br P compared to corresponding control or
***P < 0.001, compared to corresponding control (1- or 2-way ANOVA). (E) Immunoblot analyses showed that ARID3B protein levels were elevated in PANC-1 and AsPC-1 cells after transfection with bioengineered miR-1291. Both the full-length ARID3B (ARID3B-Fl, ∼61 kD) and the short-form ARID3B (ARID3B-sh, ∼28 kD) were upregulated in PANC-1 and AsPC-1 cells (72 h post-transfection), as compared to vehicle and MSA controls. β-actin was used as a loading control, and then the protein levels were normalized to vehicle group for comparison. Values are mean ± SD; ***P < 0.001, compared to vehicle; ###P < 0.001, compared to MSA (1-way ANOVA with Bonferroni posttests).
have also demonstrated that restoration of miR-1291 expression/func-tion suppresses the proliferation, migration, invasion, and tumorigen-esis of PC cells through the induction of apoptosis and 89663-86-5 arrest, as well as alteration of PC cell metabolome [17,18]. In addition, miR-1291 sensitizes PC cells to chemotherapy via direct targeting of mul-tidrug resistance-associated protein 1 (MRP1) overexpressed in the cells . Other investigators have also demonstrated that miR-1291 sup-presses renal cell carcinoma and esophageal squamous cell carcinoma growth by downregulation of glucose transporter protein type 1 (GLUT1) and mucin 1 (MUC1) [20,21]. The above reports suggest the potential of miR-1291 as a novel therapy for PC.
To explore miR-1291 based therapeutic strategies for the treatment of PC, we have successfully established a novel approach to producing large quantities of pre-miR-1291 agents in bacteria by using a sephadex aptamer tagged methionyl-tRNA (MSA) scaﬀold . Further studies demonstrated that chimeric MSA/mir-1291 or “miR-1291 prodrug” was precisely processed into mature miR-1291 in human cells, and subse-quently regulated target protein expression and suppressed the growth of PC cells . It is noteworthy that bioengineered miRNA agents produced in living cells are distinguished from conventional miRNA
agents made in test tubes by chemical synthesis or enzymatic reactions. Therefore, our biologic miRNA agents may better capture the natural characteristics of cellular RNA molecules . The objective of the current study was to investigate the utility of bioengineered miR-1291 prodrug for the control of PC and its under-lying molecular mechanisms. Following the identification of a new target AT-rich interacting domain protein 3B (ARID3B) for miR-1291, which was rather surprisingly upregulated by miR-1291, we delineated the independent actions of miR-1291, gemcitabine and nab-paclitaxel on the apoptosis, DNA damage and mitosis arrest of PC cells, respec-tively, as well as optimal eﬀects when the drugs were combined to-gether. Therapy studies in PANC-1 xenograft and three diﬀerent PC patient-derived xenograft (PDX) tumor mouse models revealed that miR-1291 prodrug alone showed comparable levels of eﬃcacy as Gem-nP in the suppression of PC tumor growth, and combination treatment with miR-1291 and Gem-nP inhibited PC tumor growth to the greatest degrees.
Fig. 2. Independent and combined actions of miR-1291 and gemcitabine plus nab-paclitaxel (Gem-nP) in human pancreatic cancer cells. (A) Gemcitabine, miR-1291 and paclitaxel may act on specific targets and thus interfere with particular cellular processes. Immunoblot (B and C) and immunofluorescence (D-F; scale bar, 20 μm) studies showed that combination (combo) treatment with miR-1291 prodrug (10 nM in PANC-1 cells, 3 nM in AsPC-1 cells) and Gem-nP exhibited the greatest degrees of DNA damage, mitosis and apoptosis in PANC-1 and AsPC-1 cells, which were indicated by γH2A.X, H3PS10, and cleaved caspase-3/7 (c-caspase-3/7), respectively. β-actin was used as a loading control. C-caspase-7 images in PANC-1 cells are provided in Supplementary Fig. S3, and individual biomarkers in AsPC-1 cells are shown in Supplementary Fig. S4.
2. Material and methods
All animal experiments were performed according to our protocol approved by the Institutional Animal Care and Use Committee at UC Davis. 5- to 6-week-old female athymic nude mice (NU/J) and NOD.CB17-Prkdcscid/J mice (The Jackson Laboratory, Bar Harbor, ME) were used to establish PANC-1 xenograft mouse models and PDX mouse models, respectively. The mice were maintained in sterile cages at constant temperature and humidity, with free access to food and water.
Materials and procedures of production of biologic miR-1291 pro-drug (MSA/mir-1291) and control RNA MSA, cell culture and treat-ment, luciferase reporter gene assay, reverse transcription quantitative real-time PCR, immunoblot analysis, immunofluorescence, animal model establishment, therapy study, and statistical analysis were de-scribed in detail in Supplementary Materials.
Our recent studies have demonstrated that miR-1291 suppresses
proliferation and tumorigenesis of PC cells . To further delineate the molecular mechanisms through which miR-1291 controls PC cell growth, computational analysis was conducted to predict potential targets of miR-1291. Among a set of putative targets, ARID3B was a top candidate consisting of four miRNA response elements (MREs) for miR-1291 within its 3’UTR (Fig. 1A). An ARID3B 3’UTR luciferase reporter plasmid was thus constructed to evaluate the interactions between miR-1291 and ARID3B 3’UTR. Surprisingly, treatment with bioengineered miR-1291 significantly increased ARID3B-3’UTR-luciferase reporter activities in AsPC-1 (Fig. 1B) and HEK293 (data not shown) cells, as compared to controls. Introduction of miR-1291 into cells with miR-1291-expressing plasmid showed the same results (Supplementary Fig. S1A), whereas ARID3B-3’UTR-luciferase reporter activities were de-creased in cells treated with miR-1291 antagomir (Supplementary Fig. S1B). These results suggest that miR-1291 targets ARID3B 3’UTR and may positively regulate the expression of ARID3B.