PCR Validation Research Articles

First Report on Development of Genome-Wide Microsatellite Markers for Stock (Matthiola incana L.)

Stock (Matthiola incana (L.) R. Br.) is a famous annual ornamental plant with important ornamental and economic value. The lack of DNA molecular markers has limited genetic analysis, genome evolution, and marker-assisted selective breeding studies of M. incana. Therefore, more DNA markers are needed to support the further elucidation of the biology and genetics of M. incana. In this study, a high-quality genome of M. incana was initially assembled and a set of effective SSR primers was developed at the whole-genome level using genome data. A total of 45,612 loci of SSRs were identified; the di-nucleotide motifs were the most abundant (77.35%). In total, 43,540 primer pairs were designed, of which 300 were randomly selected for PCR validation, and as the success rate for amplification. In addition, 22 polymorphic SSR markers were used to analyze the genetic diversity of 40 stock varieties. Clustering analysis showed that all varieties could be divided into two clusters with a genetic distance of 0.68, which were highly consistent with their flower shape (potted or cut type). Moreover, we have verified that these SSR markers are effective and transferable within the Brassicaceae family. In this study, potential SSR molecular markers were successfully developed for 40 M. incana varieties using whole genome analysis, providing an important genetic tool for theoretical and applied research on M. incana.

Identification and analysis of key microRNAs derived from osteoarthritis synovial fluid exosomes.

Identification and analysis of key microRNAs derived from osteoarthritis synovial fluid exosomes.

Circular RNA circTRPS1-2 inhibits the proliferation and migration of esophageal squamous cell carcinoma by reducing the production of ribosomes

Circular RNAs play important roles in many cancers, including esophageal squamous cell carcinoma (ESCC), but the precise functions of most circular RNAs are poorly understood. Here we detected significant downregulation of circTRPS1-2 in ESCC based on high-throughput sequencing of three pairs of ESCC tissue and adjacent normal tissue, followed by PCR validation with another 30 tissue pairs. Patients with ESCC whose circTRPS1-2 expression was below the median level for the sample showed significantly shorter median overall survival (13 months) than patients whose circTRPS1-2 expression was above the median (36 months). Overexpressing circTRPS1-2 in the human ESCC cell lines K150 and E109, which express low endogenous levels of circTRPS1-2, inhibited cell proliferation and migration. Conversely, knocking down circTRPS1-2 using short interfering RNA promoted cell proliferation and migration. Similar results were observed in mice bearing K150 xenografts in which circTRPS1-2 was overexpressed or knocked down. Several ribosomal proteins co-immunoprecipitated with circTRPS1-2 from K150 cells in culture, and K150 cells overexpressing circTRPS1-2 showed reduced numbers of ribosomes by A260 absorbance measure and electron microscopy. Our results suggest that circTRPS1-2 can inhibit ESCC proliferation and migration by reducing the production of ribosomes, establishing its potential usefulness in ESCC treatment and prediction of prognosis.

The Application of Adipose Tissue-Derived Mesenchymal Stem Cells (ADMSCs) and a Twin-Herb Formula to the Rodent Wound Healing Model: Use Alone or Together?

Our previous study reported that mesenchymal stem cells (MSCs) accelerated the wound healing process through anti-inflammatory, anti-apoptotic, and pro-angiogenetic effects in a rodent skin excision model. NF3 is a twin-herb formula, which presents similar effects in promoting wound healing. Research focusing on the interaction of MSCs and Chinese medicine is limited. In this study, we applied MSCs and the twin-herb formula to the wound healing model and investigated their interactions. Wound healing was improved in all treatment groups (MSCs only, NF3 only, and MSCs + NF3). The combined therapy further enhanced the effect: more GFP-labelled ADMSCs, collagen I and collagen III expression, Sox9 positive cells, and CD31 positive cells, along with less ED-1 positive cells, were detected; the expressions of proinflammatory cytokine IL-6 and TNF-α were downregulated; and the expression of anti-inflammatory cytokine IL-10 was upregulated. In vitro, NF3 promoted the cell viability and proliferation ability of MSCs, and a higher concentration of protein was detected in the NF3-treated supernatant. A proteomic analysis showed there were 15 and 22 proteins in the supernatants of normal ADMSCs and NF3-treated ADMSCs, respectively. After PCR validation, the expressions of 11 related genes were upregulated. The results of a western blot suggested that the TGFβ/Smad and Wnt pathways were related to the therapeutic effects of the combined treatment. Our study suggests for the first time that NF3 enhanced the therapeutic effect of MSCs in the wound healing model and the TGFβ/Smad and Wnt pathways were related to the procedure.

In silico Identification of Hypoxic Signature followed by reverse transcription-quantitative PCR Validation in Cancer Cell Lines

Hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. This study aimed to computationally identify hypoxia-related messenger RNA (mRNA) signatures in nine hypoxic-conditioned cancer cell lines and investigate their role during hypoxia. Nine RNA sequencing (RNA-Seq) expression data sets were retrieved from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in each cancer cell line. Then 23 common DEGs were selected by comparing the gene lists across the nine cancer cell lines. Reverse transcription-quantitative PCR (qRT-PCR) was performed to validate the identified DEGs. By comparing the data sets, GAPDH, LRP1, ALDOA, EFEMP2, PLOD2, CA9, EGLN3, HK, PDK1, KDM3A, UBC, and P4HA1 were identified as hub genes. In addition, miR-335-5p, miR-122-5p, miR-6807-5p, miR-1915-3p, miR-6764-5p, miR-92-3p, miR-23b-3p, miR-615-3p, miR-124-3p, miR-484, and miR-455-3p were determined as common micro RNAs. Four DEGs were selected for mRNA expression validation in cancer cells under normoxic and hypoxic conditions with qRT-PCR. The results also showed that the expression levels determined by qRT-PCR were consistent with RNA-Seq data. The identified protein-protein interaction network of common DEGs could serve as potential hypoxia biomarkers and might be helpful for improving therapeutic strategies.

Identification of

AP2/ERF transcription factors (TF) are widely involved in the biological process of Ginkgo biloba , here, the ERF gene family may regulate plant stress responses through mediating secondary metabolism. To understand the regulation mechanisms of ERF gene family during this process, two transcriptome databases of ginkgo leaves under UV-B and drought stress were constructed respectively. The conserved domains, physicochemical properties and gene family homology of AP2/ERF TFs were analyzed using online bioinformatics tools, the expression of ERF gene family under these two adversity stresses were predicted and five ERF genes involved in flavonoid pathway were selected for qRT - PCR validation analysis. The results showed that 61 AP2/ERF TFs were identified from G. biloba , and which were classified into 28 GbERF, 23 GbDREB, 5 GbAP2, and 5 GbRAV, according to their conserved domains and motifs. The Amino acid numbers, isoelectric point (pI) and some other physicochemical properties varied greatly between different members. Sequence homology analysis with Arabidopsis thaliana and Glycine max  showed that the members in the same AP2/ERF subfamily were closely related. In G. biloba , the GbERF and GbDREB subfamily together constitute the ERF gene family, transcriptome prediction analysis showed that the expression of most members of ERF gene family were increased under UV-B and drought stress. From the validation analysis, we concluded that the changes of the expression levels of these five genes were consistent with the prediction results. This study provided scientific basis for the function analysis of ERF gene family in G. biloba .

A Novel Pyroptosis-Related Gene Signature for Prediction of Disease-Free Survival in Papillary Thyroid Carcinoma.

The incidence and recurrence rate of papillary thyroid carcinoma (PTC) are high. Thus, it is critical to accurately identify patients at high risk of recurrence. Pyroptosis is a type of programmed cell death closely related to the progression and prognosis of cancer. However, the role of pyroptosis in PTC remains unclear. Transcriptome data for PTC patients were obtained from The Cancer Genome Atlas database. The expression level of pyroptosis-related genes (PRGs) in PTC and normal tissues was identified. Based on these differentially expressed genes, a risk score model of disease-free survival (DFS) was established using least absolute shrinkage and selection operator Cox regression. In-cluster and quantitative real-time PCR validations were carried out. A nomogram, in combination with clinical factors, was also established. In addition, its relationship with immune characteristics and tumor gene mutations is discussed. A risk score model with four PRGs, including CASP6, CASP9, IL-18, and NOD1, was established. The samples were divided into high- and low-risk clusters, according to the risk score, revealing significant differences in DFS between the two clusters. A nomogram was established combining age, lymph node metastasis and extrathyroidal extension. The area under the curve (AUC) of predicting one-, five-, and 10-year DFS in PTC patients was 0.745, 0.801, and 0.803, respectively. The low-risk cluster showed higher levels of immune infiltration and immune checkpoint gene expression, while the high-risk cluster demonstrated a higher tumor mutation burden. A predictive DFS model was established, based on PRGs, which may aid in identifying patients at high risk of recurrence. The present study helps to better understand the role of pyroptosis in the progression and prognosis of PTC.

A Cautionary Tale of Sexing by Methylation: Hybrid Bisulfite-Conversion Sequencing of Immunoprecipitated Methylated DNA in Chrysemys picta Turtles with Temperature-Dependent Sex Determination Reveals Contrasting Patterns of Somatic and Gonadal Methylation, but No Unobtrusive Sex Diagnostic.

Background: The gonads of Chrysemys picta, a turtle with temperature-dependent sex determination (TSD), exhibit differential DNA methylation between males and females, but whether the same is true in somatic tissues remains unknown. Such differential DNA methylation in the soma would provide a non-lethal sex diagnostic for TSD turtle hatchings who lack visually detectable sexual dimorphism when young. Methods: Here, we tested multiple approaches to study DNA methylation in tail clips of Chrysemys picta hatchlings, to identify differentially methylated candidate regions/sites that could serve as molecular sex markers To detect global differential methylation in the tails we used methylation-sensitive ELISA, and to test for differential local methylation we developed a novel hybrid method by sequencing immunoprecipitated and bisulfite converted DNA (MeDIP-BS-seq) followed by PCR validation of candidate regions/sites after digestion with a methylation-sensitive restriction enzyme. Results: We detected no global differences in methylation between males and females via ELISA. While we detected inter-individual variation in DNA methylation in the tails, this variation was not sexually dimorphic, in contrast with hatchling gonads. Conclusions: Results highlight that differential DNA methylation is tissue-specific and plays a key role in gonadal formation (primary sexual development) and maintenance post-hatching, but not in the somatic tail tissue.

Identification of miRNA-mRNA regulatory network associated with the glutamatergic system in post-traumatic epilepsy rats.

Glutamate is one of the most important excitatory neurotransmitters in the mammalian brain and is involved in a variety of neurological disorders. Increasing evidence also shows that microRNA (miRNA) and mRNA pairs are engaged in a variety of pathophysiological processes. However, the miRNA and mRNA pairs that affect the glutamatergic system in post-traumatic epilepsy (PTE) remain unknown. PTE rats were induced by injecting 0.1 mol/L, 1 μL/min FeCl2 solution. Behavioral scores and EEG monitoring were used to evaluate whether PTE was successfully induced. RNA-seq was used to obtain mRNA and miRNA expression profiles. Bioinformatics analysis was performed to screen differentially expressed mRNAs and miRNAs associated with the glutamatergic system and then predict miRNA-mRNA interaction pairs. Real-time quantitative reverse transcription PCR was used to further validate the expression of the differential miRNAs and mRNAs. The microRNA-mRNA was subject to the Pearson correlation analysis. Eight of the 91 differentially expressed mRNAs were associated with the glutamatergic system, of which six were upregulated and two were downregulated. Forty miRNAs were significantly differentially expressed, with 14 upregulated and 26 downregulated genes. The predicted miRNA-mRNA interaction network shows that five of the eight differentially expressed mRNAs associated with the glutamatergic system were targeted by multiple miRNAs, including Slc17a6, Mef2c, Fyn, Slc25a22, and Shank2, while the remaining three mRNAs were not targeted by any miRNAs. Of the 40 differentially expressed miRNAs, seven miRNAs were found to have multiple target mRNAs associated with the glutamatergic system. Real-time quantitative reverse transcription PCR validation and Pearson correlation analysis were performed on these seven targeted miRNAs-Slc17a6, Mef2c, Fyn, Slc25a22, and Shank2-and six additional miRNAs selected from the literature. Real-time quantitative reverse transcription PCR showed that the expression levels of the mRNAs and miRNAs agreed with the predictions in the study. Among them, the miR-98-5p-Slc17a6, miR-335-5p-Slc17a6, miR-30e-5p-Slc17a6, miR-1224-Slc25a22, and miR-211-5p-Slc25a22 pairs were verified to have negative correlations. Our results indicate that miRNA-mRNA interaction pairs associated with the glutamatergic system are involved in the development of PTE and have potential as diagnostic biomarkers and therapeutic targets for PTE.

Screening and characterization of sex-specific markers by NGS sequencing in Spinibarbus hollandi with implication of XY sex determination system

Sex-specific markers are critical for understanding sex determination mechanism and development of unisexual breeding in fish. Spinibarbus hollandi is an important commercial aquaculture species in southeastern China. Here, through whole genome sequencing of three female and six male individuals of S. hollandi, we first screened out a total of 88 candidate male-specific sequences. After PCR validation, three male-specific markers were proved to be able to realize male-specific amplification in S. hollandi. Subsequently, the three markers were also examined in another 24 female and 24 male individuals of S. hollandi, and the accuracy was 100%. Moreover, several male-specific genes that participate in sex determination process were identified by NR annotation, which might act as sex determining genes in S. hollandi. In general, the male-specific markers suggested an XY sex determination system in S. hollandi and would contribute a lot to understanding of sex determination mechanisms and development of unisexual breeding.

The Androgen Regulated lncRNA NAALADL2-AS2 Promotes Tumor Cell Survival in Prostate Cancer.

Castration resistance is the leading cause of death in men with prostate cancer. Recent studies indicate long noncoding RNAs (lncRNAs) to be important drivers of therapy resistance. The aim of this study was to identify differentially expressed lncRNAs in castration-resistant prostate cancer (CRPC) and to functionally characterize them in vitro. Tumor-derived RNA-sequencing data were used to quantify and compare the expression of 11,469 lncRNAs in benign, primary prostate cancer, and CRPC samples. CRPC-associated lncRNAs were selected for semi-quantitative PCR validation on 68 surgical tumor specimens. In vitro functional studies were performed by antisense-oligonucleotide-mediated lncRNA knockdown in hormone-sensitive prostate cancer (HSPC) and CRPC cell line models. Subsequently, cell proliferation, apoptosis, cell cycle, transcriptome and pathway analyses were performed using the appropriate assays. Transcriptome analysis of a prostate cancer tumor specimens unveiled NAALADL2-AS2 as a novel CRPC-upregulated lncRNA. The expression of NAALADL2-AS2 was found to be particularly high in HSPC in vitro models and to increase under androgen deprived conditions. NAALADL2-AS2 knockdown decreased cell viability and increased caspase activity and apoptotic cells. Cellular fractionization and RNA fluorescent in situ hybridization identified NAALADL2-AS2 as a nuclear transcript. Transcriptome and pathway analyses revealed that NAALADL2-AS2 modulates the expression of genes involved with cell cycle control and glycogen metabolism. We hypothesize that the nuclear lncRNA, NAALADL2-AS2, functions as a pro-survival signal in prostate cancer cells under pressure of targeted hormone therapy.

Cx3cr1 deficiency aggravates Aβ driven neurodegeneration and cognitive decline in a mouse model of Alzheimer's disease.

AbstractBackgroundDespite its identification as a key checkpoint regulator of microglial activation in Alzheimer’s disease, the overarching role of CX3CR1 signaling in modulating mechanisms of Aβ driven neurodegeneration, including accumulation of hyperphosphorylated tau in is not well understood.MethodUsing 4 ‐ and 6‐month old 5xFAD;Cx3cr1+/+ and 5xFAD;Cx3cr1‐/‐ mice, we use immunohistochemistry, western blotting, transcriptomic analysis and quantitative real time PCR to investigate accumulation of soluble and insoluble Aβ species, plaque associated microglial activation, synaptic dysregulation and neurodegeneration with disease progression. Flow cytometry based, Aβ uptake assays are used for in‐vivo characterization of the effects of CX3CR1‐signaling on microglial phagocytosis and degradation of methoxy‐X04+ fibrillar Aβ. Lastly, we use Y‐maze testing to analyze the effects of Cx3cr1 deficiency on working memory in 5xFAD mice.ResultWe show that disease progression in 5xFAD;Cx3cr1‐/‐ mice is characterized by increased deposition of filamentous plaques that display defective microglial plaque engagement, along with impaired microglial Aβ phagocytosis and lysosomal acidification in‐vivo. Interestingly, Cx3cr1 deficiency resulted in heighted accumulation of neurotoxic, oligomeric Aβ, which correlated with severe neuritic dystrophy, preferential loss of post‐synaptic densities, exacerbated tau pathology, neuronal loss and cognitive impairment. Transcriptomic analyses using cortical RNA coupled with real time PCR validation using FACS‐purified microglial cells revealed dysregulated TGFβ responses, inflammatory signaling and oxidative stress responses in 5xFAD;Cx3cr1‐/‐ mice.ConclusionIn contrast to increased Aβ uptake by Cx3cr1‐/‐ microglia during early disease, Cx3cr1 deficiency impairs microglial uptake and degradation of fibrillar Aβ, thereby triggering an accumulation of neurotoxic soluble and insoluble Aβ species with disease progression. Our results indicate that Aβ‐driven microglial dysfunction in Cx3cr1‐/‐ mice aggravates tau hyperphosphorylation, neurodegeneration, synaptic dysregulation and impairs working memory. This study underscores the importance of investigating the time‐dependent role of CX3CR1‐driven microglial responses in AD.

Virome Profiling of an Amur leopard cat Reveals Multiple Anelloviruses and a Bocaparvovirus.

As a small top predator, Amur leopard cat (Prionailurus bengalensis euptilurus) is widely distributed in northeast Asia and plays an important role in the control of small rodent populations and in the maintenance of ecological equilibrium. However, the viruses harbored by this creature have been rarely investigated. Here, we report the DNA and RNA eukaryotic virome profiling of an injured Amur leopard cat followed by PCR validation, which revealed diverse anelloviruses in multiple organs and a bocaparvovirus in the lymph, but no RNA viruses. These anelloviruses have diverse genomic structures and are classified into four phylogroups with viruses of various felines, while the bocaparvovirus is extremely similar to those recovered from diarrheal domestic cats, illustrating the transmission of the virus between domestic animals and wildlife. These data provide the first insight into the genetic diversity of Amur leopard cat viruses, highlighting the need for further investigation of wild animals.

Real-time PCR validation to estimate the number of rickettsias in the biological material under study.

Using the example of the clinical strain of R. sibirica «Bayevo 105/87», the possibility of quantitative determination of rickettsias in clinical samples from patients with Siberian tick-borne typhus by real-time polymerase chain reaction (PCR-RT) was evaluated. Cultivation was carried out in the yolk sacs of developing chicken embryos, from which a piece of the yolk sac or chorion was taken. A total of 125 samples were examined. A set of reagents "RealBest DNA Rickettsia species (kit1)" was used for PCR-RT. The obtained values of the threshold amplification cycle (Ct) were compared with the results of microscopy of smear preparations stained by the Zdrodovsky method, the values of which were divided into ranks: the I rank - single rickettsias in individual fields of vision, the II rank - single rickettsias in each field of vision, the III rank - from 10 to 25 rickettsias in each field of vision, the IV rank - from 25 to 50 rickettsias in each field of view. The median Ct value for rank I was 17.6 (16.37; 18.58), for the II - 16.0 (15.0; 16.41), for the III - 15.0 (14.0; 15.1) and for the IV - 15.0 (13.7; 14.64). A significant average correlation was established between the number of rickettsias in the preparation under microscopy and the value of the threshold cycle in PCR RT (r=-0, 4849542; p=9.968e-09). When determining the correlation between the pathomorphological characteristic and the value of the threshold cycle, its absence was established. The detection of rickettsias in the blood vessels of the chorion of developing chicken embryos was of interest. In 10 samples, the yolk sac and chorion were taken for the study, and in parallel they were examined by PCR-RT. The use of modern, more sensitive molecular biological methods allows for quantitative analysis of DNA in the chorion, while preserving the volumes of the most valuable material - the yolk sac.

Comparative transcriptomic analysis of the haustoria of Gymnosporangium yamadae and G. asiaticum

To provide a theoretical basis for controlling the spread of rust disease, cultivating disease-resistant varieties and reducing yield losses, we investigated the transcriptome differences between Gymnosporangium yamadae and Gymnosporangium asiaticum at the haustorial stage and revealed a specialized selection mechanism for Gymnosporangium species to infect host plants. We sequenced the transcriptomes of the haustoria in rust-infected leaves when basidiospores of G. yamadae and G. asiaticum infected their hosts, and obtained 21 213 and 13 015 unigenes, respectively. Real-time fluorescence quantitative PCR validation of five genes selected from G. yamadae and G. asiaticum, respectively, showed that their expression profiles were generally consistent with the results of transcriptome analysis, demonstrating the reliability of the transcriptome data. We used seven databases such as Nr, GO, KEGG, and KOG to perform gene function annotation and enrichment analysis, and found that the genes from both rusts were mainly enriched in cellular processes, translation, and metabolism-related pathways. Moreover, we used SignalP, TMHMM online website and other software such as dbCAN, BLSAT, HMMER to show that there were 343 (2.51%) and 175 (2.79%) candidate effector proteins containing 14 and 5 proteases and 10 and 3 lipases in the haustoria of G. yamadae and G. asiaticum, respectively. Furthermore, we used OrthoFinder, BLAST and KaKs Calculator software to analyze the evolutionary relationship of the two fungi. Among one-to-one homologous genes, gene pairs with > 82% alignment were considered to be under conservative selection, and 12.37% under positive selection. Five effectors of G. asiaticum were under positive selection, and one of which was a lipase. No significant differences were found in the enrichment of expressed genes between G. yamadae and G. asiaticum, indicating the biological processes involved in haustoria were relatively conserved, despite the typical host selectivity between species. The low protein similarity between the two species suggested that they were under greater host selective pressure and there was significant evolutionary divergence, which might be related to the host-specific selection mechanism. In the haustorial, the main purpose of the effectors might be to regulate physiological processes in the plants rather than attacking the host directly, and G. yamadae and G. asiaticum might use plant lipids as energy sources.

Pancreatic Cancer-Derived Exosomes Promote the Proliferation, Invasion, and Metastasis of Pancreatic Cancer by the miR-3960/TFAP2A Axis.

Background The microRNAs (miRNAs) in cancer-derived exosomes have the ability to change tumor microenvironment. This study aims to investigate the role of miRNA in cancer-derived exosomes in pancreatic cancer (PC). Methods Based on the analysis of PC-derived and healthy exosomes by bioinformatics analysis and quantitative real-time PCR validation, the miR-3960 was identified to be the most significantly different miRNA, and TFAP2A proved as its potential target gene. Besides, the exosomes were isolated from PANC-1 cells and identified. After that, PANC-1 cells were treated with the isolated exosomes or transfected with miR-3960 mimics or si-TFAP2A, the effect of PC-derived exosomes, as well as the miR-3960/TFAP2A axis in PC cells, were assessed by the CCK-8, EDU staining, Transwell, cell colony formation, and flow cytometry assays. Furthermore, the effects of exosomes and the miR-3960/TFAP2A axis on PC tumor growth were observed in tumor-bearing mice by the measurement of tumor weight and volume, and hematoxylin-eosin staining. Moreover, the expressions of TFAP2A/PTEN/AKT signaling proteins were detected by Western blot. Results PC-derived exosomes were isolated successfully and proved to have promotion effects on the proliferation, metastasis, and invasion of PC cells both in vitro and tumor growth in vivo. Also, the PC-derived exosomes upregulated the TFAP2A, Bcl-2, and p-AKT/AKT protein levels, and inhibited PTEN and Bax levels and PANC-1 cell apoptosis. Overexpression of miR-3960 antagonized the promotion effect of exosomes on PC cells and the TFAP2A/PTEN/AKT signaling pathway, inhibiting the growth of tumors. Besides, si-TFAP2A enhanced the inhibitory effect of miR-3960 in PC. Conclusion MiR-3960 antagonizes the promotion effect of tumor-derived exosomes on the proliferation, invasion, and metastasis of PC via suppressing TFAP2A.

MiRNome-transcriptome analysis unveils the key regulatory pathways involved in the tumorigenesis of tongue squamous cell carcinoma.

Tongue squamous cell carcinoma (TSCC) is considered the most common malignant tumor among the oral squamous cell carcinomas with a poor prognosis. Understanding the underlying molecular mechanisms that underpin TSCC and its treatments is the focus of the research. Deregulated expression of microRNAs (miRNAs) has recently been implicated in various biological processes linked to cancer. Therefore, in this study, we attempted to investigate miRNAs and their targets expressed in TSCC, which could be involved in its oncogenesis. We performed next-generation sequencing of small RNAs and transcriptomes in H357 TSCC cell line and human oral keratinocytes as a control to find miRNAs and mRNAs that are differentially expressed (DE), which were then supplemented with additional expression datasets from databases, yielding 269 DE miRNAs and 2094 DE genes. The target prediction followed by pathway and disease function analysis revealed that the DE targets were significantly associated with the key processes and pathways, such as apoptosis, epithelial-mesenchymal transition, endocytosis and vascular endothelial growth factor signaling pathways. Furthermore, the top 12 DE targets were chosen based on their involvement in more than one cancer-related pathway, of which 6 genes are targeted by miR-128-3p. Real-time quantitative PCR validation of this miRNA and its targets in H357 and SCC9 TSCC cells confirmed their possible targeting from their reciprocal expression, with MAP2K7 being a critical target that might be involved in oncogenesis and progression of TSCC by acting as a tumor suppressor. Further research is underway to understand how miR-128-3p regulates oncogenesis in TSCC via MAP2K7 and associated pathways.

Pentraxin 3 is an adipose tissue-related serum marker for pancreatic cancer cachexia predicting subsequent muscle mass and visceral fat loss.

Cancer cachexia, a paraneoplastic syndrome characterized by ongoing skeletal muscle mass loss, is accompanied by adipose tissue loss and strongly affects chemotherapy endurance. Our aim was to detect a serum marker reflecting pancreatic cancer cachexia and predicting subsequent loss of muscle mass and adipose tissue, focusing on adipose tissue-secreted proteins. Murine-derived pancreatic cancer cells were orthotopically injected into the mouse pancreatic tail. After 3 weeks, RNA sequencing of perigonadal fat and orthotopic tumors was carried out. We analyzed stocked sera and clinical data of metastatic pancreatic cancer patients who received chemotherapy. Perigonadal fat weight/body weight decreased in mice with orthotopic tumors compared to those without tumors. By RNA sequencing and real-time PCR validation, pentraxin 3 (PTX3) was identified as a secreted protein-encoded gene whose expression was significantly higher in the perigonadal fat of mice with orthotopic tumors than in that of mice without orthotopic tumors and was least expressed in orthotopic tumors. Serum PTX3 levels correlated with PTX3 mRNA levels in perigonadal fat and were higher in mice with orthotopic tumors than in those without tumors. In 84 patients diagnosed with metastatic pancreatic cancer, patients with high serum PTX3 levels showed a greater visceral fat loss/month and skeletal muscle mass index (SMI) decrease/month than those with low serum PTX3 levels. High serum PTX3 was an independent risk factor for visceral fat loss, decreased SMI, and poor prognosis. High serum PTX3 in pancreatic cancer patients predicts visceral fat and muscle mass loss and major clinical outcomes of cancer cachexia.

Uncovering the mechanism of anthocyanin accumulation in a purple-leaved variety of foxtail millet (Setaria italica) by transcriptome analysis.

Anthocyanin is a natural pigment that has a functional role in plants to attract pollinating insects and is important in stress response. Foxtail millet (Setaria italica) is known as a nutritional crop with high resistance to drought and barren. However, the molecular mechanism regulating anthocyanin accumulation and the relationship between anthocyanin and the stress resistance of foxtail millet remains obscure. In this study, we screened hundreds of germplasm resources and obtained several varieties with purple plants in foxtail millet. By studying the purple-leaved B100 variety and the control variety, Yugu1 with green leaves, we found that B100 could accumulate a large amount of anthocyanin in the leaf epiderma, and B100 had stronger stress tolerance. Further transcriptome analysis revealed the differences in gene expression patterns between the two varieties. We identified nine genes encoding enzymes related to anthocyanin biosynthesis using quantitative PCR validation that showed significantly higher expression levels in B100 than Yugu1. The results of this study lay the foundation for the analysis of the molecular mechanism of anthocyanin accumulation in foxtail millet, and provided genetic resources for the molecular breeding of crops with high anthocyanin content.

MicroRNA-targeting in male infertility: Sperm microRNA-19a/b-3p and its spermatogenesis related transcripts content in men with oligoasthenozoospermia.

Objective: To elucidate and validate the potential regulatory function of miR-19a/b-3p and its spermatogenesis-related transcripts content in sperm samples collected from men with oligoasthenozoospermia. Methods: Men presenting at an infertility clinic were enrolled. MicroRNA (miRNA) and target genes evaluation were carried out using in silico prediction analysis, Reverse transcription-quantitative PCR (RT-qPCR) validation, and Western blot confirmation. Results: The expression levels of miRNA-19a/b-3p were significantly up-regulated and 51 target genes were significantly down-regulated in oligoasthenozoospermic men compared with age-matched normozoospermic men as determined by RT-qPCR. Correlation analysis highlighted that sperm count, motility, and morphology were negatively correlated with miRNA-19a/b-3p and positively correlated with the lower expression level of 51 significantly identified target genes. Furthermore, an inverse correlation between higher expression levels of miRNA-19a/b-3p and lower expression levels of 51 target genes was observed. Consistent with the results of the RT-qPCR, reduced expression levels of STK33 and DNAI1 protein levels were identified in an independent cohort of sperm samples collected from men with oligoasthenozoospermia. Conclusion: Findings suggest that the higher expression of miRNA-19a/b-3p or the lower expression of target genes are associated with oligoasthenozoospermia and male infertility, probably through influencing basic semen parameters. This study lay the groundwork for future studies focused on investigating therapies for male infertility.