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There is increasing evidence that circular RNA (circRNA) are involved in cancer development, but the regulation and function of human circRNA remain largely unknown. In this study, we demonstrated that ZKSCAN1, a zinc finger family gene, is expressed in both linear and circular (circZKSCAN1) forms of RNA in human hepatocellular carcinoma (HCC) tissues and cell lines. Here, we analyzed a cohort of 102 patients and found that expression of both ZKSCAN1mRNA and circZKSCAN1 was significantly lower (P < 0.05) in the HCC samples compared with that in matched adjacent nontumorous tissues by reverse transcription PCR (RT‐PCR). The low expression level of ZKSCAN1 was only associated with tumor size (P = 0.032), while the cirZKSCAN1 levels varied in patients with different tumor numbers (P < 0.01), cirrhosis (P = 0.031), vascular invasion (P = 0.002), or microscopic vascular invasion (P = 0.002), as well as with the tumor grade (P < 0.001). Silencing both ZKSCAN1mRNA and circZKSCAN1 promoted cell proliferation, migration, and invasion. In contrast, overexpression of both forms of RNA repressed HCC progression in vivo and in vitro. Silencing or overexpression of both forms of RNA did not interfere with each other. RNA‐seq revealed a very different molecular basis for the observed effects; ZKSCAN1mRNA mainly regulated cellular metabolism, while circZKSCAN1 mediated several cancer‐related signaling pathways, suggesting a nonredundant role for ZKSCAN1mRNA and circRNA. In conclusion, our results revealed two post‐translational products (ZKSCAN1mRNA and circZKSCAN1) that cooperated closely with one another to inhibit growth, migration, and invasion of HCC. cirZKSCAN1 might be a useful marker for the diagnosis of HCC. Circular RNA have recently been shown to play a regulatory role in disease. We investigated the role of circRNA transcribed from ZKSCAN1, a member of the zinc finger family. Our results revealed two post‐translational products (ZKSCAN1 mRNA and circZKSCAN1) that cooperated closely with one another to inhibit cancer growth migration and invasion, but which affected different signaling pathways.

Although many genes may serve as reference genes, they may cause different expression patterns by selecting different reference genes because no single gene is expressed consistently in all tested tissues of an organism under all environmental and developmental conditions. Thus, it is becoming increasingly important and necessary to identify suitable reference genes before performing gene expression analysis. Currently, there are several computational tools available for evaluating the stability of candidate reference genes. These tools are based on different statistical algorithms and may produce different rankings in stability within the same reference gene study. To date, the RefFinder is the only web-based tool available for comparing and evaluating housekeeping genes as candidates to be reference genes. In this tool, we integrated the four currently available computational programs (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) into a web-based tool for evaluating the stability and reliability of reference genes. According to the gene stability rankings derived from the four programs, we assigned an appropriate weight to each gene and calculated the geometric mean of weights for the final rankings. Aside from the overall ranking, a single program or combination of the four programs can be selected for evaluating the ranking of candidate reference genes. This tool has been widely used and validated by many research laboratories around the world. You may use this tool at http://www.heartcure.com.au/reffinder/  or  https://blooge.cn/RefFinder/ . You can also download this algorithm program from https://github.com/fulxie/RefFinder and setup on your own computer. RefFinder is developed by PHP. Users can deploy it to a Php-based server (Apache + PHP) and run it.

Objectives This study was designed to investigate the role of circ_0078767/miR‐330‐3p/RASSF1A in non‐small‐cell lung cancer (NSCLC). Bioinformatic analysis was performed to screen for the differentially expressed genes in NSCLC tissues from adjacent lung tissues. Materials and Methods qRT‐PCR was used to detect the RNA expression of genes in cells and tissues, and Western blot was conducted to determine the protein levels of RASSF1A in tissues and cells. A miRanda algorithm was used to predict the targeted relationship among RNAs. A dual‐luciferase reporter gene assay was conducted to verify the targeted relationship. Flow cytometry was performed to investigate the effects of circ_0078767/miR‐330‐3p/RASSF1A on cell cycle progression and apoptosis. A CCK‐8 assay was conducted to explore the effects of circ_0078767/miR‐330‐3p/RASSF1A on cell proliferation. A transwell invasion assay was completed to study the effects of circ_0078767/miR‐330‐3p/RASSF1A on cell invasion. Lastly, an in vivo assay was conducted to investigate the effects of circ_0078767/miR‐330‐3p/RASSF1A on tumour development. Results Circ_0078767 and RASSF1A were downregulated, while miR‐330‐3p was upregulated in NSCLC tissues than that in adjacent tissues. miR‐330‐3p had a binding relationship with circ_0078767 and RASSF1A. The overexpression of circ_0078767 and RASSF1A or the underexpression of miR‐330‐3p significantly suppressed NSCLC cell viability, cell cycle progression and invasion while also significantly promoting cell apoptosis. Additionally, these modulations significantly suppressed in vivo tumour growth. Conclusions Circ_0078767 could suppress NSCLC progression by inhibiting miR‐330‐3p, which thereby increased RASSF1 levels.

Proline has been reported to play an important role in helping plants cope with several stresses, including salinity. This study investigates the relationship between proline accumulation and salt tolerance in an accession of Australian wild rice Oryza australiensis Domin using morphological, physiological, and molecular assessments. Seedlings of O. australiensis wild rice accession JC 2304 and two other cultivated rice Oryza sativa L. cultivars, Nipponbare (salt-sensitive), and Pokkali (salt-tolerant), were screened at 150 mM NaCl for 14 days. The results showed that O. australiensis was able to rapidly accumulate free proline and lower osmotic potential at a very early stage of salt stress compared to cultivated rice. The qRT-PCR result revealed that O. australiensis wild rice JC 2304 activated proline synthesis genes OsP5CS1, OsP5CS2, and OsP5CR and depressed the expression of proline degradation gene OsProDH as early as 1 h after exposure to salinity stress. Wild rice O. australiensis and Pokkali maintained their relative water content and cell membrane integrity during exposure to salinity stress, while the salt-sensitive Nipponbare failed to do so. An analysis of the sodium and potassium contents suggested that O. australiensis wild rice JC 2304 adapted to ionic stress caused by salinity by maintaining a low Na+ content and low Na+/K+ ratio in the shoots and roots. This demonstrates that O. australiensis wild rice may use a rapid accumulation of free proline as a strategy to cope with salinity stress.

Rapid and sensitive screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to limit the spread of the global pandemic we are facing. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is currently used for the clinical diagnosis of SARS-CoV-2 infection using nasopharyngeal swabs, tracheal aspirates, or bronchoalveolar lavage (BAL) samples. Despite the high sensitivity of the qRT-PCR method, false negative outcomes might occur, especially in patients with a low viral load. Here, we developed a multiplex qRT-PCR methodology for the simultaneous detection of SARS-CoV-2 genome (N gene) and of the human RNAse P gene as internal control. We found that multiplex qRT-PCR was effective in detecting SARS-Cov-2 infection in human specimens with 100% sensitivity. Notably, patients with few copies of SARS-CoV-2 RNA (<5 copies/reaction) were successfully detected by the novel multiplex qRT-PCR method. Finally, we assessed the efficacy of multiplex qRT-PCR on human nasopharyngeal swabs without RNA extraction. Collectively, our results provide evidence of a novel and reliable tool for SARS-CoV-2 RNA detection in human specimens, which allows the testing capacity to be expanded and the RNA extraction step to be bypassed.

Pancreatic cancer (PC) has posed a great health threat to a growing number of people all over the world. Detection of serum miRNAs, being sensitive, noninvasive, and easy to obtain, has a great potential of being a novel screening method for PC patients. In this study, we investigated miRNA expression levels in serum by qRT‐PCR. The study was divided into four phases: the screening, training, testing, and external validation stage. We firstly chose candidate miRNAs using Exiqon panels in the screening phase. Then, a total of 129 PC serum samples and 107 normal controls (NCs) were further analyzed in the following training and testing phases to identify differently expressed miRNAs. A cohort of 30 PC serum samples vs 30 NCs was used to confirm the diagnostic value of the identified miRNAs in the external validation phase. Moreover, miRNA expressions in additional 44 PC tumor tissue samples and the matched adjacent normal tissue samples as well as 32 pairs of serum‐derived exosomes samples were also further explored. As a result, we identified six significantly upregulated miRNAs in the serum of PC: let‐7b‐5p, miR‐192‐5p, miR‐19a‐3p, miR‐19b‐3p, miR‐223‐3p, and miR‐25‐3p. A six‐miRNA panel in serum was then established. The area under the receiver operating characteristic curves (AUC) for the panel was 0.910 for the combined training and testing phases, which showed higher diagnostic value than the individual miRNA. Prognostic value prediction using Cox's proportional hazards model and Kaplan‐Meier curves showed that increased serum miR‐19a‐3p was closely related to worse overall survival (OS). In addition, significant upregulation of miR‐192‐5p, miR‐19a‐3p, and miR‐19b‐3p was observed in both PC tissue and serum‐derived exosomes samples. In conclusion, we identified a six‐miRNA (let‐7b‐5p, miR‐192‐5p, miR‐19a‐3p, miR‐19b‐3p, miR‐223‐3p, and miR‐25‐3p) panel in the serum for PC early and noninvasive diagnosis. We identified a six‐miRNA (let‐7b‐5p, miR‐192‐5p, miR‐19a‐3p, miR‐19b‐3p, miR‐223‐3p, and miR‐25‐3p) panel in the serum by multiple‐phase validation using qRT‐PCR for PC early and noninvasive diagnosis.

Organic acids are key components that determine the taste and flavor of fruits and play a vital role in maintaining fruit quality and nutritive value. In this study, the fruits of two cultivars of passion fruit Yellow (Passiflora edulis f. flavicarpa) and purple (Passiflora edulis f. edulis) were harvested at five different developmental stages (i.e., fruitlet, green, veraison, near-mature and mature stage) from an orchard located in subtropical region of Fujian Province, China. The contents of six organic acids were quantified using ultra-performance liquid chromatography (UPLC), activities of citric acid related enzymes were determined, and expression levels of genes involved in citric acid metabolism were measured by quantitative real-time PCR (qRT-PCR). The results revealed that citric acid was the predominant organic acid in both cultivars during fruit development. The highest citric acid contents were observed in both cultivars at green stage, which were reduced with fruit maturity. Correlation analysis showed that citrate synthase (CS), cytosolic aconitase (Cyt-ACO) and cytosolic isocitrate dehydrogenase (Cyt-IDH) may be involved in regulating citric acid biosynthesis. Meanwhile, the PeCS2, PeACO4, PeACO5 and PeIDH1 genes may play an important role in regulating the accumulation of citric acid. This study provides new insights for future elucidation of key mechanisms regulating organic acid biosynthesis in passion fruit.

Breast cancer (BC) is one of the most common cancers in females. Since early detection can bring prognosis benefit, discovery of novel noninvasive biomarkers for BC diagnosis is in urgent need. In this four‐phase study, we profiled miRNA expression in plasma samples from a total of 257 BC patients and 257 normal controls (NCs). Exiqon miRNA qPCR panel was used to select candidate miRNAs in the screening phase which were further analyzed using qRT‐PCR in the following training, testing and external validation phases. Finally, we identified five plasma miRNAs (let‐7b‐5p, miR‐122‐5p, miR‐146b‐5p, miR‐210‐3p and miR‐215‐5p) whose expression levels were significantly different between BC patients and NCs. A 5‐miRNA panel in plasma with high sensitivity and specificity was then constructed to detect BC. The areas under the receiver‐operating characteristic curves (AUCs) of the panel were 0.683, 0.966, 0.978 for the training, testing and external validation sets, respectively. Expression of the identified miRNAs was further analyzed among 32 pairs of BC tissue and the adjacent normal tissue samples as well as plasma‐derived exosome samples from 32 BC patients vs 32 NCs. Let‐7b‐5p was contrarily down‐regulated in BC tissue. In exosomes samples, only miR‐122‐5p was significantly up‐regulated as in plasma for BC patients. In conclusion, we identified a 5‐miRNA plasma panel (let‐7b‐5p, miR‐122‐5p, miR‐146b‐5p, miR‐210‐3p and miR‐215‐5p) that could serve as a promising biomarker for BC detection. We identified five plasma miRNAs (let‐7b‐5p, miR‐122‐5p, miR‐146b‐5p, miR‐210‐3p and miR‐215‐5p) by multiple‐phase validation which could serve as novel noninvasive biomarkers for BC diagnosis. Our findings may contribute to the early detection of BC and be beneficial to BC patients.

Zanthoxylum armatum, a deciduous aromatic shrub has been utilized by traditional healers for treatment of various ailments. Elucidation of the transcriptome data and expression studies of the putative biosynthetic pathway gene(s) of berberine and sanguinarine production in leaf, stem, and fruit at different seasons and identification of transcription factor families involved in the biosynthesis of isoquinoline alkaloids was established in the present study. The assembled transcripts were clustered into 44254, 46402 and 46521 unigenes and a total of 32118, 27777 and 19754 CDS were predicted from unigenes in fruit, leaf and stem samples respectively. Using MISA 5576 SSRs were identified from fruit, leaf and stem samples, out of which a total of 1877 SSRs with 150 flanking regions were predicted. Putative biosynthetic pathway genes like BBE, BBE-like, SOMT, CAS, STOX, BS and SR were significantly expressed in the three samples of the plant in different seasons and at different level. Transcription factor analysis along with correlation matrix predicted abundant families like AP2/ERF family (4010), followed by MYB-related family (3010), RPL2 family (2760), MYC family (2662), DREB/CRF family (2526), and RAV (2302) to be the regulators along with WRKY. The association analysis of the metabolome and the assembled transcriptome from Zanthoxylum armatum will go a long way in re-engineering of the species by AI assisted priming. The TFs would enable artificial intelligence-based designing of efficient minipromoters for enhancing the production of the target compounds in a more efficient and predictable manner.