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Fig. 5 | Cancer Cell International

Fig. 5

From: miR-320a/SP1 negative reciprocal interaction contributes to cell growth and invasion in colorectal cancer

Fig. 5

SP1 transcriptionally suppressed miR-320a expression to promote CRC progression in vitro. a qPCR experiments showed that miR-320a is downregulated following SP1 upregulation induced by the SP1-expression vector. b A diagram showing the predicted SP1 binding sites within the miR-320a promoter. c Sequence information of the eight putative SP1 binding sites is shown. d Luciferase reporter assay was performed in SW480 cells using different versions of the miR-320a promoter created by sequential deletion, as indicated by the schematic drawing. Numbers indicate nucleotide positions with respect to + 1 as the transcriptional start site. The region 250 bp upstream of the TSS of miR-320a was responsible for the SP1-incuded decrease in luciferase activity *P = 0.000; pmiR-320a: wild-type promoter sequence of miR-320a. e The 250 bp upstream of the miR-320a promoter predictively contained three SP1 binding sites. A mutant version of the luciferase reporter created by deletion mutation of the three binding sites was generated along with its wild-type counterpart. SP1 reduced luciferase activity by recognizing the wild-type 250 bp of the miR-320a promoter (P = 0.000); this inhibition was abrogated by the mutated counterpart. n.s., not significant. Another rescue experiment was conducted by introducing miR-320a mimics into SP1-expressing SW480 and SW620 cells. f Immunoblotting assays showed that ectopic miR-320a expression successfully suppressed SP1 expression in the presence of the SP1-expressing vector. Normalized intensity of each immunoblot was analyzed and is annotated below the stripes. Consequently, SP1 promoted cell growth (g) (*P < 0.05; **P < 0.01), colony formation (h) and invasion (i) in SW480 and SW620 cells, which was partly abrogated by ectopic miR-320a expression

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