Kaempferol inhibits colorectal cancer metastasis through circ_0000345 mediated JMJD2C/ß-catenin signalling pathway.

BACKGROUND: Recurrence and metastasis are the main causes of disease deterioration in colorectal cancer (CRC) patients, yet efficient therapeutic strategies are lacking. Natural compounds for efficient antitumour therapeutics are becoming increasingly prominent. Kaempferol, one of the main components of flavonoids in plants, displays a variety of pharmacological activities. Our preliminary experiments suggested that kaempferol could inhibit CRC metastasis and is significantly associated with the ß-catenin signalling pathway. Moreover, we also defined the regulatory roles of JMJD2C in ß-catenin signalling in our previous work. PURPOSE: This study aims to reveal the mechanism by which kaempferol inhibits CRC progression and regulates the JMJD2C/ß-catenin signalling pathway. METHODS: The migratory capabilities of CRC cells after kaempferol intervention were measured by scratch wound healing and transwell assays. Circ_0000345 knockdown CRC stable cell lines were generated by lentivirus infection. The possible mechanism of kaempferol on circ_0000345 was verified by molecular-protein docking and verification program cellular thermal shift assay (CETSA). A dual luciferase reporter gene assay was carried out for the targeting relationship among circ_0000345, miR-205-5p and JMJD2C. Fluorescence in situ hybridization (FISH) was performed to determine the expression of circ_0000345 in tumour tissues. A pulmonary metastatic model of CRC in vitro was built to assess the antimetastatic effect and mechanism of kaempferol in vivo. RESULTS: In vitro, kaempferol inhibits the ability to migrate of CRC cells by reducing the activation of the JMJD2C/ß-catenin signalling pathway. MiR-205-5p is a key bridge for kaempferol to inhibit the expression of JMJD2C. The function of miR-205-5p is impeded by circ_0000345, which shows higher expression levels in human metastatic CRC tissues than nonmetastatic CRC tissues, and its formation is regulated by the RNA-binding proteins HNRNPK and HNRNPL. Mechanistically, kaempferol physically interacts with HNRNPK and HNRNPL to suppress JMJD2C by downregulating the expression of circ_0000345. In vivo, kaempferol suppresses CRC lung metastasis. Kaempferol inhibits the activation of JMJD2C/ß-catenin signalling through reducing the expression of circ_0000345 in the CRC lung metastasis model. CONCLUSION: Circ_0000345 enhances activation of the JMJD2C/ß-catenin signalling pathway through miR-205-5p to promote CRC metastasis. Kaempferol inhibits CRC metastasis through the circ_0000345-mediated JMJD2C/ß-catenin signalling pathway, and this effect is influenced as a direct consequence of the binding of kaempferol with HNRNPK and HNRNPL. This provides promising therapeutic and/or adjuvant agents for advanced CRC and sheds light on the multifaceted role of phytomedicine in cancer.

[Cangxi Tongbi Capsules promote chondrocyte autophagy by regulating circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit development of knee osteoarthritis].

To investigate the mechanism by which Cangxi Tongbi Capsules promote chondrocyte autophagy to inhibit knee osteoarthritis(KOA) progression by regulating the circRNA＿0008365/miR-1271/p38 mitogen-activated protein kinase(MAPK) pathway. The cell and animal models of KOA were established and intervened with Cangxi Tongbi Capsules, si-circRNA＿0008365, si-NC, and Cangxi Tongbi Capsules combined with si-circRNA＿0008365. Flow cytometry and transmission electron microscopy were employed to determine the level of apoptosis and observe autophagosomes, respectively. Western blot was employed to reveal the changes in the protein levels of microtubule-associated protein light chain 3(LC3)Ⅱ/Ⅰ, Beclin-1, selective autophagy junction protein p62/sequestosome 1, collagen Ⅱ, a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5), and p38 MAPK. The mRNA levels of circRNA＿0008365, miR-1271, collagen Ⅱ, and ADAMTS-5 were determined by qRT-PCR. Hematoxylin-eosin staining was employed to reveal the pathological changes of the cartilage tissue of the knee, and enzyme-linked immunosorbent assay to measure the levels of interleukin-1ß(IL-1ß) and tumor necrosis factor-alpha(TNF-α). The chondrocytes treated with IL-1ß showed down-regulated expression of circRNA＿0008365, up-regulated expression of miR-1271 and p38 MAPK, lowered autophagy level, increased apoptosis rate, and accelerated catabolism of extracellular matrix. The intervention with Cangxi Tongbi Capsules up-regulated the expression of circRNA＿0008365, down-regulated the expression of miR-1271 and p38 MAPK, increased the autophagy level, decreased the apoptosis rate, and weakened the catabolism of extracellular matrix. However, the effect of Cangxi Tongbi Capsules was suppressed after interfering with circRNA＿0008365. The in vivo experiments showed that Cangxi Tongbi Capsules dose-dependently inhibited the p38 MAPK pathway, enhanced chondrocyte autophagy, and mitigated articular cartilage damage and inflammatory response, thereby inhibiting the progression of KOA in rats. This study indicated that Cangxi Tongbi Capsules promoted chondrocyte autophagy by regulating the circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit the development of KOA.

Fuzheng Kang-Ai inhibits NSCLC cell proliferation via regulating hsa_circ_0048091/hsa-miR-378g/ARRDC3 pathway.

BACKGROUND: Current treatments for lung cancer have their own deficiencies, such as severe adverse effect. Therefore, more safe and effective drugs are needed. PURPOSE: Fuzheng Kang-Ai (FZKA for short) has been applied as an adjuvant treatment in advanced Non-Small Cell Lung Cancer (NSCLC) patients for decades in China, showing a definitive effect with minimal toxicities. However, the underlying mechanism is yet to be identified. STUDY DESIGN: Both in vitro and in vivo experiments were performed in this study to identify the exact mechanism by which FZKA inhibits NSCLC cell proliferation. METHODS: MTT and CCK-8 assays were used to detect cell viability. Xenograft model was performed for in vivo experiments. CircRNA and miRNA sequencing were used to find the differentially expressed circRNAs and miRNAs, respectively. qRT-PCR was performed to check the expression levels of circRNA, miRNA and mRNA. BaseScope was carried out to observe the expression of circRNA in situ. Actinomycin D and RNase R experiments were done to show the stability of circRNA. Nuclear-cytoplasmic fractionation and FISH were used to identify the localization of circRNA and miRNA. Pull-down, RIP, and luciferase activity assays were performed to show the biding ability of circRNA, miRNA and target proteins. Flow cytometry was done to observe cell apoptosis. Western blot and IHC were done to detect the protein expression. TCGA database was used to analyze the survival rate. RESULTS: FZKA inhibits NSCLC cell proliferation both in vitro and in vivo. Hsa_circ_0048091 and hsa-miR-378g were the most differentially expressed circRNA and miRNA, respectively, after FZKA treatment. Silencing hsa_circ_0048091 and overexpressing hsa-miR-378g promoted cell proliferation and reversed the inhibition effect of FZKA on NSCLC, respectively. Hsa-miR-378g was sponged by hsa_circ_0048091, and the overexpression of miR-378g reversed the inhibition effect of hsa_ circ_0048091 on NSCLC. ARRDC3, as a target of hsa-miR-378g, was increased by FZKA treatment. Silencing ARRDC3 reversed both the inhibition effect of FZKA and miR-378g inhibitor on NSCLC. CONCLUSION: This study, for the first time, has established the function of hsa_circ_0048091, hsa- miR-378g, and ARRDC3 in lung cancer. It also shows that FZKA inhibits NSCLC cell proliferation through hsa_circ_0048091/hsa-miR-378g/ARRDC3 pathway, uncovering a novel mechanism by which FZKA controls human NSCLC cell growth.

Deciphering the molecular mechanisms of Maxing Huoqiao Decoction in treating pulmonary fibrosis via transcriptional profiling and circRNA-miRNA-mRNA network analysis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung condition with unknown etiology and high mortality. Chinese herbal medicine has been used for more than a thousand years to treat various lung diseases. PURPOSE: The current study aimed to examine whether Chinese herbal Maxing Huoqiao Decoction (MXHQD) exerts therapeutic effects on IPF and to further uncover its underlying molecular mechanisms. METHODS: Mouse model of acute lung injury (ALI) or IPF was induced by intratracheal instillation of LPS or bleomycin, respectively. ALI mice were treated with MXHQD for 7 days, and lung tissues were taken for test after modeling 24 h. IPF mice were gavaged for 21 days after modeling. Lung tissues were subjected to whole transcriptome detection, and the differential RNAs were experimentally verified. RESULTS: The results showed that MXHQD alleviated the computed tomography (CT) and the pathological degree changes in mice with IPF, improved changes in the expression of fibrosis related genes and reduced the hydroxyproline expression in IPF mice. MXHQD also decreased the cell numbers in bronchoalveolar lavage fluids, and the expression levels of the inflammatory factors in the ALI mice lung tissues were significantly inhibited. By applying whole transcriptome analysis, results showed that MXHQD acted on 40 mRNAs, 15 miRNAs, 25 novel lncRNAs and 17 circRNAs to resist pulmonary fibrosis. The competing endogenous RNA (ceRNA) network diagram showed that the multiple components of MXHQD against fibrosis through a network of multiple targets. The differential mRNAs were mainly related to the innate immune response and the defense response to virus. Then the expression of mRNAs in the differential mRNA-miRNA-differential circRNA network in the lung tissue of IPF was verified. The expression of ZBP1 and ISG15 related to immune system and anti virus was verified at both gene and protein expressions. MXHQD could significantly inhibit the elevation of ZBP1 and ISG15 factors induced by the fibrosis model. CONCLUSION: Overall, our findings provide compelling evidence that MXHQD can alleviate IPF by modulating innate immunity. This is the first study to reveal the molecular mechanism underlying the multi-components, multi-channels and multi-targets anti-IPF immune injury of MXHQD, and supports its potential clinical application for IPF.

Evolutionary Landscape of Tea Circular RNAs and Its Contribution to Chilling Tolerance of Tea Plant.

Chilling stress threatens the yield and distribution pattern of global crops, including the tea plant (Camellia sinensis), one of the most important cash crops around the world. Circular RNA (circRNA) plays roles in regulating plant growth and biotic/abiotic stress responses. Understanding the evolutionary characteristics of circRNA and its feedbacks to chilling stress in the tea plant will help to elucidate the vital roles of circRNAs. In the current report, we systematically identified 2702 high-confidence circRNAs under chilling stress in the tea plant, and interestingly found that the generation of tea plant circRNAs was associated with the length of their flanking introns. Repetitive sequences annotation and DNA methylation analysis revealed that the longer flanking introns of circRNAs present more repetitive sequences and higher methylation levels, which suggested that repeat-elements-mediated DNA methylation might promote the circRNAs biogenesis in the tea plant. We further detected 250 differentially expressed circRNAs under chilling stress, which were functionally enriched in GO terms related to cold/stress responses. Constructing a circRNA-miRNA-mRNA interaction network discovered 139 differentially expressed circRNAs harboring potential miRNA binding sites, which further identified 14 circRNAs that might contribute to tea plant chilling responses. We further characterized a key circRNA, CSS-circFAB1, which was significantly induced under chilling stress. FISH and silencing experiments revealed that CSS-circFAB1 was potentially involved in chilling tolerance of the tea plant. Our study emphasizes the importance of circRNA and its preliminary role against low-temperature stress, providing new insights for tea plant cold tolerance breeding.

Circular RNA circ-RCCD promotes cardiomyocyte differentiation in mouse embryo development via recruiting YY1 to the promoter of MyD88.

Congenital heart disease (CHD) is the most common birth defect, affecting approximately 1% of live births. Genetic and environmental factors are leading factors to CHD, but the mechanism of CHD pathogenesis remains unclear. Circular RNAs (circRNAs) are kinds of endogenous non-coding RNAs (ncRNAs) involved in a variety of physiological and pathological processes, especially in heart diseases. In this study, three significant differently expressed circRNA between maternal embryonic day (E) E13 and E17 was found by microarray assay. Among them, the content of circ-RCCD increases with the development of heart and was enriched in primary cardiomyocytes of different species, which arouses our attention. Functional experiments revealed that inhibition of circ-RCCD dramatically suppressed the formation of beating cell clusters, the fluorescence intensity of cardiac differentiation marker MF20, and the expression of the myocardial-specific markers CTnT, Mef2c, and GATA4. Next, we found that circ-RCCD was involved in cardiomyocyte differentiation through negative regulation of MyD88 expression. Further experiments proved that circ-RCCD inhibited MyD88 levels by recruiting YY1 to the promoter of MyD88; circ-RCCD inhibited nuclear translocation of YY1. These results reported that circ-RCCD promoted cardiomyocyte differentiation by recruiting YY1 to the promoter of MyD88. And, this study provided a potential role and molecular mechanism of circ-RCCD as a target for the treatment of CHD.

Tanshinone IIA sodium sulfonate attenuates inflammation by upregulating circ-Sirt1 and inhibiting the entry of NF-κB into the nucleus.

Inflammation is a biological process that exists in a large number of diseases. NF-κB has been proven to play a pivotal role in the development of inflammation. New drugs aimed at inhibiting the expression of NF-κB have gained attention from researchers. Sirt1 has an anti-inflammatory function, and the circRNA encoded by the Sirt1 gene may also play roles in the anti-inflammatory reaction of Sirt1. In the present study, LPS-treated RAW264.7 cells were used as an inflammatory cell model, and tanshinone IIA sodium sulfonate (TSS) was used as a therapeutic drug. We found that TSS downregulated LPS-induced TNF-α and IL-1ß expression nearly threefold. LPS reduced Circ-sirt1 mRNA expression by one-third, while TSS started this phenomenon. In addition, overexpression/knockdown of Circ-sirt1 neutralized the function of TSS by regulating the translocation of NF-κB. Thus, we proved that TSS has an anti-inflammatory function by upregulating circ-Sirt1 and subsequently inhibiting the translocation of NF-κB. An in vivo experiment was also performed to confirm the protective function of TSS on inflammation. These results indicated that TSS is a potential treatment for inflammation.

YY1 and eIF4A3 are mediators of the cell proliferation, migration and invasion in cholangiocarcinoma promoted by circ-ZNF609 by targeting miR-432-5p to regulate LRRC1.

Cholangiocarcinoma is a highly aggressive malignant tumor, and its incidence is increasing all over the world. More and more evidences show that the aberrant expression of circular RNAs play important roles in tumorigenesis and progression. Current studies on the expression and function of circRNAs in cholangiocarcinoma are scarce. In this study, circ-ZNF609 was discovered as a novel circRNA highly expressed in cholangiocarcinoma for the first time. The circ-ZNF609 expression is connected with the advanced TNM stage, lymphatic invasion and survival time in cholangiocarcinoma patients, and can be used as an independent prognostic factor for the patients. Circ-ZNF609 can promote the cholangiocarcinoma cells proliferation, migration and invasion in vitro, it can also catalyze the xenograft growth in vivo. The promoting effect of circ-ZNF609 on cholangiocarcinoma is achieved via oncogene LRRC1 up-regulation through targeting miR-432-5p by endogenous competitive RNA mechanism. In addition, transcription factor YY1 can bind to the promoter of ZNF609 to further facilitate the transcription of circ-ZNF609. RNA binding protein eIF4A3 can bind to the pre-mRNA of circ-ZNF609 which promotes the circ-ZNF609 circular formation. Overall, YY1/eIF4A3/circ-ZNF609/miR-432-5p/LRRC1 have a significant role in progression of cholangiocarcinoma, and circ-ZNF609 is expected to become a novel biomarker for targeted therapy and prognosis evaluation of cholangiocarcinoma.

CRISP2, CATSPER1 and PATE1 Expression in Human Asthenozoospermic Semen.

The etiology of human asthenozoospermia is multifactorial. The need to unveil molecular mechanisms underlying this state of infertility is, thus, impelling. Circular RNAs (circRNAs) are involved in microRNA (miRNA) inhibition by a sponge activity to protect mRNA targets. All together they form the competitive endogenous RNA network (ceRNET). Recently, we have identified differentially expressed circRNAs (DE-circRNAs) in normozoospermic and asthenozoospermic patients, associated with high-quality (A-spermatozoa) and low-quality (B-spermatozoa) sperm. Here, we carried out a differential analysis of CRISP2, CATSPER1 and PATE1 mRNA expression in good quality (A-spermatozoa) and low quality (B-spermatozoa) sperm fractions collected from both normozoospermic volunteers and asthenozoospermic patients. These sperm fractions are usually separated on the basis of morphology and motility parameters by a density gradient centrifugation. B-spermatozoa showed low levels of mRNAs. Thus, we identified the possible ceRNET responsible for regulating their expression by focusing on circTRIM2, circEPS15 and circRERE. With the idea that motility perturbations could be rooted in quantitative changes of transcripts in sperm, we evaluated circRNA and mRNA modulation in A-spermatozoa and B-spermatozoa after an oral amino acid supplementation known to improve sperm motility. The profiles of CRISP2, CATSPER1 and PATE1 proteins in the same fractions of sperm well matched with the transcript levels. Our data may strengthen the role of circRNAs in asthenozoospermia and shed light on the molecular pathways linked to sperm motility regulation.

CircMEMO1 modulates the promoter methylation and expression of TCF21 to regulate hepatocellular carcinoma progression and sorafenib treatment sensitivity.

BACKGROUND: Cirrhosis is a recognized risk factor for developing hepatocellular carcinoma (HCC). Few studies have reported the expression profile of circRNAs in HCC samples compared to paratumour dysplastic nodule (DN) samples. METHODS: The Arraystar Human circRNA Array combined with laser capture microdissection (LCM) was used to analyse the expression profile of circRNAs in HCC samples compared to paratumour DN samples. Then, both in vitro and in vivo HCC models were used to determine the role and mechanism of key circRNA in HCC progression and treatment sensitivity. RESULTS: We found that circMEMO1 was significantly downregulated in HCC samples and that the level of circMEMO1 was closely related to the OS and disease-free survival (DFS) of HCC patients. Mechanistic analysis revealed that circMEMO1 can modulate the promoter methylation and gene expression of TCF21 to regulate HCC progression by acting as a sponge for miR-106b-5p, which targets the TET family of genes and increases the 5hmC level. More importantly, circMEMO1 can increase the sensitivity of HCC cells to sorafenib treatment. CONCLUSION: Our study determined that circMEMO1 can promote the demethylation and expression of TCF21 and can be considered a crucial epigenetic modifier in HCC progression.

Scutellaria barbata D.Don and Oldenlandia diffusa (Willd.) Roxb crude extracts inhibit hepatitis-B-virus-associated hepatocellular carcinoma growth through regulating circRNA expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria barbata D.Don (SB) and Oldenlandia diffusa (Willd.) Roxb are commonly known as Ban Zhi Lian and Bai Hua She Cao in Chinese herbal medicines, respectively. As a pair of herbs, they have traditionally been used as ethnomedicines for clearing away heat and toxins, removing blood stasis, and promoting blood circulation, diuresis, and detumescence. AIM OF THE STUDY: The aim of the present study was to determine the active ingredients in SB and OD extracts and to investigate whether these extracts can inhibit the growth of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) cell lines (HepG2.2.15 and Hep3B) in vitro and in vivo, as well as to explore their mechanisms of action. MATERIALS AND METHODS: We determined the levels of total flavonoids, luteolin, and apigenin in SB and OD extracts via ultraviolet-visible spectrophotometry and high-performance liquid chromatography. The effects of SB and OD extracts on HBV-associated HCC cell growth were assessed by HepG2.2.15 and Hep3B cells phenotype and RNA sequencing of Hep3B cells in vitro, and xenograft models in vivo. RESULTS: The extracts of SB and OD contained total flavonoids. There were active ingredients of luteolin and apigenin in SB, but not in OD. The extracts of SB and OD significantly inhibited HCC growth, migration, invasion, and HBV activity in vitro and in vivo, as well as altered circRNA expression in Hep3B cells. Moreover, we constructed a circRNA-miRNA-mRNA co-expression network. CONCLUSIONS: The extracts of SB and OD may inhibit HCC cell growth and HBV activity in vitro and in vivo through altering circRNA-miRNA-gene expression and that the efficacies of these extracts may be related to the presence of luteolin and apigenin.

Exosomal circRNAs contribute to intestinal development via the VEGF signalling pathway in human term and preterm colostrum.

Human breast milk (HBM) provides essential nutrients for newborn growth and development, and contains a variety of biologically active ingredients that can affect gastrointestinal tract and immune system development in breastfed infants. HBM also contains mRNAs, microRNAs and lncRNAs, most of which are encapsulated in milk-derived exosomes and exhibit various important infant development related biological functions. While previous studies have shown that exosomal circRNAs are involved in the intestinal epithelial cells' proliferation and repair. However, the effect of HBM exosomal circRNAs on intestinal development is not clear. In this study, we identified 6756 circRNAs both in preterm colostrum (PC) and term colostrum (TC), of which 66 were upregulated, and 42 were downregulated (|fold change>2|, p < 0.05) in PC. Pathway analysis showed that the VEGF signalling pathway was involved, and network analysis revealed that the differentially expressed circRNAs bound various miRNAs. Further analyses showed that has_circRNA_405708 and has_circRNA_104707 were involved in the VEGF signalling pathway, and that they all bound various mirRNAs. Exosomes found in preterm colostrum (PC) and term colostrum (TC) promoted VEGF protein expression and induced the proliferation and migration of small intestinal epithelial cells (FHCs). Exosomal circRNAs found in human colostrum (HC) binding to related miRNAs may regulate VEGF signalling, and intestinal development.

Circ-LAMP1 contributes to the growth and metastasis of cholangiocarcinoma via miR-556-5p and miR-567 mediated YY1 activation.

Dysregulation of circular RNAs (circRNAs) executes important regulatory roles in carcinogenesis. Nonetheless, few studies focused on the mechanisms of circRNAs in cholangiocarcinoma (CCA). qRT-PCR was applied to verify the dysregulated circRNAs in CCA. Fisher's exact test, Kaplan-Meier analysis and Cox regression model were utilized to investigate the clinical implications of circ-LAMP1 in the patients with CCA. The viability, apoptosis, migration and invasion of CCA cells were detected after silencing/overexpression of circ-LAMP1. Xenograft and lung metastasis assays were performed to verify the in vitro results. The regulatory networks of circ-LAMP1 were unveiled by bioinformatic analysis, RNA immunoprecipitation (RIP), RNA pulldown and luciferase reporter assays. Up-regulation of circ-LAMP1 was found in CCA tissue samples and cell lines. Enhanced level of circ-LAMP1 was linked to clinical severity, high post-operative recurrence and poor prognosis for the patients with CCA. Gain/loss-of-function assays confirmed the oncogenic role of circ-LAMP1 in mediating cell growth, apoptosis, migration and invasion. Nevertheless, the level of circ-LAMP1 had no effect on normal biliary epithelium proliferation and apoptosis. Animal study further verified the in vitro data. Mechanistically, circ-LAMP1 directly sponged miR-556-5p and miR-567, thereby releasing their suppression on YY1 at post-transcriptional level. Rescue assay indicated that the oncogenic role of circ-LAMP1 is partially dependent on its modulation of YY1 in CCA. In summary, this study suggested that circ-LAMP1 might be used as a promising biomarker/therapeutic target for CCA.

Curcumin suppresses the malignancy of non-small cell lung cancer by modulating the circ-PRKCA/miR-384/ITGB1 pathway.

BACKGROUND: Curcumin exerts a suppressive effect in tumor growth by acting as a modulator of multiple molecular targets. Circular RNA hsa_circ_0007580 (circ-PRKCA) accelerates the tumorigenesis of non-small cell lung cancer (NSCLC). However, whether curcumin can regulate circ-PRKCA to inhibit NSCLC progression is unclear. METHODS: Cell viability, colony formation, apoptosis, migration, and invasion were analyzed using Cell Counting Kit-8 (CCK-8), plate clone, flow cytometry, or transwell assay. Expression of circ-PRKCA, microRNA (miR)-384, and ITGB1 mRNA (integrin subunit beta 1) mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Curcumin repressed NSCLC growth through regulating circ-PRKCA expression was validated by xenograft assay. The targeting relationship between circ-PRKCA or ITGB1 and miR-384 was verified by dual-luciferase reporter assay. The level of ITGB1 protein was measured by western blotting. RESULTS: Circ-PRKCA and ITGB1 expression were elevated in NSCLC tissues and cells, but miR-384 had an opposing tendency. After curcumin treatment, the expression tendency of circ-PRKCA, miR-384, and ITGB1 in NSCLC cells was overturned. Furthermore, curcumin impeded viability, colony formation, migration, invasion, and accelerated apoptosis of NSCLC cells, but these impacts were partially reversed by circ-PRKCA elevation, miR-384 downregulation, or ITGB1 overexpression. Also, the inhibitory effect of curcumin on xenograft tumor was further enhanced after circ-PRKCA knockdown. Notably, circ-PRKCA regulated ITGB1 expression through sponging miR-384 in curcumin-treated NSCLC cells. CONCLUSIONS: Curcumin inhibited NSCLC growth through downregulating circ-PRKCA, which regulated ITGB1 expression by adsorbing miR-384. This study provided a new mechanism to understand how curcumin inhibited the progression of NSCLC.

Salidroside inhibits apoptosis and autophagy of cardiomyocyte by regulation of circular RNA hsa_circ_0000064 in cardiac ischemia-reperfusion injury.

Salidroside (Sal), a natural extract of Rhodiola rosea, shows a latent effect on protecting cardiovascular system. Our study explored the effect of salidroside on ischemia-reperfusion (I/R) injury in rat heart. I/R was performed on Wistar rat hearts, and Sal pretreatment was performed in I/R rats. Cardiac marker enzyme, myocardial infarct size, malondialdehyde (MDA) and superoxide dismutase (SOD) content were then measured. Compared with the untreated group, Sal pretreatment observably ameliorated the cardiac function, decreased the myocardial infarct size, reduced the levels of cardiac lactate creatine kinase-MB (CK-MB) and dehydrogenase (LDH), and inhibited the anti-oxidative stress. In addition, Sal treatment also significantly inhibited autophagy and apoptosis, which could be partially reversed by Rapamycin (RAPA), an autophagic agonist. Furthermore, Sal treatment attenuated autophagy by up-regulating the expression of hsa_circ_0000064 (circ-0000064) and Rapamycin (RAPA) treatment abolished it. Our study showed that Sal protected the heart from I/R injury, which might berelated to the upregulation of circ-0000064 and the inhibition of autophagy.

Delivery of Cas13a/crRNA by self-degradable black phosphorus nanosheets to specifically inhibit Mcl-1 for breast cancer therapy.

Mcl-1 amplification has been observed in breast cancer and demonstrated as a key determinant of breast cancer cell survival. However, the clinical use of available effective Mcl-1-specific inhibitors for breast cancer treatment remains a challenge. An RNA-guided CRISPR/Cas13a system targeting RNAs can be used to specifically knock down mRNA expression in mammalian cells. The goal of this work is to develop a self-degradable nanoplatform based on polylysine (PLL)-functionalized black phosphorus (PBP) for the delivery of Cas13a/crRNA complexes to specifically inhibit Mcl-1 at transcriptional level for breast cancer therapy. The constructed Cas13a/crRNA complex is delivered into the cytoplasm by PBP via endocytosis, followed by endosomal escape based on the biodegradation of PBP, and this efficiently knocks down the specific gene at transcriptional level up to an efficiency of 58.64%. Through designing CRISPR RNA crMcl-1, Mcl-1 can be specifically knocked down at transcriptional level in breast cancer cells, resulting in the down-regulation of the expression of Mcl-1 protein and inhibition of the cell activity. Notably, PBP/Cas13a/crMcl-1 shows an excellent tumor suppression efficacy up to 65.16% after intratumoral injection. Therefore, biodegradable PBP is an ideal nanoplatform for the delivery of CRISPR/Cas13a, which could provide a potential strategy for gene therapy.

CircAGFG1 drives metastasis and stemness in colorectal cancer by modulating YY1/CTNNB1.

Colorectal cancer (CRC) is a common malignancy with high occurrence and mortality worldwide. In recent years, the overall survival rate of CRC patients has been improved because of the advances in early diagnosis and therapy. However, the prognosis of CRC patients at the advanced stage is still poor due to high recurrence rate and metastasis. The function of circular RNA (circRNA) ArfGAP with FG repeats 1 (circAGFG1) has been explored in non-small-cell lung cancer and triple-negative breast cancer. Nevertheless, its role in CRC is not clear. In this study, circAGFG1 was upregulated in CRC cell lines. CircAGFG1 silencing significantly suppressed cell proliferation, migration, invasion, and stemness, while promoted cell apoptosis in CRC. Meanwhile, we found that circAGFG1 also accelerated CRC tumor growth and metastasis in vivo. Importantly, circAGFG1 activated Wnt/ß-catenin pathway through regulating CTNNB1. Afterwards, YY1 was found to transcriptionally activate CTNNB1. Furthermore, circAGFG1 directly sponged miR-4262 and miR-185-5p to upregulate YY1 expression. Eventually, rescue assays demonstrated that the effect of circAGFG1 silencing on CRC cell functions was observably reversed by upregulating YY1 or CTNNB1. In brief, our findings uncovered that circAGFG1 modulated YY1/CTNNB1 axis to drive metastasis and stemness in CRC by sponging miR-4262 and miR-185-5p.

Altered circular RNA expression profiles in the non-ischemic thalamus in focal cortical infarction mice.

Focal cerebral infarction leads to secondary changes in non-ischemic areas remote from but connected to the infarct site. Circular RNAs (circRNAs) are involved in the pathophysiological processes of many diseases. However, the expression and roles of circRNAs in non-ischemic remote regions after ischemic stroke remain unknown. In this study, adult male C57BL/6J mice were subjected to permanent distal middle cerebral artery occlusion (MCAO) to establish focal cortical infarction. High-throughput sequencing was used to profile the circRNA expression in the mouse ipsilateral thalamus at 7 and 14 d after MCAO. Bioinformatics analyses were conducted to predict the function of the differential expressed circRNAs' host and target genes. Compared with sham group, a total of 2659 circRNAs were significantly altered in the ipsilateral thalamus at 7 or 14 d after MCAO in mice. Among them, 73 circRNAs were significantly altered at both two time points after stroke. GO and KEGG analyses indicated that circRNAs plays important roles in secondary thalamic neurodegeneration and remodeling after focal cortical infarction. This is the first study to profile the circRNA expression in non-ischemic region of ischemic stroke, suggesting that circRNAs may be therapeutic targets for reducing post-stroke secondary remote neurodegeneration.

Circular RNA expression profiling in the nucleus accumbens: Effects of electroacupuncture treatment on morphine-induced conditioned place preference.

Electroacupuncture (EA) has been developed on the basis of traditional Chinese acupuncture. EA can suppress craving in opioid addicts and opioid-seeking responses in rodents. However, the molecular mechanism of EA on the rewarding properties of morphine and craving responses is not known. Here, we have applied a conditioned place preference paradigm in mice to measure morphine-induced rewarding effects along with EA treatment. Circular RNAs (circRNAs) can function as micro RNA (miRNA) sponges to effectively regulate gene expression levels. CircRNA profiling within the nucleus accumbens (NAc) was performed in EA-treated and sham-treated mice. Following RNAseq, data were analyzed by gene ontology (GO) and Kyoto Encyclopedia Genes and Genomes (KEGG) tools. We identified 112 significantly differentially expressed circRNAs, including 51 that were up-regulated and 61 that were down-regulated. Our bioinformatics analyses show that these differentially expressed circRNAs map into pathways that are mainly involved with renin secretion and the cGMP-PKG signaling. We further constructed a circRNA-miRNA network that predicts the potential roles of the differentially expressed circRNAs and the interaction of circRNAs with miRNAs. Our secondary sequencing and bioinformatics analysis in the NAc after EA treatment on morphine-induced CPP provides putative novel targets on molecular mechanisms involved in morphine reinforcement and possibly craving.

CircRNA_0016418 expedites the progression of human skin melanoma via miR-625/YY1 axis.

OBJECTIVE: Some circular RNAs (circRNAs) have been testified to play crucial roles in the regulation of skin melanoma, including circRNA_0016418 (circ0016418). However, the regulatory mechanism of circ0016418 in skin melanoma is undiscovered. MATERIALS AND METHODS: The RNA expression was examined through quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and associated-proteins levels were measured via Western blot. Cell counting kit-8 (CCK-8) assay was used for detecting cell proliferation. Transwell assay was conducted to assess the abilities of migration and invasion. The target relation was analyzed by Dual-Luciferase reporter assay. RESULTS: The levels of circ0016418 and Yin Yang 1 (YY1) were up-regulated in skin melanoma tissues and cells. Knockdown of both circ0016418 and YY1 had suppressive effects on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of skin melanoma cells. YY1 overexpression reverted the inhibitory effects on skin melanoma cells caused by circ0016418 knockdown. Circ0016418 negatively modulated microRNA-625 (miR-625) expression and miR-625 directly targeted YY1. Circ0016418 functioned as a competitive endogenous RNA (ceRNA) of miR-625 to regulate YY1 expression. CONCLUSIONS: Circ0016418 regulated proliferation, migration, invasion, and EMT of skin melanoma cells through miR-625/YY1 axis. Circ0016418 might be a useful indicator of the therapeutic strategies of skin melanoma.