MicroRNA expression in ileal carcinoid tumors: downregulation of microRNA-133a with tumor progression

Abstract

MicroRNAs (miRNAs) are involved in cell proliferation, differentiation, and apoptosis and can function as tumor suppressor genes or oncogenes. The role of miRNAs in neuroendocrine tumors such as ileal carcinoids is largely unknown. We examined the differential expression of 95 miRNAs by RT-PCR using the QuantiMir System in eight matching primary and metastatic carcinoid tumors from the ileum. All miRNAs chosen for the QuantiMir System array were based on their potential functions related to cancer biology, cell development, and apoptosis. The expression of miRNAs for the samples was normalized to miRNA-197, and the matching primary and metastatic tumors were compared. There was downregulation of miRNA-133a, -145, -146, -222, and -10b in all samples between the primary and matching metastatic tumors and upregulation of miRNA-183, -488, and -19a+b in six of eight metastatic carcinoids compared to the primary tumors. miRNA-133a was further analyzed by TaqMan real-time RT-PCR and northern hybridization using six additional matching primary and metastatic samples, which supported the PCR array findings. There were significant differences in miRNA-133a expression with downregulation in the metastasis compared to the primary in the eight original cases (P<0.009) and in the six additional cases used for validation (P<0.014). Laser capture microdissection and real-time RT-PCR analysis using normal ileum found miRNA-133a expression in normal enterochromaffin cells. In situ hybridization in normal ileum showed that some of the mucosal endocrine cells expressed miRNA-133a. Both primary and metastatic ileal carcinoid tumors expressed miRNA-133a by in situ hybridization. These results provide information about novel marker miRNAs that may be used as biomarkers and/or therapeutic targets in intestinal carcinoid tumors.

Figure 1

Distribution of PCR array from eight ileal carcinoid tumors as log of fold change expressed as the ratio of metastatic to primary carcinoid tumors (Figures 1A, 1B, 1C and 1D). One μg log of total RNA was used from each carcinoid tumor using a QuantiMir kit. Results were expressed relative to microRNA-197. Relative change of miRNA expression was calculated by the comparative CT method. Figures 1A, 1B, 1C and 1D show the distribution as fold change for most of the miRNAs analyzed. Sixty-two microRNAs are labeled in the four figures.

Figure 2

TaqMan qRT-PCR analysis for miRNA-133a followed by gel electrophoresis and densitometric analysis of eight matching primary and metastatic ileal carcinoid tumors (Figures 2A, 2B, 2C and 2D). The upper panel shows the relative densitometric analysis of miR133a relative to RNU48 for each sample, while the lower panels show the original gel samples. C1 through C8 are the RNU48 samples. P represents the primary tumors and M represents the metastatic samples. Results are mean ± standard error of the mean. *p<0.05 ** p<0.001.

Figure 3

TaqMan qRT-PCR analysis for miRNA-133a followed by gel electrophoresis and densitometric analysis of four samples of enterochromaffin cells harvested from 5 × 10⁴ enterochromaffin cells. The upper panel shows the relative densitometric analysis of miR133a relative to RNU48 for each of four experiments. C1 through C8 are the RNU48 samples and L represent the laser captured miRNA-133a samples. Results are expressed as mean ± standard error of the mean.

Figure 4

Northern hybridization analysis of four sets of matching primary and metastatic ileal carcinoid tumors. Twenty μg of total RNA was used for the Northern hybridization with a Signos8is Kit according to the manufacturer’s protocol. The results were developed by enhanced chemiluminescence. Results were expressed relative to small nucleolar RNA, RNU48. RNA standards (20 nucleotides and 60 nucleotides) were run with the gel.

Figure 5

In situ hybridization analysis using digoxigenin-labeled probes. A. Normal ileal mucosa showing enterochromaffin cells with a positive hybridization signal for miRNA-133a (Arrows). Formalin-fixed paraffin-embedded tissue sections. Two-hundred nM of the 133a probe labeled at the 5′ and 3′ ends were used. B. Frozen tissue sections of a primary ileal carcinoid tumor showing positive detection of miRNa-133a in the cytoplasm of tumor cells. The adjacent connective tissue is negative. C. Paraffin section of a metastatic carcinoid tumor to the liver showing a strong positive hybridization signal for miRNA-133a in the cytoplasm of the tumor cells. The adjacent liver (L) is negative. D. Paraffin section of hybridization with a scramble probe substituted for miRNA-133a used as a control. All other conditions were identical as with the miRNA-133a probe. There is no detection of miRNA-133a in the tumor or in the adjacent liver (L).