C-Myc and viral cofactor Kaposin B co-operate to elicit angiogenesis through modulating miRNome traits of endothelial cells.

Hsin-Chuan Chang,Hsei-Wei Wang,Ya-Ni Tsai,Yi-Wei Lee,Cheng-Fong Tsai,Cheng-Chung Cheng,Tsung-Han Hsieh

doi:10.1186/s12918-015-0242-3

Abstract

BackgroundMicroRNAs (miRNAs) have emerged as master regulators of angiogenesis and other cancer-related events. Discovering new angiogenesis-regulating microRNAs (angiomiRs) will eventually help in developing new therapeutic strategies for tumor angiogenesis and cardiovascular diseases. Kaposi’s sarcoma (KS), which is induced by the etiological infectious agent KS-associated herpesvirus (KSHV), is a peculiar neoplasm that expresses both blood and lymphatic endothelial markers and possesses extensive neovasculature. Using KSHV and its proteins as baits will be an efficient way to discover new angiomiRs in endothelial cells. Kaposin B is one of the latent viral genes and is expressed in all KSHV tumor cells. Since Kaposin B is a nuclear protein with no DNA-binding domain, it may regulate gene expression by incorporating itself into a transcription complex.ResultsWe demonstrated that c-Myc and Kaposin B form a transcription complex and bind to the miR-221/-222 promoter, thereby affecting their expression and anti-angiogenic ability. By small RNA sequencing (smRNA-Seq), we revealed that 72.1 % (173/240) of Kaposin B up-regulated and 46.5 % (113/243) of Kaposin B down-regulated known miRNAs were regulated by c-Myc. We also found that 77 novel miRNA were up-regulated and 28 novel miRNAs were down-regulated in cells expressing both c-Myc and Kaposin B compared with cells expressing Kaposin B only. The result was confirmed by RNA-IP-seq data.ConclusionsOur study identifies known and novel c-Myc-regulated microRNAs and reveals that a c-Myc-oriented program is coordinated by Kaposin B in KSHV-infected cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0242-3) contains supplementary material, which is available to authorized users.

Highlights

MicroRNAs have emerged as master regulators of angiogenesis and other cancer-related events
Mean expression levels of the target miRNAs are compared with the U6 control (n = 3). *: P < .05. e RT-quantitative polymerase chain reaction (qPCR) detected miR-221/miR-222 expression in Kaposin B(+) and Kaposin B(+)c-Myc(+) cells. f The biological pathway of genes which were targeted by the Kaposin B(+)c-Myc(+) induced/reduced miRNAs
In this study, we provide a global picture of KSHVand/or Kaposin B-induced microRNAs, especially those co-regulated by c-Myc

Summary

Introduction

MicroRNAs (miRNAs) have emerged as master regulators of angiogenesis and other cancer-related events. B HMEC1 cells stably expressing Kaposin B were subjected to the Transwell cell-migration assay (n = 3). Primary HUVEC stably transduced with Kaposin B or the vector control by lentivirus were used for Transwell cell-migration assays (n = 3). Five micrograms of anti-FLAG (clone M2; left panel), anti-HA (right panel) or isotype IgG control were incubated with 500 μl of cell extracts and analyzed by western blotting with indicated mAbs. g The interaction between c-Myc and Kaposin B was independent of promoter DNA. Co-immunoprecipitation assays were performed with or without DNase pre-treatment on cell lysates prepared from HMEC1 cells stably expressing Kaposin B. H-i Knockdown of endogenous c-Myc levels in Kaposin B(+) HUVECs inhibits Kaposin B-induced cell migration (h, left) and microvasculature formation (i, right) (n = 3)

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