Fig 2: Upregulation of QKI-5 promotes cell migration and invasion. (A) Transwell assay was used to detect the effect of QKI-5 on the invasion of 786-0 cells; Cells with serum-free media were placed into the upper part of an insert, and subsequently medium with 10% of FBS was added to the lower part. Once the cells had migrated through the membrane after incubation for 24 h, they were fixed with 100% methanol and stained with 0.1% crystal violet. The cells were counted in 5 randomly selected microscopic fields (×200) from each chamber. (B) Number of 786-0 cells in different groups calculated using image J. (C) Wound healing assay was used to detect the effect of QKI-5 on migration rate changes in 786-0 cells. Uniform artificial wounds were made at 2 d after transfection and the cells were cultured for another 24 h. Cell migration ability was represented by the wound gap distance in microscopic field (×40) at the time points of 0 and 24 h. The migration rates are shown in (D). (E) Effect of QKI-5 on the invasion of ACHN and Caki-2 cells with QKI-5 inhibition and overexpression (F) number of ACHN and Caki-2 cells in different groups calculated using image J. (G) Effect of QKI-5 on migration rates changes in with QKI-5 inhibition and overexpression, and the migration rates of ACHN and Caki-2 cells in different groups are shown in (H). Data are shown as mean ± SD. *, P<0.05; **, P<0.01, sh-QKI-5-NC group vs. Sh-QKI-5-1 group; ##, P<0.01, Over-QKI-5 group vs. Over-QKI-5-NC group.

Fig 3: Expression of circNRIP1 in GC can be regulated by QKI. a. We constructed a series of mutation plasmids (pZW1) consisting of either mutated I1QB or I3QB (#1, #2) or both I1QB and I3QB (#3), and we also constructed a wild-type plasmid spanning intron 1 to intron 3 (#4). b. We observed that only the wild-type plasmid (#4), and neither the I1QB nor I3QB deletion constructs (#1–3), could overexpress circNRIP1 according to northern blotting of GC cells.c. We observed that only the wild-type plasmid (#4), and neither the I1QB nor I3QB deletion constructs (#1–3), could overexpress circNRIP1 according to qRT-PCR in GC cells. d. We knocked down QKI and observed a significant reduction in circNRIP1 but not pre-mNRIP1 or mNRIP1. e. Enrichment of I1QB and I3QB was observed when we used an antibody against QKI. f. We detected higher QKI expression level in GC tumour tissues relative to adjacent normal stomach tissues among 40 patients by immunohistochemistry, scale bar = 200 µm. g. We performed qRT-PCR on these 40 patients and discovered that the expression level of circNRIP1 was positively related to the QKI histochemistry score. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 4: QKI-5 induced vascular endothelial growth factor receptor 2 (VEGFR2) activation and VEGF secretion through direct binding of the 3'UTR region of STAT3. Overexpression of QKI-5 by lentiviral gene transfer induced the secretion of VEGF on day 6 of the endothelial cell (EC) differentiation process (A) and the transcriptional activation of the VEGFR2 (B). (C): Real time polymerase chain reaction (PCR) data showing that QKI-5 leads to activation of VEGFA, JAK-1, STAT3, and AP1. (D): Western blots showing that overexpression of QKI-5 induced the expression of EC markers CD144, and CD31 in parallel to induction of JAK-1, STAT3, and phosphorylation of STAT3 (quantification in E). (F): STAT3 was knocked down by shRNA on day 3 of EC differentiation, and QKI-5 was overexpressed next day. Real data PCR data reveal that STAT3 knocked down ablated activation of EC markers CD31, CD144, eNOS, and VEGFA mediated by QKI-5. The cells were harvested on day 6 of EC differentiation. (G): Luciferase assays have shown that QKI-5 activated the 3'UTR of STAT3. QKI-5 was unable to activate the 3'UTR of STAT3 when the QKI motif was deleted. (G, lower panel) RNA binding assays have confirmed that QKI binds directly to the 3'UTR of STAT3. (H): When differentiated ECs were treated with Actinomycin D in a time point experiment from 0 to 24 hours STAT3 expression was stabilized as a decay curve is shown (data are means ± SEM [n = 3]; *, p < .05). Abbreviations: CTL, control; VEGF, vascular endothelial growth factor.

Fig 5: Upregulation of microRNA 200c (miR-200c) suppressed cell invasion and migration in 786-0 and Caki-2 cell lines. (A) Transwell assay was used to detect the effect of microRNA 200c (miR-200c) on the invasion of 786-0 and Caki-2 cell lines (stained with crystal violet, ×200); (B) Number of 786-0 and Caki-2 cells in different groups calculated using image J; (C) wound healing - assay was used to detect the effect of miR-200c on migration rate changes in 786-0 and Caki-2 cell lines, Uniform artificial wounds were made at 2 d after transfection and the cells were cultured for another 24 h. Cell migration ability was represented by the wound gap distance in microscopic field (×40) at the time points of 0 and 24 h. and the migration rates are shown in (D). Data shown as mean ± SD. **, P<0.01, miR-200c-NC mimic group vs. miR-200c-NC group; #, P<0.05; ##, P<0.01, QKI-5+miR-200c mimic group vs. QKI-5+miR-200c NC group.