Yonsei Med J.  2013 Nov;54(6):1422-1429. 10.3349/ymj.2013.54.6.1422.

Decreased Expression of Surfactant Protein Genes Is Associated with an Increased Expression of Forkhead Box M1 Gene in the Fetal Lung Tissues of Premature Rabbits

• Hahn WH
• Chang JY
• Lee KS
• Bae CW

• KMID: 1798139
• DOI: http://doi.org/10.3349/ymj.2013.54.6.1422

Abstract

PURPOSE
Recently, Forkhead box M1 (FoxM1) was reported to be correlated with lung maturation and expression of surfactant proteins (SPs) in mice models. However, no study has been conducted in rabbit lungs despite their high homology with human lungs. Thus, we attempted to investigate serial changes in the expressions of FoxM1 and SP-A/B throughout lung maturation in rabbit fetuses.
MATERIALS AND METHODS
Pregnant New Zealand White rabbits were grouped according to gestational age from 5 days before to 2 days after the day of expected full term delivery (F5, F4, F3, F2, F1, F0, P1, and P2). A total of 64 fetuses were enrolled after Cesarean sections. The expressions of mRNA and proteins of FoxM1 and SP-A/B in fetal lung tissue were tested by quantitative reverse-transcriptase real-time PCR and Western blot. Furthermore, their correlations were analyzed.
RESULTS
The mRNA expression of SP-A/B showed an increasing tendency positively correlated with gestational age, while the expression of FoxM1 mRNA and protein decreased from F5 to F0. A significant negative correlation was found between the expression levels of FoxM1 and SP-A/B (SP-A: R=-0.517, p=0.001; SP-B: R=-0.615, p<0.001).
CONCLUSION
Preterm rabbits demonstrated high expression of FoxM1 mRNA and protein in the lungs compared to full term rabbits. Also, the expression of SP-A/B was inversely related with serial changes in FoxM1 expression. This is the first report to suggest an association between FoxM1 and expression of SP-A/B and lung maturation in preterm rabbits.

Keyword

mRNA; prematurity; protein; surfactant protein-A; surfactant protein-B; FoxM1

MeSH Terms

Animals
Blotting, Western
Female
Fetus/*metabolism
Forkhead Transcription Factors/*metabolism
Lung/*metabolism
Pregnancy
Pulmonary Surfactant-Associated Protein A/genetics/*metabolism
Rabbits
Forkhead Transcription Factors
Pulmonary Surfactant-Associated Protein A

Figure

• 

  Fig. 1 Relative expression levels of surfactant protein (SP)-A and -B mRNA in rabbit lungs. Pregnant New Zealand White rabbits were grouped according to gestational age from 5 days before to 2 days after the day of expected full term delivery (F5, F4, F3, F2, F1, F0, P1, and P2). Cesarean sections were carried out and a total of 64 fetuses were enrolled. The expression levels of mRNA of SP-A and -B in fetal lung tissues were tested by quantitative reverse-transcriptase real-time PCR (qRT-PCR). qRT-PCR revealed increasing changes in endogenous SP-A and -B mRNA expression levels throughout lung development. The gene expression used a scale of fold changes compared to the mRNA level of adult rabbits, which is calculated by 2-ΔΔCT. Statistical analysis was done by one-way ANOVA. A, adult rabbit. *p<0.05 versus adult group. †p<0.05 versus F5 or preterm group. ‡p<0.05 versus F4. §p<0.05 versus F3.

• 

  Fig. 2 Relative expression levels of Forkhead box M1 (FoxM1) mRNA in rabbit lungs. Pregnant New Zealand White rabbits were grouped according to gestational age from 5 days before to 2 days after the day of expected full term delivery (F5, F4, F3, F2, F1, F0, P1, and P2). Cesarean sections were carried out and a total of 64 fetuses were enrolled. The expression levels of FoxM1 mRNA in fetal lung tissues were tested by quantitative reverse-transcriptase real-time PCR (qRT-PCR). qRT-PCR revealed progressively decreasing changes in endogenous FoxM1 mRNA expression levels throughout lung development. The gene expression used a scale of fold changes compared to the mRNA level of adult rabbits, which is calculated by 2-ΔΔCT. Statistical analysis was done by one-way ANOVA. A, adult rabbit. *p<0.05 versus adult. †p<0.05 versus F5.

• 

  Fig. 3 Relative expression levels of Forkhead box M1 (FoxM1) protein in rabbit lungs. Pregnant New Zealand White rabbits were grouped according to gestational age from 5 days before to 2 days after the day of expected full term delivery (F5, F4, F3, F2, F1, F0, P1, and P2). Cesarean sections were carried out and a total of 64 fetuses were enrolled. The expression levels of FoxM1 proteins in fetal lung tissues were tested by Western blot. The representative bands of Western blot revealed progressively decreasing changes in endogenous FoxM1 protein band intensity throughout lung development. The protein expression used a scale of fold changes compared to the FoxM1 protein level of adult rabbits. Statistical analysis was done by one-way ANOVA. A, adult rabbit. *p<0.05 versus adult. †p<0.05 versus F5. ‡p<0.05 versus F4. §p<0.05 versus F3.

• 

  Fig. 4 Univariate linear regression analysis of the expression levels of mRNA and protein of Surfactant protein (SP)-A/B and Forkhead box M1 (FoxM1) genes. The diagonal solid lines are the regression lines and the dashed lines are approximate 95% confidence intervals. Significant positive correlations between FoxM1 expression levels of mRNA and protein (A), and mRNA expression levels of SP-A and SP-B (D) were found. Moreover, significant negative correlations between mRNA expression levels of FoxM1 and SP-A/B were found (B and C).

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