Unique circulating microRNA profiles in epidemic Kaposi's sarcoma

Abstract

Background: Human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma (KS). Kaposi sarcoma in HIV/AIDS patients is referred to as epidemic KS and is the most common HIV-related malignancy worldwide. The lack of a diagnostic assay to detect latent and early-stage disease has increased disease morbidity and mortality. Serum miRNAs have previously been used as potential biomarkers of normal physiology and disease. In the current study, we profiled unique serum miRNAs in patients with epidemic KS to generate baseline data to aid in developing a miRNA-based noninvasive biomarker assay for epidemic KS.

Methods: This was a comparative cross-sectional study involving 27 patients with epidemic KS and 27 HIV-positive adults with no prior diagnosis or clinical manifestation of KS. DNA and RNA were isolated from blood and serum collected from study participants. Nested PCR for circulating HHV-8 DNA was performed on the isolated DNA, whereas miRNA library preparation and sequencing for circulating miRNA were performed on the RNA samples. The miRge2 pipeline and EdgeR were used to analyse the sequencing data.

Results: Fifteen out of the 27 epidemic KS-positive subjects (55.6%) tested positive for HHV-8 DNA, whereas only 3 (11.1%) out of the 27 HIV-positive, KS-negative subjects tested positive for HHV-8 DNA. Additionally, we found a unique miRNA expression signature in 49 circulating miRNAs in epidemic KS subjects compared to subjects with no epidemic KS, with 41 miRNAs upregulated and 8 miRNAs downregulated. Subjects with latent KS infection had a differential upregulation of circulating miR-193a compared to HIV-positive, KS-negative subjects for whom circulating HHV-8 DNA was not detected. Further analysis of serum from epidemic KS patients revealed a miRNA signature according to KS tumor status and time since first HIV diagnosis.

Conclusions: This study reveals unique circulating miRNA profiles in the serum of patients with epidemic KS versus HIV-infected subjects with no KS, as well as in subjects with latent KS. Many of the dysregulated miRNAs in epidemic KS patients were previously reported to have crucial roles in KS infection and latency, highlighting their promising roles as potential biomarkers of latent or active KS infection.

Keywords: Biomarker; Blood; Circulating microRNA; Kaposi's sarcoma; Serum; microRNA.

Fig. 1

Gel electrophoresis image after amplification of HHV 8 DNA. Legend: Lanes labeled L - 1000 bp DNA ladder. Lane NC - negative control, lane PC positive control. Lanes 1, 2, 3, 5, 7, 11, and 12 – are from samples positive for HHV 8 DNA. Lanes 4, 6, 8, 9, and 10 – are from samples negative for HHV 8 DNA.

Fig. 2

Volcano plot of miRNA differentially expressed in patients with KS verse those no KS. Legend: The plot is depicted with vertical and horizontal lines indicating the threshold for a relative expression fold change of 2 0r −2 compared with no KS (controls), at a 0.05 P value. Down regulated miRNAs are depicted in red, whereas upregulated miRNAs are depicted in green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The most variable circulating miRNAs expressed in patients with and without Epidemic KS Legend: Z score expression heatmap of the most variable circulating miRNAs expressed in patients with and without epidemic KS. Patients with epidemic KS are denoted as uci, whereas the comparative group of HIV-positive patients with no KS are denoted as iss.

Fig. 4

Differential circulating miRNAs expressed in participants testing positive or negative for HHV-8 DNA Legend: Volcano plot showing differentially expressed miRNAs in patients testing positive or negative for HHV-8 DNA. miRNAs differentially expressed with an adjusted p value < 0.005 are depicted in red for down regulation and green for up regulation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Differential miRNA expression in patients 0–2 years versus 2–5 years after index HIV diagnosis. Legend: Volcano plot showing differential miRNA expression in patients 0–2 years versus 2–5 years after index HIV diagnosis. Differentially expressed miRNAs with an adjusted p value < 0.005 are depicted in red for downregulation and green for upregulation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)