Selection and evaluation of reference genes for gene expression using quantitative real‐time PCR in Mythimna separata walker (Lepidoptera: Noctuidae)

Bai‐Zhong Zhang,Jun-Jie LIU,Xi-Ling CHEN,Guo-Hui YUAN 한국곤충학회 2018 Entomological Research Vol.48 No.5

In order to precisely assess gene expression levels, the suitable internal reference genes must be served to quantify real‐time reverse transcription polymerase chain reaction (RT‐qPCR) data. For armyworm, Mythimna separata, which reference genes are suitable for assessing the level of transcriptional expression of target genes have yet to be explored. In this study, eight common reference genes, including β‐actin (β‐ACT), 18 s ribosomal (18S), 28S ribosomal (28S), glyceraldehyde‐3‐phosphate (GAPDH), elongation fator‐alpha (EF1α), TATA box binding protein (TBP), ribosomal protein L7 (RPL7), and alpha‐tubulin (α‐TUB) that in different developmental stages, tissues and insecticide treatments of M. separata were evaluated. To further explore whether these genes were suitable to serve as endogenous controls, three software‐based approaches (geNorm, BestKeeper, and NormFinder), the delta Ct method, and one web‐based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized HSP70, and MsepCYP321A10 gene expression data. We found that the most suitable reference genes for the different experimental conditions. For developmental stages, 28S/RPL7 were the optimal reference genes, both RPL7/EF1α were suitable for experiments of different tissues, whereas for insecticide treatments, 28S/α‐TUB were suitable for normalizations of expression data. In addition, 28S/α‐TUB were the suitable reference genes because they have the most stable expression among different developmental stages, tissues and insecticide treatments. Our work is the first report on reference gene selection in M. separata, and might serve as a precedent for future gene expression studies.

Tanshinone I Enhances Neurogenesis in the Mouse Hippocampal Dentate Gyrus via Increasing Wnt-3, Phosphorylated Glycogen Synthase Kinase-3β and β-Catenin Immunoreactivities

Chen, Bai Hui,Park, Joon Ha,Cho, Jeong Hwi,Kim, In Hye,Lee, Jae Chul,Lee, Tae-Kyeong,Ahn, Ji Hyeon,Tae, Hyun Jin,Shin, Bich Na,Kim, Jong-Dai,Kang, Il Jun,Won, Moo-Ho,Lee, Yun Lyul Springer-Verlag 2016 Neurochem Res Vol.41 No.8

Down-regulation of FRα Inhibits Proliferation and Promotes Apoptosis of Cervical Cancer Cells in Vitro

Bai, Li-Xia,Ding, Ling,Jiang, Shi-Wen,Kang, Hui-Jie,Gao, Chen-Fei,Chen, Chen,Zhou, Qin,Wang, Jin-Tao Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.14

Folate receptor alpha ($FR{\alpha}$) mediates folate uptake by endocytosis, and while folate is essential to DNA methylation and synthesis and may have an important role in proliferating cells. $FR{\alpha}$ is known to be expressed in rapidly proliferating cells, including many cancer cell lines, but there has been no systematic assessment of expression in cervical cancer cell lines. The aim of the present study was to evaluate the effects of $FR{\alpha}$ on proliferation and apoptosis of cervical cells and correlation mechanism. In this study, we investigated the biological function of $FR{\alpha}$ in Hela cells using RNA interference. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK8) assay, while cell cycling and apoptosis were assessed by flow cytometry, mRNA levels by real time-PCR and protein levels of $FR{\alpha}$, c-Fos and c-Jun by Western blotting. The results revealed that $FR{\alpha}$ was highly expressed in Hela cells and its silencing with a small interfering RNA (siRNA) inhibited cell proliferation and induced cell apoptosis, arresting the cell cycle in G0/G1 stages while decreasing the proportion in S and G2/M stages, and suppressed the expression levels of c-Fos and c-Jun. In conclusion, the results of this study indicated that $FR{\alpha}$ down-regulation might be capable of suppressing cervical cancer cell proliferation and promoting apoptosis. It suggested that $FR{\alpha}$ might be a novel therapeutic target for cervical cancer.

Folate Deficiency and FHIT Hypermethylation and HPV 16 Infection Promote Cervical Cancerization

Bai, Li-Xia,Wang, Jin-Tao,Ding, Ling,Jiang, Shi-Wen,Kang, Hui-Jie,Gao, Chen-Fei,Chen, Xiao,Chen, Chen,Zhou, Qin Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.21

Fragile histidine triad (FHIT) is a suppressor gene related to cervical cancer through CpG island hypermethylation. Folate is a water-soluble B-vitamin and an important cofactor in one-carbon metabolism. It may play an essential role in cervical lesions through effects on DNA methylation. The purpose of this study was to observe effects of folate and FHIT methylation and HPV 16 on cervical cancer progression. In this study, DNA methylation of FHIT, serum folate level and HPV16 status were measured using methylation-specific polymerase chain reaction (MSP), radioimmunoassay (RIA) and polymerase chain reaction (PCR), respectively, in 310 women with a diagnosis of normal cervix (NC, n=109), cervical intraepithelial neoplasia (CIN, n=101) and squamous cell carcinoma of the cervix (SCC, n=101). There were significant differences in HPV16 status (${\chi}^2=36.64$, P<0.001), CpG island methylation of FHIT (${\chi}^2=71.31$, P<0.001) and serum folate level (F=4.57, P=0.011) across the cervical histologic groups. Interaction analysis showed that the ORs only with FHIT methylation (OR=11.47) or only with HPV 16 positive (OR=4.63) or with serum folate level lower than 3.19ng/ml (OR=1.68) in SCC group were all higher than the control status of HPV 16 negative and FHIT unmethylation and serum folate level more than 3.19ng/ml (OR=1). The ORs only with HPV 16 positive (OR=2.58) or with serum folate level lower than 3.19ng/ml (OR=1.28) in CIN group were all higher than the control status, but the OR only with FHIT methylation (OR=0.53) in CIN group was lower than the control status. HPV 16 positivity was associated with a 7.60-fold increased risk of SCC with folate deficiency and with a 1.84-fold increased risk of CIN. The patients with FHIT methylation and folate deficiency or with FHIT methylation and HPV 16 positive were SCC or CIN, and the patients with HPV 16 positive and FHIT methylation and folate deficiency were all SCC. In conclusion, HPV 16 infection, FHIT methylation and folate deficiency might promote cervical cancer progression. This suggests that FHIT may be an effective target for prevention and treatment of cervical cancer.

Rufinamide, an antiepileptic drug, improves cognition and increases neurogenesis in the aged gerbil hippocampal dentate gyrus via increasing expressions of IGF-1, IGF-1R and <i>p</i>-CREB

Chen, Bai Hui,Ahn, Ji Hyeon,Park, Joon Ha,Song, Minah,Kim, Hyunjung,Lee, Tae-Kyeong,Lee, Jae Chul,Kim, Young-Myeong,Hwang, In Koo,Kim, Dae Won,Lee, Choong-Hyun,Yan, Bing Chun,Kang, Il Jun,Won, Moo-Ho Elsevier 2018 Chemico-biological interactions Vol.286 No.-

<P><B>Abstract</B></P> <P>Rufinamide is a novel antiepileptic drug and commonly used in the treatment of Lennox-Gastaut syndrome. In the present study, we investigated effects of rufinamide on cognitive function using passive avoidance test and neurogenesis in the hippocampal dentate gyrus using Ki-67 (a marker for cell proliferation), doublecortin (DCX, a marker for neuroblast) and BrdU/NeuN (markers for newly generated mature neurons) immunohistochemistry in aged gerbils. Aged gerbils (24-month old) were treated with 1 mg/kg and 3 mg/kg rufinamide for 4 weeks. Treatment with 3 mg/kg rufinamide, not 1 mg/kg rufinamide, significantly improved cognitive function and increased neurogenesis, showing that proliferating cells (Ki-67-immunoreactive cells), differentiating neuroblasts (DCX-immunoreactive neuroblasts) and mature neurons (BrdU/NeuN-immunoreactive cells) in the aged dentate gyrus compared with those in the control group. When we examined its mechanisms, rufinamide significantly increased immunoreactivities of insulin-like growth factor-1 (IGF-1), its receptor (IGF-1R), and phosphorylated cAMP response element binding protein (<I>p</I>-CREB). However, rufinamide did not show any increase in immunoreactivities of brain-derived neurotrophic factor and its receptor. Therefore, our results indicate that rufinamide can improve cognitive function and increase neurogenesis in the hippocampus of the aged gerbil via increasing expressions of IGF-1, IGF-1R and <I>p</I>-CREB.</P> <P><B>Highlights</B></P> <P> <UL> <LI> 4-week treatment of rufinamide (Ruf) increases short-term memory in the aged gerbil. </LI> <LI> Ruf increases neurogenesis in the aged hippocampal dentate gyrus (DG). </LI> <LI> Ruf treatment increases IGF-1, IGF-1R, and <I>p</I>-CREB in the aged DG. </LI> <LI> Ruf treatment does not affect expressions of BDNF and TrkB in the aged DG. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Melatonin attenuates scopolamine-induced cognitive impairment via protecting against demyelination through BDNF-TrkB signaling in the mouse dentate gyrus

Chen, Bai Hui,Park, Joon Ha,Lee, Tae-Kyeong,Song, Minah,Kim, Hyunjung,Lee, Jae Chul,Kim, Young-Myeong,Lee, Choong-Hyun,Hwang, In Koo,Kang, Il Jun,Yan, Bing Chun,Won, Moo-Ho,Ahn, Ji Hyeon Elsevier 2018 Chemico-biological interactions Vol.285 No.-

<P><B>Abstract</B></P> <P>Animal models of scopolamine-induced amnesia are widely used to study underlying mechanisms and treatment of cognitive impairment in neurodegenerative diseases such as Alzheimer's disease (AD). Previous studies have identified that melatonin improves cognitive dysfunction in animal models. In this study, using a mouse model of scopolamine-induced amnesia, we assessed spatial and short-term memory functions for 4 weeks, investigated the expression of myelin-basic protein (MBP) in the dentate gyrus, and examined whether melatonin and scopolamine cotreatment could keep cognitive function and MBP expression. In addition, to study functions of melatonin for keeping cognitive function and MBP expression, we examined expressions of brain-derived neurotrophic factor (BDNF) and tropomycin receptor kinase B (TrkB) in the mouse dentate gyrus. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally treated for 2 and 4 weeks. Two and 4 weeks after scopolamine treatment, mice showed significant cognitive impairment; however, melatonin and scopolamine cotreatment recovered cognitive impairment. Two and 4 weeks of scopolamine treatment, the density of MBP immunoreactive myelinated nerve fibers was significantly decreased in the dentate gyrus; however, scopolamine and melatonin cotreatment significantly increased the scopolamine-induced reduction of MBP expression in the dentate gyrus. Furthermore, the cotreatment of scopolamine and melatonin significantly increased the scopolamine-induced decrease of BDNF and TrKB immunoreactivity in the dentate gyrus. Taken together, our results indicate that melatonin treatment exerts anti-amnesic effect and restores the scopolamine-induced reduction of MBP expression through increasing BDNF and TrkB expressions in the mouse dentate gyrus.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Long-term treatment of scopolamine (Sco) induces cognitive deficits. </LI> <LI> Long-term treatment of melatonin improves Sco-induced cognitive deficits. </LI> <LI> Sco significantly decreases MBP, BDNF, and TrkB in the dentate gyrus. </LI> <LI> Melatonin restores scopolamine-induced decreases in MBP, BDNF, and TrkB. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Melatonin improves vascular cognitive impairment induced by ischemic stroke by remyelination via activation of ERK1/2 signaling and restoration of glutamatergic synapses in the gerbil hippocampus

Chen, Bai Hui,Park, Joon Ha,Lee, Yun Lyul,Kang, Il Jun,Kim, Dae Won,Hwang, In Koo,Lee, Choong-Hyun,Yan, Bing Chun,Kim, Young-Myeong,Lee, Tae-Kyeong,Lee, Jae Chul,Won, Moo-Ho,Ahn, Ji Hyeon Elsevier 2018 BIOMEDICINE AND PHARMACOTHERAPY Vol.108 No.-

<P><B>Abstract</B></P> <P>Vascular dementia affects cognition by damaging axons and myelin. Melatonin is pharmacologically associated with various neurological disorders. In this study, effects of melatonin on cognitive impairment and related mechanisms were investigated in an animal model of ischemic vascular dementia (IVD). Melatonin was intraperitoneally administered to adult gerbils after transient global cerebral ischemia (tGCI) for 25 days beginning 5 days after tGCI. Cognitive impairment was examined using a passive avoidance test and the Barnes maze test. To investigate mechanisms of restorative effects by melatonin, neuronal damage/death, myelin basic protein (MBP, a marker for myelin), Rip (a marker for oligodendrocyte), extracellular signal-regulated protein kinase1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2), and vesicular glutamate transporter (VGLUT)-1 (a glutamatergic synaptic marker) in the hippocampal Cornu Ammonis 1 area (CA1) were evaluated using immunohistochemistry. Melatonin treatment significantly improved tGCI-induced cognitive impairment. Death of CA1 pyramidal neurons after tGCI was not affected by melatonin treatment. However, melatonin treatment significantly increased MBP immunoreactivity and numbers of Rip-immunoreactive oligodendrocytes in the ischemic CA1. In addition, melatonin treatment significantly increased ERK1/2 and p-ERK1/2 immunoreactivities in oligodendrocytes in the ischemic CA1. Furthermore, melatonin treatment significantly increased VGLUT-1 immunoreactive structures in the ischemic CA1. These results indicate that long-term melatonin treatment after tGCI improves cognitive deficit <I>via</I> restoration of myelin, increase of oligodendrocytes which is closely related to the activation of ERK1/2 signaling, and increase of glutamatergic synapses in the ischemic brain area.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Melatonin (Mel) improved impaired memory function after ischemia. </LI> <LI> Mel did not protect ischemia-induced (i-i) neuronal loss in the hippocampus. </LI> <LI> Mel recovered i-i damaged myelinated nerve fibers and oligodendrocytes. </LI> <LI> Mel increased ERK1/2 and p-ERK1/2 in oligodendrocytes in i-i hippocampus. </LI> <LI> Mel increased i-i damaged VGLUT-1 in the hippocampus. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Pretreated quercetin protects gerbil hippocampal CA1 pyramidal neurons from transient cerebral ischemic injury by increasing the expression of antioxidant enzymes

Chen, Bai Hui,Park, Joon Ha,Ahn, Ji Hyeon,Cho, Jeong Hwi,Kim, In Hye,Lee, Jae Chul,Won, Moo-Ho,Lee, Choong-Hyun,Hwang, In Koo,Kim, Jong-Dai,Kang, Il Jun,Cho, Jun Hwi,Shin, Bich Na,Kim, Yang Hee,Lee, Y Medknow PublicationsMedia Pvt Ltd 2017 Neural regeneration research Vol.12 No.2

<P>Quercetin (QE; 3,5,7,3′,4′-pentahydroxyflavone), a well-known flavonoid, has been shown to prevent against neurodegenerative disorders and ischemic insults. However, few studies are reported regarding the neuroprotective mechanisms of QE after ischemic insults. Therefore, in this study, we investigated the effects of QE on ischemic injury and the expression of antioxidant enzymes in the hippocampal CA1 region of gerbils subjected to 5 minutes of transient cerebral ischemia. QE was pre-treated once daily for 15 days before ischemia. Pretreatment with QE protected hippocampal CA1 pyramidal neurons from ischemic injury, which was confirmed by neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, pretreatment with QE significantly increased the expression levels of endogenous antioxidant enzymes Cu/Zn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in the hippocampal CA1 pyramidal neurons of animals with ischemic injury. These findings demonstrate that pretreated QE displayed strong neuroprotective effects against transient cerebral ischemia by increasing the expression of antioxidant enzymes.</P>

Ethanol extract of <i>Oenanthe javanica</i> increases cell proliferation and neuroblast differentiation in the adolescent rat dentate gyrus

Chen, Bai Hui,Park, Joon Ha,Cho, Jeong Hwi,Kim, In Hye,Shin, Bich Na,Ahn, Ji Hyeon,Hwang, Seok Joon,Yan, Bing Chun,Tae, Hyun Jin,Lee, Jae Chul,Bae, Eun Joo,Lee, Yun Lyul,Kim, Jong Dai,Won, Moo-Ho,Kang Medknow PublicationsMedia Pvt Ltd 2015 Neural regeneration research Vol.10 No.2

<P><I>Oenanthe javanica</I> is an aquatic perennial herb that belongs to the <I>Oenanthe genus</I> in Apiaceae family, and it displays well-known medicinal properties such as protective effects against glutamate-induced neurotoxicity. However, few studies regarding effects of <I>Oenanthe javanica</I> on neurogenesis in the brain have been reported. In this study, we examined the effects of a normal diet and a diet containing ethanol extract of <I>Oenanthe javanica</I> on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus of adolescent rats using Ki-67 (an endogenous marker for cell proliferation) and doublecortin (a marker for neuroblast). Our results showed that <I>Oenanthe javanica</I> extract significantly increased the number of Ki-67-immunoreactive cells and doublecortin-immunoreactive neuroblasts in the subgranular zone of the dentate gyrus in the adolescent rats. In addition, the immunoreactivity of brain-derived neurotrophic factor was significantly increased in the dentate gyrus of the <I>Oenanthe javanica</I> extract-treated group compared with the control group. However, we did not find that vascular endothelial growth factor expression was increased in the <I>Oenanthe javanica</I> extract-treated group compared with the control group. These results indicate that <I>Oenanthe javanica</I> extract improves cell proliferation and neuroblast differentiation by increasing brain-derived neurotrophic factor immunoreactivity in the rat dentate gyrus.</P>

Hippophae rhamnoides L. Leaves Extract Enhances Cell Proliferation and Neuroblast Differentiation through Upregulation of Intrinsic Factors in the Dentate Gyrus of the Aged Gerbil

Bai Hui Chen,Ji Hyeon Ahn,Joon Ha Park,In Hye Kim,Jeong-Hwi Cho,Jae-Chul Lee,Bich-Na Shin,Hyun-Jin Tae,Jung Hoon Choi,Yun Lyul Lee,Moo-Ho Won 한국실험동물학회 2014 한국실험동물학회 학술발표대회 논문집 Vol.2014 No.8