PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells

Abstract

The regulatory transcriptional factor PATZ1 is constantly downregulated in human thyroid cancer where it acts as a tumour suppressor by targeting p53-dependent genes involved in Epithelial-Mesenchymal Transition and cell migration. The aim of the present work was to elucidate the upstream signalling mechanisms regulating PATZ1 expression in thyroid cancer cells. The bioinformatics search for microRNAs able to potentially target PATZ1 led to the identification of several miRNAs. Among them we focused on the miR-29b since it was found upregulated in rat thyroid differentiated cells transformed by the Ha-Ras oncogene towards a high proliferating and high migratory phenotype resembling that of anaplastic carcinomas. Functional assays confirmed PATZ1 as a target of miR-29b, and, consistently, an inverse correlation between miR-29b and PATZ1 protein levels was found upon induction of Ha-Ras oncogene expression in these cells. Interestingly, restoration of PATZ1 expression in rat thyroid cells stably expressing the Ha-Ras oncogene decreased cell proliferation and migration, indicating a key role of PATZ1 in Ras-driven thyroid transformation. Together, these results suggest a novel mechanism regulating PATZ1 expression based on the upregulation of miR-29b expression induced by Ras oncogene.

Figure 1. Validation of PATZ1 as a target of miR-29b.

(a) predicted miR-29b/ PATZ1 alignment, according to microRNA.org web system. mirSVR (cutoff 0.1 or lower) and PhastCons (cutoff 0.57 or higher) are downregulation and conservation scores, respectively. (b) Western blot using anti-PATZ1 on HEK293 total cellular extracts collected 72 h after transfection with increasing amount (50–100 nM) of synthetic miR-29b precursor or scramble (100 nM) oligonucleotide. Vinculin was used for normalization. Relative expression levels, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. (c) qRT-PCR on total RNA from HEK293 cells previously transfected with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of four independent experiments performed in duplicate is reported. (d) Luciferase assay on HEK293 cells co-transfected with the Luc-PATZ1-3′UTR and pCMV renilla reporter vectors along with 100 nM synthetic miR-29b precursor or scramble oligonucleotide. Relative firefly luciferase activity levels were normalized for renilla luciferase activity and analysed relatively to scramble-transfected cells, which were set to 1. The mean ± SE of four independent experiments performed in duplicate is reported. ****P < 0,0001.

Figure 2. miR-29b targeting of PATZ1 in rat thyroid cells.

(a) Western blot using anti-PATZ1 on PC Cl3 total extracts previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. Three major specific bands were observed (arrows). Vinculin was used for normalization. Densitometric analysis by Image J software was applied on the gel: Relative expression levels of PATZ1, compared to scramble-transfected control and normalized with respect to vinculin, are indicated on the bottom. Black lines delineate the boundary between not contiguous lanes of the same gel. (b) qRT-PCR on total RNA from PC Cl3 and FRTL-5 cells previously transfected with synthetic miR-29b precursor or scramble oligonucleotide. PATZ1 mRNA levels were normalized for endogenous G6PD levels. The mean ± SE of three independent experiments performed in duplicate for each cell line is reported. *P < 0.05 compared with scramble transfected control. (c) qRT-PCR on total RNA from FRTL-5-Ras inducible cells treated with Tamoxifen at the indicated times and FRTL-5 clones V27 and V29 stably expressing the Ha-Ras V12 oncogene. miR-29b and PATZ1 expression levels were normalized for endogenous U6 and G6PD levels, respectively. The mean ± SE of one experiment performed in triplicate and three independent experiment performed in duplicate is reported for miR-29b and PATZ1, respectively. **P < 0.01; ***P < 0.001; ****P < 0.0001 compared with mock-treated or mock-transfected cells for FRTL5-Ras inducible cells and stable clones, respectively. (d) Western blot using anti-PATZ1 on total extracts from cell as in C. The three specific bands corresponding to different PATZ1 isoforms are indicated by arrows. Normalized expression levels of PATZ1, as assessed by densitometric analysis on the upper PATZ1-specific band with respect to vinculin expression, are indicated on the bottom.

Figure 3. Inhibition of proliferation in PATZ1-transfected FRTL5-Ras cells.

(a) Western blot analysis of PATZ1 in selected clones and mass populations (MP) of FRTL5-Ras-V29 cells transfected with PATZ1 (FR-PA) or the backbone vector (FR-BV). Asterisks indicate the mass populations and/or clones selected for the following experiments. (b) Growth curves on different stably expressing PATZ1 cell clones and/or mass populations of FRTL5-Ras cells (FRTL5-Ras-PATZ1) compared to controls expressing the empty vector (FRTL5-Ras-ctrl). Mean values ± SE of three clones or mass populations for each cell line are reported. FRTL5 and parental FRTL5-Ras-V29 (FRTL5-Ras) cells were also analysed for comparison of the results. (c) Flow cytometry (FACS) analysis on proliferating cells. Mean values ± SE of at least 3 independent experiments performed in two independent cell clones for each cell line are reported as percentages of the cell cycle distribution. *P < 0.05; **P < 0.01.

Figure 4. Inhibition of migration in PATZ1-transfected FRTL5-Ras cells.

(a) Representative images of a wound healing assay in control (FRTL-Ras-ctrl) and PATZ1-expressing FRTL5-Ras cells at 0, 24 h and 96 h after a confluent cell monolayer was wounded. (b) Percent of open wound, calculated as mean values ± SE of three independent cell clones or mass populations for each cell line, at 0 h, 24 h, 48 h, 72 h and 96 h after wound scratch. ***P < 0.001. (c) Representative images of a Transwell assay performed on FRTL5, FRTL5-Ras-ctrl and FRTL5-Ras-PATZ1 cells. Dark cells (stained with crystal violet) represent migrating cells. (d) Number of migrating cells expressed as mean values ± SE of three clones for each transfected construct. ***P < 0.001.