Adenosine A2A receptor blockade prevents cisplatin-induced impairments in neurogenesis and cognitive function.

Chemotherapy-induced cognitive impairment (CICI) has emerged as a significant medical problem without therapeutic options. Using the platinum-based chemotherapy cisplatin to model CICI, we revealed robust elevations in the adenosine A2A receptor (A2AR) and its downstream effectors, cAMP and CREB, by cisplatin in the adult mouse hippocampus, a critical brain structure for learning and memory. Notably, A2AR inhibition by the Food and Drug Administration-approved A2AR antagonist KW-6002 prevented cisplatin-induced impairments in neural progenitor proliferation and dendrite morphogenesis of adult-born neurons, while improving memory and anxiety-like behavior, without affecting tumor growth or cisplatin's antitumor activity. Collectively, our study identifies A2AR signaling as a key pathway that can be therapeutically targeted to prevent cisplatin-induced cognitive impairments.

3D-printed PCL framework assembling ECM-inspired multi-layer mineralized GO-Col-HAp microscaffold for in situ mandibular bone regeneration.

BACKGROUND: In recent years, natural bone extracellular matrix (ECM)-inspired materials have found widespread application as scaffolds for bone tissue engineering. However, the challenge of creating scaffolds that mimic natural bone ECM's mechanical strength and hierarchical nano-micro-macro structures remains. The purposes of this study were to introduce an innovative bone ECM-inspired scaffold that integrates a 3D-printed framework with hydroxyapatite (HAp) mineralized graphene oxide-collagen (GO-Col) microscaffolds and find its application in the repair of mandibular bone defects. METHODS: Initially, a 3D-printed polycaprolactone (PCL) scaffold was designed with cubic disks and square pores to mimic the macrostructure of bone ECM. Subsequently, we developed multi-layer mineralized GO-Col-HAp microscaffolds (MLM GCH) to simulate natural bone ECM's nano- and microstructural features. Systematic in vitro and in vivo experiments were introduced to evaluate the ECM-inspired structure of the scaffold and to explore its effect on cell proliferation and its ability to repair rat bone defects. RESULTS: The resultant MLM GCH/PCL composite scaffolds exhibited robust mechanical strength and ample assembly space. Moreover, the ECM-inspired MLM GCH microscaffolds displayed favorable attributes such as water absorption and retention and demonstrated promising cell adsorption, proliferation, and osteogenic differentiation in vitro. The MLM GCH/PCL composite scaffolds exhibited successful bone regeneration within mandibular bone defects in vivo. CONCLUSIONS: This study presents a well-conceived strategy for fabricating ECM-inspired scaffolds by integrating 3D-printed PCL frameworks with multilayer mineralized porous microscaffolds, enhancing cell proliferation, osteogenic differentiation, and bone regeneration. This construction approach holds the potential for extension to various other biomaterial types.

Risk factors and the nomogram model for malnutrition in patients with nasopharyngeal carcinoma.

BACKGROUND: For patients with nasopharyngeal carcinoma (NPC), the incidence of malnutrition is quite high, and malnutrition has severe effects on NPC patients. However, there is currently no recognized gold standard or specific nutritional assessment tool available to assess malnutrition in NPC patients. Our objective was to develop and verify a new nomogram model for NPC patients. METHODS: Data were collected from NPC patients. To evaluate risk factors for malnutrition, univariate and multivariate logistic regression analyses were used. Based on the risk factors, a new nomogram model was developed. The efficacy of the model was evaluated and validated. RESULTS: Logistic regression analysis showed that age ≥ 65 years, the number of chemotherapy cycles completed ≥ 1, a high total radiation dose received, low body mass index (BMI), low albumin, and low chloride were the risk factors. The assessment effect of the new model was good by evaluation and validation; it can be used as an assessment tool for malnutrition in NPC patients. CONCLUSIONS: Age ≥ 65 years, completing ≥ 1 chemotherapy cycles, a high total radiation dose received, low BMI, low albumin, and low chloride levels are risk factors for malnutrition in NPC patients. The assessment effect of the new model, developed based on these risk factors, is good, and it can be used as an assessment tool for malnutrition in NPC patients.

Mini Nutritional Assessment for Adult Cancer Patients: A Systematic Review and Meta-Analysis.

Early assessment of malnutrition in cancer patients is very important. The Mini Nutritional Assessment (MNA) is often used to assess malnutrition in adult cancer patients. However, the diagnostic values of MNA are controversial. We aimed to analyze the diagnostic values of MNA in assessing malnutrition in adult cancer patients. A systematic search was performed using Embase, Web of Science, PubMed, the Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang Database, and China Science and Technology Journal Database (VIP). Studies comparing MNA with other tools or criteria in cancer patients were included. The quality of the included studies was assessed by the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2). The pooled sensitivity, specificity, the area under the receiver-operating characteristic curve (AUC), and the diagnostic odds ratio (DOR) were calculated using Stata 17.0 and Meta-DiSc1.4. In addition, sensitivity, subgroup, meta-regression, and publication bias analyses were conducted. In total, 11 studies involving 1367 patients involving MNA were included. The pooled sensitivity, specificity, ROC, and DOR were 0.84 (95% CI: 0.81-0.87), 0.66 (95% CI: 0.63-0.69), 0.84 (95% CI: 0.81-0.87), and 16.11 (95% CI: 7.16-36.27), respectively. In the assessment of malnutrition in adult cancer patients, MNA has high sensitivity and moderate specificity.

A fast breeding strategy creates fragrance- and anthocyanin-enriched rice lines by marker-free gene-editing and hybridization.

As rice is a staple food for nearly half of the world's population, rice varieties with excellent agronomic traits as well as high flavor and nutritional quality such as fragrant rice and purple rice are naturally favored by the market. In the current study, we adopt a fast breeding strategy to improve the aroma and anthocyanin content in the excellent rice inbred line, F25. The strategy skillfully used the advantages of obtaining editing pure lines in T0 generation of CRISPR/Cas9 editing system and easy observation of purple character and grain shape, integrated the subsequent screening of non-transgenic lines, and the elimination of undesirable edited variants from gene-editing and cross-breeding at the same time as the separation of the progeny from the purple cross, thus expediting the breeding process. Compared with conventional breeding strategies, this strategy saves about 6-8 generations and reduces breeding costs. Firstly, we edited the OsBADH2 gene associated with rice flavor using an Agrobacterium-mediated CRISPR/Cas9 system to improve the aroma of F25. In the T0 generation, a homozygous OsBADH2-edited F25 line (F25B) containing more of the scented substance 2-AP was obtained. Then, we crossed F25B (♀) with a purple rice inbred line, P351 (♂), with high anthocyanin enrichment to improve the anthocyanin content of F25. After nearly 2.5 years of screening and identification over five generations, the undesirable variation characteristics caused by gene-editing and hybridization and the transgenic components were screened out. Finally, the improved F25 line with highly stable aroma component, 2-AP, increased anthocyanin content and no exogenous transgenic components were obtained. This study not only provides high-quality aromatic anthocyanin rice lines that meet the market demand, but also offers a reference for the comprehensive use of CRISPR/Cas9 editing technology, hybridization, and marker-assisted selection to accelerate multi-trait improvement and breeding process. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01369-1.

Method for improving the measurement accuracy of binocular stereo vision in a scattering environment.

In the scattering environment, binocular stereo vision measurement technology produces large errors due to the change of refractive index of the imaging light path and the decrease in target image contrast. To address this problem, this paper proposes a method for improving the measurement accuracy of binocular stereo vision in a scattering environment combined with polarization imaging theory. First, scattering images with different polarization directions are obtained and filtered by a Gaussian low-pass filter to calculate the degree of polarization and angle of polarization. Then, the scattered light intensity is calculated by using polarization information to obtain images after removing the scattering. Second, feature extraction and matching are carried out for the images after scattering removal. Finally, the target is measured based on the binocular stereo vision measurement model. The experimental results show that when the scattering concentration is high enough, the conventional method can no longer perform measurement, but the method proposed in this paper can still obtain the target parameters at this time, and can also improve measurement accuracy by at least 46.30%. In conclusion, the proposed method provides a valuable reference to improve the accuracy of binocular stereo vision measurement in a scattering environment by reducing the interference of scattering light.

Identification and validation of transcription factor-driven enhancers of genes related to lipid metabolism in metastatic oral squamous cell carcinomas.

BACKGROUND: The role and mechanisms of lipid metabolism in oral squamous cell carcinomas (OSCC) metastasis have not been clarified. This study aims to identify lipid metabolism-related genes and transcription factors regulated by metastasis-associated enhancers (MAEs) in OSCC. METHODS: Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were performed for lipid metabolism enrichment. TCGA data were used to analyze the differentially expressed lipid metabolism-related genes. MAEs were analyzed using GSE120634. Overlapping analysis was used to screen the MAE-regulated lipid metabolism-related genes, and the prognosis of these genes was analyzed. Transcription factor prediction was performed for the MAE-regulated lipid metabolism-related genes with prognostic value. Validation of the metastatic specificity of MAEs at ACAT1, OXSM and VAPA locus was performed using GSE88976 and GSE120634. ChIP-qPCR, qRT-PCR and Western blotting were used to verify the regulation of ACAT1, OXSM and VAPA expression by CBFB. Effects of CBFB knockdown on proliferation, invasion and lipid synthesis in metastatic OSCC cells were analyzed. RESULTS: Lipid metabolism was significantly enhanced in metastatic OSCC compared to non-metastatic OSCC. The expression of 276 lipid metabolism-related genes was significantly upregulated in metastatic OSCC, which were functionally related to lipid uptake, triacylglycerols, phospholipids and sterols metabolism. A total of 6782 MAEs and 176 MAE-regulated lipid metabolism-related genes were filtered. Three MAE-regulated lipid metabolism-related genes, ACAT1, OXSM and VAPA, were associated with a poor prognosis in OSCC patients. Enhancers at ACAT1, OXSM and VAPA locus were metastasis-specific enhancers. CBFB regulated ACAT1, OXSM and VAPA expression by binding to the enhancers of these genes. Knockdown of CBFB inhibited proliferation, invasion and lipid synthesis in metastatic OSCC cells. CONCLUSION: The MAE-regulated lipid metabolism-related genes (ACAT1, OXSM and VAPA) and the key transcription factor (CBFB) were identified. CBFB knockdown inhibited proliferation, invasion and lipid synthesis of OSCC cells. These findings provide novel candidates for the development of therapeutic targets for OSCC.

Averaging speckle patterns to improve the robustness of compressive multimode fiber imaging against fiber bend.

Fiber bend is a major challenge of multimode fiber (MMF) imaging. More robustness against fiber bend is demonstrated in compressive MMF imaging using mean speckle patterns captured at multiple potential bending configurations beforehand, rather than sticking to single patterns at initial configuration. Experiments demonstrate an overall quality improvement on recovered images than previous work, which is important for robust endoscopic application.

MicroRNA-218 inhibits type I interferon production and facilitates virus immune evasion via targeting RIG-I.

The host protective immunity against viral infection requires the effective detection of viral antigens and the subsequent production of type I interferons (IFNs) by host immune cells. Retinoic acid-inducible gene I (RIG-I) is the crucial signaling element responsible for sensing viral RNA component and initiating the downstream antiviral signaling pathways, leading to the production of type I IFNs. In this work, we identified microRNA-218 (miR-218) as a new virus-induced miRNA that dampens the expression of RIG-I in mouse and human macrophages, leading to the impaired production of type I IFNs. Interfering miR-218 expression rescued RIG-I-mediated antiviral signaling and thus protected macrophages from viral infection. Hence, our results provide new understanding of miRNA-mediated viral immune evasion and may be potentially useful for the treatment of viral infection in the future.

Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples.

BACKGROUND: Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. RESULTS: As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (< 5%), and collectively shared a "C > T|G > A" mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. CONCLUSIONS: Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Protective effects and mechanism of curcumin on myocardial injury induced by coronary microembolization.

OBJECTIVE: Coronary microembolization (CME) is a common complication during the percutaneous coronary intervention (PCI). CME-induced local myocardial inflammation and myocardial apoptosis are the primary causes of progressive cardiac dysfunction. Curcumin exerts a protective role in various cardiovascular diseases; however, its effects in CME are yet to be clarified. Therefore, the current study investigated the effects of curcumin on myocardial inflammatory responses, myocardial apoptosis, and cardiac dysfunctions induced by CME in rats. METHODS: A total of 40 Sprague-Dawley rats were randomly divided into the following groups: Sham operation (sham group), CME group, curcumin, and control with 10 rats in each group. The ascending aortas were clamped, and the CME-model group was established by injecting microspheres into the apex of the left ventricle. An equivalent amount of normal saline was injected to establish the sham group. The cardiac functions, serum c-troponin I level, and apoptotic index was examined. Also, the levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MYD88), nuclear factor κB (NF-κB) p65, BCL2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cleaved caspase-3, tumor necrosis factor α (TNF-α), and interleukin-1ß (IL-1ß) were detected. RESULTS: Myocardial dysfunction enhanced serum c-troponin I, and apoptotic index were induced following CME. Moreover, CME elevated the expression of TLR4, MyD88, NF-κB p65, cleaved caspase-3, TNF-α, and IL-1ß, while the Bcl-2/Bax ratio decreased. Curcumin reversed these effects by CME, while the gastric lavage control did not exert any effect. CONCLUSION: Curcumin was responsible for the anti-CME-induced myocardial injury. The effector mechanism might be related to the reduction of cardiomyocyte apoptosis and inhibition of myocardial inflammatory responses mediated by TLR4/MyD88/NF-κB signaling pathway.

Renal Artery Stenosis Alters Gene Expression in Swine Scattered Tubular-Like Cells.

BACKGROUND: Scattered tubular-like cells (STCs) proliferate and differentiate to support neighboring injured renal tubular cells during recovery from insults. Renal artery stenosis (RAS) induces renal ischemia and hypertension and leads to loss of kidney function, but whether RAS alters renal endogenous repair mechanisms, such as STCs, remains unknown. We hypothesize that RAS in swine modifies the messenger RNA (mRNA) profile of STCs, blunting their in vitro reparative capacity. METHODS: CD24+/CD133+ STCs were isolated from pig kidneys after 10-weeks of RAS or sham (n = 3 each) and their gene cargo analyzed using high-throughput mRNAseq. Expression profiles for upregulated and downregulated mRNAs in RAS-STCs were functionally interpreted by gene ontology analysis. STC activation was assessed by counting the total number of STCs in pig kidney sections using flow cytometry, whereas cell proliferation was assessed in vitro. RESULTS: Of all expressed genes, 1430 genes were upregulated and 315 downregulated in RAS- versus Normal-STCs. Expression of selected candidate genes followed the same fold change directions as the mRNAseq findings. Genes upregulated in RAS-STCs were involved in cell adhesion, extracellular matrix remodeling, and kidney development, whereas those downregulated in RAS-STCs are related to cell cycle and cytoskeleton. The percentage of STCs from dissociated kidney cells was higher in RAS versus Normal pigs, but their proliferation rate was blunted. CONCLUSIONS: Renal ischemia and hypertension in swine induce changes in the mRNA profile of STCs, associated with increased STC activation and impaired proliferation. These observations suggest that RAS may alter the reparative capacity of STCs.

XBSeq2: a fast and accurate quantification of differential expression and differential polyadenylation.

BACKGROUND: RNA sequencing (RNA-seq) is a high throughput technology that profiles gene expression in a genome-wide manner. RNA-seq has been mainly used for testing differential expression (DE) of transcripts between two conditions and has recently been used for testing differential alternative polyadenylation (APA). In the past, many algorithms have been developed for detecting differentially expressed genes (DEGs) from RNA-seq experiments, including the one we developed, XBSeq, which paid special attention to the context-specific background noise that is ignored in conventional gene expression quantification and DE analysis of RNA-seq data. RESULTS: We present several major updates in XBSeq2, including alternative statistical testing and parameter estimation method for detecting DEGs, capacity to directly process alignment files and methods for testing differential APA usage. We evaluated the performance of XBSeq2 against several other methods by using simulated datasets in terms of area under the receiver operating characteristic (ROC) curve (AUC), number of false discoveries and statistical power. We also benchmarked different methods concerning execution time and computational memory consumed. Finally, we demonstrated the functionality of XBSeq2 by using a set of in-house generated clear cell renal carcinoma (ccRCC) samples. CONCLUSIONS: We present several major updates to XBSeq. By using simulated datasets, we demonstrated that, overall, XBSeq2 performs equally well as XBSeq in terms of several statistical metrics and both perform better than DESeq2 and edgeR. In addition, XBSeq2 is faster in speed and consumes much less computational memory compared to XBSeq, allowing users to evaluate differential expression and APA events in parallel. XBSeq2 is available from Bioconductor: http://bioconductor.org/packages/XBSeq/.

MBDDiff: an R package designed specifically for processing MBDcap-seq datasets.

BACKGROUND: Since its initial discovery in 1975, DNA methylation has been intensively studied and shown to be involved in various biological processes, such as development, aging and tumor progression. Many experimental techniques have been developed to measure the level of DNA methylation. Methyl-CpG binding domain-based capture followed by high-throughput sequencing (MBDCap-seq) is a widely used method for characterizing DNA methylation patterns in a genome-wide manner. However, current methods for processing MBDCap-seq datasets does not take into account of the region-specific genomic characteristics that might have an impact on the measurements of the amount of methylated DNA (signal) and background fluctuation (noise). Thus, specific software needs to be developed for MBDCap-seq experiments. RESULTS: A new differential methylation quantification algorithm for MBDCap-seq, MBDDiff, was implemented. To evaluate the performance of the MBDDiff algorithm, a set of simulated signal based on negative binomial and Poisson distribution with parameters estimated from real MBDCap-seq datasets accompanied with different background noises were generated, and then performed against a set of commonly used algorithms for MBDCap-seq data analysis in terms of area under the ROC curve (AUC), number of false discoveries and statistical power. In addition, we also demonstrated the effective of MBDDiff algorithm to a set of in-house prostate cancer samples, endometrial cancer samples published earlier, and a set of public-domain triple negative breast cancer samples to identify potential factors that contribute to cancer development and recurrence. CONCLUSIONS: In this paper we developed an algorithm, MBDDiff, designed specifically for datasets derived from MBDCap-seq. MBDDiff contains three modules: quality assessment of datasets and quantification of DNA methylation; determination of differential methylation of promoter regions; and visualization functionalities. Simulation results suggest that MBDDiff performs better compared to MEDIPS and DESeq in terms of AUC and the number of false discoveries at different levels of background noise. MBDDiff outperforms MEDIPS with increased backgrounds noise, but comparable performance when noise level is lower. By applying MBDDiff to several MBDCap-seq datasets, we were able to identify potential targets that contribute to the corresponding biological processes. Taken together, MBDDiff provides user an accurate differential methylation analysis for data generated by MBDCap-seq, especially under noisy conditions.

MicroRNA-184 Modulates Doxorubicin Resistance in Osteosarcoma Cells by Targeting BCL2L1.

BACKGROUND Early metastasis of osteosarcoma (OS) is highly lethal and responds poorly to drug and radiation therapies. MicroRNAs (miRNAs) are a class of small noncoding RNAs that modulate gene expression at the post-transcriptional level. However, the detailed functions of specific miRNAs are not entirely understood. The aim of the present study was to investigate the role of miR-184 as a mediator of drug resistance in human osteosarcoma. MATERIAL AND METHODS qRT-PCR was used to analyze the expression level of miR-184 in OS cell line U-2 OS and MG-63 treated with doxorubicin. MiR-184 agomir or miR-184 antagomir was transferred into cells to regulated miR-184. The target of miR-184 was predicted by TargetScan and confirmed by luciferase reporter assay. Bcl-2-like protein 1 (BCL2L1) expression was detected by Western blot. Cell apoptosis was determined by Annexin V staining and analysis by flow cytometry. RESULTS Doxorubicin induced time-dependent expression of miR-184 in OS cell line U-2 OS and MG-63. Luciferase reporter assay identified BCL2L1 as the direct target gene of miR-184. Furthermore, doxorubicin reduced BCL2L1 expression, which was reversed by miR-184 overexpression and further decreased by miR-184 inhibition in OS cells. In addition, miR-184 agomir reduced doxorubicin-induced cell apoptosis, whereas miR-184 antagomir enhanced apoptosis in OS cells, suggesting that up-regulation of miR-184 contributes to chemoresistance of the OS cell line. CONCLUSIONS Our data show that miR-184 was up-regulated in OS patients treated with doxorubicin therapy and leads to poor response to drug therapy by targeting BCL2L1.

Differential expression analysis of RNA sequencing data by incorporating non-exonic mapped reads.

BACKGROUND: RNA sequencing (RNA-seq) is a powerful tool for genome-wide expression profiling of biological samples with the advantage of high-throughput and high resolution. There are many existing algorithms nowadays for quantifying expression levels and detecting differential gene expression, but none of them takes the misaligned reads that are mapped to non-exonic regions into account. We developed a novel algorithm, XBSeq, where a statistical model was established based on the assumption that observed signals are the convolution of true expression signals and sequencing noises. The mapped reads in non-exonic regions are considered as sequencing noises, which follows a Poisson distribution. Given measureable observed and noise signals from RNA-seq data, true expression signals, assuming governed by the negative binomial distribution, can be delineated and thus the accurate detection of differential expressed genes. RESULTS: We implemented our novel XBSeq algorithm and evaluated it by using a set of simulated expression datasets under different conditions, using a combination of negative binomial and Poisson distributions with parameters derived from real RNA-seq data. We compared the performance of our method with other commonly used differential expression analysis algorithms. We also evaluated the changes in true and false positive rates with variations in biological replicates, differential fold changes, and expression levels in non-exonic regions. We also tested the algorithm on a set of real RNA-seq data where the common and different detection results from different algorithms were reported. CONCLUSIONS: In this paper, we proposed a novel XBSeq, a differential expression analysis algorithm for RNA-seq data that takes non-exonic mapped reads into consideration. When background noise is at baseline level, the performance of XBSeq and DESeq are mostly equivalent. However, our method surpasses DESeq and other algorithms with the increase of non-exonic mapped reads. Only in very low read count condition XBSeq had a slightly higher false discovery rate, which may be improved by adjusting the background noise effect in this situation. Taken together, by considering non-exonic mapped reads, XBSeq can provide accurate expression measurement and thus detect differential expressed genes even in noisy conditions.

[Readthrough on transcription factor NKX2.5 premature stop codon by tRNA suppressors].

Human NKX2.5 (NK2 homeobox 5) premature stop codon (PTC) mutations cause congenital heart diseases such as atrial septal defect and atrioventricular block. At present, eight NKX2.5 PTC mutations were reported as E109X, Q149X, Q170X, Q187X, Q198X, Y256X, Y259X and C264X. To observe the ability of tRNA suppressors to read through NKX2.5 PTC mutations and produce functional full-length proteins, eight NKX2.5 PTC mutations were cloned into pcDNA3.1(-) vectors and four fragments (wild-type NKX2.5, E109X, Q149X and C264X) were cloned in pEGFP-N1 vectors to acquire NKX2.5-EGFP fusing plasmids. After transfection of NKX2.5-EGFP with or without corresponding tRNA suppressor into HeLa cells, the quantity of EGFP was measured to confirm the readthrough ability of the PTCs. NKX2.5 full-length and truncated protein expression levels were examined by Western blotting and the readthrough efficiency of tRNA suppressors on the PTCs was calculated respectively. The activity of NKX2.5 full-length and truncated protein was confirmed on NKX2.5 target gene-Cx43 mRNA level measured by Real-time PCR. Three tRNA suppressors were used: tRNA am, tRNA oc and tRNA op. tRNA am could suppress UAG-containing PTCs Q149X, Q170X, Q187X, Q198X and the readthrough efficiency for the latter three was above 50%. tRNA op could suppress UGA-containing PTC C264X with ~50% readthrough efficiency. tRNA oc failed to read through NKX2.5 PTC mutations. The relative Cx43 mRNA level in all readthrough samples was increased to 7%-41.7%. In conclusion, tRNA am and tRNA op could suppress NKX2.5 PTCs and induce functional protein expression. However, the effects of tRNA suppressors on cellular function are not clear yet, warranting further researches.