Fig 2: eIF5A2 silencing improves the efficacy of Dox chemotherapy in vivo. (A) Athymic nude mice (n=5 mice/group) engrafted with patient-derived xenograft tumor tissues were treated with saline, doxorubicin, sieIF5A2 or sieIF5A2 plus Dox. Xenograft tumors of hepatocellular carcinoma were imaged after 2-week treatment. (B) Tumor weight was assessed at the end of the experiments. (C) Tumor volumes and mouse body weights were monitored over time. (D) Immunohistochemical analysis of the expression of ki67 and TUNEL staining analysis in xenograft tumors dissected from mice of from the four experimental groups. The frequency of ki67 positive cells and TUNEL-positive cells was quantified. *P<0.05, **P<0.01 and ***P<0.001. eIF5A2, eukaryotic translation initiation factor 5A2; si, small interfering RNA; NC, negative control; Dox, doxorubicin.

Fig 3: miR-9 sensitizes PDAC cells to doxorubicin chemotherapy via directly targeting the eIF5A2 transcript. (A-B) PDAC cells were transfected with miR-9 or control for 48 hr. Quantitative RT-PCR analysis of the mRNA abundance of eIF5A2 (A). eIF5A2 protein expression was assessed using Western blot (B). (C) miR-9 and its supposed binding sequence of eIF5A2, and the construct of eIF5A2-luciferase reporter plasmids (eIF5A2 WT and eIF5A2 Mut). (D) HEK293T cells were co-transfected for 48 h with the eIF5A2-luciferase reporter plasmids (eIF5A2 WT and eIF5A2 Mut) along with miR-9 mimics, miR-9 inhibitor or a negative control. eIF5A2 activity was determined by the luciferase assay. Shown are relative luciferase activities after normalization to Renilla that was used as the internal control. (E) PDAC cells with miR-9 or control transfection, treated with 0.5 μg/ml doxorubicin for 48 hr. eIF5A2 protein expression was assessed using Western blot. β-actin was used as the loading control. (F) Quantitative RT-PCR analysis of eIF5A2 abundance in paired Adjacent and Tumor from PDAC patients (n=16). (G) PDAC cells with siRNA directed against eIF5A2 or control transfection, treated with 0.5 μg/ml doxorubicin for 48 hr. eIF5A2, LC3 and P62 protein expression were assessed using Western blot. β-actin was used as the loading control. (H) PDAC cells with sieIF5A2 or control transfection were incubated with indicated concentration of doxorubicin for 48 hr. Cell viability was assessed using Cell Counting Kit-8 assay. (I) PDAC cells with miR-9 or sieIF5A2 transfection were incubated with 0.5 μg/ml doxorubicin for 48 hr. LC3 and P62 protein expression were assessed using Western blot. β-actin was used as the loading control. (J) PDAC cells with sieIF5A2 or miR-9 co-transfection were incubated with indicated concentration of doxorubicin for 48 hr. Cell viability was assessed using Cell Counting Kit-8 assay. (K) PDAC cells with miR-9 or eIF5A2 overexpression plasmid (OE eIF5A2) transfection were incubated with 0.5 μg/ml doxorubicin for 48 hr. eIF5A2, LC3 and P62 protein expression were assessed using Western blot. β-actin was used as the loading control. (L) PDAC cells with eIF5A2 overexpression plasmid (OE eIF5A2) or miR-9 co-transfection were incubated with indicated concentration of doxorubicin for 48 hr. Cell viability was assessed using Cell Counting Kit-8 assay. Experiments were repeated in three times. Data are presented as the mean ± SD, and analyzed with Student's t-test or one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: eIF5A2 expression is negatively associated with prognosis of patients with liver cancer. (A) Immunohistochemical staining of eIF5A2 protein in paired tumor and adjacent tissues. (B) Composite staining scores for eIF5A2 protein from 90 paired tumor and adjacent tissues. (C) Overall survival of 90 patients with HCC. (D) RT-qPCR analysis of eIF5A2 mRNA in paired tumor and adjacent tissues from patients with HCC (n=30). (E) HCC cells were treated with the indicated doses of doxorubicin for 48 h. Cell viability was determined by the Cell Counting Kit-8 assay. (F) RT-qPCR analysis of eIF5A2 mRNA in HCC cells. (G) Association between eIF5A2 expression and IC50 values of doxorubicin in HCC cells. *P<0.05 and **P<0.01. eIF5A2, eukaryotic translation initiation factor 5A2; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR.

Fig 5: miR-9 intervention enhances doxorubicin efficacy in xenografts growth from PDXs. (A) Xenografts dissected from mice of different groups after two weeks of various treatments. (B) Body weight. (C) Tumor volume. (D) Tumor regression rates. (E) eIF5A2, LC3 and P62 protein expression in tumors were assessed using Western blot. β-actin was used as the loading control. (F) Representative TUNEL labeling images and immunohistochemistry (IHC) images of tumors stained with anti-Ki-67 or anti-eIF5A2 antibody. Scale bars, 50 μm or 100 μm. (G) Ki-67-positive, TUNEL-positive and eIF5A2-positive cells were quantified and are shown in percentages, respectively. (H) Representative ex vivo NIRF image of xenografts in the PDXs-bearing mice at 24 hr after intravenous injection of Vehicle (equal volume of PBS), PL-1-Cy5/miR-9 or SP-94dr-Cy5/miR-9 nanoparticles. Data are presented as the mean ± SD, and analyzed with Student's t-test or one-way ANOVA. *P < 0.05, **P < 0.01.