MicroRNA-33a disturbs influenza A virus replication by targeting ARCN1 and inhibiting viral ribonucleoprotein activity.

In order to explore the roles of microRNA(s) [miRNA(s)] in the influenza A virus life cycle, we compared the miRNA profiles of 293T and HeLa cell lines, as influenza A virus can replicate efficiently in 293T cells but only poorly in HeLa cells. We analysed differentially expressed miRNAs and identified five, including miR-33a, that could disturb influenza A virus replication significantly. Using TargetScan analysis, we found that ARCN1 could be a potential target of miR-33a. To confirm whether miR-33a could truly target ARCN1, we generated a luciferase reporter for the ARCN1 3' untranslated region (UTR) and performed a luciferase assay. The data indicated that miR-33a could suppress the luciferase activity of the reporter for the ARCN1 3' UTR but not a reporter in which the predicted miR-33a targeting sites on ARCN1 3' UTR were mutated. We performed immunoblotting to confirm that miR-33a could downregulate the protein level of ARCN1. Consistently, the level of ARCN1 protein in HeLa cells was significantly lower than that in 293T cells. We also demonstrated that ectopic expression of ARCN1 could partially rescue the inhibitory effect of miR-33a on virus replication. Furthermore, we demonstrated that miR-33a could impede virus replication at the stage of virus internalization, which was similar to the pattern for knockdown of ARCN1, indicating that miR-33a inhibits influenza virus infection by suppressing ARCN1 expression. In addition, we found that miR-33a could also weaken the viral ribonucleoprotein activity in an ARCN1-independent manner. In conclusion, we found that miR-33a is a novel inhibitory factor for influenza A virus replication.

Identification of the determinants of 2-deoxyglucose sensitivity in cancer cells by shRNA library screening.

Combining glycolytic inhibition with other anti-cancer therapies is a potential approach to treating cancer. In this context, we attempted to identify genes that determine sensitivity to 2-deoxyglucose (2DG), a glycolytic inhibitor, in cancer cells using pooled shRNA libraries targeting ∼15,000 genes. The screen revealed that COPB1 and ARCN1, which are essential in retrograde transport, as determinants of sensitivity to 2DG: silencing of COPB1 or ARCN1 expression sensitized cells to 2DG toxicity. To address the mechanism of potentiation of 2DG toxicity by inhibition of COPI-mediated transport, we focused on the role of lipolysis as an alternate source of energy upon inhibition of glycolysis. In the process of lipolysis, COPI-mediated transport is required for localization to lipid droplets of adipose triglyceride lipase (ATGL), a key enzyme that produces fatty acids from triacylglycerol as a substrate for ß-oxidation. The ATGL inhibitor atglistatin potentiated 2DG toxicity, consistent with a model in which a defect in COPI-mediated transport of ATGL to lipid droplets inhibits energy supply, thereby sensitizing cells to glycolytic inhibition. Collectively, our data demonstrated that a defect in COPI-mediated transport or pharmacological inhibition of ATGL potentiates 2DG toxicity in cancer cells, possibly due to a reduction in the energy supply.

COPZ1 depletion in thyroid tumor cells triggers type I IFN response and immunogenic cell death.

The coatomer protein complex zeta 1 (COPZ1) represents a non-oncogene addiction for thyroid cancer (TC); its depletion impairs the viability of thyroid tumor cells, leads to abortive autophagy, ER stress, UPR and apoptosis, and reduces tumor growth of TC xenograft models. In this study we investigated the molecular pathways activated by COPZ1 depletion and the paracrine effects on cellular microenvironment and immune response. By comprehensive and target approaches we demonstrated that COPZ1 depletion in TPC-1 and 8505C thyroid tumor cell lines activates type I IFN pathway and viral mimicry responses. The secretome from COPZ1-depleted cells was enriched for several inflammatory molecules and damage-associated molecular patterns (DAMPs). Moreover, we found that dendritic cells, exposed to these secretomes, expressed high levels of differentiation and maturation markers, and stimulated the proliferation of naïve T cells. Interestingly, T cells stimulated with COPZ1-depleted cells showed increased cytotoxic activity against parental tumor cells. Collectively, our findings support the notion that targeting COPZ1 may represent a promising therapeutic approach for TC, considering its specificity for cancer cells, the lack of effect on normal cells, and the capacity to prompt an anti-tumor immune response.

COPB2 gene silencing inhibits colorectal cancer cell proliferation and induces apoptosis via the JNK/c-Jun signaling pathway.

OBJECTIVES: Colorectal cancer (CRC) is one of the most common malignant human tumors. It is associated with high morbidity and mortality rates. In recent years, tumor gene therapy has emerged as a promising new approach for colorectal cancer therapy. Herein, we identify and analyze the role of COPB2 (coatomer protein complex, subunit beta 2) in proliferation and apoptosis of CRC cells. METHODS: To investigate the role of COPB2 in the proliferation and apoptosis of CRC cells, a shCOPB2 vector and a shCtrl vector were constructed for transfection into RKO and HCT116 cells. Cells proliferation was subsequently measured via cell counting kit-8 (CCK8) assay and Celigo cell counting assay. Apoptosis was measured via flow cytometry. The activity level of Caspase 3/7 was measured. Finally, the level of several JNK/c-Jun apoptosis pathway-related proteins were measured to characterize the mechanism of apoptosis. RESULTS: Our results showed that the proliferation rate was decreased and the apoptosis rate was increased in shCOPB2-treated RKO and HCT116 cells compared to those in controls. After the silencing of COPB2, JNK/c-Jun signal pathway activation was increased, the expression levels of apoptosis pathway-related proteins, such as Bad, p53 and Caspase 3, were also increased. CONCLUSION: COPB2 gene silencing can inhibit RKO and HCT116 cells proliferation and induce apoptosis via the JNK/c-Jun signaling pathway.

ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro.

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I-mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.

Identification of novel cancer therapeutic targets using a designed and pooled shRNA library screen.

Targeted cancer therapeutics aim to exploit tumor-specific, genetic vulnerabilities specifically affecting neoplastic cells without similarly affecting normal cells. Here we performed sequencing-based screening of an shRNA library on a panel of cancer cells of different origins as well as normal cells. The shRNA library was designed to target a subset of genes previously identified using a whole genome screening approach. This focused shRNA library was infected into cells followed by analysis of enrichment and depletion of the shRNAs over the course of cell proliferation. We developed a bootstrap likelihood ratio test for the interpretation of the effects of multiple shRNAs over multiple cell line passages. Our analysis identified 44 genes whose depletion preferentially inhibited the growth of cancer cells. Among these genes ribosomal protein RPL35A, putative RNA helicase DDX24, and coatomer complex I (COPI) subunit ARCN1 most significantly inhibited growth of multiple cancer cell lines without affecting normal cell growth and survival. Further investigation revealed that the growth inhibition caused by DDX24 depletion is independent of p53 status underlining its value as a drug target. Overall, our study establishes a new approach for the analysis of proliferation-based shRNA selection strategies and identifies new targets for the development of cancer therapeutics.

S100A13 and S100A6 exhibit distinct translocation pathways in endothelial cells.

S100 proteins have attracted great interest in recent years because of their cell- and tissue-specific expression and association with various human pathologies. Most S100 proteins are small acidic proteins with calcium-binding domains - the EF hands. It is thought that this group of proteins carry out their cellular functions by interacting with specific target proteins, an interaction that is mainly dependent on exposure of hydrophobic patches, which result from calcium binding. S100A13, one of the most recently identified members of the S100 family, is expressed in various tissues. Interestingly, hydrophobic exposure was not observed upon calcium binding to S100A13 even though the dimeric form displays two high- and two low- affinity sites for calcium. Here, we followed the translocation of S100A13 in response to an increase in intracellular calcium levels, as protein translocation has been implicated in assembly of signaling complexes and signaling cascades, and several other S100 proteins are involved in such events. Translocation of S100A13 was observed in endothelial cells in response to angiotensin II, and the process was dependent on the classic Golgi-ER pathway. By contrast, S100A6 translocation was found to be distinct and dependent on actin-stress fibers. These experiments suggest that different S100 proteins utilize distinct translocation pathways, which might lead them to certain subcellular compartments in order to perform their physiological tasks in the same cellular environment.