Fig 2: UCA1 is capable of elevating PTP1B expression through inhibiting miR-206. a Bioinformatics prediction of the interaction between UCA1 and miR-206 through complementary base-pairs was shown. The mutant sequence of UCA1 is indicated. b Luciferase activities of pGL-PTP1B were analyzed by luciferase reporter gene assay in miR-206 and/or UCA1 (or UCA1-206-mut) transfected MCF-7 cells. c In miR-206 and/or UCA1 (or UCA1-206-mut) treated cells, PTP1B expression was assessed by Western blot. **p < 0.01 and no significance (NS), Student’s t-test

Fig 3: UCA1 up-regulates the expression of PTP1B in breast cancer cells. a MCF-7 and MDA-MB-231 cells were transfected with UCA1 or vector using Lipofectamine 2000 reagent. RNA was extracted 24 h after transfection. The mRNA levels of PTP1B was measured by RT-PCR analysis. b, c MCF-7 and MDA-MB-231 cells were transfected with UCA1 or vector using Lipofectamine 2000 reagent. Protein was extracted 48 h after transfection. The protein levels of PTP1B were examined by Western blot analysis. d MCF-7 and MDA-MB-231 were transfected with siRNA-UCA1 or siRNA-control using Lipofectamine 2000 reagent. RNA was extracted 24 h after transfection. The mRNA levels of PTP1B was tested by RT-PCR analysis. e, f MCF-7 and MDA-MB-231cells were transfected with siRNA-UCA1 or siRNA-control using Lipofectamine 2000 reagent. Protein was extracted 48 h after transfection. The protein levels of PTP1B was evaluated by Western blot analysis. Statistical significant differences are indicated: **p < 0.01, Student’s t-test

Fig 4: PD-L1 directly interacts with PTP1B and inhibits its phosphatase activity.(A) PD-L1 and HA-tagged PTP1B associate with each other. HEK293T cells were transfected with PD-L1 and HA-tagged PTP1B for 48 hours and then subjected to immunoprecipitation followed by immunoblotting. (B) Ectopically expressed PD-L1 could pull down endogenous PTP1B and p38-MAPK. PD-L1 overexpressed in HEK293T was immunoprecipitated by PD-L1 antibody and the associated PTP1B were visualized by immunoblotting. (C) Endogenous PD-L1, PTP1B, and p38-MAPK form a protein complex in MDA-MB-231 cells. (D) The cytoplasmic domain of PD-L1 directly interacts with PTP1B. GST-tagged PTP1B (GST-PTP1B) and MBP-tagged PD-L1 cytoplasmic domain (MBP-PDL1-CT) were purified from E. coli. Purified proteins were analyzed by SDS-PAGE followed by Coomassie blue staining (lower panel). GST pull-down assays were performed using 0.5 g of each indicated protein. (E and F) PD-L1 inhibited the phosphatase activity of PTP1B. In vitro phosphatase assay was performed using 120 ng purified GST-PTP1B with indicated amount of MBP or MBP-PDL1-CT (E) or using endogenous PTP1B immunoprecipitated by anti-PTP1B antibody from indicated cell lysates (F). PTP1B activity in each sample was normalized against PTP1B with equal amount of MBP (E) or cells treated with control siRNA (siNC, F, immunoprecipitants pulled down from control cells by normal mouse IgG were used as negative controls of the phosphatase activity assay). (G) Inhibition of PTP1B recovered p38-MAPK activity in PD-L1–deficient cells. MDA-MB-231 cells were transfected with control (siNC) or PD-L1 (siPD-L1–1) siRNAs along with PTP1B inhibitor (20 or 40 µM) for 48 hours, then lysed for immunoblotting using indicated antibodies. The relative intensity of phosphorylated p38-MAPK (p-p38) in each sample was determined by GraphPad and normalized against the total p38-MAPK. (E–G) Results (n = 3 independent experiments) were plotted as mean ± SEM and comparisons between indicated groups were statistically analyzed using unpaired 2-tailed Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5: MiR-206/PTP1B signal mediates UCA1-accelerating cell proliferation in breast cancer. a–c Proliferation was tested by MTT and BrdU incorporation assays in MCF-7 cells treated with the indicated plasmids or siRNAs. d, e Colony formation of MCF-7 cells was counted post-transfection with the indicated plasmids or siRNAs