The Role of the 3' Untranslated Region in the Post-Transcriptional Regulation of KLF6 Gene Expression in Hepatocellular Carcinoma

Abstract

KLF6 is ubiquitously expressed in human tissues and regulates many pathways such as differentiation, development, cellular proliferation, growth-related signal transduction, and apoptosis. We previously demonstrated that KLF6 expression is altered during liver carcinogenesis. More importantly, KLF6 invalidation results in cell cycle progression inhibition and apoptosis of liver cancer cells. On the other hand, enforced expression of KLF6 variant 2 (SV2) induces cancer cell death by apoptosis. Thus, we and others demonstrated that KLF6 and its splicing variants play a critical role in liver cancer. However, little is known on the mechanisms governing KLF6 expression in HCC. In the present work, we asked whether the 3' untranslated region (3'UTR) of the KLF6 mRNA may be responsible for regulation of KLF6 expression in HCC. We found that KLF6 mRNA stability was altered in liver-derived cell lines as compared to cervical cancer-derived cell lines and human embryonic fibroblasts. Interestingly, KLF6 mRNA was highly unstable in liver cancer-derived cell lines as compared to normal hepatocytes. We next cloned the KLF6 mRNA 3'UTR into luciferase-expressing vectors and found that gene expression and activity were strongly impaired in all liver-derived cell lines tested. In addition, we found that most the KLF6 3'UTR destabilisation activity resides between nt 1,835 and nt 2,615 of the KLF6 gene. Taken together, we provide the first steps towards better understanding of the regulation of KLF6 expression in HCC. Further work is needed to identify the factors that bind to KLF6 3'UTR to regulate its expression in liver cancer-derived cell lines.

Figure 1

Organization of the KLF6 gene. (A) Schematic representation of the KLF6 gene, including untranslated regions (UTR), introns (I) and Exons (E); (B) Bioinformatic analysis of KLF6 UTR for AU-rich Elements (highlighted in grey). KLF6 UTR segment #2 is highlighted in bold. AU-rich elements were identified using [23].

Figure 2

Determination of KLF6 mRNA half life in hepatic and non-hepatic cell lines. Hela, HEK, IHH, HepG2 and Hep3B cells were treated with Actiomycin D as described in Experimental Section. KLF6 mRNA expression was quantified by qRT-PCR at the times indicated. Results are mean ± S.D. of three independent experiments. **: p < 0.01.

Figure 3

Effect of KLF6 UTR on gene expression. Hela, IHH, HepG2 and Hep3B cells were transfected for 48 h with plasmid containing KLF6 UTR. Control cells were transfected with psiCHECK2 plasmid. (A) Luciferase activity was determined as described in Experimental Section. Results are mean ± S.D. of three independent experiments. ***: p < 0.001; (B) Luciferase gene expression was measured by qRTPCR in Hela cells following plasmid transfection as described in Experimental Section. Results are mean ± S.D. of three independent experiments. **: p < 0.01.

Figure 4

Molecular dissection of KLF6 UTR. (A) Representation of the psiCHECK2-derived constructs containing segment 1 (nt 1,114–1,834), 2 (nt 1,835–2,615), 3 (nt 2,616–3,506) or 4 (3,507–4,807) of the KLF6 3'UTR. Segment size is indicated in bold. Hela, IHH, HepG2 and Hep3B cells were transfected for 48 h with plasmid encoding for the indicated KLF6 UTR segments. Control cells were transfected with psiCHECK2 plasmid; (B) Luciferase activity was determined as described in Experimental Section. Results are mean ± S.D. of three independent experiments. **: p < 0.01; (C) Luciferase gene expression was measured by qRTPCR in Hela cells following plasmid transfection as described in Experimental Section. Results are mean ± S.D. of three independent experiments. **: p < 0.01.