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. 2020 Mar;24(5):3167-3182.
doi: 10.1111/jcmm.14990. Epub 2020 Jan 28.

The expression and biological function of chemokine CXCL12 and receptor CXCR4/CXCR7 in placenta accreta spectrum disorders

Yu Long  1 , Yonghua Jiang  2 , Jingjing Zeng  3 , Yiwu Dang  3 , Yue Chen  1 , Jueying Lin  4 , Hongwei Wei  5 , Hongwei Xia  5 , Junqing Long  5 , Cuizhen Luo  4 , Zhiwei Chen  6 , Yaling Huang  7 , MuJun Li  8
Affiliations

The expression and biological function of chemokine CXCL12 and receptor CXCR4/CXCR7 in placenta accreta spectrum disorders

Yu Long et al. J Cell Mol Med. 2020 Mar.

Abstract

Objectives: Investigation of mechanism related to excessive invasion of trophoblast cells in placenta accreta spectrum disorders (PAS) provides more strategies and ideas for clinical diagnosis and treatment.

Materials and methods: Blood and placental samples were collected from included patients. The distribution and expression of CXCL12, CXCR4 and CXCR7 proteins in the paraffin of placental tissue in the included cases were analysed, and we analyse the downstream pathways or key proteins involved in cell invasion.

Results: Firstly, our results determined that CXCL12 and CXCR4/CXCR7 were increased in extravillous trophoblastic cell (CXCL12: P < .001; CXCR4: P < .001; CXCR7: P < .001), and the expression levels were closely related to the invasion depth of trophoblastic cells. Secondly, CXCL12 has the potential to become a biochemical indicator of PAS since the high expression of placental trophoblast CXCL12 may be an important source of blood CXCL12. Using lentivirus-mediated RNA interference and overexpression assay, it was found that both chemokine CXCL12 and receptor CXCR4/CXCR7 are associated with regulation of trophoblast cell proliferation, migration and invasion. Further results proved that through the activating the phosphorylation and increasing the expression of MLC and AKT proteins in the Rho/rock, PI3K/AKT signalling pathway, CXCL12, CXCR4 and CXCR7 could up-regulate the expression of RhoA, Rac1 and Cdc42 proteins to promote the migration and invasion of extravillous trophoblastic cell and ultimately formate the placenta accrete compare to the normal placenta.

Conclusions: Our research proved that trophoblasts may contribute to a PAS-associated increase in CXCL12 levels in maternal blood. CXCL12 is not only associated with biological roles of PAS, but may also be potential for prediction of PAS.

Keywords: CXCL12; CXCR4/CXCR7; Function; Invasion; Trophoblast; placenta accreta spectrum disorders.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The expression of CXCL12, CXCR4 and CXCR7 in PAS and normal pregnant placental tissues. Immunohistochemical staining (A) and Western blot (B) showed the up‐regulation of CXCL12, CXCR4 and CXCR7 in PAS and normal pregnant placental tissues (Control)
Figure 2
Figure 2
The expression of plasma CXCL12, CXCR4 and CXCR7 in PAS and normal pregnant women. (A) Plasma CXCR4 and CXCR7 levels in women with PAS and normal controls. (B) Plasma CXCL12 levels in women with PAS by depth of invasion. (C) Correlations between the immunohistochemical staining scores and plasma concentrations of CXCL12, CXCR4 and CXCR7 in women with PAS. r = Pearson's correlation coefficient. (**P ＜ .01, ***P ＜ .001)
Figure 3
Figure 3
RT‐qPCR and Western blot detected CXCL12, CXCR4 and CXCR7 expression levels in CXCL12, CXCR4 and CXCR7 silenced or overexpression HTR‐8/SVneo cells. (A) RT‐qPCR detect transcriptional levels of silenced CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo (*P ＜ .05: Silenced cells vs Control; #P ＜ .05: Silenced cells vs sh‐NC). (B) RT‐qPCR detect transcriptional levels of overexpression CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo (*P ＜ .05: Overexpressed cells vs control; #P ＜ .05: Overexpressed cells vs OE‐NC). (C) Western blot to verify protein levels of silenced CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo (*P ＜ .05: Silenced cells vs Control; #P ＜ .05: Silenced cells vs sh‐NC). (D) Western blot to verify protein levels of overexpression CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo (*P ＜ .05: Overexpressed cells vs control; #P ＜ .05: Overexpressed cells vs OE‐NC)
Figure 4
Figure 4
CXCL12, CXCR4 and CXCR7 genes promote cell proliferation of HTR‐8/SVneo. (A‐B) The effect of CXCL12, CXCR4 and CXCR7 silenced (A) or overexpression (B) in HTR‐8/SVneo on cell proliferation by CCK8 assays. (C‐F) The effect of CXCL12, CXCR4 and CXCR7 silenced (C, E) or overexpression (D, F) in HTR‐8/SVneo on cell proliferation by cloning formation experiment
Figure 5
Figure 5
CXCL12, CXCR4 and CXCR7 genes promote cell migration and invasion of HTR‐8/SVneo. (A) The effect of silenced CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo on cell migration by wound healing assays (*P ＜ .05: Silenced cells vs Control; #P ＜ .05: Silenced cells vs sh‐NC), Bar = 200 μm. (B) The effect of overexpression CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo on cell migration by wound healing assays (*P ＜ .05: Overexpressed cells vs control; #P ＜ .05: Overexpressed cells vs OE‐NC), Bar = 200 μm. (C) The effect of silenced CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo on cell invasion by transwell invasion assays. (*P ＜ .05: Silenced cells vs Control; #P ＜ .05: Silenced cells vs sh‐NC), Magnification × 100. (D) The effect of overexpression CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo on cell invasion by transwell invasion assays. (*P ＜ .05: Overexpressed cells vs control; #P ＜ .05: Overexpressed cells vs OE‐NC), Magnification × 100
Figure 6
Figure 6
CXCL12, CXCR4 and CXCR7 have no obvious effect on cell apoptosis of HTR‐8/SVneo. (A, C) The effect of silenced CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo on cell apoptosis by flow cytometric assays (*P ＜ .05: Silenced cells vs Control; #P ＜ .05: Silenced cells vs sh‐NC). (B, D) The effect of overexpression CXCL12, CXCR4 and CXCR7 in HTR‐8/SVneo on cell apoptosis by flow cytometric assays (*P ＜ .05: Overexpressed cells vs control; #P ＜ .05: Overexpressed cells vs OE‐NC)
Figure 7
Figure 7
CXCL12, CXCR4 and CXCR7 regulates cell invasion via in Rho/rock, PI3K/AKT signaling pathways. (A) Western blot assays detected RhoA, Rac1 and Cdc42 protein expressions in CXCL12, CXCR4 and CXCR7 silenced HTR‐8/SVneo cells. The intensity of Western blot bands was quantified by image J software (*P ＜ .05: Silenced cells vs Control; #P ＜ .05: Silenced cells vs sh‐NC). (B) Western blot assays detected RhoA, Rac1 and Cdc42 protein expressions in CXCL12, CXCR4 and CXCR7 overexpression HTR‐8/SVneo cells. The intensity of Western blot bands were quantified by image J software (*P ＜ .05: Overexpressed cells vs control; #P ＜ .05: Overexpressed cells vs OE‐NC). (C) The effect of silenced CXCL12, CXCR4 and CXCR7 on Rho/Rock and PI3K/AKT signaling pathways by Western blotting. (*P ＜ .05: Silenced cells vs Control; #P ＜ .05: Silenced cells vs sh‐NC), (D) The effect of silenced CXCL12, CXCR4 and CXCR7 on p‐Akt/Akt and p‐MLC/MLC ratios. (E) The effect of CXCL12, CXCR4 and CXCR7 overexpression on Rho/Rock and PI3K/AKT signaling pathways by Western blotting. (*P ＜ .05: Overexpressed cells vs Control; #P ＜ .05: Overexpressed cells vs sh‐NC), (F) The effect of CXCL12, CXCR4 and CXCR7 overexpression on p‐Akt/Akt and p‐MLC/MLC ratios
Figure 8
Figure 8
The effect of blocking Rho/Rock or PI3K/AKT pathways on the invasion of CXCL12, CXCR4 or CXCR7 stably overexpressed HTR‐8/SVneo cells by transwell invasion assay (A) and statistics (B). NVP‐BEZ235: PI3K inhibitor (#P ＜ .05: vs Control), Y‐27632: Rock inhibitor (*P ＜ .05: vs Control)
Figure 9
Figure 9
Schematic representation of CXCL12 and receptor CXCR4/CXCR7 in placenta accrete spectrum disorders (PAS). PAS increased expression of chemokine CXCL12 and receptor CXCR4/CXCR7 in extravillous trophoblastic cells. CXCL12, CXCR4 and CXCR7 could up‐regulate the expression of RhoA, Rac1 and Cdc42 proteins to promote the migration and invasion of extravillous trophoblastic cells and ultimately form PAS through increase of phospho‐MLC and phospho‐AKT in Rho/rock and PI3K/AKT signaling pathway
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