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Biochanin A (Bioch A) is a natural plant estrogen, with various biological activities such as anti-apoptosis, anti-oxidation, and suppression of inflammation. In this study, we investigated the protective effects of Bioch A on angiotensin II (AngII) - induced dopaminergic (DA) neuron damage in vivo and on molecular mechanisms. Spontaneous activity and motor ability of mice among groups was detected by open-field test and swim-test. The expression of TH, microtubule-associated proteins light chain 3B II (LC3BII)/LC3BI, beclin-1, P62, forkhead box class O3 (FoxO3), phosphorylated (p) FoxO3a/FoxO3a, FoxO3, and endophilin A2 were determined by Western blot and immunohistochemistry or immunofluorescence staining. Our results showed that AngII treatment significantly increased the behavioral dysfunction of mice and DA neuron damage. Meanwhile, AngII treatment increased the expression of LC3BII/LC3BI, beclin-1, P62, and FoxO3a and decreased the expression of endophilin A2 and p-FoxO3a/FoxO3a, however, Bioch A treatment alleviate these changes. In summary, these results suggest that Bioch A exerts protective effects on AngII-induced mouse model may be related to regulating endophilin A2, FoxO3a, and autophagy-related proteins; however, the specific mechanism is not yet clear and needs further study.

Background: Litter size is an important factor that significantly affects the development of the sheep industry. Our previous TMT proteomics analysis found that three key proteins in the ovarian steroidogenesis pathway, STAR, HSD3B1, and CYP11A1, may affect the litter size trait of Small Tail Han sheep., Objective: The purpose of this study was to better understand the relationship between polymorphisms of these three genes and litter size., Material and Method: Sequenom MassARRAY detected genetic variance of the three genes in 768 sheep. Real-time qPCR of the three genes was used to compare their expression in monotocous and polytocous sheep in relevant tissues. Finally, bioinformatics analysis predicted the protein sequences of the different SNP variants., Result: Association analysis showed that there was a significant difference in litter size among the genotypes at two loci of the CYP11A1 gene (p < 0.05), but no significant difference was observed in litter size among all genotypes at all loci of the STAR and HSD3B1 genes (p > 0.05). However, STAR expression was significantly different in polytocous and monotocous sheep in the pituitary (p < 0.01). Tissue-specific expression in the ovary was observed for HSD3B1 (p < 0.05), but its expression was not different between polytocous and monotocous sheep. Bioinformatics analysis showed that the g.33217408C > T mutation of CYP11A1 resulted in major changes to the secondary and tertiary structures. In contrast, gene polymorphisms in STAR and HSD3B1 had minimal impacts on their protein structures., Discussion: This may explain why the CYP11A1 variant impacted litter size while the others did not. The single nucleotide polymorphism of the CYP11A1 gene would serve as a good molecular marker when breeding to increase litter size in sheep. Our study provides a basis for further revealing the function of the ovarian steroidogenesis pathway in sheep reproduction and sheep breeding., (© 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The aim of this work was to identify the molecular characteristics of a chymotrypsin-like enzyme from Trichinella spiralis (Tschy) and its facilitation of larval penetration into enteral epithelial cells (EECs). The complete Tschy cDNA sequence was cloned and expressed in Escherichia coli BL21. RT-PCR, IIFA and western blotting showed that Tschy was expressed at the T. spiralis muscle larvae (ML), intestinal infective L1 larvae (IL1), adult worms (AW) and embryo stages and was primarily located in the stichosome of this parasite. The results of ELISA, IIFA and Far-western assays showed that there was a specific binding between rTschy and EECs, and the binding was dependent on the dose of both rTschy and EEC proteins. Confocal microscopy demonstrated that the binding was located in the EEC cytoplasm. rTschy facilitated T. spiralis larval penetration of EECs, and anti-rTschy antibodies impeded the larval intrusion of EECs. These results demonstrate that Tschy facilitated the larval intrusion of the host's enteral epithelium and could be a candidate molecular target for vaccine against the enteral invasive phase of T. spiralis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Among the most important sensors of extracellular danger signals, purinergic receptors have been demonstrated to play crucial roles in host defense against infection. However, the function of P2 receptors in viral infection has been little explored. Here we demonstrated that P2Y13 and its ligand ADP play an important role in protecting hosts from viral infections. First, we demonstrate that P2Y13, as a typical interferon-stimulated gene, is induced together with extracellular ADP during viral infection. Most importantly, extracellular ADP restricts the replication of different kinds of viruses, including vesicular stomatitis virus, Newcastle disease virus, herpes simplex virus 1, and murine leukemia virus. This kind of protection is dependent on P2Y13 but not P2Y1 or P2Y12, which are also considered as receptors for ADP. Furthermore, cyclic adenosine monophosphate and EPAC1 are downregulated by extracellular ADP through the P2Y13-coupled Gi alpha subunit. Accordingly, inhibition or deletion of EPAC1 significantly eliminates ADP/P2Y13-mediated antiviral activities. Taken together, our results show that P2Y13 and ADP play pivotal roles in the clearance of invaded virus and have the potential as antiviral targets., (© The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

This study aims to clarify the relationship and mechanism between expression of autophagy-related protein P62 and prognosis of patients with hepatocellular carcinoma (HCC) involving chronic hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) exposure. HCC patients who underwent resection were divided into three groups: HBV(+)/AFB1(+) (n = 26), HBV(+)/AFB1(-) (n = 68), and HBV(-)/AFB1(-) (n = 14). The groups were compared in terms of mRNA and protein levels of P62, disease-free survival (DFS), and overall survival (OS) and the expression of NRF2, Nqo1, and AKR7A3 in P62 high-expression and low-expression group. HBV(+)/AFB1(+) group has lower DFS and OS, and higher P62 expression than in the other two groups. P62 expression generally correlated with elevated NRF2 and Nqo1 expression, and reduced AKR7A3 expression. Patients expressing high levels of P62 showed significantly lower DFS and OS rates than patients expressing low levels. HCC involving HBV infection and AFB1 exposure is associated with relatively high risk of tumor recurrence, and this poor prognosis may relate to high P62 expression. High P62 expression activates the NRF2 pathway, promotes tumor recurrence. The downregulation of AKR7A3 also reduced liver detoxification of aflatoxin B1., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The Von Hippel-Lindau gene (VHL) is a tumor suppressor gene, which is widely expressed in kidney, lung, breast, ovary, and cervix. VHL gene mutations can induce VHL disease and tumorigenesis. However, whether this gene is expressed in the human fallopian tube has not been evaluated. The objectives of this study were to investigate whether the VHL gene is expressed in human fallopian tube, and to investigate its expression changes during the menstrual cycle. Twenty‑seven patients undergoing abdominal hysterectomy with adnexectomy for benign uterine disease were enrolled in the study. Human fallopian tubes were divided into proliferative stage (n=14) and secretory stage (n=13) according to the stage of the menstrual cycle they were isolated from. The expression of the VHL gene and protein was studied by reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunohistochemistry, respectively. The results revealed positive expression of the VHL protein in the cytoplasm of ciliated cells of the human fallopian tube. The mRNA and protein expression of VHL in the fallopian tubes was higher in the proliferative compared to the secretory phase of the menstrual cycle, but this difference was not significant (P>0.05). Overall, this study presents data on the VHL mRNA and protein expression in the human fallopian tube, which may be relevant to the process of differentiation of ciliated and secretory cells.

Noise-induced hearing loss (NIHL) often results from prolonged exposure to high levels of noise. Our previous study revealed that during the development of NIHL, the expression of protein kinase C γ subunit (PKCγ) and GABAB receptor (GABABR) was changed within the cochlear nuclear complex (CNC), suggesting that these molecules might be the potential targets for the treatment of NIHL. As an extending study, here we focused on puerarin, a major isoflavonoid extracted from Pueraria lobota, which has been used in the treatment of cardiovascular and cerebrovascular diseases, and investigated whether it could protect against NIHL by acting on PKCγ and GABABR. Transgenic GAD67-GFP knock-in mice were subjected to the NIHL model and their auditory functions were evaluated by the auditory brainstem response thresholds and distortion product oto-acoustic emission signals. Our results showed that 200mg/kg puerarin treatment ameliorated the thresholds of auditory brainstem response of NIHL mice significantly. Triple immunofluorescence staining and electron microscopy results revealed that GFP-positive neurons in the superficial layers of CNC expressed both PKCγ and GABABR1, and GAD67-positive terminals contacted PKCγ- or GABABR1-positive neurons. Immunoblotting and RT-PCR results showed that NIHL increased the expression of PKCγ but decreased that of GABABR1 and GABABR2 at both protein and mRNA levels in the CNC. Puerarin significantly attenuated the increased expression of PKCγ but elevated the reduced expression of GABABR1 and GABABR2 after noise exposure. Thus, we provided the first evidence that puerarin ameliorated the auditory functions of NIHL mice, and this effect may be due to its ability to regulate the expression of PKCγ and GABABR., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objectives: To study E2F3a expression and its clinical significance in children with acute lymphoblastic leukemia (ALL)., Methods: We quantified E2F3a expression at diagnosis in 148 children with ALL by real-time PCR. In the test cohort (n = 48), receiver operating characteristic (ROC) curve was used to find the best cut-off point to divide the patients into E2F3a low- and high-expression groups. The prognostic significance of E2F3a expression was investigated in the test cohort and confirmed in the validation cohort (n = 100). The correlations of E2F3a expression with the clinical features and treatment outcome of these patients were analyzed., Results: ROC curve analysis indicated that the best cut-off point of E2F3a expression was 0.3780. In the test cohort, leukemia-free survival (LFS) and event-free survival (EFS) of the low-expression group were lower than those of the high-expression group (log rank: P = 0.026 for both). This finding was verified in the validation cohort. LFS, EFS, and overall survival were also lower in the low-expression group than in the high-expression group (log rank, P = 0.015, 0.008, and 0.002 respectively). E2F3a low expression was correlated with the existence of BCR-ABL fusion. An algorithm composed of E2F3a expression and minimal residual disease (MRD) could predict relapse or induction failure more precisely than current risk stratification. These results were still significant in the ALL patients without BCR-ABL fusion., Conclusions: Low expression of E2F3a was associated with inferior prognosis in childhood ALL. An algorithm composed of E2F3a expression and MRD could predict relapse or induction failure more precisely than that of the current risk stratification., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Objective: The pathogenesis and molecular mechanism underlying asthma remain undetermined. The purpose of this study was to identify genes and pathways involved in the early airway response (EAR) phase of asthma by using serial analysis of gene expression (SAGE)., Methods: Two SAGE tag libraries of lung tissues derived from a rat model of asthma and controls were generated. Bioinformatic analyses were carried out using the Database for Annotation, Visualization and IntegratedDiscovery Functional Annotation Tool, Gene Ontology (GO) TreeMachine and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis., Results: A total of 26 552 SAGE tags of asthmatic rat lung were obtained, of which 12 221 were unique tags. Of the unique tags, 55.5% were matched with known genes. By comparison of the two libraries, 186 differentially expressed tags (P < 0.05) were identified, of which 103 were upregulated and 83 were downregulated. Using the bioinformatic tools these genes were classified into 23 functional groups, 15 KEGG pathways and 37 enriched GO categories., Conclusions: The bioinformatic analyses of gene distribution, enriched categories and the involvement of specific pathways in the SAGE libraries have provided information on regulatory networks of the EAR phase of asthma. Analyses of the regulated genes of interest may inform new hypotheses, increase our understanding of the disease and provide a foundation for future research.

The dopaminergic system is susceptible to dysfunction in numerous neurological diseases, including Parkinson's disease (PD). In addition to motor symptoms, some PD patients may experience non-motor symptoms, including cognitive and memory deficits. A possible explanation for their manifestation is a disturbed pattern of dopamine release in brain regions involved in learning and memory, such as the hippocampus. Therefore, investigating neuropathological alterations in dopamine release prior to neurodegeneration is imperative. This study aimed to characterize evoked hippocampal dopamine release and assess the impact of the neurotoxin MPP+ using a genetically encoded dopamine sensor and gene expression analysis. Additionally, considering the potential neuroprotective attributes demonstrated by apoptosis signal-regulating kinase 1 (Ask1) in various animal-disease-like models, the study also aimed to determine whether Ask1 knockdown restores MPP+-altered dopamine release in acute hippocampal slices. We applied variations of low- and highfrequency stimulation to evoke dopamine release within different hippocampal regions and discovered that acute application of MPP+ reduced the amount of dopamine released and hindered the recovery of dopamine release after repeated stimulation. In addition, we observed that Ask1 deficiency attenuated the detrimental effects of MPP+ on the recovery of dopamine release after repeated stimulation. RNA sequencing analysis indicated that genes associated with the synaptic pathways are involved in response to MPP+ exposure. Notably, Ask1 deficiency was found to downregulate the expression of Slc5a7, a gene encoding a sodium-dependent high-affinity choline transporter that regulates acetylcholine levels. Respective follow-up experiments indicated that Slc5a7 plays a role in Ask1 deficiency-mediated protection against MPP+ neurotoxicity. In addition, increasing acetylcholine levels using an acetylcholinesterase inhibitor could exacerbate the toxicity of MPP+. In conclusion, our data imply that the modulation of the dopamine-acetylcholine balance may be a crucial mechanism of action underlying the neuroprotective effects of Ask1 deficiency in PD. [ABSTRACT FROM AUTHOR]

The ethylene response factor family genes are involved in the regulation of secondary metabolism in Salvia miltiorrhiza , but the mechanism underlying this regulation remains elusive. In the present study, based on the cDNA library of S. miltiorrhiza , an AP2/ERF gene was cloned and named SmERF1b-like. This gene exhibited a significant response to exogenous ethylene supply, such that ethylene remarkably upregulated SmERF1b-like expression levels in the leaves of S. miltiorrhiza. Subcellular localization showed that SmERF1b-like is located in the nucleus. Furthermore, SmERF1b-like showed a binding affinity with a GCC-box motif in the promoter region of genes associated with tanshinone biosynthesis in S. miltiorrhiza. Overexpression of SmERF1b-like in hairy roots of S. miltiorrhiza substantially upregulated SmCPS1 and SmKSL1 expression levels, resulting in increased biosynthesis of tanshinone I and cryptotanshinone contents. This finding provides valuable theoretical support for the utilization of a plant genetic engineering strategy to enhance S. miltiorrhiza resources. [ABSTRACT FROM AUTHOR]

Background: Tamoxifen (Tam) is an effective treatment for estrogen receptor (ER) positive breast cancer. However, a significant proportion of patients develop resistance under treatment, presenting a therapeutic challenge. The study aims to determine the role of early growth response protein (EGR) 3 in tamoxifen resistance (TamR) and elucidate its molecular mechanism. Methods: TamR cell models were established and NGS was used to screening signaling alternation. Western blot and qRT-PCR were used to analysis the expression of ERα, EGR3, MCL1 and factors associated with apoptosis. CCK8, colony formation and apoptosis assay were used to analysis resistance to Tam. Immunofluorescence, chromatin immunoprecipitation, and dual luciferase assays were used to investigate mechanism of regulation. Results: We observed that EGR3, a deeply rooted ERα response factor, showed increased upregulation in response to both estrone (E1) and Tam in TamR cells with elevated level of E1 and ERα expression, indicating a potential connection between EGR3 and TamR. Mechanically, manipulating EGR3 expression revealed that it imparted resistance to Tam through increased expression of the downstream molecule MCL1 (apoptosis suppressor gene) that it regulated. Mechanismly, EGR3 directly binds to the promoter of the anti-apoptotic factor MCL1 gene, facilitating its transcription. Furthermore, apoptosis assays revealed that E1 reduces Tam induced apoptosis by upregulating EGR3 expression. Importantly, clinical public database confirmed the high expression of EGR3 in breast cancer tissue and in Tam-treated patients. Conclusions: These findings shed light on the novel estrogen/EGR3/MCL1 axis and its role in inducing TamR in ER positive breast cancer. EGR3 emerges as a promising target to overcome TamR. The elucidation of this mechanism holds potential for the development of new therapeutic modalities to overcome endocrine therapy resistance in clinical settings. [ABSTRACT FROM AUTHOR]

Auxin-responsive factors (ARFs) are an important class of transcription factors and are an important component of auxin signaling. This study conducted a genome-wide analysis of the ARF gene family in ginseng and presented its findings. Fifty-three ARF genes specific to ginseng (PgARF) were discovered after studying the ginseng genome. The coding sequence (CDS) has a length of 1092–4098 base pairs and codes for a protein sequence of 363–1565 amino acids. Among them, PgARF32 has the least number of exons (2), and PgARF16 has the most exons (18). These genes were then distributed into six subgroups based on the results obtained from phylogenetic analysis. In each subgroup, the majority of the PgARF genes displayed comparable intron/exon structures. PgARF genes are unevenly distributed on 20 chromosomes. Most PgARFs have B3 DNA binding, Auxin_resp, and PB1 domains. The PgARF promoter region contains various functional domains such as plant hormones, light signals, and developmental functions. Segmental duplications contribute to the expansion of the ARF gene family in ginseng, and the genes have undergone purifying selection during evolution. Transcriptomic results showed that some PgARFs had different expression patterns in different parts of ginseng; most PgARFs were affected by exogenous hormones, and a few PgARFs responded to environmental stress. It is suggested that PgARF is involved in the development of ginseng by regulating hormone-mediated genes. PgARF14, PgARF42, and PgARF53 are all situated in the nucleus, and both PgARR14 and PgARF53 noticeably enhance the growth length of roots in Arabidopsis. Our findings offer a theoretical and practical foundation for exploring PgARFs' role in the growth of ginseng roots. [ABSTRACT FROM AUTHOR]

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP) is an endangered species endemic to the Yangtze River in China, and it is the only freshwater whale in the genus Neophocaena. In terms of protection, three effective conservation strategies exist: in situ conservation, ex situ conservation, and artificial breeding, all of which have been implemented by the Chinese government. Of these, ex situ conservation involves the relocation of Yangtze finless porpoises to semi-natural waters with less human interference, and artificial breeding involves the relocation of Yangtze finless porpoises to a controlled environment that is more strictly managed. To compare and analyze the responsive changes in gene expression of the YFPs between the ex situ and controlled environments, we performed the RNA sequencing of blood tissues from these YFPs. A total of 1201 differentially expressed genes (DEGs) were identified, of which 423 were up-regulated in the ex situ population and 778 were up-regulated in the controlled-environment population. Gene enrichment analysis showed that 1201 DEGs between the ex situ and controlled-environment populations were generally enriched for vision-, digestion- and immune-system-related pathways. Further analysis revealed that several key immune system pathways, such as the chemokine signaling pathway and B cell receptor signal pathway, were activated in the ex situ population. In addition, the key pathways related to vision, including phototransduction and the inflammatory mediator regulation of TRP channels, as well as the pathways related to the digestive system, such as protein digestion and absorption and salivary secretion, were activated in the controlled-environment population. These results suggest that the ex situ populations may respond to complex environmental conditions in semi-natural waters by enhancing their immune function through the increased expression of immune-related genes and that the visual function and protein digestion of the YFPs were improved compared to those of the ex situ population based on the conditions of artificial feeding, such as the higher transparency of the water and regular feeding. This study provides clues for evaluating the adaptability of YFPs to different environments and is a useful reference for future ex situ conservation and artificial breeding. [ABSTRACT FROM AUTHOR]

Phytoene synthase (PSY) plays a key role in the biosynthesis and accumulation of carotenoids as the first rate-limiting enzyme in the plant carotenoid pathway. In the present study, the ‘Granny Smith’ apple PSY gene, MdPSY, was cloned using in silico cloning. The length of the MdPSY was 1500 bp, which included a complete open reading frame of 1194 bp, encoding a predicted protein of 397 amino acids. Sequence analysis revealed that the ‘Granny Smith’ apple PSY protein shared high homology with MdPSY2 (99%), MdPSY1 (93.9%), FaPSY (71.8%), CuPSY (70.7%) and NtPSY (69.7%). Phylogenetic analysis showed that the MdPSY protein was closely related to the PSY protein of Rosaceae. An expression profile analysis of different tissues indicated that MdPSY expression in the roots was higher than that in other tissues. MdPSY expression was tested throughout the fruit development period. The results showed that MdPSY plays a role in carotenoid biosynthesis in ‘Granny Smith’ apple. In addition, bagging treatment inhibits PSY gene expression, which in turn affects the synthesis and accumulation of carotenoids.

Abstract Background Abscisic acid (ABA) signaling plays a crucial role in developmental and environmental adaptation processes of plants. However, the PYL-PP2C-SnRK2 families that function as the core components of ABA signaling are not well understood in banana. Results In the present study, 24 PYL, 87 PP2C, and 11 SnRK2 genes were identified from banana, which was further supported by evolutionary relationships, conserved motif and gene structure analyses. The comprehensive transcriptomic analyses showed that banana PYL-PP2C-SnRK2 genes are involved in tissue development, fruit development and ripening, and response to abiotic stress in two cultivated varieties. Moreover, comparative expression analyses of PYL-PP2C-SnRK2 genes between BaXi Jiao (BX) and Fen Jiao (FJ) revealed that PYL-PP2C-SnRK2-mediated ABA signaling might positively regulate banana fruit ripening and tolerance to cold, salt, and osmotic stresses. Finally, interaction networks and co-expression assays demonstrated that the core components of ABA signaling were more active in FJ than in BX in response to abiotic stress, further supporting the crucial role of the genes in tolerance to abiotic stress in banana. Conclusions This study provides new insights into the complicated transcriptional control of PYL-PP2C-SnRK2 genes, improves the understanding of PYL-PP2C-SnRK2-mediated ABA signaling in the regulation of fruit development, ripening, and response to abiotic stress, and identifies some candidate genes for genetic improvement of banana.

Pumpkin (Cucurbita moschata Duch.) productivity is severely hindered by powdery mildew (PM) worldwide. The causative agent of pumpkin PM is Podosphaera xanthii, a biotrophic fungus. Pathogenesis-related protein 1 (PR1) homolog was previously identified from transcriptomic analysis of a PM-resistant pumpkin. Here, we investigated the effects of CmPR1 gene from pumpkin for resistance to PM. Subcellular localization assay revealed that CmPR1 is a cytoplasmic protein in plants. The expression of CmPR1 gene was strongly induced by P. xanthii inoculation at 48 h and exogenous ethylene (ET), jasmonic acid (JA) and NaCl treatments, but repressed by H2O2 and salicylic acid (SA) treatments. Visual disease symptoms, histological observations of fungal growth and host cell death, and accumulation of H2O2 in transgenic tobacco plants indicated that CmPR1 overexpression significantly enhanced the resistance to Golovinomyces cichoracearum compared to wild type plants during PM pathogens infection, possibly due to inducing cell death and H2O2 accumulation near infected sites. The expression of PR1a was significantly induced in transgenic tobacco plants in response to G. cichoracearum, suggesting that CmPR1 overexpression positively modulates the resistance to PM via the SA signaling pathway. These findings indicate that CmPR1 is a defense response gene in C. moschata and can be exploited to develop disease-resistant crop varieties. [ABSTRACT FROM AUTHOR]

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target—miR-335- 5p. Pathway analysis on predicted and validated miR-335- 5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335- 5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335- 5p. Blocking miR-335- 5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335- 5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335- 5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335- 5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335- 5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy. [ABSTRACT FROM AUTHOR]

Background: Panax ginseng is a perennial herb and one of the most widely used traditional medicines in China. During its long growth period, it is affected by various environmental factors. Past studies have shown that growth-regulating factors (GRFs) and GRF-interacting factors (GIFs) are involved in regulating plant growth and development, responding to environmental stress, and responding to the induction of exogenous hormones. However, GRF and GIF transcription factors in ginseng have not been reported. Results: In this study, 20 GRF gene members of ginseng were systematically identified and found to be distributed on 13 chromosomes. The ginseng GIF gene family has only ten members, which are distributed on ten chromosomes. Phylogenetic analysis divided these PgGRFs into six clades and PgGIFs into two clades. In total, 18 of the 20 PgGRFs and eight of the ten PgGIFs are segmental duplications. Most PgGRF and PgGIF gene promoters contain some hormone- and stress- related cis-regulatory elements. Based on the available public RNA-Seq data, the expression patterns of PgGRF and PgGIF genes were analysed from 14 different tissues. The responses of the PgGRF gene to different hormones (6-BA, ABA, GA3, IAA) and abiotic stresses (cold, heat, drought, and salt) were studied. The expression of the PgGRF gene was significantly upregulated under GA3 induction and three weeks of heat treatment. The expression level of the PgGIF gene changed only slightly after one week of heat treatment. Conclusions: The results of this study may be helpful for further study of the function of PgGRF and PgGIF genes and lay a foundation for further study of their role in the growth and development of Panax ginseng. [ABSTRACT FROM AUTHOR]

Cisplatin is widely used in clinical tumor chemotherapy but has severe ototoxic side effects, including tinnitus and hearing damage. This study aimed to determine the molecular mechanism underlying cisplatin-induced ototoxicity. In this study, we used CBA/CaJ mice to establish an ototoxicity model of cisplatin-induced hair cell loss, and our results showed that cisplatin treatment could reduce FOXG1 expression and autophagy levels. Additionally, H3K9me2 levels increased in cochlear hair cells after cisplatin administration. Reduced FOXG1 expression caused decreased microRNA (miRNA) expression and autophagy levels, leading to reactive oxygen species (ROS) accumulation and cochlear hair cell death. Inhibiting miRNA expression decreased the autophagy levels of OC-1 cells and significantly increased cellular ROS levels and the apoptosis ratio in vitro. In vitro, overexpression of FOXG1 and its target miRNAs could rescue the cisplatin-induced decrease in autophagy, thereby reducing apoptosis. BIX01294 is an inhibitor of G9a, the enzyme in charge of H3K9me2, and can reduce hair cell damage and rescue the hearing loss caused by cisplatin in vivo. This study demonstrates that FOXG1-related epigenetics plays a role in cisplatin-induced ototoxicity through the autophagy pathway, providing new ideas and intervention targets for treating ototoxicity. [ABSTRACT FROM AUTHOR]

At present, the role of many long non-coding RNAs (lncRNAs) as tumor suppressors in the formation and development of cervical cancer (CC) has been studied. However, lncRNA prostate cancer gene expression marker 1 (PCGEM1), whose high expression not only aggravates ovarian cancer but also can induce tumorigenesis and endometrial cancer progression, has not been studied in CC. The objective of this study was to investigate the expression and the underlying role of PCGEM1 in CC. The relative expression of PCGEM1 in CC cells was detected by real-time PCR. After the suppression of PCGEM1 expression by shRNA, the changes in the proliferation, migration, and invasion capacities were detected via CCK-8 assay, EdU assay, and colony formation assay wound healing assay. Transwell assay and the changes in expressions of epithelial-to-mesenchymal transition (EMT) markers were determined by western blot and immunofluorescence. The interplay among PCGEM1, miR-642a-5p, and kinesin family member 5B (KIF5B) was confirmed by bioinformatics analyses and luciferase reporter assay. Results showed that PCGEM1 expressions were up-regulated within CC cells. Cell viabilities, migration, and invasion were remarkably reduced after the suppression of PCGEM1 expression by shRNA in Hela and SiHa cells. N-cadherin was silenced, but E-cadherin expression was elevated by sh-PCGEM1. Moreover, by sponging miR-642a-5p in CC, PCGEM1 was verified as a competitive endogenous RNA (ceRNA) that modulates KIF5B levels. MiR-642a-5p downregulation partially rescued sh-PCGEM1's inhibitory effects on cell proliferation, migration, invasion, and EMT process. In conclusion, the PCGEM1/miR-642a-5p/KIF5B signaling axis might be a novel therapeutic target in CC. This study provides a research basis and new direction for targeted therapy of CC. [ABSTRACT FROM AUTHOR]

KT/HAK/KUP (KUP) family is responsible for potassium ion (K+) transport, which plays a vital role in the response of plants to abiotic stress by maintaining osmotic balance. However, our understanding of the functions of the KUP family in the drought-resistant crop cassava (Manihot esculenta Crantz) is limited. In the present study, 21 cassava KUP genes (MeKUPs) were identified and classified into four clusters based on phylogenetic relationships, conserved motifs, and gene structure analyses. Transcriptome analysis revealed the expression diversity of cassava KUPs in various tissues of three genotypes. Comparative transcriptome analysis showed that the activation of MeKUP genes by drought was more in roots than that in leaves of Arg7 and W14 genotypes, whereas less in roots than that in leaves of SC124 variety. These findings indicate that different cassava genotypes utilize various drought resistance mechanism mediated by KUP genes. Specific KUP genes showed broad upregulation after exposure to salt, osmotic, cold, H2O2, and abscisic acid (ABA) treatments. Taken together, this study provides insights into the KUP-mediated drought response of cassava at transcription levels and identifies candidate genes that may be utilized in improving crop tolerance to abiotic stress.

Summary: Arabidopsis RESISTANCE TO POWDERY MILDEW 8.2 (RPW8.2) is specifically induced by the powdery mildew (PM) fungus (Golovinomyces cichoracearum) in the infected epidermal cells to activate immunity. However, the mechanism of RPW8.2‐induction is not well understood.Here, we identify a G. cichoracearum effector that interacts with RPW8.2, named Gc‐RPW8.2 interacting protein 1 (GcR8IP1), by a yeast two‐hybrid screen of an Arabidopsis cDNA library.GcR8IP1 is physically associated with RPW8.2 with its REALLY INTERESTING NEW GENE finger domain that is essential and sufficient for the association. GcR8IP1 was secreted and translocated into the nucleus of host cell infected with PM. Association of GcR8IP1 with RPW8.2 led to an increase in RPW8.2 in the nucleus. In turn, the nucleus‐localized RPW8.2 promoted the activity of the RPW8.2 promoter, resulting in transcriptional self‐amplification of RPW8.2 to boost immunity at infection sites. Additionally, ectopic expression or host‐induced gene silencing of GcR8IP1 supported its role as a virulence factor in PM.Altogether, our results reveal a mechanism of RPW8.2‐dependent defense strengthening via altered partitioning of RPW8.2 and transcriptional self‐amplification triggered by a PM fungal effector, which exemplifies an atypical form of effector‐triggered immunity. [ABSTRACT FROM AUTHOR]

Mutations in the γ‐secretase complex have been well‐described in familial hidradenitis suppurativa (HS). No gene mutations have been identified in sporadic HS, which comprises 60%–70% of all HS cases. Obesity and smoking are risk factors for HS and are closely related to DNA methylation, an essential epigenetic phenomenon. Hence, we hypothesized that epigenetic modifications might be involved in sporadic HS. To investigate genes with aberrant methylation in sporadic HS cases and assess their expression in skin lesions and blood from patients with HS. Skin lesion samples and corresponding normal skin were obtained from three patients with HS and subjected to whole‐genome DNA methylation sequencing. Blood samples were collected from 20 patients with HS and 20 healthy controls (HCs). The HS mouse model was established by applying tamoxifen to NcstnΔKC mice. Target gene expression was analysed by immunohistochemistry, immunofluorescence, western blotting, enzyme‐linked immunosorbent assay (ELISA) and semiquantitative real‐time polymerase chain reaction (RT‐qPCR). Among 10 807 differentially methylated genes, we filtered 2101 genes with hypermethylated promoter regions, and following bioinformatics analyses, we focused on CXC chemokine ligand 16 (CXCL16). Subsequent functional experiments confirmed the downregulation of CXCL16 and its receptor, CXC chemokine receptor (CXCR) 6, in skin tissue from HS patients and NcstnΔKC mice. Serum CXCL16 concentrations were also significantly decreased in patients with HS. Our data revealed the downregulation of CXCL16 and CXCR6 in HS. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to decide the role of the polarization of macrophages regulated by tumor necrosis factor-α (TNF-α)-induced protein 8-like 2 (TIPE2) in meibomian gland dysfunction (MGD). Methods: Firstly, the secretory function of the meibomian gland (MG) in apolipoprotein E knockout (ApoE-/-) MGD mice and normal mice was detected by oil red staining. Then, the expression levels of markers of M1 and M2 macrophages were detected by immunofluorescence staining in MGD, normal mice, and mild and severe MGD corpses to decide the role of M1 and M2 macrophages in MGD inflammation. Meanwhile, the expression levels of TIPE2 in MGD mice and MGD patients were detected by immunofluorescence staining, and the correlations among TIPE2, M1 and M2 macrophages were analyzed by immunofluorescence double staining in MGD mice and MGD patients. Furthermore, lipopolysaccharide (LPS) and interleulkin-4 (IL-4) were used to induce M1 and M2 polarization of macrophages, and the mRNA level of TIPE2 was detected in M1 and M2 macrophages. Results: Oil red staining showed that eyelid fat congestion was more severe in (ApoE-/-) MGD mice than in normal mice, and the M1 macrophage was the primary inflammatory cell infiltrated in (ApoE-/-) MGD mice (p < 0.05). The results of the immunofluorescence staining showed that the infiltration of macrophages in MGD mice was more obvious than that in the normal group, and M1 macrophage was the dominant group (p < 0.05). Similar to the results of the MGD mouse model, more macrophage infiltration was observed in MGD patients' MG tissues, and there were more M1 cells in the severe group than in the mild group (p < 0.05). Moreover, the expression of TIPE2 was positively correlated with the expression of M2 macrophages in MGD patients and mice MG tissues (p < 0.05). The expression of TIPE2 mRNA in LPS-induced M1 macrophages declined, while the expression of TIPE2 mRNA in IL-4-induced M2 macrophages increased (p < 0.05). Conclusion: M1 macrophage was the dominant group infiltrated in the MG tissue of MGD, and TIPE2 is a potential anti-inflammatory target for preventing the development of MGD by promoting the M2 polarization of macrophages. [ABSTRACT FROM AUTHOR]

GENERAL REGULATORY FACTOR (GRF) proteins play vital roles in the regulation of plant growth, development, and response to abiotic stress. However, little information is known for this gene family in cassava (Manihot esculenta). In this study, 15 MeGRFs were identified from the cassava genome and were clustered into the ε and the non-ε groups according to phylogenetic, conserved motif, and gene structure analyses. Transcriptomic analyses showed eleven MeGRFs with constitutively high expression in stems, leaves, and storage roots of two cassava genotypes. Expression analyses revealed that the majority of GRFs showed transcriptional changes under cold, osmotic, salt, abscisic acid (ABA), and H2O2 treatments. Six MeGRFs were found to be commonly upregulated by abiotic stress, ABA, and H2O2 treatments, which may be the converging points of multiple signaling pathways. Interaction network analysis identified 18 possible interactors of MeGRFs. Taken together, this study elucidates the transcriptional control of MeGRFs in tissue development and the responses of abiotic stress and related signaling in cassava. Some constitutively expressed, tissue-specific, and abiotic stress-responsive candidate MeGRF genes were identified for the further genetic improvement of crops.

Simple Summary: Single Cell RNA Sequencing generates gene expression data at a single cell resolution. While single cell RNA has many applications in biomedical research, the high dimensionality of the data produced poses a considerable computational challenge. This study proposes a novel algorithm using penalized regression methods to analyze single cell RNA sequencing data. The proposed algorithm reduces high dimensionality of the gene expression data using a sequence feature selection methods such as Ridge regression, LASSO, Elastic Net, Drop LASSO, and Sparse Group LASSO. The proposed algorithm successfully detected highly differentiated genes, including the marker genes, for 5 different single cell RNA sequencing datasets associated with the species mouse, plant, and human. With the emergence of single-cell RNA sequencing (scRNA-seq) technology, scientists are able to examine gene expression at single-cell resolution. Analysis of scRNA-seq data has its own challenges, which stem from its high dimensionality. The method of machine learning comes with the potential of gene (feature) selection from the high-dimensional scRNA-seq data. Even though there exist multiple machine learning methods that appear to be suitable for feature selection, such as penalized regression, there is no rigorous comparison of their performances across data sets, where each poses its own challenges. Therefore, in this paper, we analyzed and compared multiple penalized regression methods for scRNA-seq data. Given the scRNA-seq data sets we analyzed, the results show that sparse group lasso (SGL) outperforms the other six methods (ridge, lasso, elastic net, drop lasso, group lasso, and big lasso) using the metrics area under the receiver operating curve (AUC) and computation time. Building on these findings, we proposed a new algorithm for feature selection using penalized regression methods. The proposed algorithm works by selecting a small subset of genes and applying SGL to select the differentially expressed genes in scRNA-seq data. By using hierarchical clustering to group genes, the proposed method bypasses the need for domain-specific knowledge for gene grouping information. In addition, the proposed algorithm provided consistently better AUC for the data sets used. [ABSTRACT FROM AUTHOR]

RNA polymerase II (Pol II) apparatuses are compartmentalized into transcriptional clusters. Whether protein factors control these clusters remains unknown. In this study, we find that the ATPase-associated with diverse cellular activities (AAA +) ATPase RUVBL2 co-occupies promoters with Pol II and various transcription factors. RUVBL2 interacts with unphosphorylated Pol II in chromatin to promote RPB1 carboxy-terminal domain (CTD) clustering and transcription initiation. Rapid depletion of RUVBL2 leads to a decrease in the number of Pol II clusters and inhibits nascent RNA synthesis, and tethering RUVBL2 to an active promoter enhances Pol II clustering at the promoter. We also identify target genes that are directly linked to the RUVBL2-Pol II axis. Many of these genes are hallmarks of cancers and encode proteins with diverse cellular functions. Our results demonstrate an emerging activity for RUVBL2 in regulating Pol II cluster formation in the nucleus. RNA polymerase II (Pol II) transcription factories play a central role in gene expression and 3D chromatin organization. Here, the authors demonstrate that RUVBL2 directly regulates Pol II clustering at active gene promoters. [ABSTRACT FROM AUTHOR]

The present study was designed to identify and understand the potential effectiveness of therapeutic target in intervertebral disc degeneration (IVDD) and its regulation mechanism. The role and mechanism of interleukin-18 (IL-18) in the disease were investigated. The IVDD degenerative nucleus pulposus (NP) tissues from the human and mouse models were used. A total of three groups of Male BALB/c mice were randomly made i.e control, IVDD, and IVDD+Ad-shIL-18 groups. After Ad-shIL-18 transfection, the expression of ECM synthesis related protein Aggrecan (ACAN) and Collagen II, apoptotic effector Caspases (Caspase-3, 8, 9, 12 and Cleaved-Caspase 3, 8, 9, 12), pro-apoptotic gene Bax and anti-apoptotic factors Bcl-2 in NP cells of the human were evaluated. The results of our study revealed that the mRNA and protein expression levels of IL-18 were notably increased in the NP tissues of IVDD patients and mice models. In the IVDD mice model, Ad-sh-IL-18 treatment reversed the IVDD progression. The levels of Aggrecan and Collagen II, contributing to ECM degradation in NP cells, were also significantly increased. Additionally, Ad-sh-IL-18 could inhibit the NP cell's apoptosis via regulating the caspase-3/9 pathway. The IL-18 knockdown via the caspase-3/9 pathway, might reduce the NP cell's death as well as the imbalance between catabolism and anabolism of ECM in IVDD. [ABSTRACT FROM AUTHOR]

Conopomorpha sinensis Bradley is the most detrimental pest to litchi and longan in China. Adult eclosion, locomotion, mating and oviposition of C. sinensis usually occur at night, regulated by a circadian rhythm. Nevertheless, our understanding of the linkages between adult circadian rhythms and clock genes remains inadequate. To address this gap, transcriptomic analysis was conducted on female and male heads (including antennae) of C. sinensis using the Illumina HiSeq 6000 platform to identify major circadian clock-related genes. The annotated sequences were analyzed by BLASTx, and candidate clock genes were classified based on conservation, predicted domain architectures, and phylogenetic analysis. The analysis revealed a higher conservation of these genes among the compared moths. Further, the expression profile analysis showed a significant spatiotemporal and circadian rhythmic accumulation of some clock genes during development. The candidate clock genes were predominantly expressed in the head, highlighting their crucial function in circadian rhythm regulation. Moreover, CsinPer , CsinTim 1, and CsinCry 1 displayed similar dynamic expressions with a peak expression level in the 4th age adults, suggesting their involvement in regulation of courtship and mating behaviors. The CsinPer and CsinTim 1 mRNA oscillated strongly with a similar phase, containing a peak expression just before the female mating peak. This work will greatly contribute to understanding the circadian clock system of C. sinensis and provide valuable information for further studies of the molecular mechanisms involved in rhythmicity in fruit-boring pests. [Display omitted] • This is the first report on the head transcriptome of C. sinensis females and males. • Clock genes encoding proteins with essential roles in the C. sinensis clock system were identified. • Phylogenetic analyses of the identified genes showed both conservation and variation. • Expression profiling in C. sinensis clock shifts strongly support the canonical clock model. • The candidate clock genes may have varied functions in regulating development and reproduction. [ABSTRACT FROM AUTHOR]

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP) is a critically endangered small odontocete species, mainly distributed in the middle and lower reaches of the Yangtze River, Poyang Lake, and Dongting Lake. Under the influence of human activities, many factors are threatening the survival and reproduction of YFPs in their natural habitat. Ex situ conservation is of great significance to strengthen the rescuing conservation of YFPs by providing suitable alternative habitats and promoting the reproduction and growth of the ex situ population. To reveal the differences in gene expression of YFPs in natural and ex situ protected waters, and to investigate the effects of environmental factors on YFPs and their mechanisms, we performed transcriptome sequencing for blood tissues of YFPs collected from natural waters and ex situ protected waters. Using RNA-seq we identified 4613 differentially expressed genes (DEGs), of which 4485 were up-regulated and 128 were down-regulated in the natural population. GO analysis showed that DEGs were significantly enriched in entries related to binding, catalytic activity, and biological regulation; KEGG analysis showed that DEGs were enriched mainly in signal transduction, endocrine system, immune system, and sensory system-related pathways. Further analysis revealed that water pollution in natural waters may affect the hormone secretion of YFPs by altering the expression pattern of endocrine genes, thus interfering with normal endocrine activities; noise pollution may induce oxidative stress and inflammatory responses in YFPs, thus impairing the auditory function of YFPs. This study provides a new perspective for further research on the effect of habitat conditions on the YFPs and suggests that improving the habitat environment may help in the conservation of YFPs. [ABSTRACT FROM AUTHOR]

Cold tolerance is improved by cold stress acclimation (CS-ACC), and the cold tolerance level is 'remembered' by plants. However, the underlying signaling mechanisms remain largely unknown. Here, the CS memory mechanism was studied by bioinformation, plant physiological and photosynthetic parameters, and gene expression. We found that CS-ACC induced the acquisition of CS memory and enhanced the maintenance of acquired cold tolerance (MACT) in cucumber seedlings. The H2O2 content and NADPH oxidase activity encoded by CsRBOH was maintained at higher levels during recovery after CS-ACC and inhibition of RBOH-dependent signaling after CS-ACC resulted in a decrease in the H2O2 content, NADPH oxidase activity, and MACT. CsRBOH2, 3, 4, and 5 showed high expression during recovery after CS-ACC. Many BZR-binding sites were identified in memory-responsive CsRBOHs promoters, and CsBZR1 and 3 showed high expression during recovery after CS-ACC. Inhibition of RBOH-dependent signaling or brassinosteroids affected the maintenance of the expression of these memory-responsive CsRBOHs and CsBZRs. The photosynthetic efficiency (PE) decreased but then increased with the prolonged recovery after CS-ACC, and was higher than the control at 48 h of recovery; however, inhibition of RBOH-dependent signaling resulted in a lower PE. Further etiolated seedlings experiments showed that a photosynthetic capacity was necessary for CS memory. Therefore, photosynthesis mediated by RBOH-dependent signaling is essential for CS memory. [ABSTRACT FROM AUTHOR]

Background: Genomic instability is one of the representative features of cancer evolution. Recent research has revealed that long noncoding RNAs (lncRNAs) play a critical role in maintaining genomic instability. Our work proposed a gene signature (GILncSig) based on genomic instability-derived lncRNAs to probe the possibility of lncRNA signatures as an index of genomic instability, providing a potential new approach to identify genomic instability-related cancer biomarkers. Methods: Lung adenocarcinoma (LUAD) gene expression data from an RNA-seq FPKM dataset, somatic mutation information and relevant clinical materials were downloaded from The Cancer Genome Atlas (TCGA). A prognostic model consisting of genomic instability-related lncRNAs was constructed, termed GILncSig, to calculate the risk score. We validated GILncSig using data from the Gene Expression Omnibus (GEO) database. In this study, we used R software for data analysis. Results: Through univariate and multivariate Cox regression analyses, five genomic instability-associated lncRNAs (LINC01671, LINC01116, LINC01214, lncRNA PTCSC3 , and LINC02555) were identified. We constructed a lncRNA signature (GILncSig) related to genomic instability. LUAD patients were classified into two risk groups by GILncSig. The results showed that the survival rate of LUAD patients in the low-risk group was higher than that of those in the high-risk group. Then, we verified GILncSig in the GEO database. GILncSig was associated with the genomic mutation rate of LUAD. We also used GILncSig to divide TP53 mutant-type patients and TP53 wild-type patients into two groups and performed prognostic analysis. The results suggested that compared with TP53 mutation status, GILncSig may have better prognostic significance. Conclusions: By combining the lncRNA expression profiles associated with somatic mutations and the corresponding clinical characteristics of LUAD, a lncRNA signature (GILncSig) related to genomic instability was established. [ABSTRACT FROM AUTHOR]

The SCN family as the encoded gens of sodium channels has been proven to participate in development of cancers including hepatocellular carcinoma (HCC), but the prognostic value of the SCN family is unclear. The results of the UALCAN database had showed that SCN2A/4A/5A/8A mRNA were highly expressed in tumour tissues, while SCN1A/7A/11A mRNA were expressed at low levels (p < 0.05), furthermore, the expression of SCN4A and SCN7A had the similar levels in microarray analysis result. The pan-tumour analysis showed that SCN7A expression was stably lower in tumours than SCN4A expression by TIMER. Both SCN4A and SCN7A were related to tumour grade, nodal metastatic status, histological subtype, patient race, individual cancer stages and TP53 mutation status to varying degrees. The Kaplan–Meier plotter demonstrated that high SCN4A mRNA expression was correlated with better overall survival (OS), disease-specific survival (DSS) and progression-free survival (PFS) and that high expression of SCN7A mRNA was associated with better OS; however, in Asians, higher SCN4A was correlated with better OS and DSS, and higher SCN7A was well correlated with better OS, recurrence-free survival (RFS), DSS and PFS. Analysis of data from cBioPortal showed that mutation of SCN7A was related to RFS and PFS. The protein expression of SCN4A and SCN7A had been detected by Immunohistochemistry. Univariate survival analysis revealed that high SCN7A protein expression was significantly linked to better OS (p = 0.001) and RFS (p = 0.003). Moreover, SCN7A displayed as an independent prognostic factor by multivariate analysis. In addition, a lower methylation level indicated a poor outcome. Pathway and functional enrichment analysis predicted a relationship between SCN7A and the PI3K pathway. In conclusion, there are significant and stable changes in SCN4A and SCN7A expression in HCC. SCN7A expression has better prognostic value and might participate in HCC progression. [ABSTRACT FROM AUTHOR]

The selection of suitable reference genes (RGs) for data normalization is a crucial step for obtaining reliable results from gene expression analysis using the quantitative real‐time polymerase chain reaction (qRT‐PCR). Expression stability of eleven putative RGs (miR‐100‐5p, miR‐92a‐3p, miR‐998‐3p, miR‐279d‐3p, miR‐305‐5p, miR‐276a‐3p, miR‐275‐3p, miR‐9a‐5p, miR‐2a‐3p, U6 snRNA and 5S rRNA) was assessed by using a web‐based tool: RefFinder, which integrates four algorithms (Delta Ct, geNorm, NormFinder, and BestKeeper), and the best combination number of RGs was calculated by geNorm. Validation of selected RGs was performed by an expression analysis of let‐7‐5p under different experimental conditions. The best combinations of RGs were shown as follows: miR‐100‐5p + miR‐275‐3p for temperature treatment, miR‐100‐5p + miR‐305‐5p for adult tissues, miR‐276a‐3p + miR‐9a‐5p for genders, miR‐2a‐3p + miR‐276a‐3p for days after eclosion, U6 snRNA + miR‐9a‐5p + miR‐2a‐3p for developmental stages, and U6 snRNA + miR‐279d‐3p for all above conditions, and there was no one RG suitable for all experimental conditions. Incorrect RG selection affected the evaluation of let‐7‐5p abundance. Our findings demonstrate that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for miRNA evaluation, and provide a basis for further studies on expression and function analysis of miRNAs and their target genes in Galeruca daurica, a new pest with great outbreaks in Inner Mongolia since 2009. [ABSTRACT FROM AUTHOR]