MicroRNA-205 inhibits Src-mediated oncogenic pathways in renal cancer - PubMed (original) (raw)

. 2011 Apr 1;71(7):2611-21.

doi: 10.1158/0008-5472.CAN-10-3666. Epub 2011 Feb 17.

Sharanjot Saini, Altaf A Dar, Hiroshi Hirata, Varahram Shahryari, Yuichiro Tanaka, Soichiro Yamamura, Koji Ueno, Mohd Saif Zaman, Kamaldeep Singh, Inik Chang, Guoren Deng, Rajvir Dahiya

Affiliations

MicroRNA-205 inhibits Src-mediated oncogenic pathways in renal cancer

Shahana Majid et al. Cancer Res. 2011.

Abstract

The Src family of protein kinases (SFK) plays key roles in regulating fundamental cellular processes, including cell growth, differentiation, cell shape, migration, and survival, and specialized cell signals in various malignancies. The pleiotropic functions of SFKs in cancer make them promising targets for intervention. Here, we sought to investigate the role of microRNA-205 (miR-205) in inhibition of Src-mediated oncogenic pathways in renal cancer. We report that expression of miR-205 was significantly suppressed in renal cancer cell lines and tumors when compared with normal tissues and a nonmalignant cell line and is correlated inversely with the expression of SFKs. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR (untranslated region) sequences complementary to either Src, Lyn, or Yes, which was abolished by mutations in these 3'-UTR regions. Overexpression of miR-205 in A498 cells reduced Src, Lyn, and Yes expression, both at mRNA and protein levels. Proliferation of renal cancer cells was suppressed by miR-205, mediated by the phospho-Src-regulated ERK1/2 pathway. Cell motility factor FAK (focal adhesion kinase) and STAT3 activation were also inhibited by miR-205. Transient and stable overexpression of miR-205 in A498 cells resulted in induction of G₀/G₁ cell-cycle arrest and apoptosis, as indicated by decreased levels of cyclin D1 and c-Myc, suppressed cell proliferation, colony formation, migration, and invasion in renal cancer cells. miR-205 also inhibited tumor cell growth in vivo. This is the first study showing that miR-205 inhibits proto-oncogenic SFKs, indicating a therapeutic potential of miR-205 in the treatment of renal cancer.

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Figures

Figure 1

Figure 1. miR-205 expression is downregulated in renal cancer and inversely correlated with expression of Src, Lyn and Yes

A) Quantitative RT-PCR analysis of miR-205 expression levels in renal cancer and nonmalignant cell lines. B) miR-205 expression in a cohort of renal cancer and normal tissue samples. C) The miR-205 seed sequence is complementary to the 3’UTR of Src, Lyn and Yes. D-E) Src, Lyn and Yes mRNA and protein expression in human renal cancer and nonmalignant cell lines. *p<0.05

Figure 2

Figure 2. Src, Lyn and Yes 3’UTRs are targets of miR-205

A) The 3’UTR sequences of Src, Lyn and Yes and mutant sequences that abolished binding. B-D) Luciferase assays showing decreased reporter activity after co-transfection of either Src-3’UTR, Lyn-3’UTR or Yes-3’UTR with miR-205 in A498 cells. The mutant 3’ UTRs of either gene had no effect on reporter activity. *p<0.05

Figure 3

Figure 3. miR-205 suppresses Src, Lyn and Yes expression and regulates the ERK1/2 pathway

A) Relative miR-205 expression level. B-C) Quantitative RT-PCR and Western blot analysis showing decreased Src, Lyn and Yes expression. D) Western analysis showing a decrease in the ERK1/2 pathway, cyclin D1, cMyc, a decrease in phosphoSTAT3, and FAK in miR-205 transfected A498 cells. E) Relative miR-205 expression in anti-miR-205 transiently transfected A498 cells F) Western blot and qRT-PCR analysis showing that Src, Lyn and Yes expression was rescued in cells transfected with anti-miR-205.

Figure 4

Figure 4. Transient transfection of miR-205 inhibits renal cancer cell proliferation, colony formation, migration, invasion, and induces apoptosis and cell cycle arrest in A498 cells

A) Proliferation of A498 cells after miR-205 transfection was significantly reduced compared to cont-miR. B) miR-205 overexpression significantly inhibits colony formation of A498 cells. C) Wound healing and migration assays of A498 cells transfected with miR-205. D) Invasion assay shows a significant decrease in the number of invading A498 cells transfected with miR-205. E) Cell cycle analysis showing an increase in the G0/G1 phase of A498 cells overexpressing miR-205. F) Apoptosis assay showing induction of apoptosis by miR-205. *p<0.05.

Figure 5

Figure 5. Knock-down of Src by small interfering RNA (siRNA)

A) Relative Src mRNA levels assessed by qRT-PCR in A498 cells transfected with 50nM siRNA duplexes (S-1, S-2 and S-3) and a nonsilencing siRNA duplex (Control). Src protein levels were assessed by Western blot in A498 cells transfected with 50nM siRNA duplexes and a nonsilencing siRNA duplex. B) Proliferation of A498 cells after S-1 transfection was significantly reduced compared to control. C) A significant decrease was observed in the migratory capability of A498 cells after siRNA (S-1) transfection compared to Control. Invasion assay shows a significant decrease in the number of invading A498 cells transfected with S-1. D) Cell cycle analysis showing an increase in the G0/G1 phase of A498 cells transfected with S-1. Apoptosis assay showing induction of apoptosis after Src knockdown by S-1. *p<0.05.

Figure 6

Figure 6. Attenuation of miR-205 expression by anti-miR-205 in HK-2 cells

A) Relative miR-205 expression. B) HK-2 cells had increased proliferation after anti-miR-205 transfection compared to the anti-miR-Control (Cont-miR). C-D) Migration and invasion assays. *p<0.05

Figure 7

Figure 7. miR-205 inhibits tumor growth in vivo. *p<0.05

A) Tumor volume following subcutaneous injection of stable A498 cells expressing miR-205 was significantly reduced. B) Tumor volume following intratumoral injection of Cont-miR or miR-205 precursor into established tumors. *p<0.05.

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