PCR Array Analyzes the Effects of Polyunsaturated Fatty Acids on Fetal Development


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PCR Array Analyzes the Effects of Polyunsaturated Fatty Acids on Fetal Development

Polyunsaturated fatty acids analysis (PUFA), including arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are essential for fetal growth. The purpose of this study was to elucidate the effects of PUFA on the expression and function of placental transporters. Placental transporters play an important role in placental function, including fetal nutritional supply, excretion of metabolites, and protection of the fetus by xenobiotics.




Human placental choriocarcinoma BeWo cells were used as a trophoblast cell model. The gene expression changes of 84 transporters induced by PUFA were studied by PCRarray. Protein levels and transporter activity were assessed by Western blotting and uptake experiments, respectively. Pregnant Wistar rats were used to analyze placental transporter expression.




1. The effects of PUFAs on the viability of BeWo cells and the expression of adipogenic differentiation-related proteins

MTT was used to assess cell viability after PUFA treatment. Treatment with AA, EPA or DHA for 24 hours did not affect the survival rate of BeWo cells. Our experiments confirmed the effect of PUFA on ADRP mRNA expression: AA, EPA, and DHA induced ADRP mRNA expression to reach 191%, 169%, and 174% of control values, respectively

2. PCR array analysis of the effect of PUFA on the expression of transporters

In this study, the expression levels of 84 transporter genes in BeWo cells were evaluated by PCRarray. Ct values of BeWo cells without PUFA treatment (control). Approximately 70% of the transporter genes in PCRarray were detected in BeWo cells. 20% of genes are highly expressed, 28% are intermediate, and 23% of genes show low expression levels. The study showed a scatter plot of the expression level of each gene in the control sample with the AA-treated or EPA-treated samples. AA and EPA significantly increased SLC7A11 expression.


3.PUFA changes the expression and function of xCT / SLC7A11


Subsequently, RT-qPCR was used to verify the change of SLC7A11 mRNA of PUFA in BeWo cells. The expression levels of SLC7A11 induced by AA and EPA reached 414% and 636% of the control values, respectively. In addition, treatment with DHA increased SLC7A11 to 294% of the control value in BeWo cells. SLC7A11 encodes the cystine / glutamate transporter xCT protein. Western blot analysis showed that PUFA (AA, EPA, and DHA) increased xCT protein levels to 209%, 222%, and 219% of the control, respectively. xCT / SLC7A11 Na +-independently mediates the uptake of cystine to the cell by glutamate efflux. The function of xCT / SLC7A11 was evaluated by [14 C] -cystine transport activity in the absence of Na +. Based on the induction of xCT protein levels, AA, EPA, and DHA significantly increased [14 C] -cystine uptake to BeWo cells to 180%, 209%, and 250% of the control, respectively.

4. Knocking down NRF2 / NFE2L2 does not affect PUFA’s changes to xCT / SLC7A11

The effect of PUFA on xCT / SLC7A11 by NRF2 in BeFA cells was investigated using siRNA. NRF2 / NFE2L2 targeted siRNA treatment for 72 hours significantly reduced NRF2 mRNA expression to approximately 20% of the negative control level. Heme oxygenase-1 (HO-1 / HMOX1) is another target gene of NRF2 and is induced by PUFA (especially AA and DHA) under negative control siRNA conditions. NRF2 siRNA treatment attenuated HO-1 induction by these PUFAs. In contrast, NRF2 siRNA treatment did not attenuate xCT / SLC7A11 induction by PUFA (AA and EPA).

5. Expression of xCT / Slc7a11 in rat placenta

XCT has been reported to be overexpressed in various cancer cells and tissues. Therefore, we studied xCT / Slc7a11 expression in normal rat placenta by RT-PCR. XCT / Slc7a11 was detected in GD12 and GD20 rat placenta. In addition, the data indicate that xCT expression of GD 20 is lower than that of GD 12.


PUFA (AA, EPA, and DHA) increases the cystine / glutamic acid transporter gene xCT / SLC7A11, which mediates cellular uptake of cystine and efflux of glutamic acid in BeWo cells. These PUFAs also increase [14 C] -cysteine uptake in BeWo cells.

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