Triptolide induces the expression of miR-142-3p: a negative regulator of heat shock protein 70 and pancreatic cancer cell proliferation

Abstract

Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits PDAC cell growth in vitro and blocks metastatic spread in vivo. Triptolide downregulates HSP70, a molecular chaperone upregulated in several tumor types. This study investigates the mechanism by which triptolide inhibits HSP70. Because microRNAs (miRNA) are becoming increasingly recognized as negative regulators of gene expression, we tested whether triptolide regulates HSP70 via miRNAs. Here, we show that triptolide as well as quercetin, but not gemcitabine, upregulated miR-142-3p in PDAC cells (MIA PaCa-2, Capan-1, and S2-013). Ectopic expression of miR-142-3p inhibited cell proliferation, measured by electric cell-substrate impedance sensing, and decreased HSP70 expression, measured by real-time PCR and immunoblotting, compared with controls. We showed that miR-142-3p directly binds to the 3'UTR of HSP70, and that this interaction is important as HSP70 overexpression rescued miR-142-3p-induced cell death. We found that miR-142-3p regulates HSP70 independently of heat shock factor 1. Furthermore, Minnelide, a water-soluble prodrug of triptolide, induced the expression of miR-142-3p in vivo. This is the first description of an miRNA-mediated mechanism of HSP70 regulation in cancer, making miR-142-3p an attractive target for PDAC therapeutic intervention.

Figure 1. Triptolide-regulated microRNAs in PDAC cells

(A) Heatmap representation of differentially expressed human miRNAs after triptolide (100 nM) treatment (0, 6, 12, 24 or 48 h) in MIA PaCa-2 and S2-013 cells as measured by microarray. Only miRNAs with strong differential expression (t-test between 0 and 24 hour replicate time points, p<0.05, average fold change>5) in at least one of the two time-courses are shown. Log-transformed expression values are shown, compared with the untreated control, for each cell line independently. (B) Triptolide (100 nM) and quercetin (100 μM) treatments induced, whereas gemcitabine (1 μM) treatment had no effect on, miR-142-3p expression (as assessed by real-time PCR) in human MIA PaCa-2, Capan-1, and S2-013 cell lines following 24 h of treatment. Expression of miR-142-3p was normalized against U6. The bars represent mean ± SEM, n=3, *p<0.02 (t test).

Figure 2. Effect of triptolide treatment or microRNA-142-3p mimic overexpression PDAC cell proliferation

(A) Triptolide (100 nM) reduced the rate of cell proliferation as measured in real-time on the Electric Cell-substrate Impedance Sensing (ECIS) instrument in MIA PaCa-2, Capan-1, and S2-013 cell lines. (B) MicroRNA-142-3p mimic (5 nM) reduced the rate of proliferation as measured in all cell lines tested by ECIS. Proliferation rates are representative of n≥4 experiments and average of 4 replicates. Because proliferation rates are normalized to 6 h following transfection, the 0 h time point represents 6 h following transfection. SEM bars (not shown) overlap from 0–15 h, * represents point after which proliferation rates differ.

Figure 3. Effect of triptolide treatment or microRNA-142-3p mimic overexpression on HSPA1B (HSP70) levels

(A) Triptolide treatment (100 nM) and miR-142-3p (5 nM) ectopic expression reduced HSPA1B (HSP70) mRNA expression (as assessed by real-time PCR) in MIA PaCa-2, Capan-1, and S2-013 cell lines following 24 h of exposure. Expression of HSPA1B (HSP70) was normalized against 18S. The bars represent mean ± SEM, n=3, *p<0.02 (t test). (B) MicroRNA-142-3p mimic (5 nM for 48 h) reduced HSP70 protein expression in MIA PaCa-2, Capan-1, and S2-013 cell lines. Expression of HSP70 was normalized against actin.

Figure 4. MicroRNA-142-3p modulates HSPA1B (HSP70) expression by binding to its 3’UTR

(A) Schematic of HSPA1B (HSP70) mRNA showing predicted miR-142-3p interaction site (upper). Seven-nucleotide interaction sequence between wildtype (wt) HSPA1B (HSP70)-3’UTR and miR-142-3p and mutant HSPA1B (HSP70)-3’UTR construct shown (lower). (B) Luciferase reporter assay using HEK-293 cells to demonstrate the direct interaction of miR-142-3p and the 3’UTR of HSPA1B (HSP70). After 24 h, miR-142-3p mimic (10 nM) reduced the ratio of renilla-to-firefly expression but not when the 3’UTR bears two point mutations in the miR-142-3p binding site. (C) HSPA1B (HSP70) ORF (lacking the 3’UTR containing the miR-142-3p binding site) transfection causes overexpression of HSPA1B (HSP70) expression (as assessed by real-time PCR) in MIA PaCa-2. (D) HSPA1B (HSP70) ORF overexpression rescued loss in cell viability caused by miR-142-3p (5 nM) overexpression for 48 h in MIA PaCa-2 cells. The bars represent mean ± SEM, n=3, *or **p<0.05 (t test).

Figure 5. HSF1 and miR-142-3p independently regulate HSPA1B (HSP70) and mediate triptolide-induced suppression of PDAC proliferation

(A) Triptolide (100 nM) treatment for 24 h decreases HSF1 levels, but ectopic expression of miR-142-3p mimic (5 nM for 24 h) has no effect (as assessed by real-time PCR) in MIA PaCa-2, Capan-1, and S2-013 cell lines. Expression of HSF1 was normalized against 18S. (B) Real-time PCR measurement of miR-142-3p following HSF1 silencing (25 nM for 24 h). Expression of miR-142-3p was normalized against U6. (C) HSF1 ORF transfection causes overexpression of HSF1 expression (as assessed by real-time PCR) in MIA PaCa-2. (D) HSF1 ORF overexpression rescues loss in cell viability caused by triptolide (100 nM for 48 h) overexpression in MIA PaCa-2 cells. The bars represent mean ± SEM, n=3, *p<0.05 (t test). (E) Model of triptolide action in PDAC cells.

Figure 6. Effect of Minnelide on miR-142-3p or HSPA1B (HSP70) expression in vivo

(A) Minnelide (0.42 mg/kg daily for seven days i.p.) induces miR-142-3p expression (as assessed by real-time PCR) in three independent human tumor xenografts grown in vivo. Expression of miR-142-3p was normalized against U6. (B) Minnelide reduces HSPA1B (HSP70) mRNA expression (as assessed by real-time PCR) of these same samples in vivo. Expression of HSPA1B (HSP70) was normalized against 18S. The bars represent mean ± SEM, n=3, *p<0.05 (t test).