A circular RNA, circSMARCA5, inhibits prostate cancer proliferative, migrative, and invasive capabilities via the miR-181b-5p/miR-17-3p-TIMP3 axis

Abstract

SMARCA5 (circSMARCA5) is involved in the occurrence of different cancers, but its role in prostate cancer carcinogenesis and metastatic transformation remains elusive. Thus, we evaluated the circSMARCA5 functional relevance in prostate cancer and its associated molecular mechanism. First, circSMARCA5 expression and function in this cancer were evaluated. To determine the miR-181b-5p/miR-17-3p target and clarify how circSMARCA5 regulates the miR-181b-5p-TIMP3 and miR-17-3p-TIMP3 axis, RNA immunoprecipitation, biotin-coupled microRNA capture, luciferase reporter, Western blot, and quantitative real-time PCR assays were employed. In primary and metastatic prostate cancer tissues, circSMARCA5 was significantly downregulated compared with normal controls. Functionally, circSMARCA5 exhibited a suppressive effect on prostate cancer cells' metastasis and growth. At the molecular level, circSMARCA5 could affect the tissue inhibitor of metalloproteinases 3 (TIMP3) expression through miR-181b-5p or miR-17-3p interactions. Moreover, lysine acetyltransferase 5 (KAT5) induced circSMARCA5 biogenesis and regulated the miR-181b-5p-TIMP3 and miR-17-3p-TIMP3 axis. These results suggested that targeting circSMARCA5-miR-181b-5p-TIMP3 and circSMARCA5-miR-17-3p-TIMP3 axis might be a novel therapeutic strategy for prostate cancer.

Keywords: TIMP3; circSMARCA5; miR-17-3p; miR-181b-5p; prostate cancer.

Figure 1

circSMARCA5 expression in prostate tumors. (A) The circSMARCA5 expression pattern in prostate cancer samples and benign controls was determined by qRT-PCR. (B) circSMARCA5 levels were measured in RWPE-1, DU145, LNCaP, and PC3 cells. *, **, *** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively. Assays were performed at least three times.

Figure 2

circSMARCA5 inhibits DU145 cell proliferative, migrative, and invasive capabilities. circSMARCA5 was upregulated or downregulated in DU145 cells. (A) circSMARCA5 expression in the cells with indicated treatments. (B) DU145 cells’ growth with different treatments. The Transwell assay results showed the migration (C, D) and migration (C, E) of DU145 cells after indicated treatment. Magnification ×100. *, **, *** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively. Assays were performed at least three times.

Figure 3

circSMARCA5 inhibits PC3 cell proliferative, migrative, and invasive capabilities. circSMARCA5 was upregulated or downregulated in PC3 cells. (A) The qRT-PCR results showed circSMARCA5 level in PC3 cells with indicated treatment. (B) PC3 cells’ growth with different treatments. The Transwell assay results showed the migration (C, D) and migration (C, E) of PC3 cells after indicated treatment. Magnification ×100. *, **, *** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively. Assays were performed at least three times.

Figure 4

circSMARCA5 suppresses tumor proliferative and invasive potential in vivo. Wild-type and circSMARCA5-overexpressed DU145 cells were injected into nude recipient mice. Every week, information about tumor size was collected. The tumor tissues were removed and the weight was determined four weeks after injection. Representative mice and resected tumors images (A, B). (C, D) Tumor weight and volume. (E) IHC staining of EMT markers, including E-cadherin, vimentin, and N-cadherin. Magnification ×20. (F) H&E staining and lung microscopic nodules quantification of each group. Magnification ×20. *, **, *** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively.

Figure 5

circSMARCA5 sponged miR-181b-5p/miR-17-3p to regulate TIMP3. (A) qRT-PCR for the pulled-down circSMARCA5. (B) qRT-PCR for circSMARCA5 enrichment in DU145 cell lysates. (C) qRT-PCR for miR-181b-5p and miR-17-3p. (D) Luciferase activities in the cells with indicated treatments. (E) Correlation between circSMARCA5 expression and miR-181b-5p, or miR-17-3p, in prostate tumor tissues. (F) Luciferase activities in the cells with indicated treatments. (G) Western blot for TIMP3 expression after overexpression or downregulation of miR-17-3p. (H) Western blot for the expression of TIMP3 after circSMARCA5 overexpression, or downregulation. *, **, *** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively. Assays were performed at least three times.

Figure 6

circSMARCA5 suppresses proliferative and invasive potential via the miR-181b-5p/miR-17-3p-TIMP3 axis. (A, B) circSMARCA5 overexpressing vectors were transfected into DU145 cells with and without miR-17-3p mimic. Cell proliferation, migration, and invasion were determined. (C, D) circSMARCA5 overexpressing vectors were transfected into DU145 cells with and without miR-181b-5p mimic. Cell proliferation, migration, and invasion were measured. *, **, *** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively. Assays were performed at least three times.

Figure 7

KAT5 induces circSMARCA5 production. (A) The KAT5 expression pattern in RWPE-1, DU145, LNCaP, and PC3 cells. (B) Detection of pulled-down circSMARCA5 by qRT-PCR. (C) qRT-PCR of KAT5, miR-181b-5p, TIMP3, and miR-17-3p in DU145 cells with and without KAT5 overexpression. (D) U145 cells were transfected with circSMARCA5-expressing vector and KAT5-specific siRNA, alone or in combination. Migration and invasion were determined by Transwell assays. *, **, *** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively. Assays were performed at least three times.