J Bacteriol Virol.  2014 Mar;44(1):84-94. 10.4167/jbv.2014.44.1.84.

Regulation of microRNA-7-5p and LRP6 by Epstein-Barr Virus-Encoded RNAs in Burkitt's Lymphoma Cell Line Akata

• Son JW
• Choi HY
• Lee HN
• Seo MK
• Lee SK

• KMID: 2135361
• DOI: http://doi.org/10.4167/jbv.2014.44.1.84

Abstract

Epstein-Barr virus (EBV)-encoded small non-coding RNAs (EBERs) are abundantly expressed in various EBV-associated malignancies, and play critical roles in cell proliferation, tumorigenesis, and apoptosis resistance. However, the mechanism how EBERs regulate cell function awaits further clarification. In this study, we investigated the effect of EBERs on the expression of cellular microRNA (miRNA) and mRNA expression. To test the effect of EBERs while unaffected by other EBV genes, we used EBERs-deleted recombinant EBV infected Burkitt's lymphoma cell line (Akata(+)EBERs(-)) as well as EBV-infected (Akata(+)) and EBV uninfected (Akata(-)) cell lines. They all have the same genetic backgrounds. First, 15 different cellular miRNAs which have reverse complementary sequences to EBERs and have reported targets were selected by bioinformatics analysis. When RT-PCR was carried out for the 16 miRNAs using RNAs from Akata(+), Akata(-), and Akata(+)EBERs(-) cells, hsa-miR-7-5p was the only one showing down-regulated expression in Akata(+) than in Akata(-) and Akata(+)EBERs(-) cells. Bioinformatics and mRNA microarray analyses for Akata(+), Akata(-), and Akata(+)EBERs(-) cell lines were then carried out to predict putative targets of hsa-miR-7-5p. Among the 6 predicted targets of hsa-miR-7-5p, only low density lipoprotein receptor-related protein 6 (LRP6) was up-regulated in EBERs-expressing cells when tested by RT-PCR and Western blot. However, luciferase reporter assay showed that the 3'-UTR of LRP6 was not directly targeted by hsa-miR-7-5p. Our data suggest that both hsa-miR-7-5p and LRP6 are regulated by EBERs in Akata cells, and these genes may partly mediate the tumorigenic function of EBERs in Burkitt's lymphoma.

Keyword

Epstein-Barr virus; EBERs; microRNA; Burkitt's lymphoma cell; LRP6; hsa-miR-7-5p

MeSH Terms

Apoptosis
Blotting, Western
Burkitt Lymphoma*
Carcinogenesis
Cell Line*
Cell Proliferation
Computational Biology
Herpesvirus 4, Human
Low Density Lipoprotein Receptor-Related Protein-6
Luciferases
MicroRNAs
RNA*
RNA, Messenger
RNA, Small Untranslated
Low Density Lipoprotein Receptor-Related Protein-6
Luciferases
MicroRNAs
RNA
RNA, Messenger
RNA, Small Untranslated

Figure

• 

  Figure 1. Verification of EBER deletion in Akata(+)EBERs(−) cell line. (A) PCR analysis was carried out to examine the absence of EBER (EBER1, 2) sequences in the EBV genome of Akata(+)-EBERs(−). For comparison, Akata(−) and Akata(+) cells were used. The presence of EBV latent genes (EBNA2, 3B, and 3C) was also examined. GAPDH was used as an internal control. (B) Expression of EBERs and two EBV BART miRNAs in Akata(−), Akata(+), and Akata(+)EBERs(−) cells were analyzed by Northern blot analysis. The expression of human miR-16 (hsa-miR-16) was also assessed as a reference. The quality and quantity of the loaded RNA were examined by reprobing the blot for U6 snRNA.

• 

  Figure 2. Down-regulated hsa-miR-7-5p expression by EBERs in Akata cells. Real-time RT-PCR was performed for 15 different cellular miRNAs to test whether EBERs can affect the expression of them. Total RNA was extracted from Akata(−), Akata(+), and Akata(+)EBERs(−) cells, and miRNA qRT-PCR was conducted using Mir-X miRNA First Strand Synthesis and SYBR qRT-PCR Kit. Relative gene expression was calculated according to the comparative CT method using U6 snRNA as a loading control. The ratio of each miRNA to U6 in Akata(+) cells was arbitrarily set as 1. (n=3, †p < 0.01, *p < 0.05)

• 

  Figure 3. Up-regulated LRP6 expression by EBERs in Akata cells. (A) Real-tizme RT-PCR was performed for six different genes to test whether EBERs can affect the expression of them. Total RNA was extracted from Akata(−), Akata(+), and Akata(+)EBERs(−) cells and quantitative real-time RT-PCR were carried out using SYBR Premix Ex Taq. Relative gene expression was calculated according to the comparative CT method using GAPDH as a loading control. The ratio of each mRNA to GAPDH in Akata(+) cells was arbitrarily set as 1. (B) The effect of EBERs on the expression of LRP6 at protein level was analyzed. Western blot analysis was carried out using cell lysate from in Akata(−), Akata(+), and Akata(+)EBERs(−) cells β-actin was used as a loading control and HeLa cell line was used as a positive control for LRP6 expression. (C) Results similar to those in panel B were obtained in two more independent experiments, and the means and standard deviation (SD) from all three independent experiments are plotted. (n=3, †p < 0.01)

• 

  Figure 4. No direct targeting of has-miR-7 for the 3′-UTR of LRP6. (A) Three seed-matched regions in the 3′-UTR of LRP6 for the has-miR-7 are shown. To test whether LRP6 expression is directly regulated by has-miR-7, the 3′-UTR fragments containing the first two or last two seed matched regions were cloned into a luciferase reporter (psiCHECK-2) vector to product psiC-LRP6 (1–2) and psiC-LRP6(2–3), respectively. (B) HEK293T cells were co-transfected with has-miR-7-5p mimic, and psiC-LRP6(1–2) or psiC-LRP6(2–3). Renillar luciferase activity was analyzed 48 h after transfection and normalized using firefly luciferase activity. Error bars indicate SD. (n=3, †p < 0.01, *p < 0.05).

• 

  Figure 5. Validation of selected mRNA from microarray data. Microarray analysis was performed for Akata(−), Akata(+), and Akata(+)EBERs(−) cells. Relative mRNA intensity was normalized by the average intensity of Akata(+) cell.

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