Overexpression of VPS11 antagonizes the promoting effect of miR-542-3p on Mycobacterium tuberculosis survival in macrophages by regulating autophagy.

Impaired autophagy is an important cause of Mycobacterium tuberculosis survival in macrophages. VPS11 is an important regulator of autophagy; decreased VPS11 expression has been observed in macrophages after tuberculosis (TB) infection. Gene ontology data revealed that various miRNAs (for example, miR-542-3p) were upregulated in macrophages upon TB infection; thus, these miRNAs were likely to reduce VPS11 expression. In this study, both TB patients and healthy subjects were enrolled, and the levels of VPS11 and some miRNAs in their blood macrophages were measured. Moreover, various macrophages were cultured and infected with M. tuberculosis. Luciferase reporter, RNA pulldown, and RNA immunoprecipitation assays were performed to determine the regulatory effect of miR-542-3p on VPS11 expression. Results showed that VPS11 expression was downregulated, whereas miR-542-3p expression was upregulated in blood macrophages after TB infection. TB infection reduced VPS11 levels in two human macrophages in vitro, but not in mouse macrophages. This might be because the seed sequence exists in the VPS11 3' untranslated region in humans, but is absent in mice and rats. miR-542-3p promoted M. tuberculosis survival in human macrophages, but VPS11 overexpression antagonized the promoting effect of miR-542-3p. Further, VPS11 was confirmed as a target of miR-542-3p. Overexpression of VPS11 or depletion of miR-542-3p promoted autophagy, which was suppressed upon TB infection. In summary, VPS11 overexpression antagonized the promoting effect of miR-542-3p on M. tuberculosis survival in macrophages by regulating autophagy.

Transcriptome profiling of a multiple recurrent muscle-invasive urothelial carcinoma of the bladder by deep sequencing.

Urothelial carcinoma of the bladder (UCB) is one of the commonly diagnosed cancers in the world. The UCB has the highest rate of recurrence of any malignancy. A genome-wide screening of transcriptome dysregulation between cancer and normal tissue would provide insight into the molecular basis of UCB recurrence and is a key step to discovering biomarkers for diagnosis and therapeutic targets. Compared with microarray technology, which is commonly used to identify expression level changes, the recently developed RNA-seq technique has the ability to detect other abnormal regulations in the cancer transcriptome, such as alternative splicing. In this study, we performed high-throughput transcriptome sequencing at ∼50× coverage on a recurrent muscle-invasive cisplatin-resistance UCB tissue and the adjacent non-tumor tissue. The results revealed cancer-specific differentially expressed genes between the tumor and non-tumor tissue enriched in the cell adhesion molecules, focal adhesion and ECM-receptor interaction pathway. Five dysregulated genes, including CDH1, VEGFA, PTPRF, CLDN7, and MMP2 were confirmed by Real time qPCR in the sequencing samples and the additional eleven samples. Our data revealed that more than three hundred genes showed differential splicing patterns between tumor tissue and non-tumor tissue. Among these genes, we filtered 24 cancer-associated alternative splicing genes with differential exon usage. The findings from RNA-Seq were validated by Real time qPCR for CD44, PDGFA, NUMB, and LPHN2. This study provides a comprehensive survey of the UCB transcriptome, which provides better insight into the complexity of regulatory changes during recurrence and metastasis.