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. 2021 May 4;25(12):5655–5670. doi: 10.1111/jcmm.16580

FIGURE 5.

FIGURE 5

QKI5 interacts with NRP1 at the 3'UTR region. A, Scatter plot of mRNA expression levels of QKI5 and NRP1 in preeclampsia placentas. Correlations were determined using Spearman's rank correlation test (N = 20, P < .0001, R 2 = 0.667). B, Relative NRP1 mRNA levels as detected by qPCR in HTR‐8/SVneo cells transfected with siRNAs against QKI5 or the QKI5 overexpression vector during hypoxia. The values of mock transfection were arbitrarily set to 1. C, Representative Western blotting and densitometric quantification of NRP1 protein expressions from cells shown in panel B. D, The half‐life of NRP1 mRNA was evaluated in QKI5 knockdown or overexpression HTR‐8/SVneo cells at 0, 3, 6, 9 and 12 h post‐treatment using a transcription inhibitor. The mRNA levels at time 0 were arbitrarily set to 100%. E, The luciferase reporter assay to evaluate 3'UTR activity. NRP1 3'UTR‐mediated expression of firefly luciferase upon binding of QKI5 was determined by harvesting QKI5 knockdown or overexpression HTR‐8/SVneo cells co‐expressing the reporter construct. Cell lysates were incubated with luciferin and RLU (relative luciferase units) indicated the luciferase activity as measured using a luminometer. (F) RNA immunoprecipitation assay. Cells transfected with siRNA against QKI5 or QKI5 overexpression vector were immunoprecipitated with IgG (control) or anti‐QKI5 antibody. Bound NRP1 RNA was detected by qPCR. Values of si‐NC or the pcDNA3.1 control were arbitrarily set to 1. G, Representative Western blotting showing immunodetection of QKI5 by RNA pull‐down against different regions of NRP1 (5'UTR, CDS and 3'UTR). Glyceraldehyde 3‐phosphate dehydrogenase was used as the input control. Shown are the means ± SD from at least three independent experiments. Statistical significance was calculated using one‐way analysis of variance for all panels except D, where the two groups were compared using Student's t test. * P < .05; ** P < .01; *** P < .001; **** P < .0001 (compared with the si‐NC group) and #### P < .0001 (compared with the OE‐NC group)