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Comparative Study
. 2013 Nov;87(22):12237-48.
doi: 10.1128/JVI.01202-13. Epub 2013 Sep 4.

Kaposi's sarcoma-associated herpesvirus microRNA single-nucleotide polymorphisms identified in clinical samples can affect microRNA processing, level of expression, and silencing activity

Soo-Jin Han  1 , Vickie Marshall, Eugene Barsov, Octavio Quiñones, Alex Ray, Nazzarena Labo, Matthew Trivett, David Ott, Rolf Renne, Denise Whitby
Affiliations
Comparative Study

Kaposi's sarcoma-associated herpesvirus microRNA single-nucleotide polymorphisms identified in clinical samples can affect microRNA processing, level of expression, and silencing activity

Soo-Jin Han et al. J Virol. 2013 Nov.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645-659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii) in vitro Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore, in vitro maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies.

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Figures

Fig 1
Fig 1
(Top) Diagram of SIREN-RetroQ-DsRed-Express retroviral vector. Large gray arrow, the U6 RNA polymerase III promoter immediately upstream of the cloned pre-microRNA; black arrow, CMV RNA polymerase II promoter expressing DsRed; LTRx, self-inactivating long terminal repeats. (Bottom) Cloned sequences of each microRNA (MiRNA), with the mature microRNA being underlined and in bold.
Fig 2
Fig 2
Box-and-whisker plots representing the CT values of transduced HEK293T cells stably expressing wild-type and variant microRNA sequences. The middle 50% of the CT values, known as the interquartile range, is inside the box. The horizontal line inside the box represents the median CT value, and the whiskers represent 1.5 times the interquartile range on each side of the box. Any points outside the whiskers are depicted with a dot. Depiction of levels of significance are as follow: not significant (ns), P ≥ 0.01; *, P < 0.01; **, P < 0.001; ***, P < 0.0001.
Fig 3
Fig 3
Applied Biosystems custom small RNA RT-PCR analysis of KSHV latently infected PEL cell lines. Mature microRNA expression levels are shown as the fold change in expression of the target genes (microRNAs) normalized to the level of expression of the internal control gene (RNU6B) and relative to the level of expression by the wild-type BCBL-1 cell line using the 2-ΔΔCT method (23). A positive fold change represents an upregulated PEL cell line, whereas a negative fold change represents a downregulated PEL cell line. The negative fold changes presented here were obtained by taking the negative inverse (i.e., −1/fold change of the result from the 2−ΔΔCT method). Standard errors were computed in the R package plotrix. These values were then used to compute interval limits (95%) on the basis of 4 degrees of freedom (3 treated sample replicates + 3 control sample replicates − 2). Values were then transformed from log2 to fold change scale by exponentiation, 2x, where x denotes the lower limit and upper limit of the standard error values for each PEL cell line of interest compared, and appropriate standard error bars are added. The standard error bars are asymmetrical due to exponentiation.
Fig 4
Fig 4
A. In vitro maturation assay for miR-K12-5 and -9. These microRNA variants have SNPs in pre-microRNA. (First and third panels) Products on urea-polyacrylamide gel. Black arrows, pre-microRNA and microRNA produced from each processing; lanes M, molecular size markers. (Second and fourth panels) Graphs of pre-microRNA quantification normalized by the levels for the wt. Results are shown as percentages ± standard deviations from three independent experiments. (B) In vitro maturation assay for miR-K12-4, miR-K12-7, and miR-K12-10. The variants of these microRNAs have SNPs in different positions in the terminal loop of pre-microRNA. (First, third, and fifth panels) Products on urea-polyacrylamide gels. Black arrows, pre-microRNA and microRNA produced from each processing; lanes M, molecular size markers. (Second, fourth, and sixth panels) Graphs of pre-microRNA quantification normalized by the levels for the wt. Results are shown as percentages ± standard deviations from three independent experiments. (C) In vitro maturation assay for miR-K12-2, miR-K12-6, and miR-K12-9mv. The variants of these microRNAs have an SNP or SNPs within mature microRNA sequences. (First, third, and fifth panels) Products on urea-polyacrylamide gels. Black arrows, pre-microRNA produced; lanes M, molecular size markers. (Second, fourth, and sixth panels) Graphs of pre-microRNA quantification normalized by the levels for the wt. Results are shown as percentages ± standard deviations from three independent experiments.
Fig 5
Fig 5
Luciferase-based microRNA repressor assay. The microRNA sensor vector and increasing amounts (0, 400, and 800 ng) of the microRNA expression vector were cotransfected with 10 ng of Renilla luciferase vector for normalization into HEK293T cells. Ratios on the x axes indicate the amount of microRNA expression vector, and the relative light unit (RLU) values are normalized to the value for Renilla luciferase and are shown as a percentage compare to the value for the wt. Empty vector was used as a negative control. Results are shown as means ± standard deviations from three independent experiments.
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