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

Abstract

Background: MicroRNAs (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.

Results: We 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.

Conclusions: Our 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.

Fig. 1

Kaposin B binds to c-Myc for regulating endothelial cell angiogenic activities. a Nuclear distribution pattern of KSHV Kaposin B protein in cells. Immunofluorescence staining for Kaposin B proteins. HMEC1 cells with stable Kaposin B (KapB) expression were fixed, and Kaposin B proteins were detected using anti-Flag mAb, followed by anti–mouse IgG secondary antibody conjugated with FITC (green). Cell nuclei were counterstained with Hoechst 33342, while actin filaments with Texas Red phalloidin (Alexa Fluor 568). b HMEC1 cells stably expressing Kaposin B were subjected to the Transwell cell-migration assay (n = 3). c Kaposin B increases cell motility in HUVEC. Primary HUVEC stably transduced with Kaposin B or the vector control by lentivirus were used for Transwell cell-migration assays (n = 3). d Kaposin B enhances microvascular formation of HUVEC in an in vitro MatriGel angiogenesis assay. Pictures were taken after 6 h of incubation (left), and tube length was then measured and compared (right). e Schematic representation of miR-221/-222 proximal promoter. Three E-boxes (E1 and E2/3, in red) were found. f Co-immunoprecipitation assays show Kaposin B and c-Myc form a protein complex. Cell lysates were prepared from HMEC1 cells stably expressing Kaposin B. 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 then 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. Anti-FLAG (clone M2) or isotype IgG control were incubated with cell extracts, and pull-down products were analyzed by western blotting with anti-HA mAb for c-Myc (upper panel) or anti-FLAG M2 mAb for Kaposin B (lower panel). 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)

Fig. 2

c-Myc cooperates with Kaposin B to regulate cellular known miRNAs. a smRNA-Seq revealed differentially expressed miRNAs between Kaposin B(+) and HUVECs (≥1.5 folds, left), and between Kaposin B(+)c-Myc(+) and HUVECs (≥1.5 folds, right). b Venn diagrams summarizing significant overlap between c-Myc and Kaposin B known miRNAs signature. (upper panel) up-regulated miRNAs; (lower panel) down-regulated miRNAs. c Gene Set Enrichment Analysis (GSEA) verified the significant overlap between c-Myc and Kaposin B miRNAs signature. d RT-qPCR detected various known miRNAs expression. Mean expression levels of the target miRNAs are compared with the U6 control (n = 3). *: P < .05. e RT-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. The P value were marked on the figure

Fig. 3

c-Myc cooperates with Kaposin B to regulate cellular novel miRNAs. a The analysis pipeline for identification of known and novel miRNAs from smRNA-seq data. Reads or sequences pass each filtration process are indicated. b Venn diagrams summarizing significant overlap between c-Myc and Kaposin B signature novel miRNAs. (upper panel) up-regulated miRNAs; (lower panel) down-regulated miRNAs. c Venn diagrams summarizing significant overlap between up-regulated or down-regulated novel miRNA candidates and Ago1/2(+) RNA-IP-seq data. d Detailed information of 5 c-Myc-promoting and -reducing novel miRNAs that expressed higher reads in different groups. e Deduced RNA secondary structures of a set of newly discovered miRNAs

Fig. 4

c-Myc enhances Kaposin B to regulate microRNA promoter activity. a Knockdown of endogenous c-Myc levels in Kaposin B(+) HUVECs rescues miR-221 (left) and miR-222 (right) expression. b ChIP analysis with immunoglobulin G control or anti-FLAG antibody (detecting Kaposin B). (upper) Schematic representation of miR-221/-222 promoter region and PCR fragments. (lower) HMEC1 cells stably expressing empty vector, Kaposin B, or Kaposin B + c-Myc were subjected into ChIP assays. The −2600 promoter region PCR product was used as a negative control. c Wild type (−1600) promoter construct or double mutant construct (upper) was transfected into a HMEC1 cell line stably expressing Kaposin B (HMEC1-Kaposin B), and ChIP-qPCR assays were performed on transfectants using anti-FLAG mAb. d Endogenous c-Myc was stably knocked down using shRNA in a HMEC1-Kaposin B stable cell line, and cells with or without c-Myc knockdown were subjected to ChIP assays and qPCR for indicated regions (n = 3). e Reporter activity of tested reporter plasmids after co-transfection with Kaposin B, c-Myc, or c-Myc + Kaposin B. Schematic representation of reporter constructs (left) and reporter assay results (n = 3) (right). *: P < .05. f Reporter assays on E1 or E2/3 single mutants, and on the E1 and E2/E3 E-boxes double mutant. Only the double mutant showed significant promoter activity restoration. (n = 3) *: P < .05

Fig. 5

A proposed model of regulation of endothelial cell activities by the Kaposin B-c-Myc circuit