MicroRNA microarray identifies Let-7i as a novel biomarker and therapeutic target in human epithelial ovarian cancer

Abstract

MicroRNAs (miRNA) are approximately 22-nucleotide noncoding RNAs that negatively regulate protein-coding gene expression in a sequence-specific manner via translational inhibition or mRNA degradation. Our recent studies showed that miRNAs exhibit genomic alterations at a high frequency and their expression is remarkably deregulated in ovarian cancer, strongly suggesting that miRNAs are involved in the initiation and progression of this disease. In the present study, we performed miRNA microarray to identify the miRNAs associated with chemotherapy response in ovarian cancer and found that let-7i expression was significantly reduced in chemotherapy-resistant patients (n = 69, P = 0.003). This result was further validated by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.015). Both loss-of-function (by synthetic let-7i inhibitor) and gain-of-function (by retroviral overexpression of let-7i) studies showed that reduced let-7i expression significantly increased the resistance of ovarian and breast cancer cells to the chemotherapy drug, cis-platinum. Finally, using miRNA microarray, we found that decreased let-7i expression was significantly associated with the shorter progression-free survival of patients with late-stage ovarian cancer (n = 72, P = 0.042). This finding was further validated in the same sample set by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.001) and in an independent sample set by in situ hybridization (n = 53, P = 0.049). Taken together, our results strongly suggest that let-7i might be used as a therapeutic target to modulate platinum-based chemotherapy and as a biomarker to predict chemotherapy response and survival in patients with ovarian cancer.

Figure 1

let-7i expression is significantly reduced in patients with chemotherapy-resistant EOC. A, microarray analysis of miRNA expression between complete response (CR) and noncomplete response (non-CR) ovarian cancer patients. B, differentially expressed miRNAs between complete response and noncomplete response patients at various statistical significance (P < 0.015, P < 0.025, and P < 0.05). C, validation of let-7i expression in complete response and noncomplete response patients by real-time reverse transcription-PCR.

Figure 2

let-7i expression regulates cis-platinum resistance of EOC cells. A, inhibition of let-7i, but not mir-509-3p or mir-509-5p, increased resistance to cis-platinum treatment in 2008 and SKOV3 cells. B, stem-loop real-time reverse transcription-PCR showed endogenous let-7i was significantly blocked by let-7i inhibitor. C, overexpression of let-7i by retroviral infection in 2008, SKOV3, and MCF7 cells increased their sensitivity to the cis-platinum treatment. Inset, stem-loop real-time reverse transcription-PCR showed that let-7i was stably overexpressed in EOC cell lines by retroviral transfection.

Figure 3

let-7i DNA copy number does not exhibit genomic alteration in human cancer. Genomic locus harboring let-7i did not exhibit alteration in EOC (n = 106). Black, deletion; gray, amplification.

Figure 4

Low let-7i expression is significantly associated with shorter survival of patients with EOC. Correlation between let-7i expression and survival of EOC patients analyzed by microarray (A, progression-free survival), real-time reverse transcription-PCR (B, progression-free survival), and tissue array (C, disease-free survival).

Figure 5

Illustration of the potential mechanism of let-7i regulating chemotherapy sensitivity in human cancer.