Oncogenic KSHV-encoded interferon regulatory factor upregulates HMGB2 and CMPK1 expression to promote cell invasion by disrupting a complex lncRNA-OIP5-AS1/miR-218-5p network

Kaposi’s sarcoma (KS), a highly disseminated tumor of hyperproliferative spindle endothelial cells, is the most common AIDS-associated malignancy caused by infection of Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV-encoded viral interferon regulatory factor 1 (vIRF1) is a viral oncogene but its role in KSHV-induced tumor invasiveness and motility remains unknown. Here, we report that vIRF1 promotes endothelial cell migration, invasion and proliferation by down-regulating miR-218-5p to relieve its suppression of downstream targets high mobility group box 2 (HMGB2) and cytidine/uridine monophosphate kinase 1 (CMPK1). Mechanistically, vIRF1 inhibits p53 function to increase the expression of DNA methyltransferase 1 (DNMT1) and DNA methylation of the promoter of pre-miR-218-1, a precursor of miR-218-5p, and increases the expression of a long non-coding RNA OIP5 antisense RNA 1 (lnc-OIP5-AS1), which acts as a competing endogenous RNA (ceRNA) of miR-218-5p to inhibit its function and reduce its stability. Moreover, lnc-OIP5-AS1 increases DNA methylation of the pre-miR-218-1 promoter. Finally, deletion of vIRF1 from the KSHV genome reduces the level of lnc-OIP5-AS1, increases the level of miR-218-5p, and inhibits KSHV-induced invasion. Together, these results define a novel complex lnc-OIP5-AS1/miR-218-5p network hijacked by vIRF1 to promote invasiveness and motility of KSHV-induced tumors.

The cellular IRFs (IRFs 1~9) are a family of cellular transcription proteins that regulate the expression of interferon and interferon-stimulating genes (ISGs) in innate immune response, among which IRF3 and IRF7 play key roles in the induction and secretion of type I interferon [12]. vIRF1 (449 amino acids), as one of the KSHV vIRFs (vIRF1 to vIRF4), is encoded by KSHV ORF-K9, which has 26.6% and 26.2% of protein homology to cellular IRF3 and IRF7, respectively [13]. vIRF1 has been shown to compete with IRF3 to interact with CBP/p300 coactivators by blocking the formation of CBP/p300-IRF3 complexes, thereby inhibiting IRF3-mediated transcription and signal transduction of type I interferon [14]. However, vIRF1 could not block IRF-7-mediated transactivation [14]. In the other hand, vIRF1 represses tumor suppressor gene p53 phosphorylation, leading to an increase of p53 ubiquitination by reducing ATM kinase activity [15]; vIRF1 could also directly bind to p53 and effectively inhibit p53-mediated apoptosis by reducing its acetylation and inhibiting the transcription of p53 activation [16,17]. In addition, vIRF1 restrains TGF-beta signaling via direct interaction with Smads (Smad3 and Smad4) to disturb Smad3/Smad4 complexes from binding to DNA and suppresses IRF-1-induced CD95/CD95L signaling-mediated apoptosis [18,19]. As the first identified oncogenic protein encoded by KSHV, vIRF1 has been reported to transform mouse embryonic fibroblasts (NIH3T3) cells [6], however, its role in KSHV-induced tumor invasiveness and motility and its underlying mechanism remains totally unclear.
Less than 2% of the human genome encodes protein-coding genes, while the vast majority of the genome is transcribed as non-coding RNAs [20]. Based on the size, non-coding RNAs could be vaguely divided into three groups: microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) [21]. Many miRNAs (~22 nucleotides in length) have been well-characterized and shown to repress gene expression by inhibiting the translation or destabilization of mRNA transcript via binding to mRNA sequences [22]. LncRNAs (>200 nucleotides in length) have indispensable roles in diverse biological processes, including chromatin remodeling, X chromosome inactivation, genomic imprinting, nuclear transport, transcription, RNA splicing and translation [23][24][25]. A growing volume of literatures support the notion that both lncRNAs and miRNAs could function as tumor suppressors or oncogenes involved in the regulation of cell proliferation, metastasis, apoptosis, and invasion [24, 26,27]. More interestingly, emerging evidence indicates that numerous lncRNAs might act as competing endogenous RNAs (ceRNAs) that competitively bind miRNAs, hence exerting influence on posttranscriptional regulation [28].
Recently, several oncogenic viruses have been shown to encode lncRNAs and are thought to participate in enhancing viral replication, promoting oncogenesis and contributing to pathogenesis [29][30][31]. KSHV encodes an lncRNA, known as polyadenylated nuclear RNA (PAN RNA). PAN is multifunctional, regulating KSHV replication, viral and host gene expression, and immune responses [32][33][34][35]. However, whether cellular lncRNAs are involved in the progression of KS is still unknown.
In the present work, we aimed to elucidate the role of vIRF1 in cell migration, invasion and proliferation. We found that vIRF1 promoted cell migration, invasion and proliferation by epigenetically silencing miR-218-5p and activating lncRNA-OIP5-AS1 transcription. Further, we uncovered that the crosstalk between miR-218-5p and lnc-OIP5-AS1 contributed to vIRF1-induced cell motility and proliferation via increasing HMGB2 and CMPK1 expression. Our novel findings illustrated a critical role of vIRF1 in the invasiveness, motility and development of KS tumor.

Exogenous vIRF1 accelerates endothelial cell motility and proliferation
Previous works showed that vIRF1, as a homologue of cellular IRFs, disrupted immune antiviral response of host cells and contributed to KSHV-induced tumorigenesis [5]. However, its role on tumor invasiveness and motility remains unclear. To determine whether vIRF1 had a role in cell motility, we transduced HUVECs with lentiviral vIRF1 at a MOI of 2. vIRF1-transduced HUVECs showed a vIRF1 mRNA expression level similar to that of KSHV-infected HUVECs (S1 Fig, Fig 1A and 1B). We then examined the effect of vIRF1 on cell migration and invasion. In transwell migration and Matrigel invasion assays, overexpression of vIRF1 enhanced cell migration and invasion (Fig 1C, 1D and 1E). In plate colony formation assay, vIRF1 clearly enhanced cell proliferation (Fig 1F and 1G). vIRF1 targets lncRNA-OIP5-AS1/miR-218-5p network to promote cell invasion

Exogenous vIRF1 expression promotes endothelial cell motility, and proliferation by negatively regulating miR-218-5p expression
To assess the mechanism mediating vIRF1 promotion of cell motility and proliferation, we performed microarray-based miRNA expression profiling and identified a set of miRNAs that were differentially expressed between vIRF1-and pHAGE-transduced HUVECs (GEO  accession number GSE119034). As a known tumor suppressor [36], miR-218-5p was significantly down-regulated in vIRF1-transduced cells, and hence was selected for further validation by qRT-PCR. As shown in Fig 1H and 1I, downregulation of miR-218-5p was observed in both vIRF1-transduced and KSHV-infected HUVECs. Then, we sought to determine whether the downregulation of miR-218-5p might contribute to vIRF1 promotion of cell motility, and proliferation. As expected, overexpression of miR-218-5p in vIRF1-transduced HUVECs reversed vIRF1-enhanced cell migration and invasion (S2 Fig, Fig 1J and 1K) as well as cell proliferation (Fig 1L).

vIRF1 enhances cell motility and proliferation by inhibiting miR-218-5p to increase the expression of its direct targets HMGB2 and CMPK1
Next, we conducted mass spectrometry analysis to investigate the direct targets of miR-218-5p. As shown in Table 1, there were a series of proteins that were up-regulated by > 1.5 folds in cells overexpressing vIRF1. Using bioinformatics analysis, we predicted four proteins that might be the potential targets of miR-218-5p, and hence chose them for further luciferase reporter assay. We confirmed that miR-218-5p decreased the 3'UTR reporter activities of both high mobility group box 2 (HMGB2) and cytidine/uridine monophosphate kinase 1 (CMPK1) (Fig 2A), which was further shown in a dose-dependent fashion ( Fig 2B). Indeed, overexpression of miR-218-5p suppressed the levels of HMGB2 and CMPK1 proteins in a dose-dependent manner ( Fig 2C). Conversely, blocking the miR-218-5p function with a specific inhibitor elevated the expression levels of HMGB2 and CMPK1 proteins in a dose-dependent manner ( Fig 2D). To further confirm that miR-218-5p directly targeted HMGB2 and CMPK1, we performed mutagenesis with miR-218-5p (Fig 2E and 2F). The mutant mimic did not have any effect on the 3'UTR reporter activities of HMGB2 and CMPK1 (Fig 2G), and the levels of HMGB2 and CMPK1 proteins ( Fig 2H). Moreover, the mRNA and protein levels of HMGB2 and CMPK1 were significantly up-regulated in cells expressing vIRF1 or infected by KSHV (Fig 3A-3D). In IHC staining, there were more HMGB2-and CMPK1-postive cells in KS lesions than in normal skin tissues (S3 Fig and Fig 3E).

Discussion
KSHV K9/vIRF1 was initially characterized as an early lytic gene. However, subsequent studies have shown that it is also expressed during viral latency. vIRF1 has two transcription start sites, one is distal to the AUG, which is active during latency in PEL, and another is a more proximal site, which is induced upon lytic reactivation [54]. Hence, vIRF1 might have a dual modes of expression during latent and lytic replication [55][56][57]. Furthermore, K9/vIRF1 mRNA is expressed in all KS tumors (total 21 KS clinical biopsies) and preferentially transcribed during latent infection of either endothelial/mesenchymal lineage cells, which strengthens the role of K9/vIRF1 in KS tumorigenesis [58]. In the current study, we found that vIRF1 promoted endothelial cell migration and invasion, as well as proliferation. Further, deletion of vIRF1 from the KSHV genome reduced KSHV-induced cell migration, invasion and proliferation. However, we could not assess the expression level of the endogenous vIRF1 protein because there is currently no vIRF1 antibody available. Despite the limitation, this work still revealed a novel role of vIRF1 in cell migration, invasion and proliferation, which is an important part of KS pathogenesis, particularly in the invasiveness and dissemination of KS tumors.
Lnc-OIP5-AS1 located at chromosome 15q15.1, known as cyrano, is~8,000 nucleotides in length and abundant in the cytoplasm. It was originally characterized in zebrafish and displayed crucial effects in embryonic nervous system development [65]. It was also reported to play a vital role in embryonic stem cells (ESCs) self-renewal maintenance [66]. With regard to its role in cancer, lnc-OIP5-AS1 exhibits multifaceted and complex features. For example, lnc-OIP5-AS1 is shown to be a tumor suppressor and inhibit HeLa cells proliferation by interacting with the RBP HuR to reduce HuR's availability for binding target mRNAs, or associating with GAK mRNA to impair GAK mRNA stability [67,68]. On the contrary, lnc-OIP5-AS1 can exert oncogenic functions in several other cancers. It was consistently up-regulated in renal cell carcinoma, glioblastoma, and gastric cancer [69][70][71]. Silencing of lnc-OIP5-AS1 repressed YAP-Notch signaling pathway activity leading to decrease of glioma cells' proliferation, migration in vitro and tumor formation in vivo [72]. Moreover, lnc-OIP5-AS1 was highly expressed in lung adenocarcinoma tissues and cells, and the loss of lnc-OIP5-AS1 inhibited lung adenocarcinoma cell proliferation, migration and invasion [73]. In our report, we found that both KSHV infection and vIRF1 expression increased the expression of lnc-OIP5-AS1 in endothelial cells. Silencing of lnc-OIP5-AS1 suppressed cell migration, invasion and proliferation. Intriguingly, we found that vIRF1 activated the transcription of lnc-OIP5-AS1, however, the precise mechanism remains unknown.
In conclusion, our study revealed that vIRF1 promoted cell migration, invasion and proliferation by a p53-and lnc-OIP5-AS1-mediated down-regulation of miR-218-5p, leading to increased expression levels of its target genes HMGB2 and CMPK1 (Fig 8G). This process was mediated by the complex crosstalk between miR-218-5p and lnc-OIP5-AS1. These novel findings extend the cross-regulatory network of cellular lncRNAs and miRNAs involved in the pathogenesis of KS.

Ethics statement
The clinical section of the research was reviewed and ethically approved by the Institutional Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (Nanjing, China; Study protocol # 2015-SR-116). Written informed consent was obtained from all participants, and all samples were anonymized. All participants were adults.

Cell culture and plasmids
The iSLK cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin-streptomycin, 1 μg/ml puromycin and 250 μg/ml G418. The established iSLK-RGB-BAC16 and iSLK-RGB-K9 mutant cells were cultivated in DMEM supplemented with 10% fetal bovine serum (FBS), 1 μg/ml puromycin, 250 μg/ml G418, and 1.2 mg/ml hygromycin B [53]. HEK293T and continuous cell lines human umbilical vein endothelial cells (catalog #CRL-1730, ATCC, Manassas, VA, USA) were maintained as previously described [78]. The latter were only used for plate colony formation assay to evaluate the ability of cell proliferation. Primary human umbilical vein endothelial cells (HUVECs), which were used for all assays except for luciferase and plate colony formation assays, were isolated and cultured as previously delineated [79].

Cell migration, invasion and plate colony formation assays
Cell migration, invasion and colony formation assays were executed as previously described [83][84][85].

Luciferase reporter assay
Luciferase reporter assay was conducted using the Promega dual-luciferase reporter assay system according to the previous study [86].

RNA pull-down assay
HUVECs were collected, washed, and re-suspended with lysis buffer (Thermo Fisher Scientific, Waltham, America). After incubating for 5 min, the lysates were precleared by centrifugation at 14,000 rpm for 10 minutes, and then were added to streptaviden magnetic beads (Thermo Fisher Scientific, Waltham, America), which were incubated with Biotin-labeled miR-218-5p, miR-218-5p mut 2, or Neg. Ctrl (Genepharma, Suzhou, China) for 4 hours. The bound RNAs in the pull-down material were quantified by qRT-PCR.

RNA Immunoprecipitation (RIP) assay
HUVECs were transfected with lnc-OIP5-AS1 Smart Silencer or its Neg. Ctrl for 48 h, and used for RIP experiments with an anti-Ago2 antibody (MERCK, Darmstadt, Germany) and the Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (MERCK, Darmstadt, Germany), according to the manufacturer's instructions. The levels of lnc-OIP5 AS1, HMGB2 or CMPK1 were examined by qRT-PCR.

Construction and identification of KSHV ORF K9 Mutant
A KSHV mutant with ORF K9 deleted was constructed as described in previous studies [52,88]. In brief, using the bacterial artificial chromosome (BAC) technology and the Escherichia coli Red recombination system, together with PCR, restriction digestion, and sequencing for strict quality control, a KSHV ORF K9 mutant (called RGB-K9-mutant) was constructed by removing K9 coding sequence (CDS) from the wild-type recombinant KSHV RGB-BAC16 [53]. RGB-BAC16 and RGB-K9 mutant DNA were transfected into iSLK cells and selected using 1 μg/ml puromycin, 250 μg/ml G418, and 1.2 mg/ml hygromycin B for 3 weeks to establish stable viral producer cell lines, iSLK-RGB-BAC16 and iSLK-RGB-K9 mutant cells. To produce virus stocks for infection, iSLK-RGB-BAC16 and iSLK-RGB-K9 mutant cells were plated at 30 to 40% confluence and induced with both Doxycycline (Dox) (1 μg/ml) and sodium vIRF1 targets lncRNA-OIP5-AS1/miR-218-5p network to promote cell invasion butyrate (NaB) (1 mM). After induction for 4 or 5 d, the supernatant was harvested, centrifuged, filtered, and concentrated by ultracentrifugation (25, 000 g at 4˚C for 3 h) using SW32 Ti rotor (Beckman Coulter Inc, USA). The pellet was resuspended, supplemented with 8 μg/ mL polybrene and then incubated with 10 5 HUVECs in a 6-well plate for 4 h. The primers for construction and identification of K9 mutant bacmid were designed as previously described [89] and the sequences of the primers could be found in S2 Table. Reverse transcription and real time quantity PCR RNA was extracted using RNA Isolator Total RNA Extraction Reagent (Vazyme Biotech Co., Ltd, Nanjing, China) from cells. Total RNA was reverse transcription by HiScript Q RT Super-Mix (Vazyme Biotech Co., Ltd, Nanjing, China). Real time quantity PCR was performed by AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China). The sequences of the primers for PCR could be found in S3 Table. The extraction of genome DNA The extraction of genome DNA was performed by using TIANamp Genomic DNA Kit (TIANGEN BIOTECH, Beijing, China) according to the user's guide. Briefly, cells were trypsinized, and neutralized by 20% FBS DMEM. The suspension was centrifuged and the supernatant was discarded.

Mass spectrometry analysis
Mass spectrometry analysis was adopted according to the previous study [86].

Immunohistochemistry (IHC)
The KS clinical specimens were kindly offered by Jiangsu Province Hospital. All samples were anonymized and all participants are provided with informed consent. IHC was carried out as previously described with specific antibodies [85,90].

Statistical analysis
All data are appeared as the means ± SD with at least three replications. Statistical analysis was on account of Student's t-test and the criterion for statistical significance was adopted as P values of < 0.05.

Accession numbers
Microarray data have been submitted and can be accessed by GEO accession number GSE119034.
Supporting information S1 Table. A list of sequences of the siRNAs mentioned in the text.