[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.

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.

[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.

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.

Dye molecule bonded titanium alkoxide: a possible new type of dye for sensitized solar cells.

An organic dye coordinated titanium iso-propoxide compound is designed and synthesized. Taking advantage of the hydrolysis of the titanium alkoxide moiety on the surface of TiO2 electrode, the dye-semiconductor surface properties, including anchoring and dispersivity, are improved, which opens a new perspective to explore dyes for DSSCs.

Effects of rhein lysinate on D-galactose-induced aging mice.

The aim of the present study was to investigate the anti-aging effects of rhein lysinate (RHL), and to explore its mechanism of action in a D-galactose-induced aging mouse model. Aging was induced by D-galactose (100 mg/kg/day) that was subcutaneously injected to animals for 8 weeks. RHL was simultaneously administered once a day by intragastric gavage. The appetite, mental condition, body weight and organ index of the mice were monitored. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined, and the levels of malondialdehyde (MDA) in the liver, kidney and serum were measured by appropriate assay kits. Western blot analysis was used to detect proteins associated with age. The results indicated that RHL may improve the appetite, mental state and organ conditions of the model mice, improve the activities of SOD and GSH-Px, reduce MDA levels and modulate the expression of age-associated proteins (Sirtuin 1, p21 and p16) in D-galactose-induced mice. Therefore, RHL may be effective at suppressing the aging process through a combination of enhancing antioxidant activity, scavenging free radicals and modulating aging-associated gene expression.

Protective effect of bicyclol against bile duct ligation-induced hepatic fibrosis in rats.

AIM: To evaluate the protective effect of bicyclol against bile duct ligation (BDL)-induced hepatic fibrosis in rats. METHODS: Sprague-Dawley male rats underwent BDL and sham-operated animals were used as healthy controls. The BDL rats were divided into two groups which received sterilized PBS or bicyclol (100 mg/kg per day) orally for two consecutive weeks. Serum, urine and bile were collected for biochemical determinations. Liver tissues were collected for histological analysis and a whole genome oligonucleotide microarray assay. Reverse transcription-polymerase chain reaction and Western blotting were used to verify the expression of liver fibrosis-related genes. RESULTS: Treatment with bicyclol significantly reduced liver fibrosis and bile duct proliferation after BDL. The levels of alanine aminotransferase (127.7 ± 72.3 vs 230.4 ± 69.6, P < 0.05) and aspartate aminotransferase (696.8 ± 232.6 vs 1032.6 ± 165.8, P < 0.05) were also decreased by treatment with bicyclol in comparison to PBS. The expression changes of 45 fibrogenic genes and several fibrogenesis-related pathways were reversed by bicyclol in the microarray assay. Bicyclol significantly reduced liver mRNA and/or protein expression levels of collagen 1a1, matrix metalloproteinase 2, tumor necrosis factor, tissue inhibitors of metalloproteinases 2, transforming growth factor-ß1 and α-smooth muscle actin. CONCLUSION: Bicyclol significantly attenuates BDL-induced liver fibrosis by reversing fibrogenic gene expression. These findings suggest that bicyclol might be an effective anti-fibrotic drug for the treatment of cholestatic liver disease.

[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.

MicroRNA-663 targets TGFB1 and regulates lung cancer proliferation.

MicroRNAs (miRNAs) play critical roles in many different cellular processes, including metabolism, apoptosis, differentiation, and development. In this study, miR-663 was shown to be highly expressed in patients with lung cancer. Furthermore, miR-663 contributed to lung cancer cell proliferation of by regulating TGFB1, P53, Bax, and Fas directly or indirectly. Our results demonstrated that miR-663 plays an important role in the biology of lung cancer and may be useful in developing therapies targeting genes.

Suppression of hepatitis B virus replication by microRNA-199a-3p and microRNA-210.

Accumulating evidence suggests that microRNAs (miRNAs) control the replication of both RNA and DNA viruses. In order to determine whether host-encoded miRNAs affect hepatitis B virus (HBV) replication, antisense oligonucleotides (ASOs) of 328 identified human miRNAs were transfected into HepG2 2.2.15 cells, respectively. ELISA and MTS assay were used to measure the expression level of HBV S protein (HBsAg), HBV e antigen (HBeAg) and cell proliferation. Compared to experimental controls, miR-199a-3p and miR-210 efficiently reduced HBsAg expression without affecting HepG2 2.2.15 cell proliferation. Quantification of HBV DNA by real-time PCR showed that both miRNAs suppressed viral replication. Bioinformatics analysis indicated a putative binding site for miR-199a-3p in the HBsAg coding region and a putative binding site for miR-210 in the HBV pre-S1 region. The direct effect of miRNAs on the target region in HBV transcripts was validated by a fluorescent reporter assay, and the suppression of HBs gene expression by both miRNAs was measured by real-time PCR and Western blot. These results suggest that up-regulation of miR-199a-3p and miR-210 in HepG2 2.2.15 cells compared to HepG2 cells may play a role in regulating HBV replication and maintenance of a suitable level of virion production in persistent infection by targeting crucial HBV genes.

[Expression of bcl-2 gene in EBV-transformed human gastric epithelial cell line GES-1].

OBJECTIVE: To detect bcl-2 gene expression in Epstein-Barr virus (EBV)-infected human gastric epithelial cell line GES-1 for understanding the role of bcl-2 gene in the carcinogenesis of EBV-associated gastric carcinoma. METHODS: Akata 1061 cells producing recombined EBV carrying neomycin resistance gene (NEOr) was used to mediate the EBV infection of human gastric epithelial cell line GES-1 via close contact, with the empty plasmid pcDNA3-transfected GES-1 cells via lipofectamine method as a control. The EBV-infected and pcDNA3-transfected cells were cloned by limited dilution and the positive clones selected with G418. Immunocytochemical staining was performed to detect the expressions of EBNA1 and Bcl-2 protein. RESULTS: Bcl-2 protein expression was detected in EBV-infected cells but not in the control cells. CONCLUSION: EBV infection can increase Bcl-2 expression in gastric epithelial cells, and such cell transformation effect of EBV is related to the overexpression of bcl-2 gene.

[A cell model of ischemia/reperfusion injury on skeletal muscle].

AIM: To establish a model of ischemia/reperfusion injury on L-6TG cell. METHODS: Cultured L-6TG cells were divided into 2 groups: control group (C), ischemia/reperfusion group (I/R), LDH in culture fluid, SOD, XOD, free calcium in L-6TG cell and mitochondria respiration were evaluated in each group, the micromorphologic changes were observed with microscope. RESULTS: Compared with control group, after L-6TG cell suffered ischemia 4 hours and reperfusion 4 hours, LDH in culture fluid, XOD, free calcium in L-6TG cell all increased significantly, while SOD in L-6TG cell and mitochondrial respiration decreased, structural damage to L-6TG cell was severe. CONCLUSION: Using mimicking ischemic solution and mimicking reperfusion solution can successfully establish a model of ischemia/reperfusion injury on L-6TG cell.