MiR-9 modulates and predicts the response to radiotherapy and EGFR-inhibition in HNSCC (2021)
Figure 4

Figure 4. miR-9 regulates Sp1 expression by targeting KLF5 transcription factor.

A. Table reporting the qRT-PCR analyses of genes expression in control (shCTR) and antimiR-9 FaDu cells, as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance.

B. On the left, WB analyses of KLF5 and Sp1 expression in control (shCTR) and antimiR-9 FaDu. Histone H3 expression was used as loading control. On the right, protein quantification analyses of KLF5 and SP1 expression normalized on H3 loading control. Data represent the mean (±SD) of three biological replicates and unpaired t test was used to verify the statistical significance.

C. Schematic design of the KLF5 3'-UTR. To test the potential miR-9 binding on KLF5 3'-UTR, four vectors were generated: WT (Wild Type containing both the seed sites), mut A or mut B (mutated for one single binding site) and mut A+B (mutated in both seed site). The predicted seed sites for miR-9 binding sites are shown in red when present and in blue when mutated.

D. Graph reporting the normalized Luciferase activity of wild type (WT) or mutated KLF5 3'-UTR described in C in control (shCTR) or antimiR-9 FaDu cells, as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance.

E. qRT-PCR analyses of KLF5 (left) and Sp1 (right) expression in FaDu cells transfected with control or KLF5 vectors. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance.

F. Upper panel shows WB analyses of KLF5 and Sp1 expression in FaDu cells. Actin expression was used as loading control. Lower graph shows the SP1 protein quantification normalized over actin expression. Data represents the mean (±SD) of three independent experiments and unpaired t test was used to verify the statistical significance.

G. Graph reporting the normalized Luciferase activity of Sp1 promoter fragments in FaDu transfected with pcDNA control or KLF5 vectors, as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance.

H. Chromatin immunoprecipitation (ChIP) assay performed on FaDu cells. Upper left panel shows WB analysis reporting KLF5 expression in the immunoprecipitation achieved using anti-KLF5 or control (IgG) antibodies used in the ChIP assay. Vinculin expression was used as loading control. Bottom left panel shows a typical image of amplified PCR fragments using ChIP DNA or genomic DNA, as indicated. Right graph reports the binding of KLF5 to the indicated region of Sp1 promoter expressed as signal relative to input in two independent immunoprecipitations (ChIP1 and ChIP2) using the KLF5 ab. IgG was used as negative control on the same chromatin. Primer pairs used to amplify the different regions of Sp1 promoter are depicted in the schema provided in Fig. 3I.

In the figure A.U. = Arbitrary Units; *p<0.05; **p<0.01; ***p<0.001.