Hsa-miR-3178/RhoB/PI3K/Akt, a novel signaling pathway regulates ABC transporters to reverse gemcitabine resistance in pancreatic cancer – Molecular Cancer

P-gp, BCRP and MRP1 proteins were overexpressed in gemcitabine-resistant pancreatic cancer tissues and cells.

ABC transporter superfamily has seven (A to G) subfamilies with a total of 48 members, and is a special class of proteins in the cell membrane [31]. Studies have shown that ABC transporters play an important role in gemcitabine chemotherapy resistance in PC [31, 32]. Among them, P-gp, BCRP and MRP1 are the recognized molecules that contributed to the development of multidrug resistance [4, 33]. In this study, IHC staining further confirmed that P-gp, BCRP, MRP1 were overexpressed in gemcitabine-resistant compare with gemcitabine-sensitive tissues (Fig. 1 A). Western blotting also showed that their expressions in gemcitabine-resistant PANC-1-GEM cells were higher than that in gemcitabine-sensitive PANC-1 cells (Fig. 1 B). The expression levels of P-gp, BCRP, and MRP1 were also markedly upregulated in pancreatic cancer tissues as compared to the pancreatic non-tumor tissues (Figs. 1I-N). These findings were validated by data from the Cancer Genome Atlas dataset (TCGA) and the Genotype-Tissue Expression (GTEx) (Fig. 1O-Q).

P-gp, BCRP and MRP1 were overexpressed in gemcitabine-resistant pancreatic cancer tissues and cells. Docking simulation revealed gemcitabine binds to the substrate binding site of P-gp, BCRP and MRP1 proteins. A IHC staining of P-gp, BCRP, MRP1 in pancreatic cancer gemcitabine-resistant and gemcitabine-sensitive tissues, 200x. Scale bar, 50 μm. B Western blotting on the expression of P-gp, BCRP, MRP1 in the indicated cells. β-actin was used as a loading control. C-H Interaction between gemcitabine and human P-gp, BCRP and MRP1 protein models. Details of the best-scoring pose of gemcitabine in the drug binding pocket of P-gp (C), BCRP (E) and MRP1 (G) binding pocket. 2D diagram of the interaction between gemcitabine and P-gp (D), BCRP (F) and MRP1 (H) binding pocket. The cavity of 20 Å was selected as the docking active region, and the docking calculation was carried out with standard parameters. Purple arrows represent hydrogen bonding, and green dash lines represent pi-pi interactions. I-N IHC staining demonstrated the P-gp (I, L), BCRP (J, M) and MRP1 (K, N) overexpression in PC tissues compared to the pancreatic non-tumor tissues. O-Q The mRNAs expression profile showed P-gp (O), BCRP (P) and MRP1 (Q) overexpression in pancreatic cancer tissue as compared to non-tumor tissue obtained from the public database (TCGA and GTEx). Data are presented as mean ± SD; *P < 0.05. IHC: immunohistochemistry

Gemcitabine binds to the substrate binding site of P-gp, BCRP and MRP1 proteins

Molecular docking simulation was performed to explore whether gemcitabine interacted with P-gp, BCRP and MRP1 proteins. The docking analysis showed that gemcitabine binds to these ABC transporter proteins. The affinity scores of interactions between gemcitabine and P-gp, BCRP and MRP1 are -8.568, -9.402 and -10.116 kcal/mol, respectively, indicating that gemcitabine has strong binding affinity with P-gp, BCRP and MRP1. It was found that the hydroxyl group of gemcitabine acts as the hydrogen bond receptor with the amino acid residue Tyr949, while the carbonyl group acts as the hydrogen bond receptor with the amino acid residue Tyr952 and forms the hydrogen bond interaction with P-gp. Moreover, the amino acid residues Leu64, Met67, Met68, Met948, Phe982 and Met985 on the binding domain of P-gp were hydrophobic with gemcitabine, which enhanced the interaction between gemcitabine and P-gp. Gemcitabine formed hydrogen bonds with amino acid residues Phe432, Thr435 and Asn436 of BCRP, and formed π-π interaction with Phe439. Amino acid residues Phe431, Phe439, Val546 and Met549 on the BCRP binding domain were hydrophobic with gemcitabine, which enhanced the interaction between gemcitabine and BCRP. Gemcitabine forms hydrogen bonds with amino acid residues Lys332, Tyr384, Thr439 and Gln450 of MRP1 receptor. Also, the amino acid residues Met339, Leu381, Phe385, Tyr440, Met443, Phe594, Pro595, Ile598 and Trp1245 on the MRP1 binding domain were hydrophobic with gemcitabine, which enhanced the interaction between gemcitabine and MRP1 (Fig. 1 C-H). These results suggested that gemcitabine possessed high binding affinity with P-gp, BCRP and MRP1.

Overexpression of hsa-miR-3178 promoted gemcitabine resistance in PANC-1 cells both in vitro and in vivo

Our previous study has shown that hsa-miR-3178 overexpression in PC is associated with poor prognosis [16]. The role of hsa-miR-3178 in gemcitabine resistance of PC was investigated. qRT-PCR analysis demonstrated the upregulation of hsa-miR-3178 in seven pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, Hs766t, PANC-1, MIA PaCa2 and SW1990) and in the PC tissues in contrast to the primary normal human pancreatic duct epithelial cell (HPDE6-C7) or adjacent non-tumor tissues (Figs. 1 A-C). We further found that hsa-miR-3178 was overexpressed in gemcitabine-resistant PANC-1-GEM cells compared with gemcitabine-sensitive PANC-1 cells (Fig. 2 D). As shown in Fig. 2 E, hsa-miR-3178 mimics increased half-inhibitory concentration (IC₅₀) of gemcitabine (8.06 ± 0.81 μmol/L) compared to control groups (un-transfected cells, 1.49 ± 0.62 μmol/L; negative control, 1.70 ± 0.18 μmol/L) in PANC-1 cells.

Over-expression of hsa-miR-3178 promoted proliferation of PANC-1 cells and conferred gemcitabine resistance. A-C qRT-PCR analysis on hsa-miR-3178 expression in seven pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, Hs766t, PANC-1, MIA PaCa2 and SW1990) and in the PC tissues compared to the primary normal human pancreatic duct epithelial cell (HPDE6-C7) or adjacent non-tumor tissues. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001. D qRT-PCR assay for relative expression of hsa-miR-3178 in PANC-1 and PANC-1-GEM cells. E CCK-8 method was used to detect IC₅₀ of Mock, MNC and hsa-miR-3178 mimic-transfected PANC-1 cells upon treatment with gemcitabine. F The CCK-8 growth curves of indicated cells upon treatment with gemcitabine (1 μmol/L). G The representative fluorescent micrograph and quantification on EdU staining of indicated cells upon treatment with gemcitabine (1 μmol/L). H Flow cytometry on cell apoptosis in indicated cells exposed to gemcitabine (1 μmol/L) for 72 h. I Tumor xenograft images from mice treated with gemcitabine (5 mg/kg, IP, q3d). J Tumor weights showed the effects of hsa-miR-3178 on the indicated groups. K Tumor volumes measured on the indicated times. Data are expressed as mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. PANC-1-GEM: gemcitabine resistant subline; IC₅₀: half-inhibitory concentration; Mock: untreated cells; MNC: Mimic negative control; PI: Propidium Iodide; FITC: Fluorescein Isothiocyanate; IHC: immunohistochemistry; IP: intraperitoneal injection

The CCK-8 and EdU assays were performed to evaluate cell viability and proliferation. It was found that PANC-1 cells with induced hsa-miR-3178 expression showed significant resistance to gemcitabine (1 μmol/L), as manifested by increased cell proliferation (Fig. 2 F, G). We further performed flow cytometry assay to evaluate whether hsa-miR-3178 was capable of inhibiting gemcitabine-induced apoptosis in PANC-1 cells. It was found that the percentage of cell apoptosis in hsa-miR-3178 transfection group was much lower than that in the control cells (Fig. 2 H).

To further verify the above results, we generated xenograft tumor models via injecting BALB/c nude mice with PANC-1 cells (with transfection of agomir-3178, agomir negative control or PBS). As expected, hsa-miR-3178 significantly enhanced xenograft tumor growth and tumor weight at the end of the experiment even with gemcitabine treatment (Fig. 2I-K). Furthermore, hsa-miR-3178 overexpression reduced gemcitabine-induced cell apoptosis, and the TUNEL⁺ cell proportion decreased compared with the control group (Figure S1 A).

Down-regulation of hsa-miR-3178 inhibited PANC-1-GEM cells proliferation and increased cell sensitivity to gemcitabine both in vitro and in vivo

To investigate the role of hsa-miR-3178 in in drug resistance in vitro, PANC-1-GEM cells with hsa-miR-3178 silencing were established by transfecting hsa-miR-3178 inhibiting vectors. As shown in Fig. 3 A, transfection of hsa-miR-3178 inhibiting vectors decreased IC₅₀ of gemcitabine (159.33 ± 0.94 μmol/L) compared to control groups (un-transfected cells, 170.77 ± 1.60 μmol/L; negative control, 170.13 ± 1.30 μmol/L) in PANC-1-GEM cells. CCK-8 and EdU assays demonstrated that PANC-1-GEM cells with reduced hsa-miR-3178 expression increased sensitivity to gemcitabine, as manifested by a significant decrease in cell proliferation (Fig. 3 B, C). Flow cytometry assays revealed that the percentage of cells apoptosis in hsa-miR-3178 inhibitor transfection group was significantly higher than that in the control cells which was the opposite of results of hsa-miR-3178 mimics transfection in PANC-1 cells (Fig. 3 D).

Down-regulation of hsa-miR-3178 inhibited proliferation of PANC-1-GEM cells and increased sensitivity of cells to gemcitabine. A CCK-8 assay on cell viability of PANC-1-GEM cells transfected with Mock, INC, and hsa-miR-3178 inhibitor and treated with gemcitabine for calculating IC₅₀. B The CCK-8 growth curves of indicated cells upon treatment with gemcitabine (150 μmol/L). (C) The representative fluorescent micrograph and quantification of EdU staining on indicated cells upon treatment with gemcitabine (150 μmol/L). D Flow cytometry on cell apoptosis in indicated cells exposed to gemcitabine (150 μmol/L) for 72 h. E Tumor xenograft images from mice treated with gemcitabine (5 mg/kg, IP, q3d). F Tumor weights showed the effects of hsa-miR-3178 inhibitor on the indicated groups. G Tumor volumes measured on the indicated times. Data are expressed as mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. PANC-1-GEM: gemcitabine resistant subline. IC₅₀: half-inhibitory concentration; Mock: untreated cells; INC: Inhibitor negative control; PI: Propidium Iodide; FITC: Fluorescein Isothiocyanate; IP: intraperitoneal injection

To confirm the above results in vivo, we generated xenograft tumor models via injecting PANC-1-GEM cells (with transfection of antagomir-3178, antagomir negative control or PBS, respectively) into BALB/c nude mice. As indicated, antagomir-3178 significantly reduced xenograft tumor weight at the end of the experiment and slowed down tumor growth following gemcitabine treatment (Fig. 3 E–G). Moreover, hsa-miR-3178 downregulation enhanced the effects of chemotherapy in vivo, and the TUNEL⁺ cell proportion increased compared with the control group (Figure S1 B).

RhoB was a direct target gene of hsa-miR-3178

By using four bioinformatics algorithms including miRWalk, miRanda, RNA22 and Targescan, and then intersecting with down-regulated genes in PANC-1-GEM cells from gemcitabine resistance dataset GSE80617 [16], we obtained two genes RhoB and HIST2H2BE. Through literature investigation, we found that RhoB plays a biological function as a cancer promoter in pancreatic cancer [21]. To explore the regulatory relationship between RhoB and hsa-miR-3178, we probed the TCGA database contained RhoB and hsa-miR-3178 expression profiles in PC patients. As shown in Fig. 4 A, we found that the expression of RhoB was negatively correlated with hsa-miR-3178.

RhoB was a direct target gene of hsa-miR-3178. A RhoB was negatively correlated with hsa-miR-3178 expression in TCGA PC patients. B Western blotting on the expression of RhoB on PANC-1 cells with hsa-miR-3178 mimics and PANC-1-GEM cells with hsa-miR-3178 inhibitor, and the expression of RhoB in PANC-1, PANC-1-GEM cells. C Illustration of putative binding sites of hsa-miR-3178 with the 3’-UTR of RhoB. D Luciferase assay showed hsa-miR-3178 targeted the 3’-UTR regions of RhoB. E–F IHC staining of RhoB and Kaplan–Meier analysis (n = 87; Log-rank test; P = 0.0037). Data are expressed as mean ± SD from three independent experiments. **P < 0.01; ***P < 0.001

Moreover, hsa-miR-3178 mimics was introduced into PANC-1 cells and hsa-miR-3178 inhibitor was introduced into PANC-1-GEM cells. Western blotting revealed that upregulation of hsa-miR-3178 inhibited RhoB expression while downregulation of hsa-miR-3178 enhanced RhoB expression (Fig. 4 B).

A dual-luciferase reporter assay was performed to validate RhoB as a direct target gene of hsa-miR-3178. To do this, hsa-miR-3178 mimics, mimics NC, and plasmids containing the wild-type 3’-UTR and mutant 3’-UTR were each constructed, and the characteristics of these vectors are presented in Fig. 4 C and supplementary-word1. The results indicated that co-transfection of hsa-miR-3178 mimics and wild-type plasmids significantly decreased luciferase activity compared with that with the transfection of mutant plasmids (Fig. 4 D). These results suggested that hsa-miR-3178 may restrain RhoB expression by directly targeting its 3’-UTR.

RhoB was reported as a cancer suppressor and loss of RhoB was strongly associated with poor survival of patients [34]. However, the effect of RhoB on survival in PC has not been studied. We performed IHC assay to explore the clinical significance of RhoB expression using a tissue microarray which contains 87 cases of PC. Kaplan–Meier survival analysis revealed that overexpression of RhoB was significantly associated with a good overall survival of PC patients (Fig. 4 E–F).

RhoB reversed hsa-miR-3178-mediated gemcitabine resistance in PC cells gemcitabine-resistant PC cells and the parental PANC-1 cells

To further confirm the role of RhoB suppression by hsa-miR-3178 in PC cell proliferation and gemcitabine resistance, we co-transfected RhoB overexpression lentivirus with hsa-miR-3178 mimics in PANC-1 cells or RhoB small interfering RNA (siRNA) with hsa-miR-3178 inhibitor in PANC-1-GEM cells. As shown in Fig. 5 A, upregulation of RhoB decreased the IC₅₀ of PANC-1 cells to gemcitabine (Vector + MNC, 1.68 ± 0.16 μmol/L; RhoB + MNC, 0.39 ± 0.02 μmol/L) and reversed hsa-miR-3178-mediated gemcitabine resistance in PANC-1 cells (Vector + Mimics, 6.87 ± 0.30 μmol/L; RhoB + Mimics, 1.44 ± 0.10 μmol/L). On the contrary, downregulation of RhoB increased the IC₅₀ of PANC-1-GEM cells to gemcitabine (Scr + INC, 168.93 ± 0.75 μmol/L; siRhoB + INC, 190.07 ± 0.60 μmol/L) and antagonized hsa-miR-3178 inhibitor-mediated gemcitabine re-sensitization in PANC-1-GEM cells (Scr + Inhibitor, 155.40 ± 0.93 μmol/L; siRhoB + Inhibitor, 169.10 ± 1.07 μmol/L) (Fig. 6 A). Furthermore, CCK-8 and EdU assay results revealed that RhoB overexpression reduced proliferation of PANC-1 cells and reversed the promotion of cell proliferation by hsa-miR-3178 in PANC-1 cells (Fig. 5 B, C). However, RhoB knockdown induced proliferation of PANC-1-GEM cells and antagonized the inhibition of cell proliferation by hsa-miR-3178 inhibitor in PANC-1-GEM cells (Fig. 6 B, C).

RhoB reversed hsa-miR-3178-mediated proliferation and gemcitabine resistance in PANC-1 cells. Co-transfection of RhoB overexpression lentivirus with hsa-miR-3178 mimics in PANC-1 cells. A CCK-8 assay for IC₅₀ of gemcitabine in PANC-1 cells with indicated transfection. B The CCK-8 growth curves of indicated cells upon treatment with gemcitabine (1 μmol/L). C The representative fluorescent micrograph and quantification of the EdU staining of indicated cells upon treatment with gemcitabine (1 μmol/L). D Flow cytometry on cell apoptosis in indicated cells exposed to gemcitabine (1 μmol/L) for 72 h. Data are expressed as mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. IC₅₀: half-inhibitory concentration; Mock: untreated cells; MNC: Mimic negative control; vector: vector-only control; PI: Propidium Iodide; FITC: Fluorescein Isothiocyanate

RhoB reversed hsa-miR-3178-mediated proliferation and gemcitabine resistance in PANC-1-GEM cells. Co-transfection of siRhoB with hsa-miR-3178 inhibitor in PANC-1-GEM cells. A CCK-8 assay for IC₅₀ of gemcitabine in PANC-1-GEM cells with indicated transfection. B The CCK-8 growth curves of indicated cells upon treatment with gemcitabine (150 μmol/L). C The representative fluorescent micrograph and quantification of the EdU staining of indicated cells upon treatment with gemcitabine (150 μmol/L). D Flow cytometry on cell apoptosis in indicated cells exposed to gemcitabine (150 μmol/L) for 72 h. Data are expressed as mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. IC₅₀: half-inhibitory concentration; Mock: untreated cells; INC: Inhibitor negative control; vector: vector-only control; siRhoB: RhoB small interfering RNA; Scr: scramble control; PI: Propidium Iodide; FITC: Fluorescein Isothiocyanate

In addition, flow cytometric analysis revealed that overexpression of RhoB promoted apoptosis of PANC-1 cells and also antagonized the suppression of cell apoptosis by hsa-miR-3178 in PANC-1 cells (Fig. 5 D). On the contrary, RhoB knockdown suppressed cell apoptotic rate compared with control group and reversed the increased apoptosis by hsa-miR-3178 inhibitor in PANC-1-GEM cells (Fig. 6 D). Taken together, these results demonstrated that RhoB could reverse hsa-miR-3178-mediated proliferation and gemcitabine resistance in PC cells.

Hsa-miR-3178/RhoB axis regulated gemcitabine resistance by PI3K/Akt signaling pathway and ABC transporters

Previous studies have reported that RhoB negatively regulates the PI3K/Akt signaling pathway [20, 35]. As shown in figure S1 A-B, IHC assay revealed that hsa-miR-3178 promoted expression of p-PI3K and p-Akt 473 in vivo. To explore the association of hsa-miR-3178, RhoB and PI3K/Akt signaling pathway, we performed Western blotting analyses to probe the protein expression involved in PI3K/Akt signaling pathway in PANC-1 cells and PANC-1-GEM cells with indicated treatments.

As shown in Fig. 7 A, RhoB overexpression decreased expression of p-PI3K, p-Akt 308 and p-Akt 473, while total PI3K and total Akt expression was not affected in PANC-1 cells. Co-transfection of RhoB and hsa-miR-3178 mimics reduced the expression of p-PI3K, p-Akt 308 and p-Akt 473 by hsa-miR-3178 in PANC-1 cells. On the contrary, RhoB knockdown increased the expression of p-PI3K, p-Akt 308 and p-Akt 473 and co-transfection of siRhoB and hsa-miR-3178 inhibitor reversed the downregulated expression of p-Akt 308 and p-Akt 473 by hsa-miR-3178 inhibitor in PANC-1-GEM cells (Fig. 7 B).

Hsa-miR-3178/RhoB axis mediated tumor growth and gemcitabine resistance by PI3K/Akt signaling pathway in PC cells. A-F Western blotting on the expression of RhoB, phosphorylated-PI3K, total PI3K, phosphorylated-Akt 308, phosphorylated-Akt 473, total Akt and P-gp, BCRP, MRP1 in the indicated cells. β-actin was used as a loading control. IC₅₀: half-inhibitory concentration

To further confirm the role of PI3K/Akt signaling pathway involved in regulating proliferation and gemcitabine resistance via hsa-miR-3178/RhoB axis in PC, PANC-1 cells and PANC-1-GEM cells were incubated with the PI3K inhibitor LY294002 and PI3K agonist 740Y-P. As expected, the stimulatory effect of hsa-miR-3178 overexpression on p-PI3K, p-Akt 308 and p-Akt 473 was inhibited by PI3K inhibitor in PANC-1 cells (Fig. 7 C). On the contrary, the inhibition of downregulated hsa-miR-3178 on p-PI3K, p-Akt 308 and p-Akt 473 was reversed by PI3K agonist in PANC-1-GEM cells (Fig. 7 D). In addition, Hsa-miR-3178 overexpression in PANC-1 cells increased expression of P-gp, BCRP and MRP1 in vitro and in vivo (Fig. 7 E, Figure S1 A), whereas, hsa-miR-3178 inhibitor decreased the expression of P-gp, BCRP and MRP1 in vitro and in vivo (Fig. 7 F, Figure S1 B). To explore the regulatory relationship between PI3K/Akt signaling pathway and ABC transporters, we probed the TCGA and GTEx data contained PIK3CA, PIK3CB, PIK3CD, PIK3CG, AKT1 and the three ABC transporters expression profiles. As shown in Figure S2, we found that the expression of PI3K/Akt was positively correlated with the ABC transporters. These results suggested that hsa-miR-3178 induces gemcitabine resistance by activating PI3K/Akt signaling pathway-mediated ABC transporters in PC cells (Fig. 8).

Schematic illustration of potential mechanisms of hsa-miR-3178/RhoB axis mediates tumor growth and gemcitabine resistance via PI3K/Akt pathway-mediated overexpression of ABC transporters in PC cells