Fig 1: A graphic for the role of circ_0009043 in suppressing the development of non-small-cell lung cancer (NSCLC) via the miR-148a-3p / DNAJB4 pathway. circ_000904 is downregulated in NSCLC cells and that, when expressed, this circRNA can serve as a molecular sponge to sequester miR-148a-3p, thereby leading to DNAJB4 upregulation and the suppression of NSCLC tumor growth
Fig 2: DNAJB4 Reversed the Regulatory Effect of miR-148a-3p on A549 and HCC827 cells. A549 and HCC827 cells were transfected with shCirc, shCirc + miR-148a-3p inhibitor negative control (Inhibitor NC), shCirc + miR-148a-3p inhibitor (Inhibitor), shCirc + miR-148a-3p inhibitor (Inhibitor) + shCTRL, or shCirc + miR-148a-3p inhibitor + shDNAJB4, respectively. A Viability in A549 and HCC827 cells at 0, 24, and 48 h. B EdU assay to detect A549 and HCC827 cell proliferation ability. C FSCS assay to detect A549 and HCC827 cell apoptosis. D Protein levels of E-cadherin, N-cadherin, Vimentin and Slug in A549 and HCC827 cells with the indicated transfection were determined by western blot. GAPDH is a loading control. Data are presented as mean ± standard deviation. **P < 0.01; *** P < 0.001
Fig 3: Overexpression of DNAJB4 inhibits the proliferation but promotes apoptosis abilities of NSCLC cells. A DNAJB4 mRNA expression in HCC827 and A549 cells transfected with pcDNA4.0 vector, pcDNA4.0- DNAJB4 vector, CTRL-shRNA, or DNAJB4 -shRNA were determined by RT-qPCR. B DNAJB4 protein expression in HCC827 and A549 cells transfected with indicated vectors, which divided into five groups, including Control, OE-CTRL, OE-DNAJB4, shCTRL and shDNAJB4. C CCK8 assay was used to compare the cell proliferation of CTRL, OE-CTRL, OE- DNAJB4, shCTRL and sh DNAJB4 groups in HCC827 and A549 cells. D Edu assay of Control, OE-CTRL, OE- DNAJB4, shCTRL and sh DNAJB4 groups in HCC827 and A549 cells, respectively. E FSC assay to detect cell apoptosis of Control, OE-CTRL, OE- DNAJB4, shCTRL and sh DNAJB4 groups in HCC827 and A549 cells. F Expression level of Bax, Bcl-2 and Cytochrome C in the HCC827 and A549 cells of the 5 groups (Control, OE-CTRL, OE- DNAJB4, shCTRL and sh DNAJB4 groups) were determined by western blotting. Control negative control, OE over expression. Data represent mean values ± SD from three replicates of each sample; **P < 0.01, ***P < 0.001, ****P < 0.001
Fig 4: Circ_0009043 inhibits tumor growth via targeting the miR-148a-3p / DNAJB4 pathway in vivo. A, C Tumors formed 6 weeks post-injection in BALB/C nude mice. Tumors in the CTRL, OE-CTRL, OE-Circ, shCTRL and shCirc groups were isolated from mice at the endpoint of experiments. B, D Tumor growth was assessed by tumor volume measurement over time in the 5 afore mentioned groups (mean ± SD; n = 5). **P < 0.01. Mice were anesthetized and sacrificed at experimental endpoints. Tumors were subsequently dissected. E, F Circ_0009043 and miR-148a-3p mRNA expression in tumors from CTRL, OE-CTRL, OE-Circ, shCTRL and shCirc groups, respectively; n = 5. G DNAJB4 protein expression in tumors from CTRL, OE-CTRL, OE-Circ, shCTRL and shCirc groups; n = 5. H Representative images of DNAJB4 IHC in CTRL, OE-CTRL, OE-Circ, shCTRL and shCirc groups, respectively. (× 200, scale bars, 100 µm). I-J TUNEL staining assay was applied to compare the cell apoptosis of CTRL, OE-CTRL, OE-Circ, shCTRL and shCirc groups in tumors (scale bar, 100 μm). K Expression level of Bax, Bcl-2, and Cytochrome C in the cells of the 5 groups (CTRL, OE-CTRL, OE-Circ, shCTRL and shCirc groups) were determined by western blotting. **P < 0.01, ***P < 0.001, ###P < 0.001; ####P < 0.0001
Fig 5: DNAJB4 is a direct target gene of miR-148a-3p. A DNAJB4 acted as a potential target of miR-148a-3p using bioinformatics analysis. B RT-qPCR was used to quantify DNAJB4 expression in NSCLC tissues as well as normal lung tissues, n = 132. C Representative images of circ_0009043 IHC staining of NSCLC and normal tissues (× 200, scale bar 100 µm), respectively. D The DNAJB4 protein expression level was significantly suppressed in NSCLC tissues compared with adjacent normal tissues in selected 24 paired tissues. E The expression level of DNAJB4 mRNA using RT-PCR in NSCLC and normal tissues (n = 24); F Correlations between DNAJB4 and circ_0009043. G Correlations between DNAJB4 and miR-148a-3p. H RT-qPCR analysis showed that circ_0009043 overexpression increased DNAJB4 level. I The mRNA level of DNAJB4 in A549 and HCC827 ells transfected with miR-148a-3p mimics or miR-148a-3p inhibitor was measured via RT-qPCR assay. J Online bioinformatics analysis showed the binding within that of miR-148a-3p in addition to 3’-UTR of DNAJB4. K The activity of DNAJB4-wt plasmid in NSCLC cells was suppressed by the mimic transfection of miR-148a-3p which was demonstrated by luciferase reporter assay. *P < 0.05, **P < 0.01, ***P < 0.001
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