Your Data: The Role of microRNAs in Cervical Cancer (TechNotes 16:1)

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TechNotes 16(1)

TaqMan® MicroRNA Assays
Ambion® Anti-miR™ miRNA Inhibitors

Your Data: The Role of microRNAs in Cervical Cancer

In a recent publication, Dr. Jeong-Won Lee and colleagues profiled microRNA (miRNA) expression in cervical cancer cells compared to normal cervical tissue. Several miRNAs were differentially expressed, including miR-199a. Inhibition of miR-199a resulted in reduced cervical cancer cell growth, and this inhibition was enhanced by cisplatin chemotherapeutic treatment. This study demonstrates the use of Applied Biosystems miRNA profiling and functional analysis workflows for the study of altered gene expression in cervical carcinogenesis.

Lee J-W, Choi CH, Choi J-J, Park Y-A, Kim S-J, Hwang SY, Kim WY, Kim T-J, Lee J-H, Kim B-G, et al. (2008) Altered microRNA expression in cervical carcinomas. Clin Cancer Res 14(9):2535-42.

Understanding Cervical Cancer
Cervical cancer involves the transformation of normal cervical epithelial cells into preneoplastic cervical intraepithelial neoplasia, which then develops into invasive cancer. The primary cause of cervical cancer is human papilloma virus (HPV), but additional factors are involved. To better understand the differences in gene expression between normal and cervical cancer cells, Dr. Jeong-Won Lee and colleages at the Sungkyunkwan University School of Medicine in Korea used Applied Biosystems tools for miRNA profiling and miRNA functional analysis to identify altered miRNA expression in cervical cancer.

The Role of microRNAs in Malignant Transformation
miRNAs are small, ~22 nt noncoding RNAs that post-transcriptionally regulate eukaryotic gene expression by binding to mRNA targets and inhibiting their translation. Increasing evidence has implicated miRNAs in the development of various cancers, in addition to their more established roles in normal developmental processes. Traditionally, microarrays have been used to generate expression profiles of miRNAs, but real-time PCR offers higher sensitivity and broader dynamic range. In this study, the authors used human TaqMan® MicroRNA Assays, which enabled the rapid profiling of 157 miRNAs, to compare miRNA expression between 10 early stage invasive squamous cell carcinomas (ISCCs) and 10 normal cervical squamous epithelial specimens obtained from patient biopsies.

Differential Expression of miRNAs
Of the 157 miRNAs profiled, 70 miRNAs were found to have significantly different expression levels between ISCC and normal epithelial tissues; 68 were upregulated, and 2 were downregulated. One of the most significantly upregulated miRNAs, miR-199a (Figure 1), was chosen for further study.

Figure 1. Differential Expression of miR-199a in Normal vs. Cervical Cancer Tissue. TaqMan® MicroRNA Assays were used to profile 157 miRNAs from 10 normal cervical samples and 10 ISCC samples. Data were normalized using let-7a as the endogenous control. miR-199a expression is shown. Data reproduced with permission from Clinical Cancer Research.

Effects of miR-199a Inhibition
To examine the role of miR-199a in cervical carcinogenesis, Ambion® Anti-miR™ miRNA Inhibitors of miR-199a were transfected into human cervical cancer cell lines (SiHa and ME-180), and TaqMan MicroRNA Assays were used to confirm that miR-199a was successfully inhibited (Figure 2A). Inhibition of miR-199a resulted in reduced cell growth in vitro (Figure 2B), and the cisplatin chemotherapeutic response was potentiated by the Anti-miR miRNA Inhibitor (Figure 2, C and D). These findings open up the exciting possibility that miR-199a represents a potential target for therapeutic intervention in patients suffering from cervical cancer.

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Figure 2. Inhibition of Cervical Cancer Cell Growth In Vitro by miR-199a Anti-miR™ miRNA Inhibitors. Transfection of Anti-miR miRNA Inhibitors of miR-199a resulted in reduced miR-199a expression (Panel A) and reduced Me-180 and SiHa cervical cancer cell growth (Panel B). miR-199a inhibition also resulted in the potentiation of the cisplatin chemotherapeutic response (Panels C and D). Shown are the averages of 3 independent experiments; bars, SE (*, P<0.05; **, P<0.01). Data reproduced with permission from Clinical Cancer Research.

TechNotes Archive

miRNA Functional Analysis Using Ambion® Pre-miR™ miRNA Precursors and Anti-miR™ miRNA Inhibitors
Ambion Pre-miR miRNA Precursors and Anti-miR miRNA Inhibitors are ideal for performing functional analyses of miRNAs, enabling researchers to tackle the fundamental questions, “Which genes do miRNAs control?” and “Which cellular processes do miRNAs regulate?” The availability of these miRNA precursor and inhibitor oligonucleotides is kept current with the Sanger miRBase Sequence Database. They are available in pre-designed formats and custom-designed products are also available.

Ambion Pre-miR miRNA Precursors and Anti-miR Inhibitors enable miRNA functional analysis by upregulation and inhibition of miRNA activity, respectively. Specific experimental designs include:
• miRNA target site identification and validation
• Screening for miRNAs that regulate the expression of a gene
• Screening for miRNAs that affect a cellular process

Ambion Pre-miR miRNA Precursors
Pre-miR miRNA Precursors are small, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNAs. These ready-to-use miRNA mimics, which are similar to, but not identical to siRNAs, can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs. They enable detailed study of miRNA biological effects via gain-of-function experiments. Positive and Negative Control Pre-miR miRNA Precursors, or the Pre-miR Precursor Starter Kit, can be used to optimize transfection of Pre-miR miRNA Precursors.

Ambion Anti-miR miRNA Inhibitors
Anti-miR miRNA Inhibitors are chemically modified, single-stranded nucleic acids designed to specifically bind to and inhibit endogenous miRNA molecules. These ready-to-use inhibitors can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs, and enable detailed study of miRNA biological effects.