Luciferase Reporter Plasmid Research Articles

Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2.

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

Abstract PS13-22: Shedding light on the dark side of chemo: Post-GWAS functional studies of rs28714259 in anthracycline-induced cardiotoxicity

Abstract Anthracyclines are commonly used chemotherapies for breast cancer treatment, but can cause dose-related cardiotoxicities and lead to congestive heart failure (CHF) in 2% of patients. Several mechanisms of anthracycline’s cardiotoxic effect have been proposed, but the molecular pathogenesis is not fully understood. In addition, there are no clinically available biomarkers to predict cardiotoxicity. Previously we have identified and validated the association of a single nucleotide polymorphism (SNP), rs28714259 (G/A), with risk of anthracycline-induced CHF through genome-wide association study (GWAS) across three large phase III adjuvant breast cancer trials. rs28714259 (G/A) locates in a putative glucocorticoid receptor (GR) response element and the risk allele (A) is predicted to disrupt GR binding. The activation of GR signaling pathway by dexamethasone is known to protect cardiomyocytes from doxorubicin-induced apoptosis in rats. To investigate the role of rs28714259 in CHF post-GWAS, we set out to determine whether rs28714259 modulates GR signaling pathway through allele-specific GR enhancer activity. We cloned a 1kb DNA sequence on both sides of rs28714259, containing either the wildtype (G) or risk allele (A), into a luciferase reporter plasmid. Luciferase assay in iPSC-derived cardiomyocytes (iPSC-CMs) with GR activation by 100nM dexamethasone showed that cardiomyocytes transfected with wildtype construct had 60% increased activity compared to control vector with no enhancer. iPSC-CMs transfected with risk allele constructs did not show increased luciferase activity, suggesting that the A-allele disrupts GR-mediated transcriptional activation. Using electrophoretic mobility shift assay (EMSA) with nuclear extract from iPSC-CMs treated with dexamethasone, we observed a prominent band shift with either G- or A-allele probes. Furthermore, a supershift band was observed with GR antibody, confirming that GR indeed binds to the rs28714259 region. Notably, the band intensity of risk allele probes decreased by 50% compared to wildtype, suggesting weaker GR binding affinity to risk allele probes, consistent with reduced transcriptional activation. Finally, to identify genes differentially regulated by rs28714259 with anthracycline exposure, we performed RNA-Seq analysis on iPSC-CMs of each genotype. RNA-Seq data revealed that the top differentially regulated network was the death receptor pathway including: FADD, FAS, and Caspase-8; these effectors are known to induce apoptosis in response to doxorubicin. Moreover, 11 genes in the GR signaling pathway were also differentially regulated by rs28714259. Taken together, these findings suggest that the rs28714259 variant may possess allele-specific GR enhancer activity and differentially regulates genes involved in doxorubicin-induced apoptosis. Citation Format: Xi Wu, Gloria Xue, Fei Shen, Guanglong Jiang, Santosh Philips, Geneva Cunningham, Erica Cantor, Bryan P Schneider. Shedding light on the dark side of chemo: Post-GWAS functional studies of rs28714259 in anthracycline-induced cardiotoxicity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-22.

Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1

Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor κB ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) in vitro by targeting ARHGAP1, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs.

Botanical formulation, TADIOS, alleviates lipopolysaccharide (LPS)-Induced acute lung injury in mice via modulation of the Nrf2-HO-1 signaling pathway

Ethnopharmacological relevanceTADIOS is an herbal formulation prepared from a mixture of Taraxacum officinale (L.) Weber ex F.H.Wigg, Dioscorea batatas Decaisne and Schizonepeta tenuifolia (Benth.) Briquet. These plants have traditionally been used in Asia to treat a variety of respiratory diseases. A bulk of literature on traditional Korean medicine describe their activities and functions for respiratory problems. Therefore, we hypothesized that the combination of these plants might be effective in alleviating respiratory symptoms. Aim of the studyIn this study, we investigated whether TADIOS ameliorates LPS-induced acute lung injury via regulation of the Nrf2-HO-1 signaling pathway. Materials and methodsThe LPS-induced acute lung injury mouse model was used to determine the anti-inflammatory and anti-oxidative stress effects of TADIOS. The amount of marker compounds contained in TADIOS was quantified using high-performance liquid chromatography (HPLC) analysis. The protein level of pro-inflammatory cytokines in culture supernatant was measured by ELISA. Changes in the RNA level of pro-inflammatory cytokines in mice lungs and RAW264.7 cells were measured by quantitative RT-PCR. The relative amounts of reactive oxygen species (ROS) were measured by DCF-DA assay. Western blot analysis was used to evaluate expression of cellular proteins. Effects of TADIOS on antioxidant responsive elements (AREs) were determined by luciferase assay. The severity of acute lung injury was evaluated by Hematoxylin & Eosin (H&E) staining. To test the effects of TADIOS on LPS-induced oxidative stress, myeloperoxidase (MPO) activity and the total antioxidant capacity were measured. ResultsTADIOS was prepared by extraction of a blend of these three plants by ethanol, and quality control was performed through quantification of marker compounds by HPLC and measurement of bioactivities using cell-based bioassays. In the murine macrophage cell line RAW264.7, TADIOS effectively suppressed the production of pro-inflammatory cytokines such as IL-6 and IL-1β, and also ROS induced by LPS. When RAW264.7 cells were transfected with a luciferase reporter plasmid containing nucleotide sequences for AREs, TADIOS treatment increased the level of relative luciferase units in a dose-dependent manner. In the LPS-induced acute lung injury mouse model, orally administered TADIOS alleviated lung damage and neutrophil infiltration induced by LPS. Consistent with the in vitro data, treatment with TADIOS inhibited the LPS-mediated expression of pro-inflammatory cytokines and oxidative stress, and activated the Nrf2-HO-1 axis. ConclusionOur data suggest the potential for TADIOS to be developed as a safe and effective therapeutics for the treatment of acute respiratory distress syndrome.

The interaction of TEA domain transcription factor 4 (TEAD4) and Yes-associated protein 1 (YAP1) promoted the malignant process mediated by serum/glucocorticoid regulated kinase 1 (SGK1)

ABSTRACT TEA domain transcription factor 4 (TEAD4) has been investigated to be implicated in the progression of various cancers, and it plays a role in the esophageal squamous cell carcinoma (ESCC). The study was designed to investigate how TEAD4 affected the progression of ESCC through Hippo signaling pathway in vitro and in vivo. The interaction of TEAD4 and Yes-associated protein (YAP) was detected though immunoprecipitation assay (IP). Following the treatment of TED-347, which was able to suppress the interaction of TEAD4 and YAP1, the malignant behaviors of cells including proliferation, invasion, and migration were assessed by EDU staining, wound healing, and transwell assay in vitro, while tumor growth was measured. Luciferase reporter plasmids containing the enhancer and promoter region of serum/glucocorticoid regulated kinase 1 (SGK1) were constructed to analyze how TEAD4 affected the transcription of SGK1. The above cell behaviors were further analyzed after the silencing of SGK1. Results showed that TED-347 hindered the promoting effect of TEAD4 overexpression on the malignant behaviors of ESCC cells, and this effect was related to the suppression of the TEAD4/YAP1 complex. Moreover, the promoter activity of SGK1 was obviously inhibited by TED-347. Decreased expression of SGK1 suppressed the above behaviors of cells and destroyed the effects of increased expression of TEAD4. Collectively, TEAD4/YAP promotes the malignant process of ESCC cells, which was inhibited by the interference of SGK1. Targeting TEAD4/YAP1 complex or SGK1 could find application in the treatment of esophageal squamous cell carcinoma.

The Myeloid Zinc Finger Gene, MZF-1, Regulates the CD34 Promoter In Vitro

MZF-1 is a C2H2 zinc finger gene encoding a putative transcriptional regulator of myeloid differentiation. The MZF-1 protein contains 13 C2H2 zinc fingers arranged in bipartite DNA binding domains containing zinc fingers through 4 and, in the carboxy-terminus, 5 through 13. We previously identified the DNA consensus binding site recognized by the two DNA binding domains. To assess the transcription regulatory function of MZF-1, the full-length MZF-1 coding region was fused to the DNA binding domain of the yeast transactivator GAL4. The expression vector was cotransfected with the chloramphenicol acetyl transferase (CAT) reporter gene regulated by the thymidine kinase promoter containing GAL4 DNA binding sites into NIH 3T3, 293, K562, and Jurkat cell lines. MZF-1 represses CAT reporter gene expression via GAL4 binding sites in the nonhematopoietic cell lines NIH 3T3 and 293. In contrast, MZF-1 activates CAT reporter gene expression in the hematopoietic cell lines K562 and Jurkat. The MZF-1 binding sites are present in the promoters of several genes expressed during myeloid differentiation, including the CD34 promoter. MZF-1 transcriptional regulation of this physiologically relevant promoter was assessed in both hematopoietic and nonhematopoietic cell lines. Recombinant MZF-1 protein specifically binds to the consensus binding sites in the CD34 promoter in mobility shift assays. MZF-1 expression vectors were cotransfected with the luciferase reporter plasmids regulated by the CD34 promoter into both nonhematopoietic and hematopoietic cell lines. As with the heterologous DNA binding domain, MZF-1 represses reporter gene expression in nonhematopoietic cell lines and activates expression in hematopoietic cell lines. Activation of CD34 expression in hematopoietic cell lines is dependent on the presence of intact MZF-1 binding sites. The cell type-specific regulation of the CD34 promoter by MZF-1 suggests the presence of tissue-specific regulators/adapters or differential MZF-1 modifications that determine MZF-1 transcriptional regulatory function.

The transcription factors SF-1 and SOX8 cooperate to upregulate Cx43 expression in mouse TM4 sertoli cells

Gap junctions made by connexins within the adult testis are essential for communication between Sertoli cells and for spermatogenesis. Sertoli cells play an important role in supporting germ cells differentiation and maturation into spermatozoa. Connexin43 (Cx43) is the most abundant and important connexin of the testis. We have shown previously that the expression of Cx43 is being regulated by SOX and AP-1 transcription factors in Sertoli cells. However, additional regulatory elements being able to recruit orphan nuclear receptors may be involved. Since SOX and SF-1 transcription factors have been shown to cooperate to regulate gene expression in Sertoli cells, we wondered if such mechanism could be involved in the activation of Cx43 expression. Thus, the activity of the Cx43 promoter was measured by co-transfections of luciferase reporter plasmid constructs with different expression vectors for transcription factors in the TM4 Sertoli cell line. The recruitment of SF-1 to the proximal region of the Cx43 promoter was evaluated by chromatin immunoprecipitation. Our results indicate that SOX8 and SF-1, as well as SOX9 and Nur77, cooperate to activate the expression of Cx43 and that SF-1 is being recruited to the −132 to −26 bp region of the Cx43 promoter. These results allow us to have a better understanding of the mechanisms regulating Cx43 expression and could explain some disturbances in communication between Sertoli cells responsible for impaired fertility.

MiR-18a Contributes to Preeclampsia by Downregulating Smad2 (Full Length) and Reducing TGF-β Signaling

The study investigated the regulation of Smad2 by miR-18a and its role in preeclampsia (PE). Bioinformatics analysis showed that both Smad2 and Smad3 were the predicted targets for miR-18a. Mass spectrum analysis showed that two mature Smad2 isoforms existed in human placenta: full length, Smad2(FL), and that lacking exon3, Smad2(Δexon3). The protein level of Smad2(FL), but not Smad2(Δexon3) or Smad3, was significantly increased in severe PE (sPE) placenta, which was inversely correlated with the level of miR-18a. Elevated Smad2(FL) phosphorylation level appeared in sPE placenta, and Smad2 was colocalized with miR-18a in various subtypes of trophoblasts in human placenta. Smad2(FL) was validated as the direct target of miR-18a in HTR8/SVneo cells. miR-18a enhanced trophoblast cell invasion, which was blocked by the overexpression of Smad2(FL). Furthermore, overexpression of miR-18a repressed Smad2 activation and the inhibition of trophoblast cell invasion by transforming growth factor-β (TGF-β). In conclusion, our results suggest that miR-18a inhibits the expression of Smad2(FL), but not Smad2(Δexon3) or Smad3, which can reduce TGF-β signaling, leading to the enhancement of trophoblast cell invasion. A lack of miR-18a, which results in the upregulation of Smad2(FL), contributes to the development of PE.

Effect and mechanism of miR-223 on fibrosis-related signaling pathway in rat cardiomyocytes

Objective: To investigate the protective effect of microRNA 223 (miR-223) on cardiac fibrosis-related signaling pathway and its regulation on expression of Twist family basic helix-loop-helix transcription factor 1 (Twist1) and transforming growth factor-β1 receptor 2 (TGFBR2) in rat cardiomyocytes. Methods: Rat cardiomyocytes (H9c2) were cultured in vitro and treated with TGF-β to induce myocardial fibrosis. The miR-223 group was transfected with miR-223 lentivirus and miR-223-NC group was transfected with miR-223-NC lentivirus. Model group and blank control group had no transfection. Immunocytochemistry staining of alpha-smooth muscle actin (α-SMA) was used to calculate myocardial fibrosis. The mRNA level of miR-223, collagen Ⅰ, collagen Ⅲ, Twist1 and TGFBR2 were detected by real-time PCR. The protein level of Twist1, TGFBR2, collagen Ⅰ, collagen Ⅲ and α-SMA were detected by Western blot. Target regulation of miR-223 on Twist1 and TGFBR2 3'UTR was verified by double luciferase reporter gene system. Results: The average optical density of α-SMA-positive cardiomyocytes in miR-223 group (0.089±0.013) was significantly lower than that in model group and miR-223-NC group (0.134±0.018, 0.132±0.016, respectively). The mRNA level of collagen Ⅰ, collagen Ⅲ, Twist1 and TGFBR2 in miR-223 group were significantly lower than that in model group and miR-223-NC group (all P<0.05). The protein level of Twist1, TGFBR2, collagen Ⅰ, collagen Ⅲ and α-SMA in miR-223 group was significantly lower than model group and miR-223-NC group (all P<0.05). Twist1, TGFBR2 3'UTR wild-type double luciferase reporter plasmids and miR-223 mimics were co-transfected in 293T cells, and luciferase activity was significantly reduced (0.48±0.06 vs 0.92±0.17 and 0.51±0.07 vs 0.94±0.12). Conclusion: MiR-223 may inhibit the activation of fibrosis-related signaling pathway in cardiomyocytes by down-regulating the expression of Twist1 andTGFBR2 genes.

Regulation of melanocortin-4-receptor (MC4R) expression by SNP rs17066842 is dependent on glucose concentration

Melanocortin-4-receptor (MC4R) gene codes for a G-protein-coupled receptor that is highly expressed in the hypothalamus and involved in the regulation of appetite. Single-nucleotide polymorphisms (SNPs) in the MC4R gene region have been associated with obesity, type 2-diabetes (T2D) and with antipsychotic-induced weight gain. Of these, rs17066842 (G>A) in the MC4R promoter region is the top variant associated with obesity and diabetes. In this study, we investigated the effect of rs17066842 on MC4R expression at various glucose concentrations using reporter gene expression in the SH-SY5Y cell line and regulation of MC4R expression in human cerebral organoids. We observed that higher glucose concentrations significantly reduced MC4R mRNA expression in SH-SY5Y cells. In addition, at high glucose concentrations, the luciferase reporter plasmid containing the MC4R promoter insert with the G-allele of rs170066842 showed significantly reduced activity compared to the A-allele carrying plasmid. The immediate early gene product, early growth-response 1 (EGR-1), was identified to bind to the sequence containing the G-allele at rs17066842 but not to the A-allele-containing sequence. Interestingly, in human induced pluripotent stem cell (hiPSC)-derived cerebral organoids, we observed increased MC4R expression in response to high glucose exposure. These opposite observations might suggest that glucose regulation is complex and may be cell-specific.This study provides evidence that rs17066842 regulates MC4R gene expression through binding of EGR-1 and that this process is influenced by glucose concentration.

Inhibition of miR-96 enhances the sensitivity of colorectal cancer cells to oxaliplatin by targeting TPM1.

Colorectal cancer (CRC) is one of the major threats to human health worldwide. In the treatment of CRC, chemoresistance affects the efficacy of platinum-based therapies. Oxaliplatin is one of the most commonly used first-line medications for the treatment of CRC; however, chemoresistance is common among patients receiving oxaliplatin treatment, which significantly decreases its therapeutic efficacy. The present study focused on the roles of microRNA (miR)-96 in the oxaliplatin resistance of CRC cells and the underlying mechanisms. First, the expression of miR-96 was compared between CRC and adjacent tissues. Furthermore, target genes of miR-96 were predicted, and a dual-luciferase reporter assay was employed to confirm whether the candidate tropomyosin 1 (TPM1) is a direct target of miR-96. In addition, CRC cells were transfected with miR-96 inhibitor, miR-negative control, small interfering RNA (siRNA) targeting TPM1 or siRNA NC, and then treated with oxaliplatin. CCK-8 assay and flow cytometry were performed to examine the proliferation and apoptosis of the CRC cell line SW480. Next, reverse transcription-quantitative PCR and western blot analysis were performed to determine the mRNA and/or protein levels of miR-96, Bcl-2, BAX and TPM1. The results indicated that miR-96 was upregulated in CRC compared with normal adjacent tissues, while TPM1 was downregulated. The luciferase activity was reduced following transfection with miR-96 mimics and luciferase reporter plasmid containing the wild-type sequence of the 3'-untranslated region of TPM1. Furthermore, knockdown of miR-96 combined with oxaliplatin reduced the viability and induced apoptosis of CRC cells, which was further verified by decreased expression of Bcl-2 and the increased expression of TPM1 and BAX. Taken together, the downregulation of miR-96 enhanced the sensitivity of CRC cells to oxaliplatin.

ARP-1 Regulates the Transcriptional Activity of the Aromatase Gene in the Mouse Brain.

An important function of aromatase in the brain is conversion of testosterone secreted from the testis into estradiol. Estradiol produced in the brain is thought to be deeply involved in the formation of sexually dimorphic nuclei and sexual behavior as a neurosteroid. We analyzed the brain-specific promoter to elucidate the control mechanisms of brain aromatase expression that may be highly involved in sexual differentiation of the brain. The 202-bp upstream region of the brain-specific exon 1 has three types of cis-acting elements, aro-AI, AII, and B. We isolated ARP-1 as an aro-AII-binding protein by yeast one-hybrid screening from a cDNA library of mouse fetal brains. ARP-1 is a member of the nuclear receptor superfamily and functions as an orphan-type transcription factor. ARP-1 protein synthesized in vitro showed the same binding property to the aro-AII site as nuclear extract from fetal brains. To determine how the promoter is involved in brain-specific transcription of the aromatase gene, we first detected the in vivo occupancy of the aro-AII site by ARP-1 using chromatin immunoprecipitation assays. Diencephalic regions of fetal brains at embryonic day 16 were analyzed, which revealed ARP-1 recruitment to the aro-AII site. To analyze the effects of ARP-1 on transcriptional regulation of the brain-specific aromatase promoter, a luciferase reporter plasmid driven by the brain-specific promoter was transfected into CV-1 cells together with a plasmid expressing ARP-1 protein. These analyses revealed that ARP-1 induced promoter activity in a dose-dependent manner. Furthermore, to determine whether ARP-1 is required for aromatase expression in neurons, ARP-1 knockdown was conducted in neuronal cell primary culture. Knockdown of ARP-1 significantly suppressed the increase in aromatase mRNA observed in cultured neurons. These results indicate that ARP-1 is involved in the transcriptional regulation of the brain-specific promoter of the aromatase gene.

Rapid and Efficient Synthetic Assembly of Multiplex Luciferase Reporter Plasmids for the Simultaneous Monitoring of Up to Six Cellular Signaling Pathways.

High-throughput cell-based screening assays are valuable tools in the discovery of chemical probes and therapeutic agents. Such assays are designed to examine the effects of small compounds on targets, pathways, or phenotypes participating in normal and disease processes. While most cell-based assays measure single quantities, multiplexed assays seek to address these limitations by obtaining multiple simultaneous measurements. The signals from such measurements should be independently detectable and cover large dynamic ranges. Luciferases are good candidates for generation of such signals. They are genetically encoded, versatile, and cost-effective, and their output signals can be sensitively detected. We recently developed a multiplex luciferase assay that allows monitoring the activity of five experimental pathways against one control simultaneously. We used synthetic assembly cloning to assemble all six luciferase reporter units into a single vector over eight stitching rounds. Because all six reporters are on a single piece of DNA, a single vector ensures stoichiometric ratios of each transcriptional unit in each transfected cell, resulting in lower experimental variation. Our proof-of-concept multiplex hextuple luciferase assay was designed to simultaneously monitor the p53, TGF-β, NF-κβ, c-Myc, and MAPK/JNK signaling pathways. The same synthetic assembly cloning pipeline allows the stitching of numerous other cellular pathway luciferase reporters. Here we present an improved three-step synthetic assembly protocol to quickly and efficiently generate multiplex hextuple luciferase reporter plasmids for other signaling pathways of interest. This improved assembly protocol provides the opportunity to analyze any five desired pathways at once much more quickly. Protocols are provided on how to prepare DNA components and destination vector plasmids, design synthetic DNA, perform assembly cloning of new transcriptional reporter elements, implement multipartite synthetic assembly cloning of single-pathway luciferase reporters, and carry out one-step assembly of final multiplex hextuple luciferase vectors. We present protocols on how to perform multiplex hextuple luciferase in an accompanying Current Protocols in Molecular Biology article. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of DNA parts and destination vectors for synthetic assembly cloning Basic Protocol 2: DNA synthesis and assembly cloning of a typical transcriptional reporter element Alternate Protocol: DNA synthesis and assembly cloning of a challenging transcriptional reporter element Basic Protocol 3: Multipartite synthetic assembly cloning of individual pathway luciferase reporters Basic Protocol 4: One step assembly into final multiplex hextuple luciferase vectors Support Protocol: Generation of home-made chemocompetent E. coli DH10B-T1R cells.

Simultaneous Examination of Cellular Pathways using Multiplex Hextuple Luciferase Assaying.

Multiplex experimentation that can assay multiple cellular signaling pathways in the same cells requires orthogonal genetically encoded reporters that report over large dynamic ranges. Luciferases are cost-effective, versatile candidates whose output signals can be sensitively detected in a multiplex fashion. Commonly used dual luciferase reporter assays detect one luciferase that is coupled to a single cellular pathway and a second that is coupled to a control pathway for normalization purposes. We have expanded this approach to multiplex hextuple luciferase assays that can report on five cellular signaling pathways and one control, each of which is encoded by a unique luciferase. Light emission by the six luciferases can be distinguished by the use of two distinct substrates, each specific for three luciferases, followed by spectral decomposition of the light emitted by each of the three luciferase enzymes with bandpass filters. Here, we present detailed protocols on how to perform multiplex hextuple luciferase assaying to monitor pathway fluxes through transcriptional response elements for five specific signaling pathways (i.e., c-Myc, NF-κβ, TGF-β, p53, and MAPK/JNK) using the constitutive CMV promoter as normalization control. Protocols are provided for preparing reporter vector plasmids for multiplex reporter assaying, performing cell culture and multiplex luciferase reporter vector plasmid transfection, executing multiplex luciferase assays, and analyzing and interpreting data obtained by a plate reader appropriately equipped to detect the different luminescences. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of vectors for multiplex hextuple luciferase assaying Basic Protocol 2: Cell culture work for multiplex hextuple luciferase assays Basic Protocol 3: Transfection of luciferase reporter plasmids followed by drug and recombinant protein treatments Basic Protocol 4: Performing the multiplex hextuple luciferase assay.

Transcription factor AP‑2α negatively regulates thymic stromal lymphopoietin expression in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection enhances the cell‑mediated immune responses of type2 helper T cells and promotes the progression of allergic inflammation and asthma by producing thymic stromal lymphopoietin (TSLP), especially long isoform TSLP (lfTSLP). However, the role of short isoform TSLP (sfTSLP) in RSV infection remains to be elucidated. The present study was designed to demonstrate the role of both lfTSLP and sfTSLP, as transcription regulators, in RSV infection. The expression of lfTSLP and sfTSLP in RSV‑infected Beas‑2B cells was analyzed. Activating protein 2 (AP‑2)α was overexpressed or knocked down to detect the changes in sfTSLP and lfTSLP expression. Luciferase reporter plasmid and chromatin immunoprecipitation experiments demonstrated that AP‑2α bound to the sfTSLP promoter region. LfTSLP and sfTSLP increased while AP‑2α decreased in RSV‑infected Beas‑2B cells. In the Beas‑2B cells, AP‑2α was found to negatively regulate the activity of the sfTSLP promoter and the mRNA level of sfTSLP. AP‑2α also negatively regulated the expression of lfTSLP at both the mRNA and protein levels. The results of the chromatin immunoprecipitation assay indicated that AP‑2α bound to the core promoter region of sfTSLP. These results confirmed that the transcription factor AP‑2α can repress the expression of lfTSLP and sfTSLP in bronchial epithelial cells in RSV infection.

Xenobiotic transcription factors CncC and maf regulate expression of CYP321A16 and CYP332A1 that mediate chlorpyrifos resistance in Spodoptera exigua

Insect cytochrome P450 s (P450 s) are associated with the metabolic detoxification of toxic xenobiotics and their constitutive upregulation is often associated with resistance to natural and synthetic toxicants. The P450 s CYP321A16 and CYP332A1 are constitutively overexpressed in an insecticide-resistant strain of beet armyworm, Spodoptera exigua. However, the function and upstream regulation of these two P450 s remain unknown. Here, we investigated the function of CYP321A16 and CYP332A1 in resistance to the organophosphate insecticide, chlorpyrifos and their regulation by the transcription factors CncC and Maf. Transgenic strains of Drosophila melanogaster expressing CYP321A16 or CYP332A1 showed higher levels of tolerance to chlorpyrifos than the control flies with the same genetic background. Furthermore, recombinant CYP321A16 and CYP332A1 proteins metabolized chlorpyrifos. Analysis of the putative promoter sequences of the genes coding for CYP321A16 and CYP332A1 revealed conserved CncC/Maf binding sites. Transfection of luciferase reporter plasmids containing the promoter of CYP450 gene together with CncC and Maf expression plasmids significantly enhanced the activity of the reporter. Promoter truncation identified a site in the promoter of CYP321A16 that is critical for the CncC/Maf binding. These data demonstrate that resistance to chlorpyrifos in S. exigua is conferred by the combined action of CYP321A16 and CYP332A1 and uncovered their regulation by the transcription factors CncC and Maf.

Potentials and pitfalls of transient in vitro reporter bioassays: interference by vector geometry and cytotoxicity in recombinant zebrafish cell lines

The water framework directive re-evaluation proposes the integration of effect-based tools, increasing the need for alternative methods. Especially within aquatic toxicology, coverage of specific toxicity pathways is scarce, and most applications are based on mammalian or bacterial models, not reflecting realistic exposure scenarios. The use of transient reporter gene assays in cells from organisms of interest could be a quick and inexpensive solution. However, interference with cellular homeostasis may impact the system beyond the function of the manipulated gene and thus lead to non-specific results. We describe how varying vector geometry and different regulatory gene elements on plasmids used for transfection in zebrafish hepatocytes and embryonic fibroblasts may lead up to a tenfold difference in potency. Cells were transiently co-transfected with an Nrf2-responsive Firefly luciferase reporter plasmid and eight different Renilla luciferase normalization plasmids. Transfected cells were exposed to two different regimes (0.1–100 µM and 7.8–250 µM) of the oxidative stress-inducing compounds, sulforaphane, tertbutylhydroquinone, and metazachlor. Nrf2 activity was measured in dual-luciferase assays. In parallel, cytotoxicity was assessed for different endpoints (energy metabolism, protein amount, membrane stability, and cell proliferation) in non-transfected cells and cells co-transfected with constructs of increasing size, to be used for normalization. Transfected cells were more susceptible to cytotoxicity in a vector size-dependent manner. Conclusively, we report that vector geometries (size, backbones, gene-regulatory units), cell line (tissue origin), applied transfection methods, and signal normalization may alter the sensitivity of reporter bioassays in a synergistic manner. Further, we propose that thorough bioassay design is needed to ensure reliability and regulatory acceptance.

Identification of integrin β6 gene promoter and analysis of its transcription regulation in colon cancer cells.

BACKGROUNDThe integrin β6 gene, which is expressed in epithelial cancer, plays a pivotal role in various aspects of cancer progression. The present research for integrin β6 regulation mainly focuses on the post-transcription and translation related regulation mechanism and its role in tumorigenesis. The mechanisms of how the integrin β6 gene is regulated transcriptionally, and the promoter and transcription factors responsible for basic transcription of integrin β6 gene remain unknown.AIMTo clone and characterize the integrin β6 promoter.METHODSSoftware analysis was used to predict the region of integrin β6 promoter. Luciferase reporter plasmids, which contained the integrin β6 promoter, were constructed. Element deletion analysis was performed to identify the location of core promoter and binding sites for transcription factors.RESULTSThe regulatory elements for the transcription of the integrin β6 gene were located between -286 and -85 and contained binding sites for transcription factors such as STAT3 and Ets-1.CONCLUSIONFor the first time, we found the region of β6 core promoter and demonstrated the binding sites for transcription factors such as Ets-1 and STAT3, which are important for integrin β6 promoter transcription activity. These findings are important for investigating the mechanism of integrin β6 activation in cancer progression.

CXCL10 mediates breast cancer tamoxifen resistance and promotes estrogen-dependent and independent proliferation

Although 70% of estrogen receptor (ER)-positive breast cancer patients can benefit from tamoxifen therapy, the rapid development of tamoxifen resistance hampers the treatment advantage. In this investigation, we found that the serum level of CXCL10 in breast cancer patients was positively correlated with tumor size and ER status. Furthermore, GSE22220 dataset analysis demonstrated that CXCL10 expression in the tumor was correlated with tumor grade and lymphatic metastasis status, and Kaplan–Meier analysis indicated that patients with high CXCL10 expression had a poor prognosis. Estrogen-deprived MCF7 cells were transfected with CXCL10 luciferase reporter plasmid and treated with 10 nM estrogen. Luciferase reporter assay confirmed that CXCL10 was regulated by estrogen. CXCL10 promoted the proliferation of both parental MCF7 cells and tamoxifen-resistant (TamR) MCF7 cells through the AKT pathway, while CXCL10 inhibition restored the sensitivity of TamR cells to tamoxifen. All of these data indicate that CXCL10 could be utilized as a biomarker to predict the prognosis of breast cancer and as a therapeutic target to treat tamoxifen resistant cases.

TRNA-derived fragments (tRFs) regulate post-transcriptional gene expression via AGO-dependent mechanism in IL-1β stimulated chondrocytes

Recent studies have shown that tRNA-derived RNA fragments (tRFs) are novel regulators of post-transcriptional gene expression. However, the expression profiles and their role in post-transcriptional gene regulation in chondrocytes is unknown. Here, we determined tRFs expression profile and explored tRF-3003a role in post-transcriptional gene regulation in IL-1β stimulated chondrocytes. We used qPCR arrays to determine tRNAs and tRFs expression in age- and sex-matched primary human OA chondrocytes and TC28/I2 cells stimulated with IL-1β. Chondrocytes were transfected with tRNA-CysGCA overexpression plasmid or tRF-3003a mimic and 3'UTR luciferase reporter plasmids of mRNAs harboring predicted tRF target "seed sequence". The AGO-RNA-induced silencing complex (AGO-RISC)-dependent repressive activity of tRF-3003a was determined by siRNA-mediated knockdown of AGO2. IL-1β increased the expression levels of specific tRNAs and of tRF-3003a, a type 3 tRF produced by the cleavage of tRNA-CysGCA. tRF-3003a "seed sequence" was identified in the 3'UTR of JAK3 mRNA and tRNA-CysGCA overexpression or transfection of a tRF-3003a mimic in chondrocytes downregulated JAK3 expression and significantly reduced the activity of the 3'UTR reporter. RIP assay showed enrichment of tRF-3003a into AGO2/RISC in IL-1β treated chondrocytes. The suppressive effect of tRF-3003a on JAK3 3'UTR reporter was abrogated with siRNA-mediated depletion of AGO2. We demonstrate that under pathological conditions chondrocytes display perturbations in the expression profile of specific tRNAs and tRFs. Furthermore, a specific tRF namely tRF-3003a can post-transcriptionally regulate JAK3 expression via AGO/RISC formation in chondrocytes. Identification of this novel mechanism may be of value in the design of precision therapies for OA.