Fig 2: Scheme of PKCβ driven nuclear localization of Lin28B connected to a TET3‐mediated positive feedback loop. KRAS promotes nuclear localization of Lin28B by PKCβ, which directly phosphorylates Lin28B. Nuclear Lin28B indirectly elevates TET3 expression by repressing the bioactivity of let‐7i, and increased TET3 can feed back to promote Lin28B expression, which maintains the stemness of PC cells.

Fig 3: Lin28B enhances TET3 expression in pancreatic cancer cells. (A) Samples from PC patients were prepared for IHC staining with antibodies against Lin28B and TET3. (B) Analysis of the expression of Lin28B correlates with TET3. The results are presented by a heat map. (C) Relative expression protein and mRNA levels of Lin28B and TET3 were assessed in PANC1, SW1990 and PaTu8988 cells. (D) TET3 mRNA and protein levels were detected by real‐time PCR or Western blotting in PANC1 with Lin28B downregulation. (E) Overexpression of Lin28B in PaTu8988 promotes the expression of TET3. All experiments were repeated three times. Each error bar represents the standard error of the mean. Statistical analysis was performed using the unpaired Student’s t‐test.

Fig 4: Lin28B maintains the stemness of pancreatic cancer cells via regulation of TET3 expression. (A–E) PaTu8988 and SW1990 cells were co‐transfected Flag‐vector and sh‐EGFP, Flag‐Lin28B and sh‐EGFP, sh‐TET3 and Flag‐vector, or Flag‐Lin28B and sh‐TET3. Relative expression of (A) mRNA levels of Lin28B and TET3, (B) cell proliferation, (C) colony formation capacity, (D) invasive ability and (E) the stem cell markers NANOG, OCT4 and SOX2 were analyzed in the two transfected cells by real‐time PCR, the CCK‐8 assay, the colony formation assay, Transwell assay, or Western blotting analysis, respectively. All experiments were repeated three times. Each error bar represents the standard error of the mean. Statistical analysis was calculated using Student’s t‐test or one‐way ANOVA test.

Fig 5: Lin28B elevates TET3 expression by repressing let‐7i. (A) Bioinformatics prediction of let‐7i binding sites in Lin28B 3′UTR or TET3 3′UTR sequence using TargetScan was shown. (B) Lin28B knockdown resulted in significant upregulation of let‐7i in PANC1. (C) The protein and mRNA levels of Lin28B and TET3 were measured in PANC1 transfected with miR‐NC or let‐7i. (D) The protein and mRNA levels of Lin28B and TET3 were assessed in PANC1 transfected with vector, Flag‐Lin28B, or Flag‐Lin28B and let‐7i. (E) The relative luciferase activities were detected by transfecting Lin28B wt or Lin28B mut and miR‐NC or let‐7i into PANC1. (F) Dual‐luciferase assays showed diminished luciferase activity when co‐transfected with TET3 wt, and let‐7i occurred in PANC1. Results represented the average of triplicate samples from three independent experiments. Each error bar represents the standard error of the mean. Statistical analysis was performed using the unpaired Student’s t‐test.