Lef1 is transcriptionally activated by Klf4 and suppresses hyperoxia-induced alveolar epithelial cell injury

Figures & data

Table 1. Characteristics of enrolled infants.

	Control group (n = 20)	BPD group (n = 20)	P value
Sex (male/female)	11/9	8/12	0.527
Gestational age (week)	30.12 ± 2. 16	29.65 ± 2.08	0.488
Birth weight (g)	1 309.56 ± 163.16	1176.45 ± 236.38	0.045
Intrauterine distress (n)	4 (20%)	7 (35%)	0.480
Mechanical ventilation time (d)	3.75 ± 1.04	15.56 ± 3.88	<0.001
Oxygen inhalation time (d)	18.16 ± 3.12	45.26 ± 7.34	<0.001
Nosocomial infection (n)	1 (5%)	7 (35%)	<0.001
Antibiotic use time 2 weeks after birth (d)	4.32 ± 3.44	9.65 ± 4.22	<0.001

Table 2. Primer sequences for qRT-PCR.

Primer name	Primer sequences
KLF4-forward	5′-TCGGACCACCTCGCCTTACA‐3′
KLF4-reverse	5′-CTGGGCTCCTTCCCTCATCG‐3′
LEF1-forward	5′-GCCACGGACGAGATGATCC‐3′
LEF1-reverse	5′-TGTCTGGCCACCTCGTGTC‐3′
ACTB-forward	5′-TGGCACCACACCTTCTACAA‐3′
ACTB-reverse	5′-CCAGAGGCGTACAGGGATAG‐3′
Klf4-forward	5′-CTATGCAGGCTGTGGCAAAACC‐3′
Klf4-reverse	5′-TTGCGGTAGTGCCTGGTCAGTT‐3′
Lef1-forward	5′-ACTGTCAGGCGACACTTCCATG‐3′
Lef1-reverse	5′-GTGCTCCTGTTTGACCTGAGGT‐3′
β-actin-forward	5′-CCCCTGAACCCTAAGGCCA‐3′
β-actin-reverse	5′-CGGAGTCCATCACAATGCCT‐3′

Figure 1. The expression of KLF4/Klf4 and LEF1/Lef1 are diminished in Clinical BPD serum samples and hyperoxia-induced MLE-12 cells. A-B. The levels of KLF4 and LEF1 in serum samples from BPD patients (n = 20) and healthy volunteers (n = 20) were evaluated utilizing qRT-PCR, male: grey, female: black; C. Klf4 and Lef1 expression were measured by qRT-PCR; D. The levels of KLF4 and LEF1 in MLE-12 cells induced by hyperoxia were evaluated utilizing western blot. Data were displayed as mean ± SD (n = 3), and analyzed utilizing t test; **, p < 0.01; ***, p < 0.001.

Figure 2. Overexpression of Lef1 suppresses hyperoxia-induced AEC injury. A. The expression of Lef1 was evaluated by qRT-PCR; B. Western blot assay for LEF1 level after transfecting oe-LEF1 into hyperoxia-induced MLE-12 cells; C. The cell viability was determined using CCK-8 assay; D. The cell proliferation was determined using EdU assay; E. Cell apoptosis was investigated by flow cytometry assay; F. The expression of Bcl-2, cleaved-caspase 3 and 9 were determined using western blot. Data were displayed as mean ± SD (n = 3) and analyzed utilizing one-way ANOVA, followed by Tukey’s multiple comparisons test; **, p < 0.01; ***, p < 0.001.

Figure 3. Klf4 activates the transcription of Lef1. A. JASPAR (https://jaspar.genereg.net/) was employed to predict probable Klf4 binding sites in the Lef1 promoter; B. The luciferase and C. ChIP assays were used to validate the prediction from JASPAR. D. Lef1 expression was determined by qRT-PCR; E. Western blot assay for LEF1 level after transfection in normoxia or hyperoxia treated MLE-12 cells with oe-Klf4. Data were displayed as mean ± SD (n = 3) and analyzed utilizing t test or one-way ANOVA followed by Tukey’s multiple comparisons test; ***, p < 0.001.

Figure 4. Klf4 impedes hyperoxia-induced alveolar epithelial cell damage via stimulating Lef1. A The cell viability was determined using CCK-8 assay after co-transfecting oe-Klf4 and si-Lef1 into hyperoxia-induced MLE-12 cells; B. The cell proliferation was determined using EdU assay; C. Cell apoptosis was investigated by flow cytometry assay; D. The expression of Bcl-2, cleaved-caspase 3 and 9 were determined using western blot. Data were displayed as mean ± SD (n = 3) and analyzed utilizing one-way ANOVA, followed by Tukey’s multiple comparisons test; *, p < 0.05; ***, p < 0.001.

Data availability statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.