ABSTRACT
Pancreatic cancer is an aggressive type of cancer with a poor prognosis, short survival rate and high mortality. Therefore, understanding the molecular mechanism underlying the aggressive growth of pancreatic cancer is of importance. An increasing number of studies suggest that numerous microRNAs (miRNAs/miRs) are associated with the tumorigenesis, progression and prognosis of tumors. In a recent study by the present authors, it was revealed that the expression of miR-221 was significantly downregulated in highly aggressive pancreatic cancer cells compared with weakly aggressive pancreatic cancer cells. In addition, miR-221 has been suggested as a novel tumor-associated miRNA, as it is involved in apoptosis, invasion, metastasis and autophagy of tumor cells. However, the function of miR-221 in pancreatic cancer remains yet to be investigated. In the present study, it was revealed that transfection with miR-221 mimic was able to significantly induce apoptosis and autophagy in pancreatic cancer cells compared with the negative control. The protein deacetylase histone deacetylase-6 (HDAC6) has emerged to be an important component in the cellular management of protein aggregates. Studies suggest that HDAC6 serves a function in the clearance of aggresomes, thereby implying a functional association between HDAC6 and autophagy. In the present study, it was revealed that transfection with miR-221 mimic was able to suppress the protein expression of HDAC6 in pancreatic cancer cells compared with the negative control. Immunofluorescence data suggested that the inhibition of HDAC6 was able to induce autophagy in pancreatic cancer cells. Additionally, the results of the present study suggest that the downregulation of miR-221 expression may serve an oncogenic function in the apoptosis and autophagy of pancreatic cancer cells by inducing the expression of HDAC6.
ABSTRACT
Pancreatic cancer is the eighth-leading cause of cancer-associated mortality worldwide. To date, the cellular and molecular mechanisms associated with the invasion and metastasis of pancreatic cancer remain unclear. To examine these mechanisms, a microRNA (miRNA/miR) microarray with 1,965 genes was hybridized with labeled miRNA probes from invasive PC-1.0 and non-invasive PC-1 cells for molecular profiling analysis. In addition, reverse transcription quantitative-polymerase chain reaction (RT-qPCR) was utilized to validate the microarray results. Online miRNA target prediction algorithms online were used to predict the target genes of the differentially expressed miRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) term enrichment analysis were performed for the potential targets of the differentially expressed miRNAs. The results demonstrated that 54 miRNAs were differentially expressed, of which 33 were upregulated and 21 were downregulated in the PC-1.0 cell line compared with the PC-1 cell line. A total of 6 upregulated miRNAs (miR-31, -34a, -181a, -181b, -193a-3p, and -193b) and 4 downregulated miRNAs (miR-221, -222, -484, and -502-3p) were selected from these 54 miRNAs and validated by RT-qPCR. The differentially expressed miRNAs were further validated by RT-qPCR in the human pancreatic cancer cell lines AsPC-1 (highly invasive) and CAPAN-2 (less invasive). The results revealed that 2 upregulated miRNAs (miR-34a and -193a-3p) and 4 downregulated miRNAs (miR-221, -222, -484, and -502-3p) exhibited a consistent expression pattern between the PC-1.0/PC-1 and AsPC-1/CAPAN-2 pancreatic cancer cells. The GO and KEGG enrichment analysis indicated that the mRNAs potentially targeted by miRNAs were involved in a range of biological functions. These results suggest that different miRNA expression profiles occur between highly and weakly invasive and metastatic pancreatic cancer cell lines, and may affect a variety of biological functions in pancreatic cancer.
ABSTRACT
This study explored the effects of butylated hydroxytoluene (BHT) and ethoxyquin (EQ) and ethyl ether extracts, ethyl acetate extracts (EAE), acetone extracts, ethanol extracts and aqueous extracts of Ginkgo biloba leaves (EGbs) on lipid oxidation in a linoleic acid emulsion, fish flesh and fish feed and in hydroxyl radical (·OH)-treated carp erythrocytes. The linoleic acid, fish flesh and fish feed were incubated with BHT, EQ and EGbs at 45°C for 8 d, respectively, except for the control group. The lipid oxidation in the linoleic acid emulsion, fish flesh and fish feed was then measured by the ferric thiocyanate method or thiobarbituric acid method. The carp erythrocytes were treated with BHT, EQ or EGbs in the presence of 40 µmol/L FeSO4 and 20 µmol/L H2O2 at 37°C for 6 h, except for the control group. Oxidative stress and apoptosis parameters in carp erythrocytes were then evaluated by the commercial kit. The results showed that BHT, EQ and EGbs inhibited lipid oxidation in the linoleic acid emulsion, fish flesh and fish feed and ·OH-induced phosphatidylserine exposure and DNA fragmentation (the biomarkers of apoptosis) in carp erythrocytes. Furthermore, BHT, EQ and EGbs decreased the generation of reactive oxygen species (ROS), inhibited the oxidation of cellular components and restored the activities of enzymatic antioxidants in ·OH-treated carp erythrocytes. Of all examined EGbs, EAE showed the strongest effects. The effects of EAE on lipid oxidation in the linoleic acid emulsion and on superoxide anion and malonaldehyde levels, catalase activity and apoptosis in ·OH-treated carp erythrocytes were equivalent to or stronger than those of BHT. Moreover, these results indicated that the inhibition order of EGbs on the generation of ROS and oxidation of cellular components in fish erythrocytes approximately agreed with that for the food and feed materials tested above. And, the antioxidative and anti-apoptotic effects of EGbs were positively correlated with their flavonoid content. Taken together, these results revealed that the fish erythrocyte system can be used as an experimental model to evaluate lipid oxidation in food and feed ingredients. The EAE can be used as a potential natural antioxidant or apoptosis inhibitor. The inhibition effects of EGbs on lipid oxidation and apoptosis may be due to the presence of flavonoid compounds.
