Tanshinones suppress AURKA through up-regulation of miR-32 expression in non-small cell lung cancer

Abstract

Tanshinone is the liposoluble constituent of Salia miltiorrhiza, a root used in traditional herbal medicine which is known to possess certain health benefits. Although it is known that tanshinones, including tanshinone I (T1), tanshinone IIA (T2A), and cryptotanshinone (CT), can inhibit the growth of lung cancer cells in vitro, the mechanism under which they act is still unclear. AURKA, an oncogene, encodes a serine-threonine kinase which regulates mitotic processes in mammalian cells. Here, we reported that tanshinones mediate AURKA suppression partly through up-regulating the expression of miR-32. We found that tanshinones could inhibit cell proliferation, promote apoptosis, and impede cell-cycle progression, thus performing an antineoplastic function in non-small cell lung cancer (NSCLC). Additionally, we demonstrated that tanshinones attained these effects in part by down-regulating AURKA, corroborating previous reports. Our results showed that in NSCLC, similar effects were obtained with knock-down of the AURKA gene by siRNA. We also verified that AURKA was the direct target of miR-32. Collectively, our results demonstrated that tanshinones could inhibit NSCLC by suppressing AURKA via up-regulating the expressions of miR-32 and other related miRNAs.

Keywords: AURKA; miRNA; non-small cell lung cancer; tanshinone.

Figure 1. Tanshinone can suppress NSCLC

A. Cell vitality of H1299 cells treated with tanshinone or DMSO was determined by CCK-8 cytotoxicity test. Results are represented as the mean±SEM of OD_450nm. Blank serves as control. In B–C. H1299 cells were respectively treated with T1 4 μM, T2A 4 μM, CT 5 μM or DMSO 5 μM (Control) for 48 h. B. Cell apoptosis situations of H1299 cells were detected by flow cytometry. C. Cell cycle distributions of H1299 cells were detected by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001, vs. control (n = 3). Representative of triplicate experiments was shown.

Figure 2. Tanshinone inhibits NSCLC by down-regulating the expression of AURKA

A. The variation of mRNA and protein of AURKA in H1299 cells respectively treated with T1 4 μM, T2A 4 μM, CT 5 μM or DMSO 5 μM (Control) for 48 h were measured by qRT-PCR and Western blot. In B–E. H1299 cells were transfected with siAURKA or siNC (100nm) and incubated at 37°C for 24 h (96 h for CCK-8 cytotoxicity test ). B. The variation of mRNA and protein of AURKA in H1299 cells were measured by qRT-PCR and Western blot. C. Cell proliferation of H1299 cells were determined by CCK-8 cytotoxicity test. D. Cell apoptosis situations of H1299 cells were detected by flow cytometry. E. Cell cycle distributions of H1299 cells were detected by flow cytometry. **P < 0.01, vs. control (n = 3). Representative of triplicate experiments was shown.

Figure 3. Tanshinone suppresses AURKA partly through up-regulating the expression of miR-32

A. Expression levels of miRNAs in H1299 cells respectively treated with T1 4 μM, T2A 4 μM, CT 5 μM or DMSO 5μM (Control) for 48 h were measured by qRT-PCR. B. Dual luciferase reporter assay. Luciferase reporter constructs containing wild-type or mutated AURKA 3′-UTR were cotransfected with miR-32 mimics or NC mimics into H1299 cells. Relative firefly luciferase expression was normalized to Renilla luciferase. C. The variation of mRNA and protein of AURKA in H1299 cells transfected with miR-32 mimics or NC mimics (100 nm) and incubated at 37°C for 24 h were measured by qRT-PCR and Western blot. *P < 0.05, **P < 0.01, ***P < 0.001, vs. control (n = 3). Representative of triplicate experiments was shown.

Figure 4. miR-32 plays a rule of tumor suppressor gene in NSCLC

In A–C, H1299 cells were transfected with NC mimics or miR-32 mimics (100 nm) and incubated at 37°C for 48 h (96 h for CCK-8 cytotoxicity test). A. Cell proliferation of H1299 cells were determined by CCK-8 cytotoxicity test. B. Cell apoptosis situations of H1299 cells were detected by flow cytometry. C. Cell cycle distributions of H1299 cells were detected by flow cytometry. D. The expression levels of miR-32 in lung cancer cell lines or pulmonary epithelial cell line (control) were measured by qRT-PCR. **P < 0.01, ***P < 0.001, vs. control (n = 3). Representative of triplicate experiments was shown.

Figure 5. Model of identified mechanism how tanshinone suppresses NSCLC

Tanshinone played a role of tumor suppressor in NSCLC by up-regulating several miRNAs to suppress genes which related to the cancer process.