br MiR upregulates the mRNA
3.2. MiR-1291 upregulates the mRNA and protein levels of ARID3B in PC cells
To define the eﬀects of miR-1291 on the expression of ARID3B, we first verified the production of high levels of mature miR-1291 from bioengineered MSA/mir-1291 in PANC-1 and AsPC-1 LipoxinA4 (Fig. 1C). We
We further conducted Western blots to examine the impact of miR-1291 on ARID3B protein levels in PC cells. As found in other types of cells by other investigators , we observed two diﬀerent ARID3B bands in both PANC-1 and AsPC-1 cells which are designated as full-length ARID3B (ARID3B-Fl, ∼61 kD) and short-form ARID3B (ARID3B-Sh, ∼28 kD), respectively (Fig. 1E). Our data showed that ARID3B-Fl protein levels significantly increased in PANC-1 cells at 72 h post-transfection with 20 nM miR-1291 prodrug. A higher degree of increase of ARID3B-Fl protein levels was found in AsPC-1 cells at both 48 h and 72 h post-treatment with 5 nM miR-1291. Interestingly, impact of miR-1291 on ARID3B-Sh protein levels appeared to follow the same pattern as ARID3B-Fl in both cell lines (Fig. 1E, Supplementary Fig. S2). These results indicate that miR-1291 upregulates ARID3B expression in PC cells.
3.3. Individual and combined actions of miR-1291 prodrug and Gem-nP on DNA damage, mitosis arrest, and apoptosis
Historically, single drug exerted very limited eﬃcacy for the treat-ment of PC. Therefore, we aimed at examining combination eﬀects (Fig. 2A) while assessing miR-1291 monotherapy and comparing it to Gem-nP, the first-line chemotherapy for PC. Individual and combined actions of miR-1291 prodrug and Gem-nP on their corresponding target or marker proteins were first investigated in PANC-1 and AsPC-1 cells by Western blots (Fig. 2B and C). Our data showed that co-adminis-tration of Gem-nP did not alter miR-1291-controlled upregulation of ARID3B in PANC-1 cells but enhanced the eﬀects in AsPC-1 cells, again suggesting distinct sensitivities of the two cell lines. Immunoblot (Fig. 2B and C) and immunofluorescence studies were further con-ducted to determine single and combined drug eﬀects on DNA damage (phosphorylated histone H2A.X , γH2A.X foci), apoptosis (cleaved caspase-3/7, c-caspase-3/7) and mitotic arrest (phosphorylated Ser10–histone H3, H3PS10) (Fig. 2D–F; Supplementary Figs. S3–4). The results showed that, in addition to the induction of apoptosis (c-cas-pase-3/7) in both PANC-1 and AsPC-1 cell lines, miR-1291 alone sur-prisingly elicited obvious DNA damage (formation of γH2A.X foci) in AsPC-1 cells. On the other hand, Gem-nP largely provoked DNA damage and mitotic arrest in both PC cell lines, as manifested by an upregula-tion of γH2A.X and H3PS10, respectively. Most importantly, combina-tion treatment with miR-1291 prodrug and Gem-nP caused the greatest extents of DNA damage, mitosis, and apoptosis in both cell lines, which were indicated by γH2A.X, H3PS10, and c-caspase-3/7, respectively. These results demonstrate that miR-1291 induces apoptosis and pos-sibly DNA damage, and suggest that combination therapy with miR-1291 and Gem-nP may produce optimal outcomes.
3.4. Bioengineered miR-1291 prodrug enhances the sensitivity of PC cells to chemotherapeutic drugs
To assess whether miR-1291 prodrug could increase the sensitivity of pancreatic cancer cells to Gem-nP, the anti-proliferative activity of Gem-nP in the presence of miR-1291 prodrug or control MSA was evaluated in PANC-1 and AsPC-1 cells by CellTiter-Glo assay. The re-sults showed that miR-1291 treated PANC-1 and AsPC-1 cells were much more sensitive to Gem-nP, as compared to MSA treated cells (Fig. 3). The enhanced sensitivity was also manifested by the lower EC50 value in miR-1291 treated PANC-1 cells (52.3 ± 20.3 nM) than that in MSA treated cells (155 ± 33 nM, *P < 0.05). In addition, miR-
3.5. Bioengineered miR-1291 prodrug monotherapy and combination therapy with Gem-nP are eﬀective to control tumor growth in PANC-1 xenograft mouse models, while they are well tolerated in mice
To determine the antitumor eﬃcacy of miR-1291 prodrug mono-therapy and combination therapy with Gem-nP in vivo, we first estab-lished PANC-1 xenograft mouse models (Fig. 4A). Systematic adminis-tration of a single dose of in vivo-jetPEI formulated miR-1291 prodrug was distributable to PANC-1 xenograft tumor tissues, as indicated by high levels of tumoral miR-1291 at 24 h after drug administration (Supplementary Fig. S5A). Compared to buﬀer or MSA treatment, miR-1291 prodrug alone significantly suppressed PANC-1 tumor growth to a similar degree as Gem-nP, while combination treatment with miR-1291 and Gem-nP inhibited tumor growth to the greatest extent (Fig. 4B). Visual inspection and weights of the dissected tumors (Fig. 4C and D) further demonstrated the remarkably optimal tumor suppressive eﬀects for combination therapy. These results demonstrate the eﬀectiveness of miR-1291 prodrug monotherapy in the control of PANC-1 xenograft tumor progression as well as an optimal outcome for combination therapy with miR-1291 and Gem-nP.