[ZEB2 Regulates the Migration and Invasion of PANC-1 Pancreatic Cancer Cells: An Experimental Study].

Objective: To investigate the effects and mechanisms of zinc finger E-box binding homeobox transcription factor-2 ( ZEB2) on the proliferation, colony formation, migration, and invasion abilities and the epithelial-mesenchymal transition (EMT) of PANC-1 cells, a human pancreatic cancer cell line. Methods: Data on the expression of ZEB2 in pancreatic cancer tissues and paracancerous tissues from The Cancer Genome Atlas (TCGA) database were analyzed. PANC-1 pancreatic cancer cells were divided into si-NC group, si- ZEB2 group, pcDNA3.1 group, and pcDNA3.1- ZEB2 group. qRT-PCR and Western blot were conducted to confirm the effectiveness of ZEB2 knockdown or overexpression. CCK-8, colony formation, wound healing, and Transwell assays were conducted to examine the effects of ZEB2 on the proliferation, colony formation, migration, and invasion of PANC-1 cells. qRT-PCR and immunofluorescence assays were performed to examine the expression of E-cadherin and vimentin, the EMT markers, in the cells. Prediction of proteins interacting with ZEB2 was made through the STRING database. Results: TCGA database analysis showed that the expression level of ZEB2 in pancreatic cancer tissues was significantly higher than that in adjacent tissues ( P<0.05). Compared with those of cells in the control group, the proliferation, colony formation, migration, and invasion of cells in the si- ZEB2 group were decreased ( P<0.05). Compared with those of cells in the pcDNA3.1 group, the proliferation, colony formation, migration and invasion of cells in the pcDNA3.1- ZEB2 group were increased (all P<0.05). According to the results of qRT-PCR and immunofluorescence assays, compared with those of the si-NC group, the expression of E-cadherin mRNA, an epithelial marker, in the si- ZEB2 group increased, while the expression of vimentin mRNA, an mesenchymal marker, and the protein decreased. Compared with those of the pcDNA3.1 group, the expression of E-cadherin mRNA in the PANC-1 cells of the pcDNA3.1- ZEB2 group decreased, while the expression of vimentin mRNA and the protein increased (all P<0.05). Analysis with the STRING database predicted that 10 proteins had close interaction with ZEB2. Conclusion: Overexpression of ZEB2 promotes the migration, invasion, and the EMT process of PANC-1 pancreatic cancer cells.

microRNA-10a-5p overexpression suppresses malignancy of colon cancer by regulating human liver cancer fibroblasts.

The crosstalk between tumor and stroma plays a critical role in cancer metastasis. However, the function of miR-10a-5p on liver fibroblasts in the metastatic microenvironment of colon cancer (CC) and the effect of activated fibroblasts on CC cells are still unclear. In our study, miR-10a-5p overexpression inhibited the proliferation, migration, and IL-6/IL-8 level of LX-2 cells and human liver cancer fibroblasts (HLCFs). Moreover, miR-10a-5p had lower expression in HLCFs than in human liver normal fibroblasts (HLNFs). The conditioned medium (CM) from LX-2 cells with miR-10a-5p overexpression or HLNFs could inhibit the invasion, migration, and stemness of CC SW480 cells, whereas HLCFs CM could promote these malignant phenotypes of SW480 cells. The present study illustrates the effect of miR-10a-5p on the liver fibroblasts and the altered liver fibroblasts in the microenvironment on CC cells induced by miR-10a-5p, which may aid the understanding of the mechanisms underlying CC liver metastasis.

[Effects of CAAP1 on Proliferation, Migration and Invasion of Hepatoma Cell Line SMMC-7721].

OBJECTIVE: To investigate the effect of caspase activity and apoptosis inhibitor 1 (CAAP1) on the proliferation, migration and invasion of hepatoma cell SMMC-7721. METHODS: pcDNA3/ CAAP1, the overexpression vector of CAAP1, and pSilencer 2.1-U6 neo/shR- CAAP1, the knockdown vector, were constructed and examined. The experiment included 4 groups of SMMC-7721 cells, pcDNA3/ CAAP1 group, pcDNA3 control group, shR- CAAP1 group and pSilencer control group. After the SMMC-7721 cells were cultured, the overexpression vector pcDNA3/ CAAP1 (the pcDNA3/ CAAP1 group), knockdown vector shR- CAAP1 (the shR- CAAP1 group) and their controls (pcDNA3 control group and pSilencer control group) were transfected into SMMC-7721 cells respectively, and the follow-up experiments were carried out 48 h later. The mRNA expression of CAAP1 in each group was examined with qRT-PCR. The protein expression level of CAAP1 and cleaved Caspase-3 were checked with Western blot. The proliferation of cells was examined with CCK-8. The colony formation ability and the motility of cells in each group were assessed with colony formation assay and wound-healing assay, respectively. The migration and invasion of cells were examined with Transwell cell chamber and the apoptosis of cells was examined with flow cytometry. The data of 75 patients with low expression of CAAP1 and 295 patients with high expression of CAAP1 were downloaded from TCGA database and the data of 48 months follow-up were analyzed. Kaplan-Meier survival curve was used to compare the correlation between different levels of CAAP1 expression and overall survival (OS) of hepatocellular carcinoma (HCC) patients. RESULTS: Double enzyme digestion analysis showed that the overexpression vector pcDNA3/ CAAP1 and knockdown vector shR- CAAP1 were constructed successfully. qRT-PCR and Western blot results showed that pcDNA3/ CAAP1 increased the mRNA and protein expression level of CAAP1 in SMMC-7721 cells (in comparison with the pcDNA3 control group, P<0.05), while shR- CAAP1 decreased the mRNA and protein expression of CAAP1 (in comparison with the pSilencer control group, P<0.05). Compared with pcDNA3 control group, the proliferation, colony formation ability, motility, migration and invasion of SMMC-7721 cells in the pcDNA3/ CAAP1 group were increased, while the apoptosis of SMMC-7721 cells was inhibited (all P<0.05). Compared with the pSilencer control group, the proliferation, colony formation ability, motility, migration and invasion ability of SMMC-7721 cells in the shR- CAAP1 group decreased, while the apoptosis increased (all P<0.05). TCGA database analysis showed that HCC patients with low CAAP1 expression had better OS than that of HCC patients with high CAAP1 expression. CONCLUSION: CAAP1 can promote the proliferation, migration and invasion of SMMC-7721 cells while it inhibit their apoptosis.

MicroRNA-185 reduces the expression of hepatitis B virus surface antigen by targeting PRKCH in HepG2 2.2.15 cells.

MicroRNAs (miRNAs) are single-stranded noncoding RNAs with 18 to 25 nucleotides and play critical roles in a wide spectrum of biological processes. We repored that miR-185 inhibited hepatitis B surface antigen (HBsAg) expression and hepatitis B virus (HBV) replication without affecting the proliferation of HepG2 2.2.15 cells, compared with the controls. We identified that protein kinase C eta (PRKCH) is a direct target gene of miR-185 that affects HBV replication and protein expression and that the miR-185 may suppress HBV replication. Our results provide more information for gene therapy in HBV infection. Keywords: miR-185; HBV; HBV surface antigen; viral replication; PRKCH.

miR-370 suppresses HBV gene expression and replication by targeting nuclear factor IA.

Hepatitis B virus (HBV) infection is a major health problem worldwide. The roles of microRNAs in the regulation of HBV expression are being increasingly recognized. In this study, we found that overexpression of miR-370 suppressed HBV gene expression and replication in Huh7 cells, whereas antisense knockdown of endogenous miR-370 enhanced HBV gene expression and replication in Huh7 cells and HepG2.2.15 cells. Further, we identified the transcription factor nuclear factor IA (NFIA) as a new host target of miR-370. Overexpression and knockdown studies showed that NFIA stimulated HBV gene expression and replication. Importantly, overexpression of NFIA counteracted the effect of miR-370 on HBV gene expression and replication. Further mechanistic studies showed that miR-370 suppressed HBV replication and gene expression by repressing HBV Enhancer I activity, and one of the NFIA binding site in the Enhancer I element was responsible for the repressive effect of miR-370 on HBV Enhancer I activity. Altogether, our results demonstrated that miR-370 suppressed HBV gene expression and replication through repressing NFIA expression, which stimulates HBV replication via direct regulation on HBV Enhancer I activities. Our findings may provide a new antiviral strategy for HBV infection. J. Med. Virol. 89:834-844, 2017. © 2016 Wiley Periodicals, Inc.

Using Next-Generation Sequencing to Detect Differential Expression Genes in Bradysia odoriphaga after Exposure to Insecticides.

Bradysia odoriphaga (Diptera: Sciaridae) is the most important pest of Chinese chive. Insecticides are used widely and frequently to control B. odoriphaga in China. However, the performance of the insecticides chlorpyrifos and clothianidin in controlling the Chinese chive maggot is quite different. Using next generation sequencing technology, different expression unigenes (DEUs) in B. odoriphaga were detected after treatment with chlorpyrifos and clothianidin for 6 and 48 h in comparison with control. The number of DEUs ranged between 703 and 1161 after insecticide treatment. In these DEUs, 370-863 unigenes can be classified into 41-46 categories of gene ontology (GO), and 354-658 DEUs can be mapped into 987-1623 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expressions of DEUs related to insecticide-metabolism-related genes were analyzed. The cytochrome P450-like unigene group was the largest group in DEUs. Most glutathione S-transferase-like unigenes were down-regulated and most sodium channel-like unigenes were up-regulated after insecticide treatment. Finally, 14 insecticide-metabolism-related unigenes were chosen to confirm the relative expression in each treatment by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The results of qRT-PCR and RNA Sequencing (RNA-Seq) are fairly well-established. Our results demonstrate that a next-generation sequencing tool facilitates the identification of insecticide-metabolism-related genes and the illustration of the insecticide mechanisms of chlorpyrifos and clothianidin.

HBx-induced MiR-1269b in NF-κB dependent manner upregulates cell division cycle 40 homolog (CDC40) to promote proliferation and migration in hepatoma cells.

BACKGROUND: Occurrence and progression of hepatocellular carcinoma (HCC) are associated with hepatitis B virus (HBV) infection. miR-1269b is up-regulated in HCC cells and tissues. However, the regulation of miR-1269b expression by HBV and the mechanism underlying the oncogenic activity of miR-1269b in HCC are unclear. METHODS: Reverse transcription quantitative PCR (RT-qPCR) was used to measure the expression of miR-1269b and target genes in HCC tissues and cell lines. Western blot analysis was used to assess the expression of miR-1269b target genes and related proteins. Using luciferase reporter assays and EMSA, we identified the factors regulating the transcriptional level of miR-1269b. Colony formation, flow cytometry and cell migration assays were performed to evaluate the phenotypic changes caused by miR-1269b and its target in HCC cells. RESULTS: We demonstrated that the expression levels of pre-miR-1269b and miR-1269b in HBV-positive HepG2.2.15 cells were dramatically increased compared with HBV-negative HepG2 cells. HBx was shown to facilitate translocation of NF-κB from the cytoplasm to the nucleus, and NF-κB binds to the promoter of miR-1269b to enhance its transcription. miR-1269b targets and up-regulates CDC40, a cell division cycle 40 homolog. CDC40 increases cell cycle progression, cell proliferation and migration. Rescue experiment indicated that CDC40 promotes malignancy induced by miR-1269b in HCC cells. CONCLUSION: We found that HBx activates NF-κB to promote the expression of miR1269b, which augments CDC40 expression, contributing to malignancy in HCC. Our findings provide insights into the mechanisms underlying HBV-induced hepatocarcinogenesis.

VEGF-mediated suppression of cell proliferation and invasion by miR-410 in osteosarcoma.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. The aberrant expression of miRNA has become a major focus in cancer research. This study aimed to investigate the importance of miR-410 in the diagnosis and therapy of osteosarcoma (OS). Western blot analysis showed that the expression of VEGF was higher in Saos-2 and MG-63 cells than that in three other OS cell lines. We also found that miR-410 was lowly expressed and inversely correlated with VEGF expression in OS specimens. Over-expression of miR-410 had a greater repression on VEGF expression than other candidate VEGF-targeting miRNAs. Luciferase reporter assay demonstrated that miR-410 directly decreased VEGF expression by targeting its 3'-untranslated region. Further investigation demonstrated the regulation of miR-410 in OS cells via VEGF. In vitro MTT assay, Transwell, and flow cytometry showed that transfection of the miR-410 expression plasmid inhibited cell proliferation and contributed to apoptosis in OS cells. Moreover, restoration of VEGF reversed the effect of miR-410 on OS cells, and upregulated the expression of phosphorylated AKT. Finally, overexpression of miR-410 also showed a negative effect on tumor growth in vivo. Our findings suggest a cooperative relationship between miR-410 and VEGF in OS cell regulation. This information may help researchers to better understand miRNA regulation in cancer and provide a rationale for developing miRNA-based strategies for OS treatment.

Influence of Constant Temperature on Reproductive Parameters of Holotrichia oblita (Coleoptera: Scarabaeidae).

Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) is a major pest both in field crops and forests because the larvae could eat the roots of most crops in the field, and the adults damage the leaves of trees and field crops. In this study, we focused on the effects of temperature on H. oblita reproductive parameters. The results indicated H. oblita female adults at 25 °C could lay more eggs (84.0 eggs per female) and have the shortest preoviposition period (19.1 d), the greatest oviposition rate (2.8 eggs per female per 3 d), and largest percentage of life span spent in oviposition (59.5%). The longevity and the time to 50% egg laying decreased with increasing temperature, and female longevity was always longer than male longevity. The preoviposition and postoviposition period decreased with increasing temperature from 15 to 25 °C and then increased when the temperature increased from 25 to 30 °C. These results show that 25 °C is the optimal temperature for reproduction of H. oblita.

A complex system health state assessment method with reference value optimization for interpretable BRB.

Health condition assessment is the basis for formulating and optimizing maintenance strategies of complex systems, which is crucial for ensuring the safe and stable operation of these systems. In complex system health condition assessment, it is not only necessary for the model to handle various uncertainties to ensure the accuracy of assessment results, but also to have a transparent and reasonable assessment process and interpretable, traceable assessment results. belief rule base (BRB) has been widely used as an interpretable modeling method in health condition assessment. However, BRB-based models currently face two issues: (1) inaccuracies in expert-provided parameters that can affect the model's accuracy, and (2) after model optimization, interpretability may be reduced. Therefore, this paper proposes a new method for complex system health condition assessment called interpretable BRB with reference value optimization (I-BRB). Firstly, to address the issue of inaccurate reference values, a reference value optimization algorithm with interpretability constraints is designed, which optimizes the reference values without compromising expert knowledge. Secondly, the remaining parameters are optimized using the projection covariance matrix adaptation evolution strategy (P-CMA-ES) with interpretability constraints to improve the model's accuracy. Finally, a case study evaluating the bearing components of a flywheel system is conducted to validate the proposed method. Experimental results demonstrate that I-BRB achieves higher accuracy in health condition assessment.

[Application of urinary proteomics in early diagnosis of bladder urothelial carcinoma].

OBJECTIVE: To investigate the difference in urinary proteome between patients with bladder urothelial carcinoma (BUC) and healthy volunteers and to provide a basis for the early diagnosis of BUC. METHODS: The urine samples from BUC patients and healthy volunteers (controls) were treated by 25% ethanol precipitation and two-dimensional gel electrophoresis (2-DE), and the obtained urinary proteins were subjected to Coomassie brilliant blue staining and analysis by PDQuest 8.0 (2-DE image analysis software); the differentially expressed proteins were sequenced by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry and identified using the Swiss-Prot database; the differential expression of these proteins was verified by western blot. RESULTS: High-resolution and high-reproducibility 2-DE images were obtained from the urine samples of BUC patients and controls, with 789 ± 18 and 762 ± 14 protein spots, respectively. Compared with the control group, the BUC grouP had significantly decreased expression of 6 protein spots and significantly increased expression of 11 protein spots. The mass spectrometry revealed five proteins with increased expression in the BUC group, including fibrinogen, lactate dehydrogenase B, apolipoprotein A1, clusterin, and haptoglobin, and the results were confirmed by western blot. CONCLUSION: There is significant difference in urinary proteome between BUC patients and healthy volunteers; the identification of differentially expressed proteins in urine lays the foundation for identifying potential molecular markers in early diagnosis of BUC.

A new stock market analysis method based on evidential reasoning and hierarchical belief rule base to support investment decision making.

Stock market analysis is helpful for investors to make reasonable decisions and maintain market stability, and it usually involves not only quantitative data but also qualitative information, so the analysis method needs to have the ability to deal with both types of information comprehensively. In addition, due to the inherent risk of stock investment, it is necessary to ensure that the analysis results can be traced and interpreted. To solve the above problems, a stock market analysis method based on evidential reasoning (ER) and hierarchical belief rule base (HBRB) is proposed in this paper. First, an evaluation model is constructed based on expert knowledge and ER to evaluate stock market sentiment. Then, a stock market decision model based on HBRB is constructed to support investment decision making, such as buying and selling stocks and holding positions. Finally, the Shanghai Stock Index from 2010 to 2019 is used as an example to verify the applicability and effectiveness of the proposed stock market analysis method for investment decision support. Experimental research demonstrates that the proposed method can help analyze the stock market comprehensively and support investors to make investment decisions effectively.

G-quadruplex-enhanced circular single-stranded DNA (G4-CSSD) adsorption of miRNA to inhibit colon cancer progression.

BACKGROUND: Chromosomal heterogeneity leads to the abnormal expression and mutation of tumor-specific genes. Drugs targeting oncogenes have been extensively developed. However, given the random mutation of tumor suppressor genes, the development of its targeted drugs is difficult. METHODS: Our early research revealed that artificial circular single-stranded DNA (CSSD) can restore multiple tumor suppressor genes to inhibit tumor malignant progression by adsorbing miRNA. Here, we improved CSSD to a fully closed single-stranded DNA with G quadruplex DNA secondary structure (G4-CSSD), which made G4-CSSD with higher acquisition rate and decreased degradation. The Cancer Genome Atlas (TCGA) and Human Protein Atlas database were used to predict tumour suppressor genes in colon cancer. Cellular and animal experiments were performed to validate the role of G4-CSSD in cancer cell progression. RESULTS: In colon cancer, we observed the simultaneous low expressions of chloride channel accessory 1 (CLCA1), UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 6 (B3GNT6) and UDP glucuronosyltransferase family 2 member A3 (UGT2A3), which indicated an favourable prognosis. After repressing miR-590-3p with G4-CSSD590, the upregulation of CLCA1, B3GNT6 and UGT2A3 inhibited the proliferation and metastasis of colon cancer cells. CONCLUSIONS: This study may provide basis for new treatment methods for colon cancer by restoration of tumor suppressor genes.

A miR-340/SPP1 axis inhibits the activation and proliferation of hepatic stellate cells by inhibiting the TGF-ß1/Smads pathway.

BACKGROUND: Hepatic fibrosis (HF) is a common pathological complication of liver cirrhosis which affects human health. It is well established that microRNAs (miRNAs) regulate the proliferation, activation and apoptosis of hepatic stellate cells (HSCs). OBJECTIVES: To determine the function and molecular mechanism of miR-340-5p/secreted phosphoprotein 1 (SPP1) axis in HF and identify potential therapeutic targets. MATERIAL AND METHODS: The HF model in cholestatic rats was induced by ligating the common bile duct. The histological sections of the liver tissues were stained with hematoxylin and eosin (H&E), Masson's trichrome or Sirius Red. The differential expression of mRNAs in the liver tissues was examined using the microarray analysis. The expression levels of miR-340-5p, SPP1, alpha-smooth muscle actin (α-SMA), Collagen I, phosphorylated Smad2 (p-Smad2), and p-Smad3 were determined using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell proliferation was quantified using cell counting kit-8 (CCK-8) assays. The regulatory effect of miR-340-5p on SPP1 was determined with fluorescent reporter assay. RESULTS: The bile duct ligation (BDL) rat model was successfully induced, and SPP1 was upregulated in liver tissue from the BDL group compared to that of the sham group. The expression level of miR-340-5p was decreased in activated human primary normal fibroblasts (NFs) and activated LX-2 cells, and the mRNA and protein expression levels of SPP1 were increased in activated LX-2 cells. The SPP1 was the target of miR-340-5p, and the overexpression of SPP1 increased the proliferation of LX-2 cells, the expression of HF markers α-SMA and Collagen I, and key factors p-Smad2 and p-Smad3 (all p < 0.05). However, reverse results were obtained with the overexpression of miR-340-5p in LX-2 cells. CONCLUSIONS: Our findings provide evidence that SPP1 targeted by miR-340-5p promotes LX-2 cell proliferation and activation through the TGF-ß1/Smads signaling pathway. Therefore, miR-340-5p and SPP1 may be possible therapeutic targets for HF.

HSC-derived exosomal miR-199a-5p promotes HSC activation and hepatocyte EMT via targeting SIRT1 in hepatic fibrosis.

Exosomes have been implicated in inflammation-related diseases, such as hepatic fibrosis (HF) and renal fibrosis, via transferring bioactive cargoes to recipient cells. This study aimed to investigate the possible effect of hepatic stellate cell (HSC)-derived exosomes on the initiation and development of HF by delivering microRNA (miR)-199a-5p. In HF rats with cholestasis induced by ligating the common bile duct, miR-199a-5p was upregulated while SIRT1 was downregulated in liver tissues from bile duct ligation (BDL) rats compared with that of sham rats. Furthermore, miR-199a-5p expression was upregulated, but the mRNA and protein expression levels of SIRT1 were downregulated in TGF-ß1-activated LX-2. miR-199a-5p promoted the proliferation and further activation of LX-2 and enhanced the expression levels of the HF markers COL1A1 and α-SMA. Subsequently, the binding of miR-199a-5p to the 3'UTR of SIRT1 mRNA was predicted by bioinformatics websites and evidenced by fluorescent reporter assay. Knocking down SIRT1 enhanced the abilities of LX-2 cell proliferation, migration, and colony formation and increased the expression levels of the HF markers α-SMA and COL1A1. LX-2-derived exosomal miR-199a-5p transferred to LX-2 and THLE-2, inhibited the proliferation of THLE-2, and promoted the epithelial mesenchymal transition (EMT) and senescence of THLE-2. Furthermore, in vivo results suggested that miR-199a-5p overexpression aggravated HF in BDL rats; increased miR-199a-5p, α-SMA, and COL1A1 expression levels; and significantly upregulated the serum ALT, AST, TBA, and TBIL levels. However, reverse results were obtained with inhibited miR-199a-5p expression. In conclusion, HSC-derived exosomal miR-199a-5p may promote HF by accelerating HSC activation and hepatocyte EMT by targeting SIRT1, suggesting that miR-199a-5p and SIRT1 may serve as potential therapeutic targets for HF.

Research on the Impact of Pro-Environment Game and Guilt on Environmentally Sustainable Behaviour.

Game strategies are widely used by companies to attract users and increase their stickiness. At the same time, the protection of the ecological environment is also an important expression of corporate social responsibility. This paper explores the integration of social responsibility with gaming strategies from the psychological perspective of game withdrawal, and explores the incorporation of social responsibility as an element in gamification design to reduce user withdrawal behaviour, thereby increasing individual's environmentally sustainable behaviour. We evidenced our hypothesis through two studies. Study one proved our hypothesis by recruiting 106 university undergraduates (from Wuhan University, mean age 20, of whom 47 were female and 59 were male) to prove our hypothesis by recalling previous experiences with different types of games. Study two further tested our hypothesis by manipulating participants' guilt through randomly recruiting 196 participants (mean age 35, of whom 88 were female and 108 were male, 35 of them were students, 107 were office workers and 54 were from other sectors) from different industries through the questionnaire research website Credamo. The findings show that incorporating social responsibility elements into the design of games can make users engage in pro-social behaviour while playing the game, and the guilt that users feel because of the game will be compensated by pro-social behaviour, thus reducing the game frequency and duration and improving the intent of pro-social behaviour. At the same time, players' self-control moderates the effect of guilt on game play volume under a socially responsible gamification design.

Transcriptome analysis of Holotrichia oblita reveals differentially expressed unigenes related to reproduction and development under different photoperiods.

Holotrichia oblita (Faldermann) (Coleoptera: Scarabaeidae) is an insect whose feeding and mating behaviors occur at night. A scotophase is necessary for H. oblita reproduction. We used RNA sequencing (RNA-seq) to compare the expression patterns of H. oblita at five photoperiods (0:24, 8:16, 12:12, 16:8, and 24:0 h) (L:D). Compared to the control (24:0) (L:D), 161-684 differentially expressed unigenes (DEUs) were found in female samples, while 698-2322 DEUs were found in male samples. For all DEUs, a total of 92-1143 DEUs were allocated to 116-662 categories of gene ontology (GO), and 81-1116 DEUs were assigned into 77-286 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The iPath diagram showed that the DEUs generated by comparing female and male samples with photoperiods of 0:24 and 24:0, respectively, involved multiple metabolic pathways, such as carbohydrate metabolism, lipid metabolism, nucleotide metabolism, purine metabolism and glutathione metabolism. Most of these DEUs were upregulated. Finally, 13 DEUs related to reproduction and development were selected to confirm the consistency of relative expression between RNA-Seq and quantitative real-time polymerase chain reaction (qRT-PCR). Most of these comparison results agreed well, except for some qRT-PCR results that were not detected in male samples due to their low expression. These results provide useful information for understanding the dark-induced reproduction of H. oblita.

MiR-22-3p and miR-29a-3p synergistically inhibit hepatic stellate cell activation by targeting AKT3.

Hepatic fibrosis (HF) is a worldwide health problem for which there is no medically effective drug treatment at present, and which is characterized by activation of hepatic stellate cells (HSCs) and excessive extracellular matrix (ECM) deposition. The HF model in cholestatic rats by ligating the common bile duct was induced and the differentially expressed miRNAs in the liver tissues were analyzed by microarray, which showed that miR-22-3p and miR-29a-3p were significantly downregulated in bile-duct ligation (BDL) rat liver compared with the sham control. The synergistic anti-HF activity and molecular mechanism of miR-22-3p and miR-29a-3p by targeting AKT serine/threonine kinase 3 (AKT3) in HSCs were explored. The expression levels of miR-22-3p and miR-29a-3p were downregulated in activated LX-2 and human primary normal hepatic fibroblasts (NFs), whereas AKT3 was found to be upregulated in BDL rat liver and activated LX-2 cells. The proliferation, colony-forming, and migration ability of LX-2 were inhibited synergistically by miR-22-3p and miR-29a-3p. In addition, cellular senescence was induced and the expressions of the LX-2 fibrosis markers COL1A1 and α-SMA were inhibited by miR-22-3p and miR-29a-3p synergistically. Subsequently, these two miRNAs binding to the 3'UTR of AKT3 mRNA was predicted and evidenced by the luciferase reporter assay. Furthermore, the proliferation, migration, colony-forming ability, and the expression levels of COL1A1 and α-SMA were promoted and cellular senescence was inhibited by AKT3 in LX-2 cells. Thus, miR-22-3p/miR-29a-3p/AKT3 regulates the activation of HSCs, providing a new avenue in the study and treatment of HF.

Effect of Photoperiod on Longevity, Food Consumption, and Reproduction of Holotrichia oblita (Coleoptera: Scarabaeidae).

Holotrichia oblita (Faldermann) (Coleoptera: Scarabaeidae) is a major soil insect pest that damages forest trees, crops, and lawns. Adults of H. oblita fly, forage, and mate at night but remain underground during the day. We studied the effect of photoperiod on H. oblita reproduction. H. oblita females laid more eggs at 8:16 (L:D) h and 0:24 (L:D) h than other photoperiods. As the scotophase increased, the preoviposition period decreased and the oviposition period increased. Female longevity exceeded that of males at all photoperiods, and both males and females at 0:24 (L:D) h had the shortest longevity. The number of eggs laid per female increased with increasing food consumption. Females at 8:16 (L:D) h had the greatest food consumption and laid the most eggs, while females at 24:0 (L:D) h had the lowest food consumption and laid few eggs. The food intake of adults increased gradually and decreased slowly after reaching a peak. Females began to lay eggs when their food consumption reached a maximum. These results indicate that a scotophase is necessary for the reproduction of H. oblita. A long scotophase promotes greater oviposition. The effect of photoperiod on reproduction is affected by food intake.

Evaluation of reference genes for quantitative real-time PCR normalization in the scarab beetle Holotrichia oblita.

Quantitative real-time polymerase chain reaction (qPT-PCR) is commonly used to analyze gene expression, however, the accuracy of the normalized results is affected by the expression stability of reference genes. Holotrichia oblita (Coleoptera: Scarabaeidae) causes serious damage to crops. Reliable reference genes in H. oblita are needed for qRT-PCR analysis. Therefore, we evaluated 13 reference genes under biotic and abiotic conditions. RefFinder provided a comprehensive stability ranking, and geNorm suggested the optimal number of reference genes for normalization. RPL13a and RPL18 were the most suitable reference genes for developmental stages, tissues, and temperature treatments; RPL13a and RPS3 were the most suitable for pesticide and photoperiod treatments; RPS18 and RPL18 were the most suitable for the two sexes. We validated the normalized results using odorant-binding protein genes as target genes in different tissues. Compared with the selected suitable reference genes, the expression of OBP1 in antennae, abdomen, and wings, and OBP2 in antennae and wings were overestimated due to the instability of ACTb. These results identified several reliable reference genes in H. oblita for normalization, and are valuable for future molecular studies.