MiR-371-5p regulates trophoblast cell proliferation, migration, and invasion by directly targeting ZNF516.

Despite its prevalence, preeclampsia (PE) remains unclear as to its etiology. Here, we aimed to investigate the mechanisms regulating differences in the gene expression of zinc-finger protein 516 (ZNF516) in the placenta. The expression of the placental ZNF516 gene and its association with critical clinical markers were verified, and a rigorous correlation analysis was conducted. With a dual-luciferase reporter gene assay, microRNA targeting the ZNF516 gene was predicted and confirmed. Finally, the molecular processes associated with ZNF516 were explored via microarray and bioinformatic analyses. In hypoxic conditions, miR-371-5p expression was reduced, resulting in ZNF516 expression being induced. Moreover, ZNF516 was shown to hinder trophoblast cell migration and invasion while enhancing trophoblast cell death in various in vitro cellular assays, such as cell counting kit-8, colony formation, wound healing, and Transwell assays. Our findings reveal a new regulatory network facilitated by ZNF516. ZNF516 overexpression inhibits trophoblast growth, movement, and penetration, potentially causing problems with placenta formation with the help of miR-371-5p suppression.

Whole transcriptome sequencing reveals core genes related to spermatogenesis in Bactrian camels.

Bactrian camels survive and reproduce better in extreme climatic conditions than other domestic animals can. However, the reproductive efficiency of camels under their natural pastoral conditions is low. Several factors affect mammalian reproductive performance, including testicular development, semen quality, libido, and mating ability. Testis is a main reproductive organ of the male and is responsible for producing spermatozoa and hormones. However, our understanding of the expression patterns of the genes in camel testis is minimal. Thus, we performed total RNA-sequencing to investigate the gene expression pattern. As a result, 1,538 differential expressed mRNAs (DEmRNAs), 702 differential expressed long non-coding RNAs (DElncRNAs), and 61 differential expressed microRNAs (DEmiRNAs) were identified between pubertal and adult Bactrian camel testes. Then the genomic features, length distribution, and other characteristics of the lncRNAs and mRNAs in the Bactrian camel testis were investigated. Target genes of the DEmiRNAs and DEmRNAs were further subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Genes, such as AMHR2, FGF1, ACTL7A, GATA4, WNT4, ID2, LAMA1, IGF1, INHBB, and TLR2, were mainly involved in the TGF-ß, PI3K-AKT, Wnt, GnRH, and Hippo signaling pathways which relate to spermatogenesis. Some of the DEmiRNAs were predicted to be associated with numerous DElncRNAs and DEmRNAs through competing endogenous RNA (ceRNA) regulatory network. At last, the candidate genes were validated by RT-qPCR, dual fluorescent reporter gene, and a fluorescence in situ hybridization (FISH) assay. This research provides high-throughput RNA sequencing data of the testes of Bactrian camels across different developmental stages. It lays the foundation for further investigations on lncRNAs, miRNAs, and mRNAs that involved in Bactrian camel spermatogenesis.

Bactrian camel breeding has a long history and has played an extremely important role in desert and semi-desert management and grassland culture, economy, and ecological development. As a precious livestock resource, the Bactrian camel has developed into an important part of China's grassland livestock industry. However, due to their biological characteristics, camels have lower fertility than other livestock. Fertility is one of the most important factors affecting camel productivity. Maintaining a high level of fertility is essential to improve their performance and genetic improvement. Fertility is mainly related to testicular development and regulation of gene expression during spermatogenesis. Therefore, the study of genes related to testicular development and spermatogenesis and the elucidation of their molecular mechanisms are important for improving and protecting male fertility and preventing male reproductive disorders. This study provided a theoretical foundation for further research into the molecular mechanisms of testis development and spermatogenesis in Bactrian camels by constructing the lncRNA-miRNA-mRNA regulatory interactions network.

LncRNA SNHG12 Decreases Non-Small Cell Lung Cancer Cell Sensitivity to Cisplatin by Repressing miR-525-5p and Promoting XIAP.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is recognized as one of the primary causes of global cancer-related mortality. Long noncoding RNAs (lncRNAs) participate in NSCLC cell progression. This study probed the potential mechanism of lncRNA small nucleolar RNA host gene 12 (SNHG12) in cisplatin (DDP)-resistance in NSCLC cells. METHODS: The intracellular expressions of SNHG12, miR-525-5p, and XIAP were examined via reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Afterwards, small interfering RNAs (siRNAs) of SNHG12, microRNA (miR)-525-5p inhibitor, and X-linked inhibitor of apoptosis (XIAP) pcDNA3.1 were transfected into NSCLC cells. Subsequently, changes in half-maximal (50%) inhibitory concentration (IC50) of NSCLC cells to DDP were detected through the cell counting kit-8 (CCK-8) method. NSCLC proliferative ability and apoptosis rate were determined with the help of colony formation and flow cytometry assays. The subcellular localization of SNHG12 was analyzed by nuclear/cytosol fractionation assay and binding relationships between miR-525-5p and SNHG12 or XIAP were analyzed via dual-luciferase reporter gene assay. Furthermore, rescue experiments were designed to detect the effects of miR-525-5p and XIAP on NSCLC sensitivity to DDP. RESULTS: SNHG12 and XIAP were up-regulated in NSCLC cells while miR-525-5p was down-regulated. After DDP treatment and SNHG12 repression, NSCLC proliferative ability was decreased whereas apoptosis rate was increased, and NSCLC sensitivity to DDP was enhanced. Mechanically, SNHG12 repressed miR-525-5p expression, and miR-525-5p could targeted inhibit XIAP transcription level. miR-525-5p repression or XIAP overexpression reduced NSCLC sensitivity to DDP. CONCLUSION: SNHG12 was overexpressed in NSCLC cells and promoted XIAP transcription by repressing miR-525-5p expression, enhancing DDP-resistance in NSCLC cells.

Machine learning and experimental validation to construct a metastasis-related gene signature and ceRNA network for predicting osteosarcoma prognosis.

OBJECTIVE: Osteosarcoma (OS) is more common in adolescents and significantly harmful, and the survival rate is considerably low, especially in patients with metastatic OS. The identification of effective biomarkers and associated regulatory mechanisms, which predict OS occurrence and development as well as improve prognostic accuracy, will help develop more refined protocols for OS treatment. METHODS: In this study, genes showing differential expression in metastatic and non-metastatic types of OS were identified, and the ones affecting OS prognosis were screened from among these. Following this, the functions and pathways associated with the genes were explored via enrichment analysis, and an effective predictive signature was constructed using Cox regression based on the machine learning algorithm, least absolute shrinkage and selection operator (LASSO). Next, a correlative competing endogenous RNA (ceRNA) regulatory axis was constructed after verification by bioinformatics analysis and luciferase reporter gene experiments conducted based on the prognostic signature. RESULTS: Overall, 251 differentially expressed genes were identified and screened using bioinformatics and double luciferase reporter gene experiments. An effective prognostic signature was constructed based on 15 genes associated with OS metastasis, and upstream non-coding RNAs were identified to construct the "NBR2/miR-129-5p/FKBP11" regulatory axis based on the ceRNA networks, which helped identify candidate biomarkers for the OS clinical diagnosis and treatment, drug research, and prognostic prediction, among other applications. The findings of this study provide a novel strategy for determining the mechanism underlying OS occurrence and development and the appropriate treatment.

The expression profile of miR-222b-5p/MAPK10 in spleens of SPF chickens infected with REV-SNV at 28-42 dpi.

Reticuloendotheliosis virus (REV) is an avian oncogenic retrovirus that causes atrophy of immune organs, such as the spleen, thymus, and bursa of Fabricius, leading to severe immunosuppression. However, there is limited information describing the genes or microRNAs (miRNAs) that play a role in replicating REV-spleen necrosis virus (SNV). Our previous miRNA and RNA sequencing data showed that the expression of gga-miR-222b-5p was significantly upregulated in REV-SNV-infected chicken spleens of 7, 14, and 21 dpi compared to non-infected chicken spleens, but mitogen-activated protein kinase 10 (MAPK10), which is related to innate immunity, had the opposite expression pattern. To understand chicken cellular miRNA function in the virus-host interactions during REV infection, we used quantitative reverse transcription PCR (qRT-PCR) to determine whether the expression of gga-miR-222b-5p and MAPK10 in the spleen of specific-pathogen-free chickens at 28, 35, and 42 dpi was consistent with the first 3 time points, and dual-luciferase reporter assay was used to determine the targeting relationship between gga-miR-222b-5p and MAPK10. Results show that MAPK10 was downregulated at all 3 time points; however, significant difference (p⟨0.01) was noted only at 35 dpi. Moreover, the expression of gga-miR-222b-5p was upregulated; however, significant difference (p⟨0.01) was observed only at 28 and 35 dpi. A dual-luciferase reporter assay showed that MAPK10 is a direct target of gga-miR-222b-5p. This study suggests that gga-miR-222b-5p may target MAPK10 to promote the REV-SNV-induced tumorigenesis via the RLRs signaling pathway.

Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale.

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

MiR-518a-5p Targets GZMB to Extenuate Vascular Endothelial Cell Injury Induced by Hypoxia-Reoxygenation and Thereby Improves Myocardial Ischemia.

To probe the function of miR-518a-5p/Granzyme B (GZMB) in hypoxia/reoxygenation (H/R) -induced vascular endothelial cell injury.The key genes of myocardial infarction were screened by bioinformatic methods. The upstream micro RNAs (miRNAs) of GZMB were predicted by TargetScan. The binding of miR-518a-5p to GZMB was verified with luciferase reporter assay. The H/R model was constructed with human vascular endothelial cell (HUVEC) in vitro. Cell Counting Kit-8 (CCK8) assay was performed to detect cell proliferation. Western blot was utilized to evaluate the levels of indicated proteins.GZMB was up-regulated in patients with myocardial infarction and identified as the key gene by the bioinformatics analysis. Then the prediction from TargetScan indicated that miR-518a-5p, which is down-regulated in myocardial infarction patients, might be the potential upstream miRNA for GZMB. The following experiments verified that miR-518a-5p could bind to the 3'UTR of GZMB and negatively modulates GZMB expression. More importantly, the miR-518a-5p mimic enhanced cell proliferation and repressed apoptosis of H/R-injured HUVEC cells by inhibiting GZMB expression.We proved that miR-518a-5p could partly attenuate H/R-induced HUVEC cell injury by targeting GZMB, and perhaps the miR-518a-5p/GZMB axis could be potential therapeutic targets for myocardial infarction.

Association between the group III metabotropic glutamate receptor gene polymorphisms and attention-deficit/hyperactivity disorder and functional exploration of risk loci.

Existing evidence suggests that the group III metabotropic glutamate receptor (mGluR) gene variations are involved in attention-deficit/hyperactivity disorder (ADHD), but few studies have fully explored this association. We conducted a case-control study with 617 cases and 636 controls to investigate the association between functional single-nucleotide polymorphisms (SNPs) from the group III mGluR gene polymorphisms (GRM4, GRM7, GRM8) and ADHD in the Chinese Han population and initially explored the function of positive SNPs. The GRM4 rs1906953 T genotype showed a significant association with a decreased risk of ADHD (TT:CC, OR = 0.55, 95% CI = 0.40-0.77; recessive model, OR = 0.58, 95% CI = 0.43-0.78). GRM7 rs9826579 C showed a significant association with an increased risk of ADHD (TC:TT, OR = 1.81, 95% CI = 1.39-2.36; dominant model, OR = 1.74, 95% CI = 1.35-2.24; additive model, OR = 1.56, 95% CI = 1.24-1.97). In addition, compared with subjects with the rs1906953 TT genotype, subjects with of the CC genotype showed more obvious attention deficit behaviours and hyperactivity/impulsive behaviours. Dual-luciferase reporter gene assays showed that a promoter reporter with the rs1906953 TT genotype significantly decreased luciferase activity compared with the CC genotype. According to electrophoretic mobility shift assays, the binding capacity of rs1906953 T probe with nucleoprotein was lower than that of the rs1906953 C probe. Our results revealed the association of GRM4 rs1906953 and GRM7 rs9826579 with ADHD. Moreover, we found that rs1906953 disturbs the transcriptional activity of GRM4.

Signaling in TNFSF15-mediated Suppression of VEGF Production in Endothelial Cells.

Vascular endothelial growth factor (VEGF) plays a pivotal role in promoting neovascularization. Tumor necrosis factor superfamily 15 (TNFSF15) is an antiangiogenic cytokine prominently produced by endothelial cells in a normal vasculature. In this study, Western blot, quantitative polymerase chain reaction (qPCR), and dual luciferase reporter gene assay were used to validate the mechanisms of TNFSF15-mediated suppression of VEGF production in endothelial cells. We report that TNFSF15 inhibits VEGF production via microRNA-29b (miR-29b) targeting the 3'-UTR of VEGF transcript in mouse endothelial cell line bEnd.3. Neutralizing antibody against TNFSF15, 4-3H, inhibits the level of miR-29b and reinvigorates VEGF. In addition, TNFSF15 activates the JNK signaling pathway as well as the transcription factor GATA3, resulting in enhanced miR-29b production. SP600125, an inhibitor of JNK, eradicates TNFSF15-induced GATA3 expression. Moreover, GATA3 siRNA suppressed TNFSF15-induced miR-29b expression. Together, this study provides evidence and method of activation of the JNK-GATA3 signaling pathway by TNFSF15 that suppresses VEGF gene expression, which gives rise to upregulation of miR-29b.

MiR-24 Protects Cardiomyocytes Against Hypoxia/Reoxygenation-Induced Injury Through Regulating Mitogen-Activated Protein Kinase 14.

This study aimed to explore the function of miR-24 in hypoxia/reoxygenation (H/R) -induced cardiomyocyte injury.We constructed a cardiomyocyte model of H/R using the primary cardiomyocytes isolated from Sprague-Dawley rats. To explore the role of miR-24, cells were transfected with a miR-24 mimic or miR-24 inhibitor. The RNA expression levels of miR-24 and Mapk14 were determined using qRT-PCR. The proliferation and apoptosis of cells were determined using a CCK8 assay and a flow cytometer. The TargetScan website was used to predict the targets of miR-24. A dual-luciferase reporter gene assay was conducted to verify whether Mapk14 is indeed a target of miR-24. A Western blot was applied for protein detection.H/R exposure decreased the expression of miR-24 in rat cardiomyocytes. Transfection of the miR-24 mimic into cardiomyocytes reduced H/R-induced injury as evidenced by an increase in proliferation and a decrease in the apoptotic rate. By contrast, transfection of the miR-24 inhibitor aggravated H/R-induced injury. The expression of Bcl-2 was increased while the levels of Bax and Active-caspase 3 were reduced in the H/R+miR-24 mimic group compared to those in the H/R group. H/R+miR-24 inhibitor group showed the opposite results. Mapk14 was identified as a target of miR-24. The mRNA level of Mapk14 and its protein (p38 MAPK) level were negatively affected by miR-24. Furthermore, we discovered that depletion of Mapk14 reduced the promoting effect of the miR-24 inhibitor on cell apoptosis.Overall, our results illustrated that miR-24 could attenuate H/R-induced injury partly by regulating Mapk14.

[MicroRNA-29a-3p regulates osteoblast differentiation and peri-implant osseointegration in a rat model of hyperlipidemia by modulating Frizzled 4 expression].

OBJECTIVE: This work aimed to study and identify the influence and target gene of microRNA-29a-3p (miR-29a-3p) in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a high-fat environment in vitro and in vivo. METHODS: 1) In vitro: BMSCs were randomly allocated into two groups and were then induced to undergo osteogenic differentiation in a normal or high-fat environment. Next, a miR-29a-3p mimic/inhibitor was transfected into the two groups of cells. The mRNA expression levels of alkaline phosphatase (ALP), Runt related gene 2 (Runx2), and miR-29a-3p and the protein expression levels of ALP and Runx2 were detected before and after transfection through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Moreover, Frizzled (Fzd) 4 was predicted as the target gene of miR-29a-3p by using an online database (Target Scan, MiRNA.org). The interactive relationship between miR-29a-3p and Fzd4 was confirmed through dual-luciferase assays. 2) In vivo: Rats were randomly divided into two groups and fed with a standard or high-fat diet. Titanium implants were grown in rats. Then, the expression levels of miR-29a-3p, ALP, and Runx2 were detected in bone tissues surrounding implants. Moreover, hard tissue sections were subjected to methylene blue-acid magenta staining and observed under microscopy to study bone formation around implants. In addition, miR-29a-3p-overexpressing lentiviral vectors were transfected into rats, and the expression levels of ALP, Runx2, and miR-29a-3p in bone tissues surrounding implants were detected at 3 and 10 days after transfection. RESULTS: The expression levels of ALP, Runx2, and miR-29a-3p and the osteogenic differentiation of BMSCs were suppressed in high-fat groups in vitro and in vivo. CONCLUSIONS: MiR-29a-3p plays a positive role in the regulation of BMSCs in a high-fat environment. It can increase ALP and Runx2 expression levels in bone tissues surrounding implants in hyperlipidemia models. This result implies that miR-29a-3p can promote implant osseointergration in a rat model of hyperlipidemia.

Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays.

OBJECTIVE: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. METHODS: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3'-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3'-UTR and pGL3 Control LDLR mutant-3'-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3'-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. RESULTS: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3'-UTR-driven (p<0.01), but not mutant LDLR-3'-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = -0.656, p<0.05). CONCLUSION: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3'-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

MicroRNA-29a-3p regulates osteoblast differentiation and peri-implant osseointegration in a rat model of hyperlipidemia by modulating Frizzled 4 expression / 华西口腔医学杂志

OBJECTIVE@#This work aimed to study and identify the influence and target gene of microRNA-29a-3p (miR-29a-3p) in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a high-fat environment in vitro and in vivo.@*METHODS@#1) In vitro: BMSCs were randomly allocated into two groups and were then induced to undergo osteogenic differentiation in a normal or high-fat environment. Next, a miR-29a-3p mimic/inhibitor was transfected into the two groups of cells. The mRNA expression levels of alkaline phosphatase (ALP), Runt related gene 2 (Runx2), and miR-29a-3p and the protein expression levels of ALP and Runx2 were detected before and after transfection through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Moreover, Frizzled (Fzd) 4 was predicted as the target gene of miR-29a-3p by using an online database (Target Scan, MiRNA.org). The interactive relationship between miR-29a-3p and Fzd4 was confirmed through dual-luciferase assays. 2) In vivo: Rats were randomly divided into two groups and fed with a standard or high-fat diet. Titanium implants were grown in rats. Then, the expression levels of miR-29a-3p, ALP, and Runx2 were detected in bone tissues surrounding implants. Moreover, hard tissue sections were subjected to methylene blue-acid magenta staining and observed under microscopy to study bone formation around implants. In addition, miR-29a-3p-overexpressing lentiviral vectors were transfected into rats, and the expression levels of ALP, Runx2, and miR-29a-3p in bone tissues surrounding implants were detected at 3 and 10 days after transfection.@*RESULTS@#The expression levels of ALP, Runx2, and miR-29a-3p and the osteogenic differentiation of BMSCs were suppressed in high-fat groups in vitro and in vivo.@*CONCLUSIONS@#MiR-29a-3p plays a positive role in the regulation of BMSCs in a high-fat environment. It can increase ALP and Runx2 expression levels in bone tissues surrounding implants in hyperlipidemia models. This result implies that miR-29a-3p can promote implant osseointergration in a rat model of hyperlipidemia.

[Establishment of a suitable control reporter plasmid of a dual luciferase reporter gene system for hormone research in silkworm cell lines].

The dual luciferase reporter gene system provides sensitive readout, while it relies on a constitutively-expressed control gene for readout normalization. However, most standard control reporter genes are not constitutively expressed under all conditions. Here, we report an effective method to construct a control reporter plasmid for the dual luciferase reporter gene system that would be suitable for hormone research in silkworm cell lines. First, we modified BmVgP78M, a stably-expressed constitutive promoter in silkworm cells by mutating its hormone-related element. Then, we constructed the pRL-VgP78M control reporter plasmid by replacing the SV40 promoter and chimeric intron sequences in pRL-SV40 with the BmVgP78M sequence. Finally, we confirmed that the pRL-VgP78M control reporter plasmid could be stably expressed in silkworm cell lines via cell transfection experiments, and it was unresponsive to the induction of ecdysone, juvenile hormone, or their transcription factors. We thus obtained a control reporter plasmid pRL-VgP78M that could be expressed stably and moderately in silkworm cells. It can be readily used as the control reporter plasmid of the dual luciferase reporter gene system for hormone research in silkworm cell lines. It will also provide a reference for construction of control reporter plasmids of dual luciferase reporter gene systems that are adaptable to cell lines isolated from other species.

[Expression and Regulatory Effect of miR-30b on Dynamin in Cochlear Hair Cells].

OBJECTIVE: To determine the expression of miR-30b in hair cells of mice,and its regulatory effect on the target gene DNM1 and expression of Dynamin,the key protein of synaptic endocytosis in inner hair cells. METHODS: The basilar membrane of cochlear in adult C57 mice was obtained. The expression of miR-30b in the hair cells was detected by in situ hybridization. Luciferase vector was constructed and transfected into 293T cells with miR-30b. Changes in luciferase activity were measured to verify whether DNM1 was the target gene of miR-30b. Adeno-associated virus carrying miR-30b were micro-injected into cochlear via the round window membrane. mRNA expressions of DNM1 and miR-30b were detected by RT-PCR 14 days later. The expression of Dynamin was detected by Western blot. RESULTS: miR-30b expressed in the inner and outer hair cells scattered in the region of the nucleus and cytoplasm. miR-30b reduced luciferase activity from the reporter vector containing DNM11 (P<0.05),but not in its mutants. Increased expressions of miR-30b and decreased mRNA expressions of DNM1 and Dynamin were observed following transfection of AAV-miR-30b. CONCLUSION: miR-30b expresses in inner and outer hair cells,which is consistent with the morphological orientation of dynamin. miR-30b inhibits the expression of Dynamin by targeting DNM11 gene.

Prediction and identification of transcriptional regulatory elements at the lung cancer-specific DKK1 locus.

The glycoprotein dickkopf 1 (DKK1) is highly expressed in lung cancer cell lines and tissues. Our previous study demonstrated that DKK1 promoter activity is low in lung cancer cell lines. This may be because it lacks the necessary transcriptional regulatory elements (TREs) required for higher activity levels. However, it is difficult to computationally predict functionally significant TREs, as TREs from different locations can affect large segments of distant DNA. The Encyclopedia of DNA Elements project features multiple integrated technologies and approaches for the discovery and definition of functional elements, including enhancer elements and enhancer-blocking insulators. In the present study, DNase I hypersensitive sites and histone modifications of DKK1 were investigated in the A549 lung cancer cell line using the UCSC Genome Browser. A set of cis-acting enhancer elements were identified by a dual-luciferase reporter gene assay system to increase activity of the DKK1 promoter with lung cancer specificity. To the best of our knowledge, these data provide the first insight into the role of the DKK1 locus in lung cancer, and confirm the contribution of intronic cis-acting elements to the regulation of DKK1 expression, providing a new insight into gene regulation in lung cancer, which could inform the development of targeted therapy.

Long noncoding RNA PVT1 enhances the viability and invasion of papillary thyroid carcinoma cells by functioning as ceRNA of microRNA-30a through mediating expression of insulin like growth factor 1 receptor.

OBJECTIVE: Invasion and metastasis of papillary thyroid carcinoma (PTC) significantly affects prognosis and quality of life of patients. Herein, we explored the binding relationship of long noncoding RNA PVT1 as ceRNA to microRNA-30a (miR-30a), and their effect on the development of PTC through regulating insulin like growth factor 1 receptor (IGF1R). METHODS: PTC and adjacent normal tissues were collected, where the qRT-PCR and western blot assay were employed to evaluate the expression levels of PVT1, miR-30a and IGF1R. The correlation between PVT1 expression and clinicopathological characteristics of PTC patients was observed. PTC cell lines with the most/least significant difference from normal thyroid cells were selected and treated with siRNA PVT1 or overexpression PVT1 plasmids, miR-30a mimics or miR-30a inhibitors. Nucleus and cytoplasm segmentation was used to identify subcellular fractionation of PVT1. The binding relationship of PVT1 to miR-30a and the targeting relationship of miR-30a to IGF1R were confirmed by using bioinformatic prediction program, dual-luciferase reporter gene assay and RNA-pull down. Cell viability, cell cycle and apoptosis, invasion and migration capacities were assessed by MTT, flow cytometry, Transwell assay and scratch test, respectively. Western blot assay was employed to examine protein expression of IGF1R, apoptosis-related factors (caspase-3, cleaved capase-3) and epithelial-mesenchymal transition (EMT)-related factors (E-cadherin, Vimentin). RESULTS: In the PTC tissues and cells, PVT1 and IGF1R were highly expressed and miR-30a was poorly expressed. PVT1 exerted its effects on PTC mainly in the cytoplasm. The PVT1 expression was correlated with TNM staging, LNM and tumor infiltration of PTC. The competitive binding of PVT1 to miR-30a enhanced expression of IGF1R. In the in vitro experiments, BCPAP and TPC-1 cells were selected. When subjected to siRNA PVT1 or miR-30a mimics, BCPAP and TPC-1 cells exhibited inhibited proliferation, cell cycle progression, invasion, migration, EMT (increased E-cadherin and reduced Vimentin) and promoted apoptosis (reduced caspase-3 and increased cleaved capase-3), and moreover, the expression of IGF1R was reduced. CONCLUSION: This study provides evidence that long noncoding RNA PVT1 enhances the expression of IGF1R through competitive binding to miR-30a, whereby PVT1 facilitates the development of PTC.

Establishment of a suitable control reporter plasmid of a dual luciferase reporter gene system for hormone research in silkworm cell lines / 生物工程学报

The dual luciferase reporter gene system provides sensitive readout, while it relies on a constitutively-expressed control gene for readout normalization. However, most standard control reporter genes are not constitutively expressed under all conditions. Here, we report an effective method to construct a control reporter plasmid for the dual luciferase reporter gene system that would be suitable for hormone research in silkworm cell lines. First, we modified BmVgP78M, a stably-expressed constitutive promoter in silkworm cells by mutating its hormone-related element. Then, we constructed the pRL-VgP78M control reporter plasmid by replacing the SV40 promoter and chimeric intron sequences in pRL-SV40 with the BmVgP78M sequence. Finally, we confirmed that the pRL-VgP78M control reporter plasmid could be stably expressed in silkworm cell lines via cell transfection experiments, and it was unresponsive to the induction of ecdysone, juvenile hormone, or their transcription factors. We thus obtained a control reporter plasmid pRL-VgP78M that could be expressed stably and moderately in silkworm cells. It can be readily used as the control reporter plasmid of the dual luciferase reporter gene system for hormone research in silkworm cell lines. It will also provide a reference for construction of control reporter plasmids of dual luciferase reporter gene systems that are adaptable to cell lines isolated from other species.

Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons.

Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor (GDNF) in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin (sh)RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 shRNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region (GDNF-luciferase) were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-shRNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-shRNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.

A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic ß-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.