFβR1 expression in fibroblasts. In addition, miR-10b-5p inhibitor blocked the secretion of TGFβ1 in GC cells and the directional migration of fibroblasts. Therefore, up-regulated exosomal miR-10b-5p is involved in the interaction of GC cells and fibroblasts in tumor microenvironment via participating in the regulation of TGFβ signaling pathway.

LincRNA-p21 Inhibits Cisplatin-Induced Apoptosis of Human Renal Proximal Tubular Epithelial Cells by Sponging miR-449a

Introduction: LincRNA-p21 is predicted to interact with miR-449a, which plays a protective role in cisplatin-induced acute kidney injury (CIA). Objective: This study aimed to analyze the involvement of lincRNA-p21 in breast cancer patients with CIA. Methods: Levels of lincRNA-p21 in plasma from CIA, triple negative breast cancer, and control groups were measured by performing RT-qPCR. The potential interaction between lincRNA-p21 and miR-449a was first predicted by RT-qPCR. The relationship between lincRNA-p21 and miR-449a was analyzed by overexpression experiment. Results: We found that lincRNA-p21 is downregulated in CIA. Dual luciferase activity assay showed that lincRNA-p21 and miR-449a can interact with each other, while overexpression of lincRNA-p21 and miR-449a failed to affect the expression of each other. In human renal proximal tubular epithelial cells (HRPTEpCs), cisplatin led to the upregulated miR-449a but downregulated lincRNA-p21. Interestingly, lincRNA-p21 overexpression led to reduced enhancing effects of miR-449a on the cisplatin-induced apoptosis of HRPTEpCs. Conclusion: Therefore, lincRNA-p21 is downregulated in CIA and may sponge miR-449a to inhibit cisplatin-induced apoptosis of HRPTEpCs.

Knockdown of miR-1275 protects against cardiomyocytes injury through promoting neuromedin U type 1 receptor

ABSTRACT The present study aimed to assess the role of miR-1275 in cardiac ischemia reperfusion injury. H9 human embryonic stem cell (hESC)-derived cardiomyocytes stimulated by oxygen-glucose deprivation/reoxygenation (OGD/R) were used to simulate myocardial injury in vitro. miR-1275 expression levels in cells were measured by RT-qPCR. The release of lactate dehydrogenase (LDH) and creatine kinase (CK) was examined through LDH and CK ELISA kits. Cell apoptosis was detected through flow cytometry. A Fura-2 Calcium Flux Assay Kit and a Fluo-4 assay kit were used to determine the Ca2+ concentration. Expression levels of proteins were tested by Western blotting. The binding effect of miR-1275 and neuromedin U type 1 receptor (NMUR1) was detected by dual-luciferase activity assay. The results showed that miR-1275 was upregulated in OGD/R-stimulated cardiomyocytes. Inhibition of miR-1275 suppressed the increased activity of LDH and CK, cell apoptosis, reactive oxygen species (ROS) production, intracellular Ca2+ concentration and sarcoplasmic reticulum (SR) Ca2+ leak induced by OGD/R treatment in cardiomyocytes. miR-1275 directly targets 3ʹUTR of NMUR1 and negatively regulates NMUR1 expression. Silence of NMUR1 abolished the protecting effect of the miR-1275 antagomir on myocardial OGD/R injury. Our study indicated that the miR-1275 antagomir protects cardiomyocytes from OGD/R injury through the promotion of NMUR1.

Metabolomic and Transcriptomic Analyses of Anthocyanin Biosynthesis Mechanisms in the Color Mutant Ziziphus jujuba cv. Tailihong.

The purplish-red color of "Tailihong" jujube fruit skins is caused primarily by anthocyanin accumulation, but the mechanisms that underlie anthocyanin biosynthesis in jujube fruit have rarely been studied. We performed metabolomic and transcriptomic analyses of jujube fruit skins at different developmental stages and identified a total of 158 flavonoids, among which cyanidin-3-O-rutinoside and peonidin-3,5-O-diglucoside were the primary anthocyanins. During fruit development and maturation, the anthocyanin content was strongly correlated with the expression of ZjANS and ZjUGT79B1, suggesting that these are key genes in the anthocyanin biosynthesis process. Transcriptomic analysis indicated that the transcription factors ZjMYB5, ZjTT8, and ZjWDR3 regulated anthocyanin biosynthesis in jujube fruit skins. Subcellular localization experiments confirmed that ZjANS and ZjUGT79B1 were localized to the nucleus and the endoplasmic reticulum. ZjMYB5 and ZjTT8 were found only in the nucleus, whereas strong fluorescence signals from ZjWDR3 were observed in the nucleus and cytoplasm. Prokaryotic expression and in vitro enzyme activity assays showed that the recombinant ZjANS protein catalyzed the formation of cyanidin from (+)-catechin. Secondary glycosylation by ZjUFGT79B1 modified cyanidin-3-O-glucoside to produce cyanidin-3-O-rutinoside, and ZjCCoAOMT readily catalyzed the production of the methylated anthocyanin peonidin-3,5-O-diglucoside from cyanidin 3,5-O-glucoside. Dual-Luciferase and GUS activity assays showed that the ZjANS and ZjUGT79B1 promoters were activated by ZjMYB5, ZjTT8, and ZjWDR3. All data indicated that these three transcription factors played important roles in anthocyanin biosynthesis in the color mutant Ziziphus jujuba cv. Tailihong, contributing to anthocyanin accumulation by enhancing the expression of ZjANS and ZjUGT79B1.

Knockdown of miR-130a-3p alleviates spinal cord injury induced neuropathic pain by activating IGF-1/IGF-1R pathway

BackgroundRecent studies have elucidated the instrumental role of microRNAs (miRNAs) in neuropathic pain (NP) progression. As one member of miRNAs, miR-130a-3p has been proved as a mediator in inflammation and neuronal maturation. The present study attempted to elucidate what effect miR-130a-3p exerts on NP. Materials and methodsThe miR-130a-3p expression in the spinal cord tissues of rat with spinal cord compression injury (SCI) and LPS-induced BV2 microglia was determined with RT-PCR, which was further applied to analyze the clinical relevance between miR-130a-3p and neuropathic pain. Besides, the expression of IGF-1, IL-1β, IL-6, and TNF-α in the spinal cord tissues of rats was measured using RT-PCR and ELISA after intrathecal injection of miR-130a-3p inhibitors and tail vein injection of IGF-1 low-expression lentivirus (Lv-shIGF-1). Further, neuronal apoptosis (labeled by Caspase3) and microglial activation (labeled by Iba1) were examined by immunohistochemistry (IHC), and the levels of IGF-1, IGF-1R, NF-κB were determined by western blot. Additionally, bioinformatic was employed to analyze the potential target genes of miR-130a-3p. Furthermore, the dual luciferase activity assay and RNA immunoprecipitation assay were conducted to further substantiate whether miR-130a-3p targets IGF-1. ResultsIn comparison with the sham group, the miR-130a-3p expression was remarkably up-regulated in the spinal cord lesions of SCI rats. The ELISA results showed that inhibiting the miR-130a-3p significantly reduced NP symptoms of SCI rats, mitigated neuronal apoptosis, microglial activation, repressed NF-κB phosphorylation and the IL-1β, IL-6 and TNF-α expressions in SCI rats. Contrarily, downregulation of miR-130a-3p increased IGF-1 and IGF-1R expression. What's more, we observed the same effects in BV2 microglia. In addition, the bioinformatics analysis showed that miR-130-3p targeted at the 3′-untranslated region of IGF-1 and inhibiting its expression. However, abolishing IGF-1 not only promoted the inflammatory responses in the SCI lesions, but also aggravated NP of SCI rats, while those effects were attenuated by the downregulation of miR-130a-3p. ConclusionThe inhibition of miR-130a-3p expression up-regulates the IGF-1/IGF-1R signaling pathway, thus reducing neuropathic pain caused by spinal cord injury.

LncRNA RUNX1-IT1 is Downregulated in Endometrial Cancer and Binds to miR-21 Precursor to Suppress Its Maturation.

BackgroundRUNX1-IT1 suppresses colorectal cancer and liver cancer, while its role in other cancers is unknown. This study was performed to investigate the role of RUNX1-IT1 in endometrial cancer (EC).MethodsEC and paired non-tumor tissues were collected from 62 EC patients, and the expression of RUNX1-IT1, mature miR-21 and miR-21 precursor in these tissue samples were determined by RT-qPCR. Correlations were analyzed by linear regression. Overexpression of RUNX1-IT1 was achieved in EC cells and the expression of mature miR-21 and miR-21 precursor were analyzed by RT-qPCR. CCK-8 assay was used for cell proliferation analysis.ResultsWe found that RUNX1-IT1 was downregulated in EC and inversely correlated with mature miR-21 but not miR-21 precursor. RUNX1-IT1 was predicted to bind with miR-21 precursor. The interaction between them was verified by dual-luciferase activity assay and RNA pull-down assay. In EC cells, overexpression of RUNX1-IT1 downregulated mature miR-21, but not miR-21 precursor. Overexpression of RUNX1-IT1 suppressed the role of miR-21 in increasing cell proliferation.ConclusionRUNX1-IT1 is downregulated in EC and inhibits cancer cell proliferation by suppressing the maturation of miR-21.

Circular RNA Circ-03955 Promotes Epithelial-Mesenchymal Transition in Osteosarcoma by Regulating miR-3662/Metadherin Pathway.

Osteosarcoma is the most common primary malignant tumor, especially in children and adolescents. Circular RNAs (circRNAs) are found to play roles in the progression of osteosarcoma. However, the exact functions of circRNAs in osteosarcoma development still need to be clarified. We obtained differentially expressed circRNAs and miRNAs from a GSE99671 data set (GEO database). The gene co-expression network of ceRNAs and osteosarcoma-related genes was analyzed using the STRING database. qRT-PCR was used to detect the expression of circ-03955 and miR-3662. Transwell assays and flow cytometry were performed to detect phenotypic changes in cell function. A xenograft tumor model was established using BALB/c nude mice. Dual luciferase activity and RNA immunoprecipitation assays were performed to assess the relationship between circ-03955, miR-3662, and metadherin (MTDH). Immunohistochemistry, immunofluorescence, and Western blotting were used to assess protein expression levels. Circ-03955 was significantly upregulated, and miR-3662 was downregulated in osteosarcoma. Circ-03955 silencing inhibited the growth and metastasis of osteosarcoma. Mechanism analysis revealed that circ-03955 could bind to miR-3662, and the latter could target MTDH, leading to its suppressed expression and facilitating epithelial-mesenchymal transition (EMT). All these findings demonstrate that the presence of circ-03955 promotes EMT in osteosarcoma by acting as miR-3662 sponge-mediated MTDH expression.

Upregulated ox40l Can Be Inhibited by miR-146a-5p in Condylar Chondrocytes Induced by IL-1β and TNF-α: A Possible Regulatory Mechanism in Osteoarthritis

Introduction: Osteoarthritis (OA) is a common musculoskeletal disease characterized by pain, stiffness, limited activity, occasional effusion, and local inflammation. MiR-146 is one of the noncoding RNA closely related to OA, but the role of miR-146 in OA remains controversial. The tumour necrosis factor receptor OX40 is activated by its cognate ligand OX40L (TNFSF4) and functions as a T-cell costimulatory molecule. The T-cell functions, including cytokine production, expansion, and survival, are enhanced by the OX40 costimulatory signals. Methods: We established an inflammatory model of condylar chondrocytes induced by IL-1β and TNF-α and detected the expression of miRNA by miRNA sequencing. Then, cell transfection was used to study the role of miR146a-5p in OA. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and database analysis were used to screen out potential target genes of miR-146a-5p. A dual luciferase activity assay tested whether ox40l is the target gene of miR-146a-5p. Results: MiR-146a-5p and OX40L was upregulated after induced by IL-1β and TNF-α, miR-146a-5p reduced the production of inflammatory factors but had no effect on chondrophenotypic factors, and ox40l was targeted by miR-146a-5p. Conclusion: OX40L and miR-146a-5p of condylar chondrocytes in the inflammatory environment (induced by IL-1β and TNF-α) were significantly increased, miR-146a-5p is a protective factor in the inflammatory response, which can reduce the production of inflammatory factors, and miR-146a-5p may regulate T-cell-mediated immunity through targeting of ox40l in OA.

LINC00460 Enhances Bladder Carcinoma Cell Proliferation and Migration by Modulating miR-612/FOXK1 Axis

Introduction: LincRNA (long intergenic noncoding RNA) has been indicated as a mediator in tumorigenesis of bladder carcinoma. This study was performed to evaluate the role of LINC00460 in bladder carcinoma progression. Methods: Expression levels of LINC00460 in bladder carcinoma tissues and cell lines were analyzed via qRT-PCR. MTT, EdU (5-ethynyl-2′-deoxyuridine) staining, and colony formation assays were utilized to evaluate cell viability and proliferation. The wound healing assay was performed to evaluate bladder cancer cell migration, and the transwell assay was used to evaluate cell invasion. The microRNA (miRNA) target of LINC00460 and the corresponding target gene were validated via the dual luciferase activity assay. The tumorigenic function of LINC00460 was determined via establishment of a xenotransplanted tumor model. Results: LINC00460 was elevated in bladder carcinoma tissues and cell lines. Elevated LINC00460 was associated with shorter overall survival of bladder carcinoma patients. Overexpression of LINC00460 promoted cell viability, proliferation, invasion, and migration, while silencing of LINC00460 indicated the opposite effect on bladder carcinoma progression. LINC00460 could directly bind to miR-612 and inhibit miR-612 expression. Moreover, LINC00460 expression was negatively correlated with miR-612 in patients with bladder carcinoma. FOXK1 (Forkhead Box K1) was identified as the target of miR-612 and upregulated in patients with bladder carcinoma. Overexpression of FOXK1 attenuated interference of LINC00460-inhibited bladder carcinoma progression. Knockdown of LINC00460 suppressed in vivo bladder carcinoma growth. Conclusions: LINC00460 promoted bladder carcinoma progression via sponging miR-612 to facilitate FOXK1 expression, suggesting that LINC00460 might have the potential of being explored as a therapeutic target for treatment of bladder carcinoma.

Hypoxic Non-Small-Cell Lung Cancer Cell-Secreted Exosomal microRNA-582-3p Drives Cancer Cell Malignant Phenotypes by Targeting Secreted Frizzled-Related Protein 1.

BackgroundHypoxic environment and exosomes (exos)-mediated intercellular communication are crucial for cancer invasion and metastasis, but the mechanisms are not yet fully understood. In this study, we investigated the regulatory effect of hypoxic tumor cell-secreted exosomal miR-582-3p on non-small-cell lung cancer (NSCLC) cell malignant phenotypes.MethodsThe concentration and diameters of exos were evaluated by nanosight particle tracking analysis. microRNA-582-3p (miR-582-3p) expression was detected by quantitative real-time PCR. The fluorescent dye PKH26 was used to label exos. The direct interaction between miR-582-3p and secreted frizzled-related protein 1 (SFRP1) was determined by dual-luciferase activity assay. NSCLC cell proliferation, migration, and invasion abilities were assessed by cell count kit-8 assay, wound healing assay, and transwell migration and invasion assay. Western blot analysis was performed to detect the protein expression level.ResultsHypoxic NSCLC cell-derived exos promoted the proliferation, migration, and invasion of normoxic NSCLC cells. miR-582-3p expression was upregulated in hypoxic NSCLC cells and hypoxic NSCLC cell-secreted exos. Hypoxic NSCLC cell-derived exos transmitted miR-582-3p to normoxic NSCLC cells. Hypoxic NSCLC cell-secreted exosomal miR-582-3p promoted the proliferation, migration, and invasion of normoxic NSCLC cells. miR-582-3p inhibited the expression of SFRP1 protein by binding to its 3ʹ-UTR. In addition, enforced expression of SFRP1 restrained malignant phenotypes of normoxic NSCLC cells, which was abrogated by hypoxic NSCLC cell-secreted exosomal miR-582-3p.ConclusionHypoxic NSCLC cell-secreted exosomal miR-582-3p drives cancer cell malignant phenotypes by targeting SFRP1, which provides a better understanding of cancer metastasis and may facilitate the development of therapeutics against human NSCLC.

SOX4 promotes the growth and metastasis of breast cancer

PurposeIncreasing evidence has shown that the transcription factor SOX4 is closely associated with the development and progression of many malignant tumors. However, the effect of SOX4 on breast cancer is unclear. In this study, we purposed to investigate the role of SOX4 in the growth and metastasis in breast cancer and the underlying mechanism. Moreover, the effect of SOX4 on cancer cell resistance to chemotherapeutic agents was also evaluated in vitro and in vivo.MethodsWe used lentivirus technique to ectopically express SOX4 in MDA-MB-231 and SUM149 cells or knockdown SOX4 in BT474 cells, and examined the effect of these changes on various cellular functions. MTT assay was used to determine the cell viability as well as resistance to chemotherapeutic agents. The regulation of SOX4 on epithelial-mesenchymal transition (EMT)-related genes was analyzed using qRT-PCR. The binding of SOX4 to the CXCR7 gene was demonstrated using chromatin immunoprecipitation assay and dual-luciferase reporter activity assay. The effect of SOX4/CXCR7 axis on metastasis was examined using Transwell migration and Matrigel invasion assays. The expression of SOX4/CXCR7 in primary tumors and metastatic foci in lymph nodes was assessed using immunohistochemistry. Cellular morphology was investigated under phase contrast microscope and transmission electron microscopy. Moreover, the effect of SOX4 on tumor growth, metastasis, and resistance to chemotherapy was also studied in vivo by using bioluminescent imaging.ResultsSOX4 increased breast cancer cell viability, migration, and invasion in vitro and enhanced tumor growth and metastasis in vivo. It regulated EMT-related genes and bound to CXCR7 promoter to upregulate CXCR7 transcription. Both SOX4 and CXCR7 were highly expressed in human primary tumors and metastatic foci in lymph nodes. Treatment of breast cancer cells with the CXCR7 inhibitor CCX771 reversed the SOX4 effect on cell migration and invasion. Ectopic expression of SOX4 increased the susceptibility of cells to paclitaxel.ConclusionsSOX4 plays an important role in the growth and metastasis of breast cancer. SOX4/CXCR7 may serve as potential therapeutic targets for the treatment. Paclitaxel may be a good therapeutic option if the expression level of SOX4 is high.

LncRNA UASR1 sponges miR-107 in colorectal cancer to upregulate oncogenic CDK8 and promote cell proliferation.

lncRNA UASR1 (UASR1) has been characterized as an oncogenic lncRNA in breast cancer. UASR1 was predicted to interact with miR-107, which serves tumor suppressive roles mainly by targeting CDK8. The present study was performed to investigate the interactions among UASR1, miR-107 and CDK8 in colorectal cancer (CRC). A total of 62 patients with CRC, including 40 males and 22 females (age range, 38–67 years; mean age, 57.2±7.6 years) were enrolled at the Second Hospital of Shandong University between July 2012 and July 2014. The expression of UASR1 in tissues and cells were detected by reverse transcription-quantitative polymerase chain reaction. The interaction between UASR1 and miR-107 was investigated by performing dual luciferase activity assay, and the effects of overexpression of UASR1, miR-107 and CDK8 on the proliferation of CR4 cells were analyzed by performing cell proliferation analysis. It was observed that UASR1 is upregulated in CRC and its high expression levels predicted poor survival in patients with CRC. RNA-RNA interaction prediction demonstrated that UASR1 may interact with miR-107. In CRC cells, overexpression of UASR1 and miR-107 did not affect each other. However, the expression of CDK8, a target of miR-107, was upregulated following overexpression of UASR1. Notably, overexpression of UASR1 decreased the inhibitory effects of miR-107 on cell proliferation and the expression of CDK8. Therefore, UASR1 may sponge miR-107 to upregulate oncogenic CDK8, thereby promoting CRC cell proliferation.

AXL Inactivation Inhibits Mesothelioma Growth and Migration via Regulation of p53 Expression.

Simple SummaryPrevious studies have shown that AXL is a crucial protein that is activated in mesothelioma to cause growth and invasiveness. p53 is the tumor suppressor protein that is most frequently inactivated by mutations in human cancers, but these p53 inactivating mutations are not common in mesothelioma. In the studies reported herein we demonstrate that AXL activation causes p53 functional inactivation in mesothelioma. Specifically, we show that AXL suppresses p53 expression by binding to DNA sequences upstream from the p53 gene, thereby blocking transcription of p53 DNA into RNA. We also show that AXL inhibition by a selective drug inhibits mesothelioma cell viability, migration, and invasion. p53 inactivation attenuates the impact of the AXL inhibitor, providing further support for interplay between AXL and p53 in mesothelioma oncogenesis. These studies demonstrate a novel feedback regulation loop between AXL and p53, and provide a rationale for mesothelioma therapies targeting AXL/p53 signaling.Malignant mesothelioma is a locally aggressive and highly lethal neoplasm. Dysregulation and activation of Gas6/AXL tyrosine kinase signaling are associated with mesothelioma progression, but the mechanisms of these AXL tumorigenic roles are poorly understood. p53 mutants in lung carcinoma upregulate AXL expression by binding and acetylating the AXL promoter. Although TP53 mutations are uncommon in mesothelioma, we hypothesized that these tumors might have alternative feedback mechanisms between AXL and p53. In the current report, we investigated AXL regulation of TP53 transcription, expression, and biological function in mesothelioma. AXL expression was stronger in mesothelioma than most of the other tumor types from the TCGA gene expression profile dataset. AXL knockdown by shRNA induced wild-type and mutant p53 expression in mesothelioma cell lines, suggesting that AXL pro-tumorigenic roles result in part from the suppression of p53 function. Likewise, induced AXL inhibited expression of wild type p53 in COS-7 cells and 293T cells. Immunofluorescence staining showed nuclear colocalization of AXL and p53; however, association of AXL and p53 was not demonstrated in immunoprecipitation complexes. The AXL effects on p53 expression resulted from the inhibition of TP53 transcription, as demonstrated by qRT-PCR after AXL silencing and TP53 promotor dual luciferase activity assays. Chromatin immunoprecipitation-qPCR and sequencing showed that AXL bound to the initial 600 bp sequence at the 5′ end of the TP53 promoter. AXL inhibition (shRNA or R428) reduced mesothelioma cell viability, migration, and invasion, whereas TP53 shRNA knockdown attenuated antiproliferative, migration, and invasive effects of AXL silencing or AXL inactivation in these cells. These studies demonstrate a novel feedback regulation loop between AXL and p53, and provide a rationale for mesothelioma therapies targeting AXL/p53 signaling.

MiR-140 ameliorates neuropathic pain in CCI rats by targeting S1PR1

Objectives Neuropathic pain, with lots of clinical conditions in various diseases, whose physiopathology is implicated in inflammation. MicroRNAs (miRNAs) have largely been shown to exert anti-inflammatory effects against chronic diseases. We then evaluated the effects and regulatory mechanism of miR-140 on neuropathic pain. Methods Rats model with neuropathic pain were established via chronic constriction injury (CCI) and verified by determination of mechanical withdrawal threshold (MWT) and paw withdrawal latency (PWL). The expression level of miR-140 was determined via qRT-PCR (quantitative real-time polymerase chain reaction). Intrathecal injection of miR-140 agomiR was conducted to evaluate the influence of miR-140 on CCI rats via evaluation of MWT, PWL and inflammatory factors secretion. The binding target of miR-140 was predicted and characterized via dual luciferase activity assay. Results Decreased MWT and PWL, as well as increased inflammatory factor secretion, including IL (interleukin)-1β, IL-6 and interferon-γ (IFN-γ), were found in rats under CCI compared with sham rats. MiR-140 was decreased in rats under CCI. Intrathecal injection of miR-140 agomiR increased MWT and PWL, thus attenuating mechanical and thermal hyperalgesia in CCI rats. Moreover, decreased inflammatory factor secretion in rats under CCI injected with miR-140 agomiR, suggesting a negatively regulatory role of miR-140 on neuroinflammation. MiR-140 could bind with Sphingosine-1-phosphate receptor 1 (S1PR1). S1PR1 agonist, SEW2871, could reverse the suppression of miR-140 on neuropathic pain. Conclusions MiR-140 could mollify CCI-stimulated neuropathic pain via targeting S1PR1, suggesting a potential therapeutic target in the treatment of neuropathic pain.

LncRNA THRIL is upregulated in sepsis and sponges miR-19a to upregulate TNF-\u03b1 in human bronchial epithelial cells

BackgroundLong non-coding RNAs (lncRNAs) have been demonstrated to play critical roles in various diseases. Our bioinformatics analysis showed that lncRNA TNFα and heterogenous nuclear ribonucleoprotein L (hnRNPL) related immunoregulatory LincRNA (THRIL) may interact with miR-19a, which targets TNF-α. This study aimed to explore the role of THRIL, an enhancer of LPS-induced inflammatory, in sepsis.MethodsResearch subjects of the present study included 66 sepsis patients and 66 healthy volunteers. The expression levels of THRIL, miR-19a and TNF-α in plasma samples from these participants were determined by RT-qPCR. The interaction between THRIL and miR-19a was explored by performing overexpression experiments in human bronchial epithelial cells (HBEpCs). The roles of THRIL, miR-19a and TNF-α in regulating the apoptosis of HBEpCs were analyzed by cell apoptosis assay.ResultsWe found that THRIL was upregulated in sepsis patients. THRIL is predicted to interact with miR-19a, and the interaction was confirmed by dual-luciferase activity assay. However, THRIL and miR-19a did not affect the expression of each other. Instead, overexpression of THRIL resulted in the increased expression levels of TNF-α, a downstream target of miR-19a in HBEpCs. In HBEpCs, LPS treatment induced the overexpression of THRIL. Cell apoptosis analysis showed that overexpression of THRIL and TNF-α promoted the apoptosis of HBEpCs induced by LPS, while overexpression of miR-19a played an opposite role. Overexpression of THRIL attenuated the effects of overexpression of miR-19a.ConclusionTherefore, THRIL is upregulated in sepsis and may sponge miR-19a to upregulate TNF-α, thereby promoting lung cell apoptosis.

MiR-215 Targeting Novel Genes EREG, NIPAL1 and PTPRU Regulates the Resistance to E.coli F18 in Piglets

Previous research has revealed that miR-215 might be an important miRNA regulating weaned piglets’ resistance to Escherichia coli (E. coli) F18. In this study, target genes of miR-215 were identified by RNA-seq, bioinformatics analysis and dual luciferase detection. The relationship between target genes and E. coli infection was explored by RNAi technology, combined with E. coli stimulation and enzyme linked immunosorbent assay (ELISA) detection. Molecular regulating mechanisms of target genes expression were analyzed by methylation detection of promoter regions and dual luciferase activity assay of single nucleotide polymorphisms (SNPs) in core promoter regions. The results showed that miR-215 could target EREG, NIPAL1 and PTPRU genes. Expression levels of three genes in porcine intestinal epithelial cells (IPEC-J2) in the RNAi group were significantly lower than those in the negative control pGMLV vector (pGMLV-NC) group after E. coli F18 stimulation, while cytokines levels of TNF-α and IL-1β in the RNAi group were significantly higher than in the pGMLV-NC group. Variant sites in the promoter region of three genes could affect their promoter activities. These results suggested that miR-215 could regulate weaned piglets’ resistance to E. coli F18 by targeting EREG, NIPAL1 and PTPRU genes. This study is the first to annotate new biological functions of EREG, NIPAL1 and PTPRU genes in pigs, and provides a new experimental basis and reference for the research of piglets disease-resistance breeding.

LncRNA TUG1 reduces inflammation and enhances insulin sensitivity in white adipose tissue by regulating miR-204/SIRT1 axis in obesity mice.

Prevalence of obesity becomes an important health issue worldwide, but the management of obesity remains unsatisfied. This study aimed to explore the mechanism of long non-coding RNA TUG/miR-204/SIRT1 axis, which was involved in the pathogenesis of obesity. Obesity mouse model was induced by high-fat diet and treated with taurine upregulated gene1 (TUG1) virus via tail intravenous injection. Then, body weight, serum glucose, insulin tolerance, testicular fat weight were detected, as well as the expression of TUG1, microRNA-204 (miR-204), sirtuin1 (SIRT1), and inflammation and fatty accumulation associated proteins and cytokines. Regulatory relationship between TUG1/SIRT1 and miR-204 was confirmed by dual-luciferase reporter activity assay. A high-glucose-induced 3T3-L1 cell model was also constructed to explore the regulatory mechanism of TUG/miR-204/SIRT1 axis in the pathogenesis of obesity at cell level after altering the expression of TUG1, miR-204, and SIRT1. Overexpression of TUG1 could significantly attenuate the weight, serum glucose, glucose, insulin tolerance, fatty accumulation, and inflammation in obesity mice, as well as the elevation of miR-204, but increase the expression of SIRT1, phosphorylated AKT (p-AKT), glucose transporter4 (GLUT4), and peroxisome proliferator activated receptorγ (PPARγ). Both TUG1 and SIRT1 were targets of miR-204 and could be negatively regulated by miR-204. Overexpression of TUG1 could suppress the inflammation in adipocytes via downregulating miR-204 and promote GLUT4/PPARγ/AKT pathway high-glucose-induced inflammation in 3T3-L1 cells. miR-204 inhibitors could also suppress high-glucose-induced inflammation in 3T3-L1 cells via promoting SIRT1/ GLUT4/PPARγ/AKT pathway. LncRNA TUG1 could negatively regulate miR-204 to alleviate inflammation and insulin tolerance via promoting SIRT1/GLUT4/PPARγ/AKT pathway.