Fig 2: Proliferation of ovarian cancer cells is inhibited by either siRNA to BCAR3 or mimics of tRF5-Glu(A) PEO4 cells transfected with either a control siRNA, siRNAs targeting BCAR3 or mimics of tRF5-Glu were grown in culture for up to six days and then fixed and assessed by SRB assay. Proliferation measures the overall cell number resulting from the balance of cell division and cell death over time. Values are shown as percent of control (control siRNA transfected cells; set to 100%) at day one, day three and day six. (B) 2008 cells were transfected and grown as described above for PEO4 cells. Cell proliferation experiments are replicates of three independent experiments and error bars refer to +/- standard error. p-value was less than 0.05 for PEO4 and 2008 cells treated with siRNAs to BCAR3 and tRF5-Glu mimic on day six.

Fig 3: Direct regulation of BCAR3 by tRF5-Glu is predicted in TargetScan 6. 2 which shows a binding site for miR-2476/tRF5-Glu in the BCAR3 3’UTRMimics and inhibitors of tRF5-Glu were used to determine if BCAR3 is regulated by tRF5-Glu. (A) Western analysis was conducted on protein extracts from PEO4, PEO1, OVCAR3, 2008 and SKOV3 cells, estrogen starved and transfected with either a control mimic, a control inhibitor, a tRF5-Glu mimic or a tRF5-Glu inhibitor. Protein lysates were collected at 48 hours post transfection. Densitometry of each band was measured and the relative ratio of BCAR3/β-Actin is shown. (B) A schematic of the 50 base pair region of the BCAR3 3’UTR and predicted binding site for tRF5-Glu is shown with the wild type and mutant seed sequences listed below. Luciferase reporter plasmids were constructed containing the predicted binding site intact or mutated at two base pairs within the predicted seed region (designated by underlined letters). (C) Luciferase constructs with the wild type or mutant 50bp region of BCAR3 3’UTR were transfected into HEK293T, PEO4 and 2008 cells with a control mimic or a mimic of tRF5-Glu. Transfections were performed in triplicate. The result was compared to a control mimic and error bars refer to +/- standard error (**** p-value <.001).

Fig 4: tRF5-Glu and its potential target BCAR3 are expressed in ovarian cancer cell linesOvarian cancer cell lines were grown under estrogen starved (NE) or estrogen stimulated (E) conditions to determine the best conditions to study the potential relationship between tRF5-Glu and BCAR3. (A) Expression of BCAR3 mRNA in cDNA from PEO4 and 2008 cells estrogen starved (NE) or estrogen stimulated (E) as assayed by qRT-PCR. qRT-PCR experiments are replicates of three independent experiments and error bars refer to +/- standard error (** p-value <.01 and *** p-value<.005). (B) Western analysis of BCAR3 protein expression at 48 hours of estrogen starvation (NE) or estrogen stimulation (E) in the two ovarian cancer cell lines. Densitometry of each band was measured and the relative ratio of BCAR3/β-Actin is shown. (C) Expression of tRF5-Glu amplified from cDNA of PEO4 and 2008 cells estrogen starved (NE) or estrogen stimulated (E) as assayed by ligation PCR. (D) Northern analysis of tRF5-Glu in estrogen starved (NE) or estrogen stimulated (E) PEO4 and 2008 cells. Cells were grown for 48 hours in estrogen depleted media and then the media was replaced by new estrogen depleted media (NE) or media supplemented with estrogen for an additional 48 hours (E). Cells were then lysed by direct addition of Qiazol for RNA extraction. The tRF5-Glu bands are shown within a box.

Fig 5: Binding of the BCAR3 mRNA by tRF5-Glu is demonstrated using miR-Catch and luciferase assays(A) Secondary structure of the BCAR3 mRNA with placement of the DNA capture probe in relationship to the tRF5-Glu binding site is shown with arrows. (B) Triplicate samples of PEO4 cell lysates probed with either BCAR3 or a random probe were amplified by RT-PCR for BCAR3 and tRF5-Glu expression. The BCAR3 mRNA in the flow-through was amplified and is shown for each sample. (C) A schematic of the BCAR3 3’UTR and predicted binding site for tRF5-Glu is shown with the wild type and mutant seed sequences listed below. Luciferase reporter plasmids were constructed containing the predicted binding site intact or mutated at two base pairs within the predicted seed region (designated by underlined letters). (D) Luciferase constructs with the wild type or mutant BCAR3 3’UTR were transfected into HEK293T, PEO4 and 2008 cells with a control mimic or a mimic of tRF5-Glu. Transfections were performed in triplicate. The result was compared to a control mimic and error bars refer to +/- standard error (* p-value <.05 and **** p-value <.001).