Figures & data
Figure 1. Silence of C2CD4D-AS1 hinders cell malignant phenotype in LUAD a. The overexpression of C2CD4D-AS1 in LUAD tissues was showed in GEPIA database. b. Differences of C2CD4D-AS1 expression between LUAD cell lines and normal cell line were evaluated with help of RT-qPCR analysis. c. The knockdown efficiency of sh-C2CD4D-AS1#1/2/3 was detected. d-e. Cell proliferation capacity was examined by CCK8 and colony formation assays. F-G. Transwell assays were applied to measure the migration and invasion of PC-9 and H1299 cells. h. Cell apoptosis was observed with the employment of TUNEL assay. *P < 0.05, **P < 0.01
Figure 2. ETV4 stimulates the up-regulation of C2CD4D-AS1 a. Venn diagram demonstrated transcription factors with high binding scores in HumanTFDB overlapped with up-regulated transcription factors in LUAD. b-c. RT-qPCR tested the efficiency of ETV4 inhibition or overexpression, as well as the variations in C2CD4D-AS1 expression caused by ETV4 down-regulation or up-regulation. d. The binding sequence between ETV4 and C2CD4D-AS1 promoter was presented. e. The affinity of ETV4 and C2CD4D-AS1 promoter was examined through ChIP assay. f. In DNA pull down assay, the enrichment of ETV4 in Bio-C2CD4D-AS1 promoter was measured by western blot. g. The binding of ETV4 and C2CD4D-AS1 promoter was further confirmed by luciferase reporter assay. **P < 0.01, n.s.: no significance
Figure 3. C2CD4D-AS1 serves as a miR–3681–3p sponge a. The distribution of C2CD4D-AS1 in nucleus and cytoplasm of LUAD cells was detected via subcellular fractionation. b. FISH assay was done for the confirmation of C2CD4D-AS1 distribution in LUAD cells. C. RNA pull down revealed the strong interaction between C2CD4D-AS1 and miR–3681–3p. d. Binding sequences between C2CD4D-AS1 and miR–3681–3p, along with mutated C2CD4D-AS1 sequence were displayed. e. MiR–3681–3p expression in LUAD cells with miR–3681–3p mimics transfection was quantified via RT-qPCR. f. The luciferase activity of C2CD4D-AS1-Wt/Mut was assessed by luciferase reporter assay in response to the transfection of miR–3681–3p mimics. **P < 0.01, n.s.: no significance
Figure 4. NEK2 is negatively regulated by miR–3681–3p a. 3 potential mRNAs were predicted by utilizing starBase (CLIP Data ≥ 5) and GEPIA (log2 fold change ≥ 2). b. The expression of these mRNAs was examined after overexpressing miR–3681–3p. c. Bioinformatics tools analyzed the binding sequence between miR–3681–3p and NEK2 3ʹUTR. d. RIP assay was performed for investigation into the combination among C2CD4D-AS1, miR–3681–3p and NEK2. e. The binding of miR–3681–3p and NEK2 3ʹUTR was validated by luciferase reporter assay. f-g. The mRNA and protein levels of NEK2 in sh-NC group, sh-C2CD4D-AS1#1 group and sh-C2CD4D-AS1#1+ miR–3681–3p inhibitor group was measured via RT-qPCR and western blot. **P < 0.01, n.s.: no significance
Figure 5. C2CD4D-AS1 accelerates LUAD cell proliferation, migration, invasion and suppresses cell apoptosis by modulating miR–3681–3p/NEK2 axis a. RT-qPCR analyzed NEK2 expression in response to NEK2 augment. b-c. Cell proliferation in sh-NC group, sh-C2CD4D-AS1#1 group, sh-C2CD4D-AS1#1+ miR-3681–3p inhibitor group and sh-C2CD4D-AS1#1+ pcDNA3.1/NEK2 group was detected by CCK8 and colony formation assays. d-e. Transwell assay was implemented for observation of cell migration and invasion in different groups. f. The apoptotic capacity of LUAD cells transfected with indicated plasmids was examined by TUNEL assay. **P < 0.01
