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. 2021 Jan 1;11(4):1732–1752. doi: 10.7150/thno.45302

Figure 3.

Figure 3

Knockdown of Estrogen-induced circPGR inhibits ER-positive breast cancer cell growth and tumorigenesis. (A) Diagram showing the two circPGRs, circPGR (3) and circPGR (5), with designable siRNAs targeting junction regions. (B, C) MCF7 cells cultured in stripping medium were transfected with control siRNA (si-CTL) or siRNA specifically targeting circPGR (3) (si-circPGR (3)) (B) or circPGR (5) (si-circPGR (5)) (C) and treated with or without estrogen (E2, 10-7 M, 6 h) followed by standard PCR and DNA electrophoresis to visualize the knockdown efficiency of si-circPGR (3) and si-circPGR (5). Actin was served as a loading control. The size of PCR products was shown as indicated. bp: base pair. (D, E) MCF7 cells cultured in stripping medium were transfected with control siRNA (si-CTL) or siRNA specifically targeting circPGR (3) (si-circPGR (3)) or circPGR (5) (si-circPGR (5)) and treated with or without estrogen (E2, 10-7 M, 6 h) for duration as indicated, followed by cell proliferation assay (D) and FACS analysis (E) (± s.e.m., *P < 0.05, **P < 0.01). (F) MCF7 cells were infected with lenti-viral control shRNA (sh-CTL) or two independent shRNAs specifically targeting circPGR (sh-circPGR#1 and sh-circPGR#2) followed by colony formation assay. (G) Quantification of the crystal violet dye as shown in (F) (± s.e.m., ***P < 0.001). (H) MCF7 cells as described in (F) was subjected to RNA extraction and RT-qPCR analysis to examine the expression of circPGR. Data shown was the relative fold change compared to control samples (sh-CTL) after normalization to actin (± s.e.m., **P < 0.01, ***P < 0.001). (I, K) MCF7 cells transfected with control siRNA (si-CTL) or two independent siRNAs specifically targeting circPGR (si-circPGR#1 and si-circPGR#2) for 48 h were both re-seeded at full confluence and then subjected to wound healing (I) or trans-well (K) assay. (J, L) Quantification of wound closure (J) and number of colonies (L) as shown in (I) and (K), respectively (± s.e.m., *P < 0.05, ***P < 0.001). (M) MCF7 cells infected with lenti-viral control shRNA (sh-CTL) or two independent shRNAs specifically targeting circPGR (sh-circPGR#1 and sh-circPGR#2) were injected subcutaneously into female BALB/C nude mice for xenograft experiments. (N) Tumor weight as shown in (M) (± s.e.m., *P < 0.05, **P < 0.01).