Overexpression of ribonucleotide reductase subunit M2 (RRM2), involved in deoxyribonucleotide synthesis, drives the chemoresistance of pancreatic cancer to nucleoside analogs (e.g., gemcitabine). While silencing RRM2 by synthetic means has shown promise in reducing chemoresistance, targeting endogenous molecules, especially microRNAs (miRNAs), to advance chemotherapeutic outcomes has been poorly explored. Based on computational predictions, we hypothesized that the let-7 tumor suppressor miRNAs will inhibit RRM2-mediated gemcitabine chemoresistance in pancreatic cancer. Reduced expression of the majority of let-7 miRNAs with an inverse relationship to RRM2 expression was identified in innately gemcitabine-resistant pancreatic cancer cell lines. Direct binding of let-7 miRNAs to the 3′ UTR of RRM2 transcripts identified post-transcriptional regulation of RRM2 influencing gemcitabine chemosensitivity. Intriguingly, overexpression of human precursor-let-7 miRNAs led to differential RRM2 expression and chemosensitivity responses in a poorly differentiated pancreatic cancer cell line, MIA PaCa-2. Defective processing of let-7a precursors to mature forms, in part, explained the discrepancies observed with let-7a expressional outcomes. Consistently, the ratios of mature to precursor let-7a were progressively reduced in gemcitabine-sensitive L3.6pl and Capan-1 cell lines induced to acquire gemcitabine resistance. Besides known regulators of let-7 biogenesis (e.g., LIN-28), short hairpin RNA library screening identified several novel RNA binding proteins, including the SET oncoprotein, to differentially impact let-7 biogenesis and chemosensitivity in gemcitabine-sensitive versus -resistant pancreatic cancer cells. Further, LIN-28 and SET knockdown in the cells led to profound reductions in cellular proliferation and colony-formation capacities. Finally, defective processing of let-7a precursors with a positive correlation to RRM2 overexpression was identified in patient-derived pancreatic ductal adenocarcinoma (PDAC) tissues. These data demonstrate an intricate post-transcriptional regulation of RRM2 and chemosensitivity by let-7a and that the manipulation of regulatory proteins involved in let-7a transcription/processing may provide a mechanism for improving chemotherapeutic and/or tumor growth control responses in pancreatic cancer.
PurposeWe endeavored to understand the contribution of canonical TGFβ signaling on expression of a 13‐gene microRNA (miR) signature of erlotinib response.BackgroundWe identified a 13‐gene miRNA signature predictive of response to the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in Non‐Small Cell Lung Cancer (NSCLC) cell lines. Bioinformatic analysis of the signature showed a functional convergence on TGFβ canonical signaling. We hypothesize that TGFβ signaling controls expression of the miR genes comprising an erlotinib response signature in NSCLC.MethodsTwo signature miR genes (miR‐140‐3p and miR‐141) were chosen for study. Promoters regions of both genes harbor putative Smad Binding Elements (SBEs), DNA binding domains for TGFβ‐responsive Smad transcription factors. TGFβ signaling impact on miR‐140 and ‐141 expression was assessed in erlotinib‐resistant (A549) and –sensitive (PC9) NSCLC cells by quantitative real‐time PCR (qRT‐PCR), luciferase reporter assays, and chromatin immunoprecipitation (ChIP).Results(1) qRT‐PCR showed endogenous miR 140/141 expression decreased after short term TGFβ1 treatment in both cell lines. After 7 days of treatment, the trend continued. (2) The promoter region of miR‐141, containing 6 SBEs, was cloned into a luciferase reporter vector. The miR‐141 luciferase construct is responsive to TGFβ stimulation like that seen in qRT‐PCR. (3) ChIP experiments will assess specific Smad complexes binding SBE sites.ConclusionWe expected that canonical TGFβ signaling molecules, Smads, bind to and control miR genes with SBEs in A549 cells. In PC9 cells, some miR genes likely control Smad protein expression while a non‐canonical TGFβ response may regulate signature miR expressionCollege funds were used.
Purpose We endeavored to understand the contribution of canonical TGFβ signaling on expression of a 13‐gene microRNA (miR) signature of erlotinib response. Background We identified a 13‐gene miRNA signature predictive of response to the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in Non‐Small Cell Lung Cancer (NSCLC) cell lines. Bioinformatic analysis of the signature showed a functional convergence on TGFβ canonical signaling. We hypothesize that TGFβ signaling controls expression of the miR genes comprising an erlotinib response signature in NSCLC. Methods Two signature miR genes (miR‐140‐3p and miR‐141) were chosen for study. Promoters regions of both genes harbor putative Smad Binding Elements (SBEs), DNA binding domains for TGFβ‐responsive Smad transcription factors. TGFβ signaling impact on miR‐140 and ‐141 expression was assessed in erlotinib‐resistant (A549) and –sensitive (PC9) NSCLC cells by quantitative real‐time PCR (qRT‐PCR), luciferase reporter assays, and chromatin immunoprecipitation (ChIP). Results (1) qRT‐PCR showed endogenous miR 140/141 expression decreased after short term TGFβ1 treatment in both cell lines. After 7 days of treatment, the trend continued. (2) The promoter region of miR‐141, containing 6 SBEs, was cloned into a luciferase reporter vector. The miR‐141 luciferase construct is responsive to TGFβ stimulation like that seen in qRT‐PCR. (3) ChIP experiments will assess specific Smad complexes binding SBE sites. Conclusion We expected that canonical TGFβ signaling molecules, Smads, bind to and control miR genes with SBEs in A549 cells. In PC9 cells, some miR genes likely control Smad protein expression while a non‐canonical TGFβ response may regulate signature miR expression College funds were used.
ObjectiveTo bolster the Science, Technology, Engineering, and Math (STEM) education of K‐12 students by providing dynamic, hands‐on experiences aligned with national education standards while providing professional‐development for university students.MethodsUniversity of Kentucky (UK) students in the allied‐sciences programs volunteer to work with an elementary school class for a calendar year. Graduate students collaborate with UK professors and their assigned teachers to design lesson plans in line with the core standards tailored to be palatable to specific grade levels. Lessons are led by UK students in order to offer advanced science students an experience in teaching and conveying difficult concepts at a lay level. Gifted high‐school students also work on developing exciting experiments for primary school students.ResultsElementary student science interest increased drastically in the first four trial months of this initiative. Interest in science for students that originally reported that they did not like science has also increased. UK graduate students involved in the program have noticeably enhanced communication skills over students who have not yet chosen to participate in the program. Post‐secondary science students are not commonly taught how to teach, and participation in this program allows them to learn, practice, and perfect the art of teaching and communicating with the mentorship of a professional <a>educator.</a>ConclusionParticipation in the program increases scientific education of K‐12 students. UK students who participate in the program gain the teaching and communication skill, organization and preparedness, and the community‐outreach commitment qualities valued in academic and industrial researchers alike.
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