Four microRNAs promote prostate cell proliferation with regulation of PTEN and its downstream signals in vitro

Abstract

Background: Phosphatase and tensin homologue (PTEN), as a tumor suppressor, plays vital roles in tumorigenesis and progression of prostate cancer. However, the mechanisms of PTEN regulation still need further investigation. We here report that a combination of four microRNAs (miR-19b, miR-23b, miR-26a and miR-92a) promotes prostate cell proliferation by regulating PTEN and its downstream signals in vitro.

Methodology/principal findings: We found that the four microRNAs (miRNAs) could effectively suppress PTEN expression by directly interacting with its 3' UTR in prostate epithelial and cancer cells. Under-expression of the four miRNAs by antisense neutralization up-regulates PTEN expression, while overexpression of the four miRNAs accelerates epithelial and prostate cancer cell proliferation. Furthermore, the expression of the four miRNAs could, singly or jointly, alter the expression of the key components in the phosphoinositide 3-kinase (PI3K)/Akt pathway, including PIK3CA, PIK3CD, PIK3R1 and Akt, along with their downstream signal, cyclin D1.

Conclusions: These results suggested that the four miRNAs could promote prostate cancer cell proliferation by co-regulating the expression of PTEN, PI3K/Akt pathway and cyclin D1 in vitro. These findings increase understanding of the molecular mechanisms of prostate carcinogenesis and progression, even provide valuable insights into the diagnosis, prognosis, and rational design of novel therapeutics for prostate cancer.

Figure 1. Identification of miRNA targets in the PTEN 3’ UTR.

A. Diagram of the PTEN mRNA. Predicted binding sites of four miRNAs (miR-19b, miR-23b, miR-26a and miR-92a) in the 3’ UTR region of PTEN. B. Schematic map of luciferase reporter vectors for measuring the efficacy of the miRNA regulation. C. PNT1B cells were co-transfected the luciferase reporter vectors, which harbored either miR-target positive control (PC) or full-length PTEN 3’ UTR (PU), with relevant anti-miRNA inhibitors or negative control (NC) for the luciferase reporter assay. D. Luciferase reporter vectors containing relevant truncated fragments (PUA, PUB, PUC or PUD) of PTEN 3’ UTR were co-transfected with specific anti-miRNA inhibitors or negative control for the luciferase reporter assay. *indicates a significant difference from the control (p<0.01).

Figure 2. Identification of the endogenous expression of four miRNAs and PTEN in multiple prostate cell lines.

A. The endogenous expression of four miRNAs in prostate cancer, BPH and normal prostate epithelial cell line was measured by qRT-PCR. B. The endogenous expression of PTEN in prostate cancer, BPH and normal prostate epithelial cell line was measured by qRT-PCR and western blot. The relative quantification of PTEN protein was measured by densitometry.

Figure 3. The four miRNAs co-regulate PTEN expression.

Overexpression of miR-19b, miR-23b, miR-26a or miR-92a repressed PTEN expression in DU145 (A) and PNT1B (C), while neutralization of each miRNA alone up-regulated PTEN expression in DU145 (B) and PNT1B (D). The expression of PTEN was measured by qRT-PCR and western blot. The relative quantification of PTEN protein was measured by densitometry.

Figure 4. Endogenous expression of p110α, p100β, p110δ, p85 and Akt in DU145 and PNT1B cell lines.

A. The mRNA expressional level of the 5 genes was measured by qRT-PCR. B. the protein expressional level was measured by western blot. The relative quantification of PTEN protein was measured by densitometry.

Figure 5. The alteration of mRNA expression level in miRNA overexpressed DU145 and PNT1B cells.

The mRNA expression level of p110δ (A), p110α (B), p85 (C) and Akt (D) was measured by qRT-PCR and appeared to be altered when the relevant miRNA was overexpressed in DU145 or PNT1B cells.

Figure 6. The alteration of mRNA expression level in miRNA inhibited DU145 and PNT1B cells.

The mRNA expression level of p110δ (A), p110α (B), p85 (C) and Akt (D) was measured by qRT-PCR and appeared to be altered when the relevant miRNA was inhibited in DU145 or PNT1B cells.

Figure 7. The four miRNAs co-regulate cyclin D1 expression.

Overexpression of miR-19b, miR-23b, miR-26a or miR-92a up-regulated cyclin D1 expression in DU145 (A) and PNT1B (C), while neutralization of each miRNA alone repressed the cyclin D1 expression in DU145 (B) and PNT1B (D). The expression of cyclin D1 was measured by qRT-PCR and western blot. The relative quantification of PTEN protein was measured by densitometry.

Figure 8. Cell proliferation assay of DU145 and PNT1B cells after overexpression of the four miRNAs.

Overexpression of miR-19b, miR-23b, miR-26a, or miR-92a stimulated cell proliferation in DU145 (A) and PNT1B (B). *indicates a significant difference from the control (p<0.01).

Figure 9. Schematic model of a putative mechanism for prostate cancer cell proliferation via microRNA regulation of PTEN and its downstream signals.

Increased amount of miR-19b, miR-23b, miR-26a and miR-92a may impose inhibitory effects on PTEN, PI3K/Akt signaling components, and cyclin D1, respectively. The expression level of PI3-kinase, Akt and cyclin D1 was associated with the expression level of relevant upstream regulators and the regulation of the relevant miRNAs.