Rapid Amplification Of cDNA Ends Research Articles

HcCnAα regulates NF-κB signaling in Hyriopsis cumingii by interacting with HcIKK and facilitating IκB phosphorylation.

Calcineurin (CN), a serine/threonine protein phosphatase regulated by Ca2+ and calmodulin, plays a crucial role in the immune response of bivalves. In this study, we examined the effects of gene silencing of the CN subunit (HcCnAα) in Hyriopsis cumingii on the expression of genes associated with the NF-κB signaling pathway, as well as the phosphorylation of the inhibitor of NF-κB (IκB), through RNA interference (RNAi), quantitative PCR (qPCR), and Western blot (WB) analyses. The IκB kinase (HcIKK) and B-cell CLL/lymphoma 10 (HcBcl10) genes of H. cumingii were cloned using rapid amplification of cDNA ends, and protein interactions with HcCnAα were investigated through yeast two-hybrid assays. The results demonstrated that RNAi-mediated knockdown of HcCnAα significantly reduced the expression of IKK, p65, and downstream immune-related genes in the NF-κB signaling pathway-Lys, The, Def, α2-M, TNF-α, and IL-17-all showing significant decreases (P<0.05). Additionally, phosphorylation of IκB was inhibited. These findings suggest that HcCnAα plays a regulatory role in the NF-κB signaling pathway, influencing the expression of downstream immune response-related genes and defense proteins. Furthermore, yeast two-hybrid assay results indicated a direct protein-protein interaction between HcIKK and HcCnAα, while no interaction was observed between HcBcl10 and HcCnAα. This study elucidates the specific molecular mechanisms by which HcCnAα regulates the immune response in H. cumingii, providing a foundational basis for further exploration of immune regulation mechanisms and the development of disease prevention strategies in bivalves.

Molecular characterisation and expression profiles of an odorant-binding proteins gene (FoccOBP9) from Frankliniella occidentalis.

Insect odorant-binding proteins (OBPs) are the key proteins in insect olfactory perception and play an important role in the perception and discrimination of insects. Frankliniella occidentalis is a polyphagous pest and seriously harms the quality and yield of fruits, flowers and crops worldwide. Therefore, the discovery of OBPs has greatly improved the understanding of behavioural response that mediates the chemoreception of F. occidentalis. To identify the OBP gene of F. occidentalis and its sequence and expression, rapid amplification cDNA ends (RACE) and qRT-PCR reaction system were performed. The results showed that the sequence of FoccOBP9 gene was 846 bp and the reading frame was 558 bp, encoding 185 amino acid residues, a 3' non-coding region of 195 bp and a 5' non-coding region of 93 bp.The molecular weight of the protein was about 20.08 kDa, and the isoelectric point was 8.89. FoccOBP9 was similar to AtumGOBP and CnipOBP2 (30%), followed by BdorGOBP, DficGOBP, DsuzGOBP, AalbOBP38, CmarOBP6 and SexiOBP. Phylogenetic analysis of the FoccOBP9 demonstrated that the FoccOBP9 had a relatively close evolutionary relationship with SgreOBP1, AtumGOBP, HeleOBP3, CbowOBP17, CnipOBP2 and CpalOBP2. The prediction of secondary structure showed that FoccOBP9 protein contained 135 amino acid residues forming α-helix, 91 amino acid residues forming β-sheets and 24 amino acid residues forming β-turning. However, three-dimensional structure prediction showed that the FoccOBP9 protein skeleton was composed of six α-helices and the loops connecting these helices. Dynamic observation of the three-dimensional structure revealed that five α-helices (α1, α2, α4, α5, α6) were found in the structure. The expression profiles analysis revealed that FoccOBP9 are highly abundant in antenna significantly, followed by the head and belly, and almost no expression in the chest and foot. Therefore, the identification and analysis of OBP may be useful for monitoring and limiting the damage of F. occidentalis.

Comprehensive analysis of small RNA, transcriptome, and degradome sequencing: Mapping the miRNA-gene regulatory network for the development of sweet potato tuber roots.

As an important starch crop, sweet potato has significant practical importance for maintaining food security worldwide. This study identified differential expressed genes associated with the expansion of tuberous roots by comparing the transcriptome across tuberous roots at the initial period (initiated tuberous roots (ITRs), rapid expansion period (tuberous roots (TRs), fibrous roots (FRs) at the seedling stage, and fibrous roots at the adult stage (unexpanded FRs (UFRs)). sRNA-seq and degradome analyses were performed to reveal the role of miRNAs in tuberous root development in sweet potato. A total of 29,633 genes and 510 miRNAs were differential expressed among FRs, ITRs, TRs, and UFRs. Integrated analyses of these data revealed genes involved in metabolism, hormone response, and signal transduction that might participate in the induction of tuberous root formation, while genes involved in carbohydrate and energy metabolism that might participate in the tuberous root swelling. A joint analysis of miRNAs and DEGs related to tuber development revealed by degradome-seq identified twelve miRNA-target gene pairs involved in gene expression process, hormone response, and metabolism of secondary metabolites that might be key regulators of root tuber development in sweet potato. Moreover, the functions of many miRNA-target gene pairs involved in the initiation of root tuber were related to auxin signaling response, and an exogenous hormone treatment experiment was further performed. The results indicated that auxin treatment had the most significant effect on increasing sweet potato yield, suggesting a dominant role of the auxin pathway in the regulation of sweet potato tuberous root development. Additionally, two miRNA-target pairs, miR319-TCP4 and miR172-AP2, which were identified from the degradome, were verified via 5' RNA ligase-mediated rapid amplification of cDNA ends (RLR-RACE) and tobacco transient cotransformation tests, and their expression was impacted by auxin treatment, which further validated the reliability of our multiomics analysis results. Our research provides new insights into the role of miRNAs in sweet potato root tuber development.

Characterization and expression analysis of heat shock proteins HfHspc1 and HfHspc4 in Haemaphysalis flava ticks.

In this study, the cDNAs of heat shock cognate 90 protein 1 (HfHspc1) and heat shock cognate 90 protein 4 (HfHspc4) from Haemaphysalis flava (Acari: Ixodidae) were obtained using the rapid amplification of cDNA ends (RACE) approach, and the expression patterns of HfHspc1 and HfHspc4 in different developmental stages, engorgement stages and tick organs were analyzed by qPCR. The full length of HfHspc1 was 2411 bp, and its open reading frame (ORF) was 2196 bp, encoding a protein of 732 aa, containing five HSPC family signatures, with MEEVD motif at its extreme C-terminal. The full length of HfHspc4 was 2800 bp, and its ORF was 2364 bp, encoding a protein of 789 aa, containing a signal peptide and five family signatures, with HEEL motif at its extreme C-terminal. The expression of HfHspc1 and HfHspc4 was the highest in males, while it was significantly the highest in the ovaries of fully engorged females, potentially implying the roles of these proteins in the successful digestion of blood and development of eggs.

Complete genome sequence of a novel iflavirus from wheat sawfly (Dolerus tritici).

Little is known about the insect viruses in wheat sawfly, Dolerus tritici, which is an important agricultural insect feeding on wheat leaves. Here, we used RNA sequencing to identify a novel single positive-strand RNA virus from the larvae of wheat sawfly collected in northern China and then determined its complete genome sequence by rapid amplification of cDNA ends. The complete genome is 9,594 nt in length, including a polyA tail at its 3' terminus, and it is predicted to encode a 326.3-kDa polyprotein. Phylogenetic analysis based on deduced amino acid sequences of the polyprotein revealed that this RNA virus clustered in a clade with deformed wing virus of the genus Iflavirus, family Iflaviridae. The full genome of this RNA virus shows 42.0-50.0% sequence identity with other iflaviruses. Comparisons of amino acid sequences showed that the coat protein of this RNA virus is most similar to that of slow bee paralysis virus, with 33.6% identity, suggesting that this virus is a new member in the genus Iflavirus. Thus, we have tentatively designated it as "Dolerus tritici iflavirus 1" (DtIV1). To our knowledge, this is the first report of an insect virus in wheat sawfly.

Comparative study of two Rift Valley fever virus field strains originating from Mauritania.

Rift Valley fever (RVF) is one of the major viral arthropod-borne diseases in Africa. In recent decades, RVF virus (RVFV), the causative agent of RVF, has been responsible for multiple outbreaks in West Africa with important consequences on human and animal health. In particular, an outbreak occurred in 2010 after heavy rains in the desertic region of Adrar, Mauritania. It was characterized by the appearance of severe clinical signs among dromedary camels. Another one occurred in 2013-2014 across Senegal and the southern part of Mauritania. In this study, we characterized two RVFV field strains isolated during these two outbreaks. The first strain, MRU25010-30, was isolated from a camel (2010) while the second, MRU2687-3, was isolated from a goat (2013). By deep-sequencing and rapid amplification of cDNA-ends by polymerase chain reaction, we successfully sequenced the complete genome of these two RVFV strains as well as the reference laboratory strain ZH548. Phylogenetic analysis showed that the two field viruses belong to two different RVFV genetic lineages. Moreover, we showed that MRU25010-30 replicates more efficiently in various in vitro cell culture models than MRU2687-3 and ZH548. In vivo, MRU25010-30 caused rapid death of BALB/c mice and proved to be more virulent than MRU2687-3, regardless of the route of inoculation (subcutaneous or intranasal). The virulence of MRU25010-30 is associated with a high viral load in the liver and serum of infected mice, while the death of mice infected with MRU2687-3 and ZH548 correlated with a high viral load in the brain. Altogether, the data presented in this study provide new avenues to unveil the molecular viral determinants that modulate RVFV virulence and replication capacity.

Identification of the unique beginning of the 5’-end of the RNA1 of the Iranian squash mosaic virus genome using the 5’- RACE (Rapid amplification of cDNA ends) technique

Identification of the unique beginning of the 5’-end of the RNA1 of the Iranian squash mosaic virus genome using the 5’- RACE (Rapid amplification of cDNA ends) technique

The novel role of LOC344887 in the enhancement of hepatocellular carcinoma progression via modulation of SHP1-regulated STAT3/HMGA2 signaling axis.

Pseudogene-derived long non-coding RNAs (lncRNAs) have become crucial regulators in cancer progression. Extensive research highlights the pivotal role of signal transducer and activator of transcription 3 (STAT3) in promoting hepatocellular carcinoma (HCC) progression. As a result, targeting aberrant STAT3 activation presents a promising therapeutic strategy for HCC. Our study aims to identify the key pseudogene-derived lncRNA involved in modulating STAT3 activation and driving HCC progression. Our study is the first to identify a significant upregulation of LOC344887, a pseudogene-derived lncRNA, in HCC tissues. Elevated LOC344887 levels correlated with poor overall survival (OS) and recurrence-free survival (RFS), highlighting its potential as a biomarker for HCC. The rapid amplification of cDNA ends (RACE) and RT-PCR experiments revealed the expression of a novel LOC344887 transcript, named LOC344887-v2, alongside the annotated RefSeq transcript NR_151491 (LOC344887-v1) in both HCC tissues and hepatoma cell lines. Functional assays demonstrated that LOC344887 enhances cellular migration and invasion, with its variant LOC344887-v2 exhibiting a more pronounced effect. Further, LOC344887 mechanistically regulates STAT3 phosphorylation at tyrosine 705, which is crucial for maintaining STAT3 activation in HCC. Our findings unravel that LOC344887 not only physically interacts with p-STAT3 but also prevents its dephosphorylation by src homology region 2 domain-containing phosphatase 1 (SHP-1), thereby sustaining oncogenic signaling. In addition, we identified HMGA2 as a target of the LOC344887/SHP-1/STAT3 axis, with higher HMGA2 expression correlating with poorer prognosis in HCC patients. The ability of LOC344887 to regulate HMGA2 through direct binding of STAT3 to its promoter underlines its role in HCC progression. Collectively, these findings elucidate a novel oncogenic role of LOC344887 in HCC and suggest that targeting this lncRNA and its associated pathways may provide novel therapeutic strategies for improving patient outcomes in HCC.

The Molecular Characterization and Antioxidant Defense of a Novel Nrf2 from the Pacific Abalone Haliotis discus hannai Ino.

The Nrf2/ARE pathway is considered the most important endogenous antioxidant signaling pathway in mammals, playing a crucial role in defending against external damage. This study investigated the functional characteristics of Nrf2 in the abalone, Haliotis discus hannai. The full-length cDNA sequence of the HdhNrf2 gene was cloned using rapid amplification of cDNA ends (RACE) technology and consists of 4568 base pairs encoding a protein of 694 amino acids. The predicted theoretical molecular weight was 77 kDa, with an isoelectric point of 4.72. Multiple sequence alignment analysis revealed the relative conservation of the HdhNrf2 amino acid sequence in H. discus hannai. The tissue expression pattern of the HdhNrf2 gene was analyzed using real-time fluorescence quantitative PCR, which showed the highest expression in the gills, followed by hemocytes, with the lowest levels in the foot and mantle. The inducible expression of HdhNrf2 and antioxidant genes in abalone under H2O2 stress was investigated at various time points. Furthermore, an expression vector, pET-28a(+)-rHdhNrf2, was constructed, and the recombinant protein rHdhNrf2 was obtained through induced expression and purification. These findings indicated that HdhNrf2 plays a crucial role in the defense of abalones against oxidative stress.

A new cancer/testis long noncoding RNA, the OTP-AS1 RNA

The orthopedia homeobox (OTP) gene encodes a homeodomain-containing transcription factor involved in brain development. OTP is mapped to human chromosome 5q14.1. Earlier we described transcription in the second intron of this gene in wide variety of tumors, but among normal tissues only in testis. In GeneBank these transcripts are represented by several 300–400 nucleotide long AI267901-like ESTs. We assumed that the AI267901-like ESTs belonged to the longer transcript(s). We used the Rapid Amplification of cDNA Ends (RACE) approach and other methods to find the full-length transcript. The transcript we found was a 2436 nucleotide polyadenylated sequence in antisense to OTP gene. The corresponding gene consisted of two exons separated by an intron of 2961 bp. The first exon was found to be 91 bp long and located in the third exon of OTP. The second exon was 2345 bp long and located in the second intron of OTP. We have shown the expression of this gene in many human tumors but as few as a single sample of normal testis. The transcript lacked significant ORFs suggesting that we discovered a new antisense cancer/testis (CT) sequence OTP-AS1 (OTP—antisense RNA 1), which belongs to the class of long noncoding RNAs (lncRNAs). According to our findings we assume that OTP-AS1 and OTP genes may be a CT-coding gene/CT-ncRNA pair, or sense-antisense gene pair involved in regulatory interactions.

Use of 3' Rapid Amplification of cDNA Ends (3' RACE)-Based Targeted RNA Sequencing for Profiling of Druggable Genetic Alterations in Urothelial Carcinomas.

Targeted treatment of advanced or metastatic urothelial carcinomas (UCs) requires the identification of druggable mutations. This study describes the development of a 3' Rapid Amplification of cDNA Ends (3' RACE)-based targeted RNA sequencing panel which accounts for the status of all genes relevant to UC treatment, namely, FGFR1-4, KRAS, NRAS, BRAF, ERBB2 (HER2), CD274 (PD-L1) and PIK3CA. FGFR2/3-activating point mutations or fusions were found in 54/233 (23.2%) tumors. FGFR3 rearrangements were identified in 11 patients, with eight of them being undetectable by commonly used PCR kits. In addition, one tumor contained a high-copy FGFR2 gene amplification accompanied by strong overexpression of the gene. Mutations in RAS/RAF genes were present in 30/233 (12.9%) UCs and were mutually exclusive with alterations affecting FGFR2/3 genes. On the contrary, activating events in the HER2 oncogene (point mutations and overexpression), as well as PIK3CA mutations, which were relatively common, occurred with similar frequencies in RAS/RAF- or FGFR2/3-positive vs. negative samples. High PD-L1 mRNA expression was associated with advanced disease stage and was not observed in tumors with increased HER2 mRNA expression or in UCs with evidence for FGFR2/3 activation. Three of the studied carcinomas had high-level microsatellite instability (MSI). Overall, more than half of the UCs had potentially druggable genetic alterations. The proposed NGS panel permits comprehensive and cost-efficient analysis of UC-specific molecular targets and may be considered in clinical routine.

CTIM-18. HIGH LEVEL OF MUTATIONAL LOAD AND DIVERSITY OF TUMOR INFILTRATING T LYMPHOCYTES ARE FAVORABLE PROGNOSTIC MARKERS FOR CANCER SURVIVAL IN CHILDREN WITH RECURRENT MEDULLOBLASTOMA DURING ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive cellular therapy (ACT) has shown remarkable efficacy in some patients with solid tumors, resulting in durable complete responses. A key mechanism behind this success is the targeting of neoantigens by T cells. There is culminating evidence that tumor-infiltrating lymphocytes (TILs) have a major effect on the clinical attributes of human cancer and can influence the tumor response to various therapy regimens. We have been sequencing RNA isolated from patient’s tumors to evaluate their mutational burden and identify tumor-specific neoantigens. Additionally, we have identified the entire repertoire of tumor-infiltrating lymphocytes in patients with recurrent medulloblastoma. METHODS Total tumor RNA was extracted from resected brain tumor specimens and after we applied in-house bulk RNA-Seq and TCR-Seq techniques using rapid amplification of cDNA ends (RACE) technology with the addition of a common adapter at the 5’ end. RESULTS &amp; CONCLUSIONS Analysis of the 22 tumor tissues revealed that the frequency of non-synonymous mutations varied dramatically more than 10-fold among patients with recurrent medulloblastoma undergoing adoptive cellular therapy (Re-MATCH protocol, FDA IND BB-14058). Patients with shorter overall survival exhibited non-synonymous mutation frequencies between 0.113 and 0.335 mutations per megabase (Mb). Children with prolonged overall survival had higher mutation frequencies, ranging from 0.6 to 1.1 mutations per Mb. These findings highlight the potential of using mutational burden as a biomarker for patient selection and treatment customization, ultimately aiming to improve outcomes in ACT for recurrent medulloblastoma. Up to 2000 unique TCR clones were found among TIL populations in patients who responded well to ACT. Patients with poor outcomes had significantly lower TCR diversity, with only 10-40 unique TCR clones among TILs. The correlation between TCR diversity among TILs and clinical outcomes in pediatric brain tumors underscores the importance of a diverse T cell repertoire for the success of ACT.

A novel ITGB8 transcript variant sustains ovarian cancer cell survival through genomic instability and altered ploidy on a mutant p53 background

BackgroundTranscript variants and protein isoforms are central to unique tissue functions and maintenance of homeostasis, in addition to being associated with aberrant states such as cancer, where their crosstalk with the mutated tumor suppressor p53 may contribute to genomic instability and chromosomal rearrangements. We previously identified several novel splice variants in ovarian cancer RNA-sequencing datasets; herein, we aimed to elucidate the biological effects of the Integrin Subunit Beta 8 variant (termed pITGB8-205).MethodsResolution of the full-length sequence of pITGB8-205 through rapid amplification of cDNA ends (RACE-PCR). Cell cycle analysis and karyotype studies were performed to further explore genomic instability. RNA-seq and proteomics analyses were used to identify the differential expression of the genes.ResultsThis full-length study revealed a unique 5’ sequence in pITGB8-205 that differed from the reported ITGB8-205 sequence, suggesting differential regulation of this novel transcript. Under a p53 mutant background, overexpression of pITGB8-205 triggered genetic instability reminiscent of oncogene-induced replicative stress with extensive abnormal mitoses and chromosomal and nuclear aberrations indicative of chromosomal instability, leading to near whole-genome duplication that imposes energy stress on cellular resources. Micronuclei and aneuploidy are striking features of pITGB8-205-overexpressing p53-mutant cells but are not enhanced in p53 wild-type (WT) cells. RNA-seq and proteomics analyses further suggested that p53 inactivation in ovarian cancer provides a permissive intracellular molecular niche for pITGB8-205 to mediate its effects on genomic instability. This observation is pivotal considering that most high-grade serous ovarian carcinoma (HGSC) tumors express mutant p53. The resulting aneuploid clones with enhanced self-renewal and survival capabilities disrupt clonal dominance under stress yet maintain a balance between replicative stress and prosurvival advantages.ConclusionpITGB8-205-overexpressing clones sustain ovarian tumor cell survival, achieve homeostasis and are formidable opponents of therapy.

Identification and Characterization of Four Novel Viruses in Balclutha incisa.

Balclutha incisa (Cicadellidae: Deltocephalinae), a leafhopper prevalent in tropical and temperate regions, is notably abundant in grasses and rice. The virome of B. incisa was investigated using deep transcriptome sequencing, leading to the first identification of four viruses belonging to the families Aliusviridae, Iflaviridae, and Totiviridae in B. incisa. These viruses have been provisionally named B. incisa ollusvirus 1 (BiOV1), B. incisa ollusvirus 2 (BiOV2), B. incisa iflavirus 1 (BiIV1), and B. incisa totivirus 1 (BiTV1). The complete genome sequences of these viruses were obtained through rapid amplification of cDNA ends (RACE). BiOV1 has a linear genome of 15,125 nucleotides (nt), while BiOV2 possesses a circular genome of 14,853 nt. The BiIV1 genome, excluding the poly(A) tail, is 10,903 nt in length and encodes a single open reading frame (ORF) for a polyprotein consisting of 3194 amino acids (aa). The BiTV1 genome is 4357 nt long and contains two overlapping ORFs, with the viral RNA-dependent RNA polymerase (RdRp) translated via a -1 ribosomal frameshift. Phylogenetic and sequence identity analyses suggest that all these viruses are novel members of their respective families. This study significantly expands our understanding of the virome associated with B. incisa by reporting and characterizing these novel viruses.

Structural and functional characterization of haemoglobin genes in Labeo catla: Insights into hypoxic adaptation and survival

Haemoglobin (HB) protein comprises four subunits: two identical α-subunits (HBA) and two identical β-subunits (HBB), encoded by the HBA and HBB genes. In this investigation, 5′/3′ RACE PCR (Rapid Amplification of cDNA Ends) was used to obtain complete coding sequences (CDSs) of both the genes from farmed Labeo catla. The resulting CDSs were 432 base pairs and 447 base pairs for HBA and HBB, respectively, corresponding to 143 and 148 amino acids. Phylogenetic analysis revealed close relationships with other cyprinids, with Labeo rohita being the closest relative. Functional analysis and protein structure prediction were conducted using bioinformatics tools. Expression profiling of both genes was checked in various tissues under control (C) and hypoxic (H) conditions. Notably, under hypoxia, HBA and HBB genes were significantly upregulated (P < 0.05) initially, followed by a return to normal expression levels. Similar trends were observed for Hif1α (Hypoxia-inducible factor one alpha) and EPO (Erythropoietin) genes. Additionally, haematological indices also significantly increased corresponding to the gene expressions. However, with the decrease in the expression of these genes an onset of mortality was observed in the hypoxia (H) treated groups. The results of the current study explored the role of haemoglobin genes in adaptation to the hypoxic condition.

Characterization and functional analysis of Litopenaeus vannamei Na+/K+/2Cl− cotransporter 1 under nitrite stress

The function of Litopenaeus vannamei Na+/K+/2Cl− cotransporter 1 (NKCC1) under nitrite stress was investigated. The full-length cDNA sequence of the L. vannamei NKCC1 gene was cloned using the rapid amplification of cDNA ends (RACE) technique, and the sequence was analysed using bioinformatics tools. Expression and localisation of NKCC1 in tissues were assessed using real-time quantitative PCR and in situ hybridisation, respectively. The impact of nitrite stress on the survival, physiology, biochemistry and tissue structure of L. vannamei was investigated following silencing of NKCC1 by RNA interference. The 3143 bp cDNA sequence of L. vannamei NKCC1 encodes a polypeptide of 918 amino acids. It is evolutionarily conserved. NKCC1 expression was highest in gill tissue, particularly within cuticle and gill epithelial cells. After silencing NKCC1, an increase in shrimp survival was observed, accompanied by a significant reduction in nitrite entry into the body (P < 0.05). Moreover, the oxidative stress enzyme system remained unaffected and damage to gill tissue was alleviated. The results suggest that NKCC1 is involved in regulating nitrite uptake, and plays a crucial role in facilitating nitrite entry into the organism through gill tissue. The findings provide a vital experimental basis for addressing concerns related to nitrite toxicity.

Regression of renal cell carcinoma by T cell receptor-engineered T cells targeting a human endogenous retrovirus

BackgroundWe discovered a novel human endogenous retrovirus (CT-RCC HERV-E) that was selectively expressed in most clear cell renal cell carcinomas (ccRCC) and served as a source of antigens for T...

Long non-coding RNA MAGEA4-AS1 binding to p53 enhances MK2 signaling pathway and promotes the proliferation and metastasis of oral squamous cell carcinoma

Long non-coding RNAs (lncRNAs) regulate the occurrence, development and progression of oral squamous cell carcinoma (OSCC). We elucidated the expression features of MAGEA4-AS1 in patients with OSCC and its activity as an OSCC biomarker. Furthermore, the impact of up-regulation of MAGEA4-AS1 on the cellular behaviors (proliferation, migration and invasion) of OSCC cells and intrinsic signal mechanisms were evaluated. Firstly, we analyzed MAGEA4-AS1 expression data in The Cancer Genome Atlas (TCGA) OSCC using a bioinformatics approach and in 45 pairs of OSCC tissues using qPCR. Then CCK-8, ethynyl deoxyuridine, colony formation, transwell and wound healing assays were conducted to assess changes in the cell proliferation, migration and invasion protential of shMAGEA4-AS1 HSC3 and CAL27 cells. The RNA sequence of MAGEA4-AS1 was identified using the rapid amplification of cDNA ends (RACE) assay. And whole-transcriptome sequencing was used to identify MAGEA4-AS1 affected genes. Additionally, dual-luciferase reporter system, RNA-binding protein immunoprecipitation (RIP), and rescue experiments were performed to clarify the role of the MAGEA4-AS1-p53-MK2 signaling pathway. As results, we found MAGEA4-AS1 was up-regulated in OSCC tissues. We identified a 418 nucleotides length of the MAGEA4-AS1 transcript and it primarily located in the cell nucleus. MAGEA4-AS1 stable knockdown weakened the proliferation, migration and invasion abilities of OSCC cells. Mechanistically, p53 protein was capable to activate MK2 gene transcription. RIP assay revealed an interaction between p53 and MAGEA4-AS1. MK2 up-regulation in MAGEA4-AS1 down-regulated OSCC cells restored MK2 and epithelial-to-mesenchymal transition related proteins’ expression levels. In conclusion, MAGEA4-AS1-p53 complexes bind to MK2 promoter, enhancing the transcription of MK2 and activating the downstream signaling pathways, consequently promoting the proliferation and metastasis of OSCC cells. MAGEA4-AS1 may serve as a diagnostic marker and therapeutic target for OSCC patients.

Discovery and genomic characterization of three double-stranded RNA viruses coinfecting Conidiobolus taihushanensis.

Conidiobolus sensu lato, a genus within the family Ancylistaceae, encompasses a diverse range of fungal species that are widely distributed in plant debris and soil. In this study, we identified three double-stranded RNA (dsRNA) viruses coinfecting a strain of Conidiobolus taihushanensis. These viruses were identified as Conidiobolus taihushanensis totivirus 1 (CtTV1), Conidiobolus nonsegmented RNA virus 1-2 (CNRV1-2), and Conidiobolus taihushanensis virus 1 (CtV1). Through high-throughput sequencing and RNA-ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we determined their complete genome sequences. The genome of CtTV1 is 6,921 nucleotides in length, containing two open reading frames (ORFs). ORF1 encodes a 1,124-amino-acid capsid protein (CP) with a molecular weight of 125.07 kDa, and ORF2 encodes a 780-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular weight of 88.05 kDa. CNRV1-2, approximately 3.0 kb in length, also contains two ORFs, which are predicted to encode a 186-amino-acid hypothetical protein (HP) and a 758-amino-acid RdRp. CtV1 has a smaller genome consisting of 3,081 base pairs (bp) with two ORFs: one encoding a 244-amino-acid HP (26.85 kDa) and the other encoding a 707-amino-acid RdRp (80.64 kDa). Phylogenetic analysis based on RdRp sequences revealed that CtTV1 shows the highest similarity to Phytophthora pluvialis RNA virus 1, with 38.79% sequence identity, and clusters with members of the family Orthototiviridae, and it is most closely related to Utsjoki toti-like virus. In contrast, CtV1 formed a unique branch and might represent a new genus. The genome sequence of CNRV1-2 is 99.74% identical to that of the previously described Conidiobolus non-segmented RNA virus 1 (CNRV1). Our findings indicate that CtTV1 and CtV1 are distinct novel viruses, while CNRV1-2 appears to be a variant of CNRV1. This study enhances our understanding of the genetic diversity and evolutionary relationships among mycoviruses associated with C. taihushanensis.

Discovery and Genomic Analysis of Three Novel Viruses in the Order Mononegavirales in Leafhoppers.

Leafhoppers are economically important pests and may serve as vectors for pathogenic viruses that cause substantial crop damage. In this study, using deep transcriptome sequencing, we identified three novel viruses within the order Mononegavirales, including two viruses belonging to the family Rhabdoviridae and one to the family Lispiviridae. The complete genome sequences were obtained via the rapid amplification of cDNA ends and tentatively named Recilia dorsalis rhabdovirus 1 (RdRV1, 14,251 nucleotides, nt), Nephotettix virescens rhabdovirus 1 (NvRV1, 13,726 nt), and Nephotettix virescens lispivirus 1 (NvLV1, 14,055 nt). The results of a phylogenetic analysis and sequence identity comparison suggest that RdRV1 and NvRV1 represent novel species within the family Rhabdoviridae, while NvLV1 is a new virus belonging to the family Lispiviridae. As negative-sense single-strand RNA viruses, RdRV1 and NvRV1 contain the conserved transcription termination signal and intergenic trinucleotides in the non-transcribed region. Intergenomic sequence and transcriptome profile analyses suggested that all these genes were co-transcriptionally expressed in these viral genomes, facilitated by specific intergenic trinucleotides and putative transcription initiation sequences.