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. 2019 Dec 21;7(4):1900949. doi: 10.1002/advs.201900949

Figure 5.

Figure 5

Circ‐MALAT1 acts as miR‐6887‐3p sponge to up‐regulate JAK2. A) Top five miRNAs with the highest binding free energy were analyzed in circ‐MALAT1 overexpressed (circ‐MALAT1 O/E) or empty vector (Vector) Huh7 cells by qRT‐PCR. Control primers, U6. B) In vivo circ‐MALAT1 pull‐down using circ‐MALAT1 specific probes (Probe 1 and Probe 2) was performed in circ‐MALAT1 overexpressed Huh7 cells, followed by qRT‐PCR to detect circ‐MALAT1 (left panel) and miR‐6887‐3p (right panel). C) After treatment with miR‐6887‐3p mimic or its scrambled version (control mimic), both miR‐6887‐3p level and JAK2 expression at mRNA were detected by qRT‐PCR (top panel), and JAK2 expression at protein level was detected by western blot (bottom panel), respectively. U6 and GAPDH were used as control primers. D) After treatment with the miR‐6887‐3p inhibitor or its scrambled version (the control inhibitor), the level of both the miR‐6887‐3p and JAK2 mRNA was detected by qRT‐PCR (top panel), and JAK2 expression at the protein level was detected by western blot (bottom panel). U6 and GAPDH were used as control primers. E) The luciferase activities of pMIR‐JAK2‐3'UTR wild type (WT) and mutated type (MUT) were detected when cells were cotransfected with miR‐6887‐3p mimic (top panel) or miR‐6887‐3p inhibitor (bottom panel). F) The key molecules of JAK2/STAT3 pathway were analyzed in circ‐MALAT1 overexpressed (circ‐MALAT1 O/E) or empty vector (Vector) cells by western blot. p‐JAK2, phospho‐JAK2; p‐STAT3, phospho‐STAT3. G) The key protein molecules in JAK2/STAT3 signaling pathway were analyzed in CSCs and adherent cells of Hep3B cell line by western blot. p‐JAK2, phospho‐JAK2; p‐STAT3, phospho‐STAT3. CSCs were enriched by the tumorsphere assay. Columns, means from three independent experiments; bars, SD. ** p < 0.01, * p < 0.05.