Changes in miRNAs Signal High-Risk HPV Infections
HPV18 E6 and E7 oncoproteins regulate the expression of host miRNAs. Eight of thirteen host miRNAs with altered expression among all four platforms (venn diagram) were examined by RT-qPCR in 11-day raft cultures of HFKs transduced with HPV18 E6, E7 or E6E7 retrovirus or an empty control retrovirus vector. miR-34a was used as a positive control for HPV18 E6 activity (Wang, X., et al. RNA 15:637-47, 2009) while U6 RNA was used as an internal loading control. The bar graph shows the relative fold increase (above 0) or decrease (below 0) (mean + SD) of each miRNA in the raft tissues expressing E6, E7, or E6E7 over the empty vector after being normalized to U6 RNA from two independent experiments.
microRNAs (miRNAs) are approximately 21 nucleotide long, non-coding RNAs that regulate the expression of certain proteins. As part of the RNA-induced silencing complex or RISC, miRNAs bind to complementary sequences in the 3’ untranslated regions of target messenger RNAs, blocking protein synthesis and sometimes leading to the destruction of the target RNA. Numerous studies have shown that the levels of cellular miRNAs can be altered in diseased tissues, and these changes potentially could be used for diagnosis or disease monitoring.
Cervical cancer, which is caused by persistent infection with high-risk human papilloma viruses (HPVs), such as HPV16 and HPV18, is the second most common cancer among women worldwide, with higher incidence in areas with low uptake of Pap testing. Prior studies conducted by Zhi-Ming Zheng, M.D., Ph.D., Head of the Tumor Virus RNA Biology Section in CCR’s Gene Regulation and Chromosome Biology Laboratory, and other researchers have identified a handful of miRNAs that seem to be regulated by HPV infection and important for cancer cell behavior. However, the varied experimental systems and methods of analysis led to distinct observations. To overcome these differences, Zheng and his colleagues decided to comprehensively examine changes in miRNA expression associated with high-risk HPV infection and progression of cervical disease by using miRNA array analysis and small RNA sequencing (miRNA-seq).
The researchers began their studies by determining the miRNA profiles of human foreskin and vaginal keratinocyte raft cultures infected with HPV16 or HPV18 or left untreated. HPV infection increased the expression of some miRNAs and decreased the abundance of others. The investigators confirmed these results with Northern blots of a subset of the miRNAs. Although the two cell types had slightly different baseline miRNA expression profiles, the scientists identified 13 miRNAs with altered expression after HPV infection that were detected by both miRNA array and miRNA-seq. HPV infection increased the expression of eight of the miRNAs and decreased the expression of the remaining five. However, the researchers did not detect any HPV18 viral miRNAs by miRNA-seq in either cell type.
To verify that the changes in miRNA expression they observed are specific to HPV infection, the investigators analyzed the miRNA profiles of HPV18-infected raft cultures at different times over the course of the infection. Compared to uninfected cells, they found that the altered miRNAs could be divided into four distinct groups: those with higher expression throughout infection; those with reduced expression throughout infection; those with early increased expression but decreased expression later; and those with decreased expression early but increased expression later. The latter two groups included miRNAs that are associated with cell proliferation and skin differentiation, indicating they are regulated by infection but are also a result of the model system. In contrast, nine of the previously identified 13 miRNAs were in the first two groups, suggesting they are truly regulated by HPV infection. The scientists selected eight of the miRNAs for further study: miR-16, miR-22, miR-25, miR-27a, miR-29a, miR-92a, miR-100, and miR-378.
The researchers then asked whether the altered miRNAs were regulated by the oncoproteins E6 and E7 because of their important roles in HPV-mediated transformation. The scientists infected raft cultures with a control retrovirus or retroviruses expressing E6, E7, or both and examined miRNA levels using reverse transcription quantitative PCR (RT-qPCR). Encouragingly, they found that E7 could alter the expression of all eight miRNAs while E6 affected all but miR-25.
To see whether the results from their model system matched those in HPV infected tissue, the investigators used RT-qPCR to evaluate the levels of their eight identified miRNAs in 158 cervical tissue samples (38 from normal cervix without HPV infection, 13 cervical intraepithelial neoplasia (CIN) 1 and 2, 39 CIN3, and 68 cervical cancer samples). In agreement with the raft culture results, they found significant increases in miR-25, miR-92a, and miR-378 in the HPV infected tissues. miR-16 expression showed a trend toward increased levels in the CIN3 and cancer samples but may have been distorted by very high expression in four samples. In contrast, the researchers observed no significant change in miR-22, miR-29a, or miR-100 in the HPV- infected tissues. Likewise, miR-27a, which had reduced expression after HPV infection in the cultures, was significantly elevated in the cancer samples but showed no difference in the CIN tissues.
Based on mean values and statistical analysis, the scientists combined miR-25 and miR-92a into one group and miR-22 and miR-29a into another and averaged their expression levels. They saw a step-wise increase in miR-25/92a from normal to CIN1+2, CIN3, and cancer but observed no trend for the miR-22/29a group. Since miRNA levels between samples varied, the researchers converted the expression levels into a ratio between miR-25/92a and miR-22/29a. Using 1.5 as a possible cutoff to diagnose CIN and cervical cancer, they found that only 13 percent of normal samples had a ratio of 1.5 or higher while 88 percent of the cervical cancers, 49 percent of the CIN3, and 38 percent of the CIN1+2 had ratios of 1.5 or higher. Together these data suggest that the expression ratio between miR-25/92a and miR-22/29a could be useful in diagnosing and monitoring the progression of cervical lesions caused by infections with high-risk HPVs.Summary Posted: Sat, 03/01/2014
Wang X, Wang HK, Li Y, Hafner M, Banerjee NS, Tang S, Briskin D, Meyers C, Chow L, Xie X, Tuschl T, and Zheng ZM. miRNAs are biomarkers of oncogenic HPV infections. PNAS. February 10, 2014 PubMed Link