Differential expression of the miR-200 family microRNAs in epithelial and B cells and regulation of Epstein-Barr virus reactivation by the miR-200 family member miR-429

Abstract

The miR-200 microRNA family is important for maintaining the epithelial phenotype, partially through suppressing ZEB1 and ZEB2. Since ZEB1 inhibits Epstein-Barr virus (EBV) reactivation, we hypothesized that expression of miR-200 family members in epithelial cells may partly account for higher levels of EBV reactivation in this tissue (relative to nonplasma B cells). Here we show that, whereas miR-200 family members are expressed in epithelial cells, their expression is low in latently infected B cells. Furthermore, the miR-200 family member miR-429 shows elevated expression in plasma cell lines and is induced by B-cell-receptor activation in Akata cells. Lastly, expression of miR-429 can break latency.

FIG. 1.

Expression of miR-200 family miRNAs in EBV-trophic cell lines. (A) miR-200 family genomic loci. miR-200b, miR-200a, and miR-429 are transcribed as a single polycistronic transcript from chromosome (Chr.) 1. miR-200c and miR-141 are carried by a single transcript from chromosome 12. Chromosomal coordinates are based on the 2009 version of the public human genome assembly (GRCh37). (B) Quantitative RT-PCR of the miR-200b-miR-200a-miR-429 primary transcript (Pri). Primer set for the miR-200b-miR-200a-miR-429 primary transcript: 5′-AGTGGGGCTCACTCTCCAC-3′ and 5′-AGGAGGAGGAGGAGGAGAAA-3′ (1). Primer set for glyceraldehyde 3-phosphate dehydrogenase (G3PDH): 5′-GCCAAGGTCATCCATGACAACTTTGG-3′ and 5′-GCCTGCTTCACCACCTTCTTGATGTC-3′. Expression of the miR-200b-miR-200a-miR-429 primary transcript was determined by the comparative threshold cycle (C_T) method (2^−ΔΔCT). Mutu I, EBV-positive type I latency Burkitt's lymphoma cell line; JY, Jijoye, and X50-7, EBV-positive type III latency B-cell lines; OKF6/TERT2, EBV-negative telomerase immortalized human keratinocyte cell line; CNE1 and CNE1-EBV, EBV-negative and -positive nasopharyngeal carcinoma cell lines, respectively; AGS, EBV-negative human gastric adenocarcinoma cell line; MCF7, EBV-negative human breast cancer cell line. (C) Mature miR-429 expression was quantified using a TaqMan microRNA assay (Applied Biosystems). RNU6B was analyzed as a reference. The expression of miR-429 was determined by the comparative C_T method (2^−ΔΔCT). DG75, EBV-negative Burkitt's lymphoma cell line; Rael and Akata, EBV-positive type I latency Burkitt's lymphoma cell lines; AGS-EBV, AGS cells infected with a recombinant EBV. Other cells used are as described in the legend to panel B. (D) Mature miR-200c expression was assessed using a mirVana (quantitative RT-PCR [qRT-PCR]) microRNA detection kit (AM1558; Ambion) with the mirVana qRT-PCR miR-200c primer set (AM30096; Ambion) and the mirVana qRT-PCR 5S primer set (AM30302; Ambion), according to the manufacturer's protocol. The expression of miR-200c was determined by the comparative C_T method (2^−ΔΔCT). Cell lines are defined in the legends to panels B and C.

FIG. 2.

Induction of EBV lytic reactivation in miR-429-transduced EBV-293 cells. (A) A 441-bp region spanning the miR-429 hairpin sequence (chromosome 1: positions 1,104,265 to 1,104,705 of the GRCH37 assembly) was isolated from JY genomic DNA by PCR and cloned into the plasmid pMSCV-puro-GFP-miR (2). Retroviral infection was conducted as described previously (19), and cells were selected with puromycin for 14 days prior to analysis. Mature miR-429 expression was analyzed by quantitative RT-PCR, and values were determined by the comparative C_T method (2^−ΔΔCT), using RNU6B as a control (Cntl). (B) Two separate miR-429-transduced EBV-293 cell cultures show morphological changes relative to two separate control-transduced EBV-293 cell cultures. (C) Western blot analysis of ZEB1 expression in retrovirally transduced EBV-293 cells. (D) Immunofluorescence assay of Zta in retrovirally transduced EBV-293 cells. The assay was performed as previously described (13). The number of Zta-positive cells on coverslips containing 12,000 total cells was counted for each culture. The averages and standard errors from the results of triplicate coverslips are shown for each cell culture.

FIG. 3.

Induction of EBV reactivation in miR-429-transduced B-cell lines. (A) Generation of retrovirally transduced Mutu I cell lines and analysis of mature miR-429 expression were performed as described in the legend to Fig. 2A. ZEB1 and actin were detected using polyclonal antibodies, as in Fig. 2C. (B) Retrovirally transduced Akata cell lines were generated in the same manner as the Mutu I cell lines, and analysis of mature miR-429 expression was performed as described in the legend to Fig. 2A. ZEB1 and actin were detected using polyclonal antibodies, as in Fig. 2C. (C) Mature miR-429 expression was quantified using a TaqMan microRNA assay (Applied Biosystems). RNU6B was analyzed as a reference. The expression of miR-429 was determined by the comparative C_T method (2^−ΔΔCT). Akata cells were treated with anti-human Ig (I5260; Sigma), and cells were harvested at 0 h, 8 h, 12 h, and 24 h posttreatment. Human plasma cell lines U266 and CCL-155 were obtained from the ATCC.

FIG. 4.

miR-429 induces the Zta promoter. (A) 293 cells stably infected with a Zta knockout EB virus [EBV(Zk/o)/293] (6) were transfected using a modified calcium phosphate precipitation method (13). Cells were cotransfected with either a miR-429 expression vector (pMSCV-puro-GFP-miR-429) or a control vector (pMSCV-puro-GFP-miRcntl) plus either a Zta expression vector [BS(SVp/e)-Zta] or its control vector [BS(SVp/e)]. Cells were harvested 72 h after transfection, reporter activity was analyzed by a luciferase assay, and protein levels were assessed by Western blot analysis. Fold changes are relative to cells transfected with Zta control vector plus miR-429 control vector. (B) EBV(Zk/o)/293 cells were transfected with the indicated plasmids. Zp-Luc is a luciferase reporter plasmid containing Zp sequences (positions −221 to +13). ZVmut-Luc is a luciferase reporter plasmid bearing a defective ZV-ZEB1 binding site that was generated by changing the A at position −12 (relative to the Zp transcriptional initiation site) into a C. BG-Luc, which contains a minimal beta-globin promoter, was used as a control reporter construct. RLU, relative light units. (C) Fold changes are relative to Zta-induced reporter activity in the absence of miR-429. Statistical significance of the difference in fold change between the Zp-Luc group and the ZVmut-Luc group was analyzed by Student's t test.