miR-183 inhibits TGF-beta1-induced apoptosis by downregulation of PDCD4 expression in human hepatocellular carcinoma cells

Abstract

Background: In recent years, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. In our previous studies, a set of miRNAs were revealed to be dysregulated in HCC tissues. However, the functions of these miRNAs in HCC remain largely undefined.

Methods: The expression profiles of miR-183 were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was used to screen the potential target genes of miR-183. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-183 in hepatoma cells was examined.

Results: Among the 25 HCC samples analyzed, microRNA-183 was significantly up-regulated (twofold to 367-fold) in 17 samples compared with the matching nontumoral liver tissues. Programmed cell death 4 (PDCD4) was identified as the target gene of miR-183. Moreover, PDCD4 is a proapoptotic molecule involved in TGF-beta1-induced apoptosis in human HCC cells, we found that miR-183 transfectants were resistant to apoptosis induced by TGF-beta1.

Conclusions: We conclude that miR-183 can inhibit apoptosis in human HCC cells by repressing the PDCD4 expression, and miR-183 may play an important role in HCC development.

Figure 1

miR-183 inhibits the mRNA and protein expression of PDCD4. (A) HepG2 cells transfected with miR-183 compared with negative control transfected cells for 48 hours, the mRNA expression levels of three potential target genes were detected by qRT-PCR. (B) PDCD4 protein was detected by western blot using anti-PDCD4 antibody and anti-β-actin antibody as a control. (C) Correlation between expression levels of miR-183 and PDCD4. U indicates up-regulation of miR-183 in HCC tissues, and NU indicates down-regulation and no significant change of miR-183 in HCC tissues. The mean value is shown as a horizontal line. Statistical analysis was performed with Student's t-test.

Figure 2

miR-183 can act on the 3'-UTR of PDCD4 directly. (A) The target site of miR-183 in PDCD4 3'UTR is conserved (shown in red) in many species. The PDCD4 3'-UTR mutant is identical to the wild-type, except that it has four point substitutions (red) disrupting pairing to miR-183 seed. (B) and (C) Luciferase assays indicated that miR-183 downregulated the expression of PDCD4 by targeting putative target site. NC RNA, miR-183, and miR-183 inhibitor were cotransfected with a modified pGL-3 control vector containing wild-type PDCD4 3'-UTR or mutant, respectively.

Figure 3

miR-183 inhibits TGF-β1-induced PDCD4 expression in Huh7 cells. Cells were transfected with NC RNA, siPDCD4, or miR-183 respectively, after 24 hours, TGF-β1 (5 ng/mL) was added. Cells were harvested 24 hours later, and were subsequently processed for qRT-PCR (A) and Western blot (C) analysis by hybridization with antibodies against PDCD4 and β-actin. At the same time, the effect of TGF-β1 on miR-183 was examined (B). Huh7 cells were treated with TGF-β1 (5 ng/mL) for 48 h, then harvested and processed for qRT-PCR analysis.

Figure 4

miR-183 transfectants were resistant to apoptosis induced by TGF-β1. (A) DAPI assay. Huh7 cells were transfected with NC RNA, siPDCD4, or miR-183 respectively, after 24 hours, TGF-β1 (5 ng/mL) was added. Cells were further incubated for 48 hours, fixed, and stained with DAPI. *P < 0.05 compared with Huh7 cells transfected with NC RNA. (B) Annexin-V assays. Huh7 cells were transfected and cultured as described in (A), then collected by trypsinization followed by centrifugation and stained with FITC conjugated Annexin-V and PI. (C) miR-183 and PDCD4 expression vector were cotransfected into Huh7 cells, after 24 hours, TGF-β1 (5 ng/mL) was added. Cells were further incubated for 48 hours, fixed, and stained with DAPI. * P < 0.05 compared with Huh7 cells transfected with control pCMV vector.