Dual-luciferase Reporter Analysis Research Articles

Analysis of the Association of Two SNPs in the Promoter Regions of the PPP2R5C and SLC39A5 Genes with Litter Size in Yunshang Black Goats.

Simple SummaryThe protein phosphatase 2 regulatory subunit B’gamma (PPP2R5C) and solute carrier family 39 member 5 (SLC39A5) genes are essential in mammalian growth and development. Here, KASP genotyping was used to analyze the association between polymorphisms of PPP2R5C and SLC39A5 and litter size in Yunshang black goats, a cultivated goat breed with high prolificacy in China. The results show that PPP2R5C g.65977743C>T and SLC39A5 g.50676693T>C were significantly associated with litter size of the third parity in Yunshang black goats. Luciferase assays and RT-qPCR showed that individuals with the CC genotype of PPP2R5C and TT genotype of SLC39A5 showed higher expression of these genes in goat ovarian tissues than those with other genotypes. Transcription factor predictions showed that SOX18, ZNF418, and ZNF667 and NKX2-4 and TBX6 bind to the polymorphisms of two respective genes. These two polymorphisms provide new insights into the study of fertility in goats.Screening for candidate genes and genetic variants associated with litter size is important for goat breeding. The aim of this study was to analyze the relationship between single nucleotide polymorphisms (SNPs) in PPP2R5C and SLC39A5 and litter size in Yunshang black goats. KASP genotyping was used to detect the SNP genetic markers in the PPP2R5C and SLC39A5 in a population of 569 Yunshang black goats. The results show that there were two SNPs in the PPP2R5C and SLC39A5 promoter regions. Association analysis revealed that the polymorphisms PPP2R5C g.65977743C>T and SLC39A5 g.50676693T>C were significantly associated with the litter size of the third parity of Yunshang black goats (p < 0.05). To further explore the regulatory mechanism of the two genes, the expression of different genotypes of PPP2R5C and SLC39A5 was validated by RT-qPCR and Western blotting. The expression of PPP2R5C was significantly higher in individuals with the TT genotype than in those with the TC and CC genotypes (p < 0.05). The expression of SLC39A5 was also significantly higher in individuals with the TT genotype than in TC and CC genotypes (p < 0.05). Dual luciferase reporter analysis showed that the luciferase activity of PPP2R5C-C variant was significantly higher than that of PPP2R5C-T variant (p < 0.05). The luciferase activity of SLC39A5-T variant was significantly higher than that of SLC39A5-C variant (p < 0.05). Software was used to predict the binding of transcription factors to the polymorphic sites, and the results show that SOX18, ZNF418, and ZNF667 and NKX2-4 and TBX6 might bind to PPP2R5C g.65977743C>T and SLC39A5 g.50676693T>C, respectively. These results provide new insights into the identification of candidate genes for marker-assisted selection (MAS) in goats.

Therapeutic Potential of POU3F3, a Novel Long Non-coding RNA, Alleviates the Pathogenesis of Osteoarthritis by Regulating the miR-29a- 3p/FOXO3 Axis

Osteoarthritis (OA) is the predominant threat to the health of the elderly, and it is crucial to understand the molecular pathogenetic mechanisms involved in it. This study aims to investigate the role of a well-studied cancer-related long non-coding RNA (lncRNA)-POU3F3 in OA and its implicated molecular mechanisms. The expression of POU3F3 and miR-29a-3p was examined in osteoarthritis patients, as well as destabilization of the medial meniscus (DMM) mouse OA model and IL- 1β induced chondrocytes cell OA model, by quantitative real-time PCR. The interaction between POU3F3, miR-29a-3p and transcription factor forkhead box O3 (FOXO3) was verified via dual-luciferase reporter analysis and RNA immunoprecipitation analyses. Cell proliferation and apoptosis were evaluated by cell viability assay and flow cytometry, respectively. Cartilage extracellular matrix (ECM) degradation was investigated with ELISA and western blotting. In addition, the in vivo regulation of POU3F3 in OA was verified by intra-articular injection of lentivirus overexpression POU3F31 in mice models. The expression level of POU3F3 was decreased in OA patients/animal cartilage tissues and IL-1β-stimulated in vitro chondrocyte model. POU3F3 overexpression inhibited IL-1β-induced injury of chondrocytes, enhancing cell viability, suppressing apoptosis and inflammatory cytokine secretion, rescuing metabolic dysfunction, and restraining autophagy in vitro. Mechanistically, Luciferase reporter and RNA immunoprecipitation (RIP) assays indicated that miR-29a-3p could directly bind to POU3F3, and FOXO3 was a target gene of miR-29a-3p. Functional rescue assays confirmed this POU3F3/miR-29a-3p/FOXO3 axis in chondrocytes during OA occurrence. Furthermore, intraarticularly delivery of lentivirus containing POU3F3 alleviates the damage in mouse OA model in vivo. In conclusion, this work highlights the role of the POU3F3/miR-29a-3p/FOXO3 axis in the OA pathogenesis, suggesting this axis as a potential therapeutic target for OA.

MiR-92a-3p promotes breast cancer proliferation by regulating the KLF2/BIRC5 axis.

Breast cancer remains the most common malignancy in females around the world. Recently, a growing number of studies have focused on gene dysregulation. In our previous study, Krüppel-like factors (KLFs) were found to play essential roles in breast cancer development, among which KLF2 could function as a tumor suppressor. Nevertheless, the underlying molecular mechanism remains unclear. miR-92a-3p was identified as the upstream regulator of KLF2 by starBase v.3.0. The regulation of KLF2 by miR-92a-3p was verified by a series of in vitro and in vivo assays. Further exploration revealed that Baculoviral IAP Repeat Containing 5 (BIRC5)was the target of KLF2. ChIP assay, dual-luciferase reporter analysis, quantitative real-time PCR, and western blot were performed for verification. miR-92a-3p functioned as a tumor promoter by inhibiting KLF2 by binding to its 3'-untranslated region (3'-UTR). In addition, KLF2 could transcriptionally suppress the expression of BIRC5. Collectively, our results uncovered the miR-92a-3p/KLF2/BIRC5 axis in breast cancer and provided a potential mechanism for breast cancer development, which may serve as promising strategies for breast cancer therapy.

Circ_0042986 Presence Restrains Cervical Cancer Development via Upregulating PEG3 by Directly Targeting miR-582-3p.

The participation of circular RNAs (circRNAs) in carcinogenesis is widely established. Numerous circRNAs with aberrant expression in cervical cancer (CC) are identified by RNA sequencing, whereas the function of these circRNAs remains unclear. We thus aimed to unveil the effects and mechanisms of circ_0042986 in CC. Circ_0042986 with aberrant downregulation in CC was obtained from GSE10286 dataset. qPCR and western blotting were employed for the detection of circ_0042986, miR-582-3p, and paternally expressed 3 (PEG3) expressions. EdU assay, colony formation assay, wound healing assay, transwell assay, tube formation assay, and flow cytometry assay were applied for functional analyses. The potential binding site was ensured by dual-luciferase reporter analysis, and their binding relationship was verified by pull-down assay. The transplanted tumor models were constructed for in vivo function verification of circ_0042986. Our findings exposed that the downregulation of circ_0042986 was verified in clinical CC samples. Circ_0042986 overexpression largely attenuated CC cell growth, invasiveness, angiogenesis, and survival. MiR-582-3p was targeted by circ_0042986, and the inhibitory roles of circ_0042986 presence were partly abolished by miR-582-3p enrichment. MiR-582-3p combined with PEG3, and circ_0042986 increased PEG3 expression via decoying miR-582-3p. The interaction between miR-582-3p and PEG3 on CC cell functions was confirmed, evidenced by the reversal effects of PEG3 knockdown on miR-582-3p deficiency-inhibited cancer cell malignant behaviors. Circ_0042986 overexpression also limited tumorigenesis of CC in animal models. In summary, circ_0042986 overexpression decoyed miR-582-3p to increase PEG3 expression, thereby blocking the malignant progression of CC.

MiR-299-3p Inhibits Nasopharyngeal Carcinoma Cell Proliferation and Migration by Targeting MMP-2

Purpose. Nasopharyngeal carcinoma (NPC) is a type of squamous cell carcinoma that originated from the epithelial cells of the nose and throat, and its incidence ranks the first among head and neck tumors. However, NPC has a unique and complex etiology that is not completely understood. MiR-299-3p was discovered to be abnormally expressed in cancers. However, the involvement of miR-299-3p in the incidence and progression of nasopharyngeal cancer remains unknown. Methods. The miR-299-3p expression in nasopharyngeal cancer samples and cell lines was identified using quantitative PCR (qPCR). Nasopharyngeal cancer cells were evaluated for proliferation, migration, and invasion using MTT, colony formation assay, and Transwell invasion assay. MiRBase and TargetScan databases identified the possible miR-299-3p target genes that were confirmed using a dual-luciferase reporter analysis. Additionally, the miR-299-3p target genes were validated by Western blot, colony formation assay, and Transwell assays. Results. It was found that miR-299-3p expression was low in nasopharyngeal cancer tissues and cell lines, according to qPCR data. Cell proliferation, colony formation, and migration were considerably reduced by miR-299-3p overexpression. Furthermore, matrix metalloproteinase 2 (MMP-2) expression was regulated by miR-299-3p, whereas MMP-2 knockdown significantly inhibited the capacity of nasopharyngeal cancer cells to form colonies and migrate. Overexpression of MMP-2 substantially reduced the miR-299-3p inhibitory impact on nasopharyngeal cancer cell migration and colony formation. Conclusion. The miR-299-3p acts as a tumor suppressor gene to suppress the growth and spread of nasopharyngeal cancer by regulating MMP-2 expression. Therefore, miR-299-3p and MMP-2 could be important therapeutic targets for suppressing nasopharyngeal cancer growth and metastasis.

MiR-210-3p targets CELF2 to facilitate progression of lung squamous carcinoma through PI3K/AKT pathway.

This study examined the internal mechanism of miR-210-3p/CELF2 in LUSC. Expression data of mRNAs and miRNAs in LUSC were acquired from TCGA and subjected to differential expression analysis. qRT-PCR was applied to examine miR-210-3p and CELF2 expression. Besides, western blot was utilized to evaluate protein expression of CELF2 and PI3K/AKT pathway-related proteins. Dual-luciferase reporter analysis was conducted to validate targeting relationship between miR-210-3p and CELF2. Additionally, CCK-8, colony formation, transwell and flow cytometry were employed to respectively test proliferation, migration, invasion abilities and cell cycle distribution. Xenograft tumor models were used to evaluate the influence of miR-210-3p and CELF2 on tumor growth. MiR-210-3p was highly expressed, while CELF2 was less expressed in LUSC cells. Besides, miR-210-3p could downregulate CELF2 expression. Cell functional assay verified that miR-210-3p accelerated aggressive behaviors of LUSC cells. Additionally, rescue assay suggested that miR-210-3p downregulated CELF2 level to stimulate LUSC cell phenotypes and cell cycle progression through PI3K/AKT pathway. Moreover, miR-210-3p/CELF2 stimulated the tumor growth in vivo. To sum up, miR-210-3p modulated CELF2 expression, thus affecting cell phenotypes and cell cycle distribution in LUSC through PI3K/AKT pathway.

Circular RNA hsa_circ_0002360 Promotes Proliferation and Invasion and Inhibits Oxidative Stress in Gastric Cancer by Sponging miR-629-3p and Regulating the PDLIM4 Expression.

Many studies have found that circRNA hsa_0002360 (circ0002360) plays an important role in cancer onset and progression. However, its role in gastric cancer (GC) remains uncertain. Circ0002360 was found to be upregulated in GC cells using QRT-PCR. Furthermore, miR-629-3p, a target miRNA of circ0002360, was the most suppressed miRNA following circ0002360 overexpression. RNA immunoprecipitation (RIP), dual-luciferase reporter analyses, clone formation, transwell, DCFH-DA, and ELISA assays demonstrated that circ0002360-targeted miR-629-3p promotes cell proliferation and migration while inhibiting oxidative stress. GC-related mRNA microarrays from the GEO and TCGA databases, including GSE103236, GSE79973, GSE33429, GSE22804, GSE84437, and TCGA-STAD datasets, were used to find hub biomarkers between normal and gastric cancer samples. WGCNA and uni-Cox analysis were used to identify 27 survival-related risk genes, which were then used to build a risk model for prognosis prediction. Following that, all patients from the GSE84437 and TCGA-STAD datasets with 27 survival-related genes and enough data on survival status and time were randomly assigned to train (n = 433) and test (n = 375) cohorts. Furthermore, ROC and Kaplan-Meier (KM) analyses were used to validate the risk model for both cohorts. randomForest analysis indicated that PDLIM4 was the target gene of miR-629-3p, whose level was increased by circ0002360 but reversed by miR-629-3p mimics. Finally, this study confirmed that circ0002360 sponged miR-629-3p and then upregulated PDLIM4 expression. As a result, circ0002360 may be a useful marker for predicting GC prognosis and an anti-GC treatment target.

Transcriptional repression of MaRBOHs by MaHsf26 is associated with heat shock-alleviated chilling injury in banana fruit

Both heat shock factors (Hsfs) and reactive oxygen species (ROS) are widely perceived to be involved in chilling stress response. However, the particular mechanism of Hsfs mediated-ROS metabolism related to chilling stress remains largely unknown, especially during chilling injury development of postharvest economically cold-sensitive fruit such as banana. In this study, we found that heat shock treatment (HST) could effectively alleviate postharvest banana chilling injury and reduce ROS accumulation during storage. The expression levels of several respiratory burst oxidase homolog (RBOH) genes, encoding ROS-producing enzymes, increased consistently with the progression, whereas were inhibited upon HST. Significantly, we identified an Hsfs MaHsf26, which exhibited a opposite expression pattern as that of MaRBOHs, as the putative binding protein of MaRBOHB-like1, MaRBOHE-like and MaRBOHF-like promoters using yeast one-hybrid (Y1H) screening analysis. Further electrophoretic mobility shift assay (EMSA) and dual luciferase reporter (DLR) analysis displayed MaHsf26 suppressed the transcriptions of MaRBOHB-like1, MaRBOHE-like and MaRBOHF-like through binding to the heat shock elements (HSE) in their promoters, respectively. In general, our results suggested that MaHsf26 plays as a transcriptional repressor of ROS generation by suppressing the transcriptions of MaRBOHs and thereby involving in the HST-mitigated chilling injury in postharvest banana fruit.

Circ_0093887 regulated ox-LDL induced human aortic endothelial cells viability, apoptosis, and inflammation through modulating miR-758-3p/BAMBI axis in atherosclerosis.

Compelling evidence demonstrated that circular RNAs (circRNAs) were involved in the progression of atherosclerosis (AS). However, the role of circ_0093887 in the progression of AS is unclear. The purpose of this study was to explore the role and mechanism of circ_0093887 in oxidized-low density lipoprotein (ox-LDL)-induced human aortic endothelial cells (HAECs). HAECs were stimulated by ox-LDL to simulate AS-like injury in vitro. Circ_0093887, microRNA-758-3p (miR-758-3p), and BMP And Activin Membrane-Bound Inhibitor (BAMBI) levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). PCNA, Bax, Bcl-2, and BAMBI protein levels were detected by western blot. Cell viability and apoptosis were examined by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Tube formation assay was used to assess tube formation. The levels of inflammatory factors TNF-α and IL-1β were detected by corresponding ELISA kits. The relationship between miR-758-3p and circ_0093887 or BAMBI was tested via dual-luciferase reporter analysis and RNA immunoprecipitation. Oxidative stress related indexes (ROS and MDA) were detected by corresponding kits. The expression levels of circ_0093887 and BAMBI were prominently downregulated in ox-LDL-induced HAECs compared with control, whereas the expression of miR-758-3p was upregulated. Overexpression of circ_0093887 promoted HAECs viability and tube formation, and restrained cell apoptosis in ox-LDL-induced HAECs compared with untreated HAECs. Mechanistically, circ_0093887 regulated the expression of BAMBI through miR-758-3p. Further experiments showed that upregulation of miR-758-3p reversed changes in cell function induced by circ_0093887. In addition, reduced BAMBI salvaged miR-758-3p knockdown mediated effects on cell function. Circ_0093887 demonstrated its diagnostic and therapeutic value in AS by promoting the role of the miR-758-3p/BAMBI axis in the ox-LDL-induced endothelial injury of HAECs.

MicroRNA-22-3p ameliorates Alzheimer’s disease by targeting SOX9 through the NF-κB signaling pathway in the hippocampus

BackgroundStudies have suggested that many down-regulated miRNAs identified in the brain tissue or serum of Alzheimer’s disease (AD) patients were involved in the formation of senile plaques and neurofibrillary tangles. Specifically, our previous study revealed that microRNA-22-3p (miR-22-3p) was significantly down-regulated in AD patients. However, the molecular mechanism underlying the down-regulation of miR-22-3p has not been comprehensively investigated.MethodsThe ameliorating effect of miR-22-3p on apoptosis of the Aβ-treated HT22 cells was detected by TUNEL staining, flow cytometry, and western blotting. The cognition of mice with stereotaxic injection of agomir or antagomir of miR-22-3p was assessed by Morris water maze test. Pathological changes in the mouse hippocampus were analyzed using hematoxylin and eosin (HE) staining, Nissl staining, and immunohistochemistry. Proteomics analysis was performed to identify the targets of miR-22-3p, which were further validated using dual-luciferase reporter analysis and western blotting analysis.ResultsThe miR-22-3p played an important role in ameliorating apoptosis in the Aβ-treated HT22 cells. Increased levels of miR-22-3p in the mouse hippocampus improved the cognition in mice. Although the miR-22-3p did not cause the decrease of neuronal loss in the hippocampus, it reduced the Aβ deposition. Proteomics analysis revealed Sox9 protein as the target of miR-22-3p, which was verified by the luciferase reporter experiments.ConclusionOur study showed that miR-22-3p could improve apoptosis and reduce Aβ deposition by acting on Sox9 through the NF-κB signaling pathway to improve the cognition in AD mice. We concluded that miR-22-3p ameliorated AD by targeting Sox9 through the NF-κB signaling pathway in the hippocampus.

Circ_0061395 functions as an oncogenic gene in hepatocellular carcinoma by acting as a miR-1182 sponge

ABSTRACT Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in liver cancer, with a high rate of metastasis and recurrence. Circular RNA_0061395 (circ_0061395) has been shown to be involved in the advance of HCC. However, the interaction between circ_0061395 and microRNA (miRNA) in HCC has not been studied. Quantitative real-time polymerase-chain reaction (qRT-PCR) was used to detect the expression of related genes in liver cancer tissues and cells. The stability of circ_0061395 was verified by RNase R digestion. Through detection of cell malignant behavior and apoptosis, the capping experiment was carried out to verify the regulatory relationship between miR-1182 and circ_0061395 or SPARC/osteonectin, CWCV and Kazal-like domains proteoglycan 1 (SPOCK1). The expression of related proteins was detected by western blot. The interaction of miR-1182 with circ_0061395 or SPOCK1 has been notarized by Dual-luciferase reporter analysis and RNA immunoprecipitation (RIP) assay. Xenotransplantation experiments using BALB/C nude mice were used to confirm the function of circ_0061395 in vivo. Circ_0061395 and SPOCK1 were significantly expressed in liver cancer tissues and cells. Silencing circ_0061395 reduced the proliferation, migration, invasion, tube formation and tumor spheroid formation rate of Huh-7 and SNU-387 cells. MiR-1182 was a target of circ_0061395. Silencing circ_0061395 inhibited the malignant behavior of HCC cells by releasing miR-1182. In addition, SPOCK1 was the target of miR-1182. Overexpression of SPOCK1 partially restored the inhibitory effect of miR-1182 on cell proliferation. Animal experiments confirmed the anti-tumor effect of silence circ_0061395. Circ_0061395 induced the changes of the expression of SPOCK1 by regulating miR-1182, thereby mediating the process of HCC, and at least partially promoting the development of HCC cells, providing a novel targeted therapy for HCC.

Analysis of circRNAs and circRNA-associated competing endogenous RNA networks in β-thalassemia

The involvement of circRNAs in β-thalassemia and their actions on fetal hemoglobin (HbF) is unclear. Here, the circRNAs in β-thalassemia carriers with high HbF levels were comprehensively analyzed and compared with those of healthy individuals. Differential expression of 2183 circRNAs was observed and their correlations with hematological parameters were investigated. Down-regulated hsa-circRNA-100466 had a strong negative correlation with HbF and HbA2. Bioinformatics was employed to construct a hsa-circRNA-100466‑associated competing endogenous RNA (ceRNA) network to identify hub genes and associated miRNAs. The hsa-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified using both present and previously published data. The ceRNA network was verified by qRT-PCR analysis of β-thalassemia samples, RNA immunoprecipitation of K562 cell lysates, and dual-luciferase reporter analysis. qRT-PCR confirmed that hsa-circRNA-100466 and SOX6 were significantly down-regulated, while miR-19b-3p was up-regulated. Hsa-circRNA-100466, miR-19b-3p, and SOX6 were co-immunoprecipitated by anti-argonaute antibodies, indicating involvement with HbF induction. A further dual-luciferase reporter assay verified that miR-19b-3p interacted directly with hsa-circRNA-100466 and SOX6. Furthermore, spearman correlation coefficients revealed their significant correlations with HbF. In conclusion, a novel hsa-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified, providing insight into HbF induction and suggesting targets β-thalassemia treatment.

MicroRNA-4701-5p protects against interleukin-1\u03b2 induced human chondrocyte CHON-001 cells injury via modulating HMGA1

BackgroundmiRNA-4701-5p has been reported to be a vital regulator in many diseases, including rheumatoid arthritis, and miRNA-4701-5p is evidenced to be participated in synovial invasion and joint destruction. In our report, we investigated the roles of miRNA-4701-5p in osteoarthritis (OA) and analyzed the molecular mechanism.MethodsInterleukin-1β (IL-1β) was applied for stimulating human chondrocyte CHON-001 cells to establish an OA injury model. mRNA levels and protein expression were measured using qRT-PCR and western blot assay, respectively. The proliferation ability and cytotoxicity of CHON-001 cells were checked using MTT assay and lactate dehydrogenase activity. The inflammation of chondrocytes was accessed by the secretion levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF)-α. The apoptosis of chondrocytes was determined by flow cytometry assay. Bioinformatics software Starbase v2.0 analyzed the functional binding sites between miRNA-4701-5p and HMGA1 and the interaction was further confirmed using dual luciferase reporter analysis. Results: miRNA-4701-5p was down-regulated in the IL-1β-stimulated chondrocytes and HMGA1 directly targeted miRNA-4701-5p. Up-regulation of miRNA-4701-5p could alleviate IL-1β-treated CHON-001 cells inflammation and apoptosis, and reversed the cell proliferation decrease and cytotoxicity increase after IL-1β treatment. Nevertheless, all the roles of miRNA-4701-5p overexpression in CHON-001 cells could be reversed by HMGA1 up-regulation.ConclusionsmiRNA-4701-5p could alleviate the inflammatory injury of IL-1β-treated CHON-001 cells via down-regulating HMGA1, indicating that miRNA-4701-5p/HMGA1 is a promising therapeutic target for OA.

Silencing of LncRNA KCNQ1OT1 confers an inhibitory effect on renal fibrosis through repressing miR-124-3p activity

ABSTRACT LncRNA have been increasingly shown that plays pivotal roles in the development of various diseases, including renal fibrosis. Nevertheless, the pathological function of Long non-coding RNA KCNQ1OT1 (KCNQ1OT1) in the renal fibrosis remains obscure. Unilateral ureteral obstruction (UUO) was used to induce renal fibrosis. We detected the expression levels of KCNQ1OT1 in the TGF-β1-induced HK-2 cells via RT-qPCR analysis. The functions of KCNQ1OT1 on the progression of renal fibrosis were examined by CCK-8, EdU, dual-luciferase reporter, and immunofluorescence analyses. In the present study, we found that sh-KCNQ1OT1 obviously attenuated UUO-induced renal fibrosis. Moreover, production of extracellular matrix (ECM), including α-SMA and Fibronectin levels, was significantly increased in kidney and HK-2 cells after UUO or TGF-β stimulation. Knockdown of KCNQ1OT1 inhibited cell proliferation and inhibits the α-SMA and Fibronectin expression of TGF-β1-induced HK-2 cells. In addition, bioinformatics analysis and dual-luciferase reporter assay indicated that miR-124-3p was a target gene of KCNQ1OT1. Mechanistically, silencing miR-124-3p abolished the repressive effects of KCNQ1OT1 on TGF-β1-induced HK-2 cells. In conclusion, KCNQ1OT1 knockdown plays an anti-fibrotic effect through promotion of miR-124-3p expression in renal fibrosis, which provides a promising therapeutic target for the treatment of renal fibrosis.

CircRNA hsa_circ_0018289 exerts an oncogenic role in cervical cancer progression through miR-1294/ICMT axis.

BackgroundcircRNA hsa_circ_0018289‐mediated growth and metastasis of CC cells were investigated, as well as the mechanistic pathway.MethodsQuantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) was carried out to examine the expression of hsa_circ_0018289, microRNA (miR)‐1294, and isoprenylcysteine carboxyl methyltransferase (ICMT). CC cell proliferation, migration, and invasion were evaluated by 5‐ethynyl‐2’‐deoxyuridine (EdU) incorporation, colony formation, transwell assays, Western blot analysis of ICMT, and glycolysis‐associated proteins. Dual‐luciferase reporter or RNA pull‐down analysis of the target interaction between miR‐1294 and hsa_hsa_circ_0018289 or ICMT. Xenograft model assay was implemented to assess the role of hsa_circ_0018289 in vivo. Immunofluorescence (IHC) was employed to detect the level of Ki‐67.ResultsHsa_circ_0018289 was elevated in CC tissues and cells, its deficiency could repress growth, metastasis, and glycolysis of CC cells in vitro, as well as hamper tumor growth in vivo. Hsa_circ_0018289 sponged miR‐1294 while miR‐1294 bound with ICMT, and the inhibition of miR‐1294 or addition of ICMT could partially relieve the effect caused by hsa_circ_0018289 depletion.ConclusionHsa_circ_0018289 contributes to malignant development by regulating the miR‐1294/ICMT axis, affording novel insight into CC therapy.

Overexpression of miR-493-3p suppresses ovarian cancer cell proliferation, migration and invasion through downregulating DPY30

Accumulating evidence has verified that the aberrant expression level of miR-493−3p is often associated with the occurrence of numerous cancers. Nevertheless, the expression level and effect of this microRNA in ovarian cancer (OC) remain largely unclear. Therefore, the molecular function of miR-493−3p in OC progression was systematically investigated in this study.The expression of miR-493−3p and DPY30 was assessed by qRT-PCR. The protein expression level of DPY30 in cell lines was further assessed by western blot. Cell viability was respectively examined in vitro functional experiments including CCK-8 assay, EdU assay, wound healing assay, colony formation and apoptosis assays as well as the scratch test and transwell assay. Bioinformatics analysis and luciferase reporter assays were performed to predict and clarity of the correlation between miR-493−3p and DPY30.The expression of miR-493−3p was significantly reduced in OC tissues and cells. Functional experimental results showed that miR-493−3p suppressed cellular proliferation, migration, invasion, but promoted apoptosis in OC cells. Mechanistically, we also confirmed that DPY30 could be directly targeted by miR-493−3p based on bioinformatics and dual-luciferase reporter analysis. Rescue experiments results indicated that the inhibitory effect of miR-493−3p on cellular proliferation, migration and invasion and the promotive effect of miR-493−3p on apoptosis was abolished by DPY30 overexpression.Our findings demonstrated the antitumor effect of miR-493−3p through targeting DPY30 in ovarian cancer, indicating that miR-493−3p might represent a promising target for ovarian cancer diagnosis and treatment.

Downregulation of the long non-coding RNA MALAT1 in tenofovir-treated pregnant women with hepatitis B virus infection promotes immune recovery of natural killer cells via the has-miR-155-5p/HIF-1α axis

BackgroundCurrently, tenofovir disoproxil fumarate (TDF) treatment in pregnant women with hepatitis B virus (HBV) infection in the second trimester of pregnancy to reduce the HBV DNA load and block the mother-to-child transmission of HBV has become a consensus. An increasing number of studies have shown that lncRNAs play an important role in immune regulation; however, there are only a few studies on how lncRNAs affect the immune function of TDF-treated pregnant women with HBV infection. MethodsThe peripheral blood of pregnant women with HBV infection was collected before and after TDF treatment for whole-transcriptome sequencing; the differential expression of lncRNA MALAT1 in PBMCs and natural killer (NK) cells was verified by qRT-PCR. ELISA and flow cytometry were used to detect the effect of MALAT1 on NK-92 cells on IFN-γ secretion. Dual-luciferase reporter, qRT-PCR, and western blot analyses were used to verify the relationship between the expression levels of MALAT1, has-miR-155-5p and HIF-1α. ResultsAfter TDF treatment, the MALAT1 expression in the PBMCs of pregnant women with HBV infection, especially in NK cells, was significantly reduced. MALAT1 overexpression decreased IFN-γ secretion in NK-92 cells, while IFN-γ secretion increased after MALAT1 knockdown. Mechanistically, we identified MALAT1 as a competitive endogenous RNA that regulates HIF-1α expression by sponging has-miR-155-5p. Low HIF-1α expression resulted in increased IFNG expression in, and IFN-γ secretion by, NK cells. ConclusionsThus, MALAT1 may play an important role in NK cell-mediated immunity in TDF-treated pregnant women with HBV infection by regulating the has-miR-155-5p/HIF-1α axis.

Identification and Characterization of MicroRNAs Involving in Initial Sex Differentiation of Chlamys farreri Gonads.

Simple SummarySex formation of gonads encompasses two ancient and highly conserved biological processes, sex determination and sex differentiation. The processes are strictly regulated by a complex of gene networks. There is increasing evidence that miRNAs play key roles in many biological processes. however, information is limited in their contribution to sex differentiation in animals. In the present study, we identified the novel miRNAs involved in sex-related genes regulation and explored the miRNA–mRNA networks underlying the posttranscriptional regulation during the initial sex differentiation in Zhikong scallop, Chlamys farreri. Our findings provide an important basis for studying the sex differentiation mechanisms, as well as developing sex control techniques in bivalves.Research on expressional regulation of genes at the initial sex differentiation of gonads will help to elucidate the mechanisms of sex determination and differentiation in animals. However, information on initial sex differentiation of gonads is limited in bivalves. MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that can regulate the target gene expression at the posttranscription level by degrading the mRNA or repressing the mRNA translation. In the present study, we investigated the small RNAs transcriptome using the testes and ovaries of Zhikong scallop Chlamys farreri juveniles with a shell height of 5.0 mm, a critical stage of initial sex differentiation of gonads. A total of 75 known mature miRNAs and 103 novel miRNAs were identified. By comparing the expression of miRNAs between the ovary and testis, 11 miRNAs were determined to be differentially expressed. GO annotations and KEGG analyses indicated that many putative target genes that matched to these differentially expressed miRNAs participated in the regulation of sex differentiation. Furthermore, two selected miRNAs, cfa-novel_miR65 and cfa-miR-87a-3p_1, were confirmed to downregulate expressions of Foxl2 (a female-critical gene) and Klf4 (a male-critical gene), respectively, using a dual-luciferase reporter analysis. Our findings provided new insights into the initial sex differentiation of gonads regulated by miRNAs in bivalves.

Circular RNA Circ_0008043 promotes the proliferation and metastasis of hepatocellular carcinoma cells by regulating the microRNA (miR)-326/RAB21 axis

ABSTRACT Circular RNAs (circRNAs) are non-coding RNAs with covalently closed structures that modulate the progression of hepatocellular carcinoma (HCC). Here, we explored whether circ_0008043 regulated the biological function of HCC cells. Quantitative real-time polymerase chain reaction (qPCR) was used to detect circ_0008043, microRNA (miR)-326, and RAB21 levels. Expression of E-cadherin, N-cadherin, and vimentin was assessed using qPCR. Cell proliferation, migration, and invasion were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and transwell assays. Xenograft tumors were used to evaluate cell growth in vivo. The interaction between miR-326 and circ_0008043 or RAB21 was assessed using dual-luciferase reporter analysis and RNA pull-down analysis. The data illustrated that circ_0008043 and RAB21 were highly expressed, while miR-326 was expressed at less levels in HCC tissues and cells. Interfering with circ_0008043 suppressed cellular proliferation, migration, invasion, and cell growth. Circ_0008043 was confirmed to be an miR-326 sponge that targets RAB21. Rescue experiments showed that inhibiting miR-326 abrogated the effect induced by knockdown of circ_0008043, and overexpressed RAB21 abolished the effect induced by miR-326 overexpression. In summary, silencing of circ_0008043 impeded HCC progression by regulating the miR-326/RAB21 axis. These data suggest that circ_0008043 may have clinical value in the treatment of HCC.

MiR-526b-3p Inhibits the Resistance of Glioma Cells to Adriamycin by Targeting MAPRE1.

Background Cell resistance is the main reason for the high mortality in glioma. Adriamycin (ADR) is a treatment drug for glioma and often leads to chemoresistance. Previous studies have confirmed that the abnormal expression of microRNA (miRNA) affects the resistance of glioma cells. Methods RT-qPCR and western blot were conducted for detecting miR-526b-3p levels and related protein expressions. CCK8 assay, colony formation, flow cytometry, and Transwell were adopted to assess cell viability, proliferation, apoptosis, and metastasis. Moreover, downstream targets of miR-526b-3p were identified through a dual-luciferase reporter and RNA pull-down analysis. Results Nevertheless, miR-526b-3p functions on glioma cell resistance to ADR are not well characterized. This study demonstrated that miR-526b-3p levels were decreased within glioma cells and further decreased within ADR-resistant glioma cells. Then, miR-526b-3p overexpression repressed glioma cell proliferation and invasion while inducing cell apoptosis. Overexpression of miR-526b-3p within ADR-resistant glioma cells obtained similar results, which suggested miR-526b-3p suppressed glioma cell resistance to ADR. Mechanistically, miR-526b-3p targeted MAPKE1 and negatively regulated MAPKE1 expressions. Restoration of MAPKE1 levels reversed miR-526b-3p effects on the glioma process and resistance to ADR. Conclusion These results suggest that miR-526b-3p acts as a diagnostic marker in glioma development and therapeutic target of the glioma resistance to ADR.