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. 2009 Apr;5(4):e1000389.
doi: 10.1371/journal.ppat.1000389. Epub 2009 Apr 17.

Pre-micro RNA signatures delineate stages of endothelial cell transformation in Kaposi sarcoma

Andrea J O'Hara  1 , Pauline Chugh, Ling Wang, Eduardo M Netto, Estrella Luz, William J Harrington, Bruce J Dezube, Blossom Damania, Dirk P Dittmer
Affiliations

Pre-micro RNA signatures delineate stages of endothelial cell transformation in Kaposi sarcoma

Andrea J O'Hara et al. PLoS Pathog. 2009 Apr.

Abstract

MicroRNAs (miRNA) have emerged as key regulators of cell lineage differentiation and cancer. We used precursor miRNA profiling by a novel real-time QPCR method (i) to define progressive stages of endothelial cell transformation cumulating in Kaposi sarcoma (KS) and (ii) to identify specific miRNAs that serve as biomarkers for tumor progression. We were able to compare primary patient biopsies to well-established culture and mouse tumor models. Loss of mir-221 and gain of mir-15 expression demarked the transition from merely immortalized to fully tumorigenic endothelial cells. Mir-140 and Kaposi sarcoma-associated herpesvirus viral miRNAs increased linearly with the degree of transformation. Mir-24 emerged as a biomarker specific for KS.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Pre-miRNA profiling of all samples.
Shown is a heatmap, with blue indicating low levels of signal, white intermediate, and red high levels of signal, when comparing across all primer pairs. The horizontal axis indicates samples: PEL indicates PEL, B indicates KSHV-negative B cell lymphoma, T indicates T cell lymphoma, Tonsil indicates tonsil and Endothelial indicates all endothelial culture cell models, including SLK, E1 and L1 TIVE, and infected and uninfected HUVEC and HMVEC cells. Additionally, viral status is shown, with + indicating KSHV-positive and – indicating KSHV-negative. The vertical axis indicates the pre-miRNA. Groups I and II represent the miRNAs that are unchanged across all samples. Group III indicates cellular and KSHV miRNAs induced upon KSHV infection. Group IV represents the miRNAs that are downregulated in KSHV-positive infection. Group V represents 11 cellular miRNAs highly expressed in the HUVEC and HMVEC cells, both uninfected and infected. Group VI contains cellular miRNAs that are downregulated in all B-cell lymphomas, including PEL.
Figure 2
Figure 2. Pre-miRNA profiling of primary KS biopsies and endothelial derived cell cultures.
(A) Shown is a heatmap, with blue indicating low levels of signal, white intermediate, and red high levels of signal, when comparing across all primer pairs. The horizontal axis indicates samples: KS indicates primary KS biopsies and Endothelial indicates all endothelial culture cell models, including SLK, E1 and L1 TIVE, and infected and uninfected HUVEC and HMVEC cells. Additionally, viral status is shown, with + indicating KSHV-positive and – indicating KSHV-negative. The vertical axis indicates the pre-miRNA. Group I and II represent the miRNAs that are unchanged in all samples. Group III shows the cellular and viral miRNAs that are upregulated upon KSHV infection. Group IV indicates viral and cellular miRNAs upregulated specifically in KS, group V for E1 and L1 TIVE, and group VI for uninfected and infected HUVEC and HMVEC cells. Group VII are cellular miRNAs downregulated in uninfected and infected HUVEC and HMVEC cells. Bar graphs representative of each group described are shown on the left (B–H). All samples are shown in the same order as the clustering, with KS representing primary KS biopsies and Endothelial indicating all endothelial cell culture models profiled. Again, viral status is also listed. In the bar graphs, each cell type is divided by a black bar. All bar graphs are dCT U6 normalized and Z-standardized. (B) Lana expression is high in all KSHV-positive cell lines and not present in KSHV-negative cell lines. (C) KSHV-mir-K2 is upregulated in all KSHV-positive samples, with the highest level of expression in primary KS biopsies, intermediate in E1 and L1 TIVE cells and lowest in infected HUVEC and HMVEC cells. (D) Cellular miRNA let-7a-3 shows a similar expression pattern of increased expression upon KSHV infection. (E) Mir-24-2 has increased expression in primary KS biopsies, as compared to other cell types. (F) Mir-32 has increased expression in E1 and L1 TIVE cells, as compared to other cell types. (G) Mir-29a has increased expression in both infected and uninfected HUVEC and HMVEC cells while mir-370 (H) shows reduced expression in this group, as compared to other cell types.
Figure 3
Figure 3. Validation of 4 individual biomarkers for endothelial cell transformation.
(A–C) Shown are relative transcription levels (dCT) on the vertical and sample class on the horizontal axis. Dots indicate individual data points (>2 for each sample) for mir-221, mir-140, mir-24-2. (D) Decision tree of clustering based on just these 3 biomarkers. (E–G) Densityplots of dCT values for these four genes. Relative frequency (in %) is shown on the vertical axis and relative levels (dCT) on the horizontal axis. The dotted line indicates the cut-off value for class determination.
Figure 4
Figure 4. Model of KSHV-dependent progressive transformation of endothelial cells as identified by pre-miRNA clustering.
Upon KSHV infection, KSHV miRNAs and cellular miRNAs are induced. Through various stages of transformation, additional cellular miRNAs are induced and KSHV miRNA expression increases. E indicates primary endothelial cells. EK indicates endothelial cells, which can grow in reduced serum due to KSHV infection. ET indicates KSHV infected endothelial cells, which can form tumors in nude mice, such as E1 and L1 TIVE cells. KS indicates Kaposi sarcoma, which is the only cell type to experience notable levels of lytic reactivation with KSHV infection.
Figure 5
Figure 5. QPCR quality control.
Agilent Bioanalyzer™ RNA chip results are shown for two common extraction methods of RNA. The BC2 cell line was used. (A) The RNA nanochip on Triazol™ extracted total RNA has a calculated concentration of 237.5 ng/ul, using peak comparison, and a RNA integrity value of 9.7, while (B) the high molecular weight deleted sample (HMWD) is depleted for the rRNA and tRNA necessary for these calculations. (C) The small RNA chip on the total RNA sample has a concentration of 20.5 pg/µl as measured by area between 20–40 nucleotide while (D) the HMWD depleted sample has only 10.5 pg/µl in the same region. Average U6 values of all samples for the DNA (E) and RNA (F) are shown in box and whisker plots. C indicates endothelial cell culture models, both infected and uninfected, KS indicates primary KS biopsies, L indicates B and T cell lymphoma cell lines, P indicates PEL, and T indicates Tonsil Tissue. Outliers are shown in open dots. Histograms of individual QPCR reactions are shown for all reaction types, with frequency shown on the y-axis and CT value shown on the x-axis. A CT value ≤38.00 is considered positive. (G) For all RT negative reactions (n = 5,920), 4.83% are positive. (H) For all RT positive reactions (n = 20,000), 41.50% are positive. (I) For all DNA (n = 13,920), reactions 90.28% are positive. (J) For all non-template water control reactions (n = 5,120), 3.96% are positive.
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