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. 2016 May 31;7(22):33363-73.
doi: 10.18632/oncotarget.9221.

Kaposi's sarcoma herpesvirus (KSHV) microRNA K12-1 functions as an oncogene by activating NF-κB/IL-6/STAT3 signaling

Mingqing Chen  1   2   3 , Fan Sun  1   2 , Lei Han  1   2 , Zhaoxia Qu  1   2
Affiliations

Kaposi's sarcoma herpesvirus (KSHV) microRNA K12-1 functions as an oncogene by activating NF-κB/IL-6/STAT3 signaling

Mingqing Chen et al. Oncotarget. .

Abstract

The human oncogenic virus Kaposi's sarcoma herpesvirus (KSHV) is the most common cause of malignancies among AIDS patients. KSHV possesses over hundred genes, including 25 microRNAs (miRNAs). The roles of these miRNAs and many other viral genes in KSHV biology and pathogenesis remain largely unknown. Accordingly, the molecular mechanisms by which KSHV induces tumorigenesis are still poorly defined. Here, we identify KSHV miRNA K12-1 (miR-K12-1) as a novel viral oncogene by activating two important transcription factors, nuclear factor-κb (NF-κB) and signal transducer and activator of transcription 3 (STAT3). Interestingly, miR-K12-1 activates STAT3 indirectly through inducing NF-κB activation and NF-κB-dependent expression of the cytokine interleukin-6 (IL-6) by repressing the expression of the NF-κB inhibitor IκBα. Accordingly, expression of ectopic IκBα or knockdown of NF-κB RelA, IL-6 or STAT3 prevents expression of cell growth genes and suppresses the oncogenicities of both miR-K12-1 and KSHV. These data identify miR-K12-1/NF-κB/IL-6/STAT3 as a novel oncogenic signaling underlying KSHV tumorigenesis. These data also provide the first evidence showing that IL-6/STAT3 signaling acts as an essential mediator of NF-κB oncogenic actions. These findings significantly improve our understanding of KSHV pathogenesis and oncogenic interaction between NF-κB and STAT3.

Keywords: IL-6; KSHV/HHV8; NF-κB; STAT3; miR-K12-1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. KSHV miR-K12-1 induces NF-κB and STAT3 activation and cell transformation
A. miR-K12-1 represses RNA and protein expression of IκBα. Real-time RT-PCR and immunoblotting analyses were performed to examine the IκBα RNA and protein levels, respectively, in Hela cells transfected with miR-K12-1 or an empty vector. B. miR-K12-1 induces RelA nuclear translocation. Immunofluorescence assay was performed to examine the nuclear translocation of RelA protein in Hela cells transfected with miR-K12-1 or an empty vector. Scale bar: 10 μm. C. miR-K12-1 induces transcriptional activation of NF-κB. Luciferase assay was performed to measure NF-κB transcriptional activity in Hela cells transfected with miR-K12-1 or an empty vector, together with NF-κB luciferase reporter. D. miR-K12-1 induces RelA and STAT3 nuclear expression. Nuclear fraction immunoblotting was performed to examine the nuclear expression of RelA and STAT3 proteins in Hela cells transfected with miR-K12-1 or an empty vector. E. miR-K12-1 induces STAT3 nuclear translocation. Immunofluorescence assay was performed to examine the nuclear translocation of STAT3 protein in Hela cells transfected with miR-K12-1 or an empty vector. Scale bar: 10 μm. F. miR-K12-1 induces transcriptional activation of STAT3. Luciferase assay was performed to measure STAT3 transcriptional activity in Hela cells transfected with miR-K12-1 or an empty vector, together with STAT3 luciferase reporter. G. miR-K12-1 promotes cell growth. Cell growth rates were determined for Rat-1 cells stably expressing miR-K12-1 or an empty vector. H. miR-K12-1 induces cell transformation. Soft agar colony formation assay was performed for Rat-1 cells stably expressing miR-K12-1 or an empty vector.
Figure 2
Figure 2. KSHV miR-K12-1 activates STAT3 via activating IκBα/NF-κB/IL-6 signaling
A. miR-K12-1 induces IL-6 expression. Real-time RT-PCR analysis was performed to examine the RNA levels of IL-6 in Hela cells transfected with miR-K12-1 or an empty vector. B. miR-K12-1 induces a delayed STAT3 activation compared to NF-κB activation. The dynamics of NF-κB and STAT3 transcriptional activation induced by miR-K12-1 were determined using luciferase assay for Hela cells co-transfected with miR-K12-1 and NF-κB or STAT3 luciferase reporter, respectively. C. IκBα expression or IL-6 knockdown inhibits STAT3 nuclear expression induced by miR-K12-1. Subcellular fraction immunoblotting was performed to examine the nuclear expression of RelA and STAT3 proteins in Hela cells transfected with the indicated constructs. D. IκBα expression or IL-6 knockdown prevents STAT3 nuclear translocation induced by miR-K12-1. Immunofluorescence assay was performed to examine the nuclear translocation of RelA and STAT3 proteins in Hela cells transfected with the indicated constructs. Scale bar: 10 μm. E. IκBα expression or IL-6 knockdown represses STAT3 transcriptional activation induced by miR-K12-1. Luciferase assay was performed to measure NF-κB and STAT3 transcriptional activity in Hela cells transfected with the indicated constructs together with NF-κB or STAT3 luciferase reporter, respectively.
Figure 3
Figure 3. The oncogenicity of KSHV miR-K12-1 depends on the activation of IκBα/NF-κB/IL-6/STAT3 signaling
A. IκBα expression, IL-6 knockdown, or STAT3 knockdown has no obvious effect on miR-K12-1-expressing cells cultured in medium containing 10% FBS. Cell growth rates were determined for Rat-1 cells stably expressing an empty vector, miR-K12-1, or miR-K12-1 together with IκBα, IL-6 shRNA or STAT3 shRNA cultured in medium containing 10% FBS. B. IκBα expression, IL-6 knockdown, or STAT3 knockdown suppresses miR-K12-1-driven cell growth when the supplemented FBS is decreased to 5%. Cell growth rates were determined for Rat-1 cells stably expressing an empty vector, miR-K12-1, or miR-K12-1 together with IκBα, IL-6 shRNA or STAT3 shRNA cultured in medium containing 5% FBS. C. IκBα expression, IL-6 knockdown, or STAT3 knockdown suppresses miR-K12-1-driven cell transformation. Soft agar colony formation assay was performed for Rat-1 cells stably expressing an empty vector, miR-K12-1, or miR-K12-1 together with IκBα, IL-6 shRNA or STAT3 shRNA.
Figure 4
Figure 4. KSHV oncogenicity involves activation of miR-K12-1/NF-κB/IL-6/STAT3 signaling
A. RelA knockdown decreases STAT3 nuclear expression in BCBL-1 cells. Nuclear fraction immunoblotting assay was performed to examine the nuclear expression of RelA and STAT3 proteins in BCBL-1 cells stably expressing RelA shRNA or an empty vector. B. RelA knockdown suppresses IL-6 but not STAT3 expression in BCBL-1 cells. Real time PCR analysis was performed to detect RelA, IL-6 and STAT3 RNA expression levels in BCBL-1 cells stably expressing RelA shRNA or an empty vector. C. IL-6 knockdown blocks STAT3 nuclear expression in BCBL-1 cells. Nuclear fraction immunoblotting assay was performed to examine the nuclear expression of RelA and STAT3 proteins in BCBL-1 cells stably expressing IL-6 shRNA or an empty vector. D. KSHV requires miR-K12-1 to induce NF-κB and STAT3 activation. Luciferase reporter activity assay was performed to examine the activation of NF-κB and STAT3 in 293FT cells with an empty vector, KSHVBac36, KSHVBac36 ΔmiRNA, or KSHVBac36 ΔmiRNA plus miR-K12-1. E. Knockdown of RelA, IL-6 or STAT3 suppresses the oncogenicty of BCBL-1 cells. Soft agar colony formation assay was performed for BCBL-1 cells stably expressing RelA shRNA, IL-6 shRNA, STAT3 shRNA, or an empty vector. F. RelA knockdown suppresses expression of cell survival and proliferation genes in BCBL-1 cells. Real time PCR analysis was performed to detect RNA expression levels of the indicated genes in BCBL-1 cells stably expressing RelA shRNA or an empty vector. G. STAT3 knockdown suppresses expression of cell survival and proliferation genes in BCBL-1 cells. Real time PCR analysis was performed to detect RNA expression levels of the indicated genes in BCBL-1 cells stably expressing STAT3 shRNA or an empty vector.
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