The role of microRNA-221 and microRNA-222 in androgen-independent prostate cancer cell lines

Abstract

Androgen-dependent prostate cancer typically progresses to castration-resistant prostate cancer (CRPC) after the androgen deprivation therapy. MicroRNAs (miR) are noncoding small RNAs (19-25nt) that play an important role in the regulation of gene expression. Recent studies have shown that miR expression patterns are significantly different in normal and neoplastic prostate epithelial cells. However, the importance of miRs in the development of CRPC has not yet been explored. By performing genome-wide expression profiling of miRs, we found that expression levels of several miRs, in particular miR-221 and miR-222, were significantly increased in CRPC cells (the LNCaP-derived cell line LNCaP-Abl), compared with those in the androgen-dependent prostate cancer cell line (LNCaP). Overexpression of miR-221 or miR-222 in LNCaP or another androgen-dependent cell line, LAPC-4, significantly reduced the level of the dihydrotestosterone (DHT) induced up-regulation of prostate-specific antigen (PSA) expression and increased androgen-independent growth of LNCaP cells. Knocking down the expression level of miR-221 and miR-222 with antagonist miRs in the LNCaP-Abl cell line restored the response to the DHT induction of PSA transcription and also increased the growth response of the LNCaP-Abl cells to the androgen treatment. Changing the expression level of p27/kip1, a known target of miR-221 and miR-222, alone in LNCaP cells affected the DHT-independent cell growth but did not significantly influence the response of PSA transcription to the DHT treatment. In conclusion, our data suggest the involvement of miR-221 and miR-222 in the development or maintenance of the CRPC phenotype.

Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1. Comparison of miR expression patterns in LNCaP and LNCaP-Abl

Fold changes (LNCaP-Abl versus LNCaP) of the miRs are presented. LNCaP-1/LNCaP-Abl-1 and LNCaP-2 /LNCaP-Abl-2 represent two independently performed experiments. The tree graphs display the log2 transformation of the average fold changes. Arrays were mean centered and normalized by Gene Cluster 2.0. Average linkage clustering was performed by using uncensored correlation metric. The scale bar on the right upper corner displays the color range and the corresponding log2 transformation of average fold changes.

Figure 2. The relative expression of selected miRs in LNCaP and LNCaP derived CRPC cell lines

25 ng of total RNA from each cell line was used to measure miR expression levels by qRT-PCR. All of the data was normalized by the U6 expression level and presented as the relative expression level. The relative expression level of each miR in LNCaP was arbitrarily set as 1.0.

Figure 3. The impact of miR-221 and -222 expression levels on the AR mediated transcription in response to the DHT treatment

(A, B) Quantitative analysis of the expression level of PSA and AR in LNCaP (A) and LNCaP-Abl (B) with (+) or without androgen, DHT (-). Total RNAs were isolated from LNCaP that were mock-transfected (−), and transfected with pre-miR-221 (221), pre-miR-222 (222), pre-miR-221&-222 (221&222), or pre-miR-Neg RNA (NegRNA), and from LNCaP-Abl that were mock-transfected (−) and transfected with anti-miR-221 (221), anti-miR-222 (222), anti-miR221&-222 (221&222) or anti-miR-Neg RNA (Neg RNA). Left panels confirm the expression levels of miR-221 and -222 by RT-PCR in each transfected LNCaP (A) and LNCaP-Abl (B) cell lines. The expression of U6 was used as a control. Middle panels show the PSA expression levels analyzed by qRT-PCR. Right panels show the AR expression in each transfected cell line measured by qRT-PCR. (C) Comparison of the DHT induced PSA expression in LNCaP and LAPC-4. Cells were transfected with (+) or without (−) pre-miR-221 or pre-miR-Neg. (D) Effect of anti-androgen treatment on the PSA expression inducted by DHT. LNCaP and LNCaP-Abl were first transfected with Pre-miR-Negative (Neg RNA) or Pre-miR-221 and Anti-miR-Negative (Neg RNA) or Anti-miR-221 or without transfection (Mock) as indicated respectively, and then treated with (+) or without (−) DHT, Casodex and Flutamide. In all experiments, the relative expression levels of PSA and AR in each sample were normalized by the expression level of GAPDH. Values represent the fold differences relative to those in cells without any drug treatment or transfection (Mock), which were set as 1.0. *s indicate that the fold changes of those tranfected samples compared with their corresponding negative controls show a P-value < 0.05 in one-way ANOVA.

Figure 4. Effect of the expression level of miR-221 on the growth of LNCaP and LNCaP-Abl

(A) WST-1 analysis of the growth of LNCaP cells that were transfected with Pre-miR-221, miRNA-precursor-negative control (Neg), mocked transfected (lipo 2000) and kept in medium with (+DHT, solid lines) or without androgen (broken lines). (B) WST-1 analysis of the growth of LNCaP-Abl cells that were transfected with Anti-miR-221, Anti-miRNA inhibitors negative control (Neg), mocked transfected (lipo 2000) and kept in medium with (+DHT, solid lines) or without androgen (broken lines). Triplicate experiments were performed for each set. The data represents mean ± S.D. (n = 3).

Figure 5. Effect of p27/kip1 expression on the DHT induction of the PSA expression in LNCaP

(A). Western blot analysis of p27/kip1 in LNCaP and LNCaP derived CRPC cell lines. The amount of β-actin in each lane is used as a loading control. (B). Western blot analysis of p27/kip1 in LNCaP cells that were mock-transfected (mock) or transfected with Pre-miR-221 (Pre-221), Pre-miR-222 (Pre-222), miRNA precursor-negative control (Neg RNA), p27/kip1 siRNA (p27 siRNA) or pCMV-SOPRT 6-p27/kip1 vector (pCMV-p27). Total proteins were extracted from cells 48 hours after transfection. (C). The impact of p27/kip1 siRNA on the growth of LNCaP. WST-1 assay was used to measure the cell growth. LNCaP cells that were transfected with p27/kip1 siRNA (p27siRNA, open squares), anti-miRNA inhibitors negative control (Neg, triangles), and mocked transfected (lipo 2000, black squares) were cultured in medium with (+DHT, solid lines) or without DHT (broken lines). Triplicate experiments were performed for each set. The data represents mean ± S.D. (n = 3). (D). QRT-PCR of the relative PSA expression level in transfected LNCaP cells as those described in (B). The relative expression level of PSA in each sample was normalized by the expression level of GAPDH. Values represent the fold differences relative to that in mock transfected cells without any drug treatment, which was arbitrarily set as 1.0. *s indicate that the fold changes of those transfected samples compared with their corresponding negative control exhibited a P-value < 0.05 in one-way ANOVA analysis.