Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.
Treatment of MCF-7 human breast cancer cells with 10 nM 17 beta-estradiol (E2) resulted in a 2-fold induction of heat shock protein (Hsp) 27 mRNA levels, and this response persisted for up to 24 h. The 5'-promoter region of the gene was further investigated to identify genomic sequences associated with E2 responsiveness. An Sp1 and half-palindromic estrogen response element (ERE) separated by 10 nucleotides, GGGCGGG(N)10GGTCA, were identified at -105 to -84, and formation of the Sp1/estrogen receptor (ER) complex was investigated by in vitro assays using synthetic Hsp 27-[32P]Sp1/ERE oligonucleotides in a gel mobility shift assay and transient transfection studies using short (-108/-84) and long (-108/+23) 5'-promoter sequences linked to a thymidine kinase promoter and the bacterial chloramphenicol acetyl transferase (CAT) reporter gene (Hsp-CATs and Hsp-CATl, respectively). Incubation of nuclear extracts from MCF-7 cells with an Hsp 27-[32P]Sp1/ERE oligonucleotide results in formation of an Sp1/ER complex. The formation of this complex was inhibited by coincubation with unlabeled Sp1/ERE, ERE, and Sp1 oligonucleotides and by preincubation with ER or Sp1 antibodies (immunodepletion). In addition, the complex was supershifted by coincubation with ER antibodies. Mutation of either Sp1 or ERE sites also decreases formation of the retarded band. E2 induced CAT activity in MCF-7 cells transiently transfected with either Hsp-CATs or Hsp-CATl plasmids. It was also demonstrated that E2 did not significantly induce CAT activity in MCF-7 cells transiently transfected with Hsp-CATl-containing mutations in both the Sp1 and ERE sites. The results of this study demonstrate that an Sp1/ER complex is involved in E2-induced Hsp 27 gene expression.
MicroRNAs (miRNAs) that exert function by posttranscriptional suppression have recently brought insight in our understanding of the role of non-protein-coding RNAs in carcinogenesis and metastasis. In this study, we described the function and molecular mechanism of miR-139-5p in colorectal cancer (CRC) and its potential clinical application in CRC. We found that miR-139-5p was significantly downregulated in 73.8% CRC samples compared with adjacent noncancerous tissues (NCTs), and decreased miR-139-5p was associated with poor prognosis. Functional analyses demonstrated that ectopic expression of miR-139-5p suppressed CRC cell migration and invasion in vitro and metastasis in vivo. Mechanistic investigations revealed that miR-139-5p suppress CRC cell invasion and metastasis by targeting AMFR and NOTCH1. Knockdown of the two genes phenocopied the inhibitory effect of miR-139-5p on CRC metastasis. Furthermore, the protein levels of the two genes were upregulated in CRC samples compared with NCTs, and inversely correlated with the miR-139-5p expression. Increased NOTCH1 protein expression was correlated with poor prognosis of CRC patients. Together, our data indicate that miR-139-5p is a potential tumor suppressor and prognostic factor for CRC, and targeting miR-139-5p may repress the metastasis of CRC and improve survival.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0093-5) contains supplementary material, which is available to authorized users.
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