Correlation of miRNA expression profiling in surgical pathology materials, with Ki-67, HER2, ER and PR in breast cancer patients.

BACKGROUND: New molecular markers related to prognosis and/or clinical outcome have been extensively studied in breast cancer. In particular, microRNA (miRNA) has attracted the interest of both basic and clinical investigators as one of the promising molecular markers of breast cancer patients. MiRNAs are a class of short noncoding RNAs that regulate mRNAs at posttranscriptional level and are deregulated in various human malignancies. Previous studies have reported that miRNAs were stably conserved in 10% formalin-fixed paraffin-embedded tissues without significant degradation, in contrast to more fragile RNA. METHODS: Therefore, in this study, we examined 21 surgical breast cancer specimens using the Human Cancer microRNA PCR Array system (QIAGEN) to explore potential molecular targets of miRNAs. RESULTS: Profiling of miRNA expression in archival materials demonstrated that a group of deregulated miRNAs was associated with clinicopathological parameters of the patients, such as Ki-67, HER2, ER and PR. For instance, an abundant expression of multiple let-7 miRNA family, also known as tumor suppressor, was detected in low Ki-67 and HER2 groups. Elevated expression of 8 miRNAs overlapped between Ki-67+/HER2+/ER+/PR+ groups, including several known oncogenic miRNAs such as miR-148b, miR-15b, miR-200c, miR-150, miR-191, miR-96, miR-25 and miR-21. CONCLUSIONS: These results all indicated that when analyzing miRNAs in surgical pathology specimens of breast cancer as a biomarker, they should be examined as a cluster through miRNA profiling, rather than relying on the analysis of a single miRNA.

Identification of androgen-responsive microRNAs and androgen-related genes in breast cancer.

BACKGROUND: Androgen receptors (ARs) are expressed in many breast cancer cells, but the mechanism of action of androgens is not as well-characterized as in other cell types. The study of microRNAs has recently provided with important insights into the biology of hormone-dependent cancer. MATERIALS AND METHODS: We attempted to identify microRNAs induced by dihydrotestosterone in an AR-positive cell line. We examined a possible correlation among microRNAs, target genes, and ARs in breast cancer tissues using immunohistochemistry and laser capture microscopy. RESULTS: Our analysis demonstrated that miR-363 and its possible target IQ motif and WD repeats-1 (IQWD1) are involved in a microRNA-mRNA pathway related to the mechanism of action of androgens. Our analyses showed that a high tumor level of IQWD1 in patients with breast cancer was significantly associated with adverse clinical outcomes. CONCLUSION: Our findings indicated that the recruitment of IQWD1 to ARs may be a prerequisite for the growth stimulation by androgens through ARs in breast cancer cells.

An induction of microRNA, miR-7 through estrogen treatment in breast carcinoma.

BACKGROUND: Estrogen plays an important role in the development of estrogen-dependent breast carcinoma. Recently, several studies demonstrated a possible involvement of several micro RNAs (miRNAs) in the development of resistance to endocrine therapy in breast cancer patients, but the correlation between estrogen actions and miRNA expression in breast carcinoma still remains largely unknown. Therefore, in this study, we examined the in vitro effects of estrogen upon miRNA expression profiles in breast carcinoma. METHODS: We first screened the miRNA expression profiles induced by 17ß-Estradiol (E2) using RT2 miRNA PCR Array in the ER-positive breast carcinoma cell line MCF-7. We identified miR-7 as the important miRNA associated with estrogen actions in these cells and further examined the changes of estrogen-dependent EGFR expression by miR-7 in ER-positive or -negative breast carcinoma cell lines including MCF-7. We also evaluated the correlation between miR-7 and EGFR expression in breast carcinoma cells derived from 21 patients using laser capture microdissection combined with quantitative reverse transcriptase-PCR. RESULTS: Seventeen miRNAs were significantly induced by E2 treatment in the MCF-7 cell line. Among 17 miRNAs induced by estradiol treatment, only miR-7 expression was significantly decreased by subsequent ICI treatment. The expression of miR-7 was up-regulated 2.94-fold by E2 treatment. miR-7 was reported to suppress epidermal growth factor receptor (EGFR) expression in several human malignancies. Transfection of miR-7 significantly suppressed EGFR mRNA levels in MCF-7 cells. Depletion of E2 from cell culture media also increased the expression level of EGFR mRNA in MCF-7 and T-47D cells but not in ER-negative, MDA-MB-231 and SK-BR-3 cells. We also evaluated the status of miR-7 in breast carcinoma tissues, but the correlation between the status of miR-7 and EGFR in carcinoma cells isolated by laser capture microscopy was not detected. CONCLUSIONS: These results suggest that miR-7 may play a role in the development of resistance to endocrine therapy in breast cancer patients through regulating EGFR expression of carcinoma cells.

Phosphodiesterase type 9 (PDE9) in the human lower urinary tract: an immunohistochemical study.

OBJECTIVES: To examine the roles of the one of the cyclic guanosine monophosphate (cGMP)-specific phosphodiesterases (PDEs), PDE9, in human lower urinary tract (LUT), we performed immunohistochemistry (IHC) using autopsy specimens. To demonstrate the potential functional differences between PDE5 and PDE9 in human LUT, the localization of PDE5 and PDE9 was also compared using laser-capture microdissection (LCM)/reverse transcriptase-polymerase chain reaction (RT-PCR) method. MATERIALS AND METHODS: • We immunolocalized PDE9 in human bladder (60 cases) and prostate (40) specimens using IHC. We performed LCM/RT-PCR evaluation in six human bladders to determine the differential expression patterns of PDE5 and PDE9. RESULTS: PDE9 immunoreactivity was detected in the bladder urothelium of all the cases and in 85% of the prostatic urethral urothelium. PDE9 immunoreactivity was detected in the perikaryon of the intramural ganglia. LCM/RT-PCR evaluation showed that PDE5 mRNA was exclusively detected in smooth muscle cells but PDE9 mRNA was mainly detected in the urothelium of the human bladder. CONCLUSION: PDE9 is widely distributed in the urothelial epithelium of the human LUT and its potential roles may be different from those of PDE5.

LIN28: a regulator of tumor-suppressing activity of let-7 microRNA in human breast cancer.

A tumor-suppressor gene, let-7 microRNA (miRNA) family, is often inactivated in various human malignancies. LIN28 is a RNA-binding protein that has been well characterized for regulation of let-7 maturation in undifferentiated embryonic stem cells at post-transcriptional level. Oncogenic regulation of let-7 miRNAs has been demonstrated in several human malignancies but their correlation with LIN28 has not been studied in breast cancer. We therefore explored a possible mechanism of tumorigenesis in breast carcinoma tissue via an alternation of let-7 miRNA precursor processing by LIN28 in this study. A total of 26 breast cancer surgical pathology specimens were evaluated for LIN28 and LIN28B expression using immunohistochemistry. We then isolated carcinoma cells in 21 cases using laser capture microdissection, and the miRNAs from these samples were profiled using PCR array analysis. LIN28 status was positively correlated with ERα, PR, and Ki-67 status and inversely correlated with HER2 status. These results suggest the possible involvement of LIN28 in regulation of sex steroid dependent cell proliferation of breast carcinoma cells. We further demonstrated that expression of let-7a, let-7c, let-7d (P=0.026) and let-7f (P=0.016) were inversely correlated with those of LIN28. These results also suggest that LIN28 promotes tumorigenic activity by suppressing let-7 miRNA maturation in breast carcinoma cells.