The Mechanism of HIF-1α in Migration and Invasion of Dairy Cow Placental Trophoblast Cells

doi:10.7668/hbnxb.20193290

Abstract

Abstract:

To investigate the effect of HIF-1α on the migration and invasion of trophoblast cells in placenta of dairy cows affected by cobalt dichloride(CoCl₂),and reveal the formation mechanism of placenta of dairy cows under hypoxia conditions,bovine placental trophoblast cells(BTCs)were isolated and purified from the placenta of early pregnancy cows.The cell morphology was observed by giemsa staining method,and the expression of cell markers cytokeratin 7(CK7)and Thy/CD90 were detected by immunofluorescence method.pCI-neo-hTERT plasmid was then transfected into BTCs,qPCR and Western Blot methods were used to detect the TERT mRNA and protein expression in BTCs.Proliferation rate and placental lactogen(PL)secretory ability were analyzed with CCK8 method and ELISA,respectively.siRNA was further used to silence the expression of Hypoxia inducible factor-1α(HIF-1α),vascular endothelial growth factor A(VEGFA)secretion ability in BTCs was detected by ELISA and the ability of migration and invasion of BTCs was detected by scratch test and transwell.According to the above all results,the isolated and purified BTCs were typical epithelioid cells with a certain number of binucleated cells and expressed epithelial marker protein CK7,but Thy/CD90 expression was not observed.The transfected cells could stably express telomerase reverse transcriptase(TERT)mRNA and protein.No cell aging was observed after 50 generations of continuous passage,and the proliferation rate was significantly higher than these primary BTCs(P<0.05),and the PL secretion ability showed no significantly different from that of primary BTCs(P>0.05).The cell viability of BTCs treated with CoCl₂ decreased with the increase of CoCl₂ concentration and time(P<0.05),and HIF-1α mRNA and protein expression decreased with the increase of CoCl₂ concentration(P<0.05).BTCs was treated with 400 μmol/L CoCl₂ for 24 h to establish a hypoxia model,and VEGFA secretion and invasion ability were significantly increased under BTCs hypoxia(P<0.05),while HIF-1α silencing decreased VEGFA secretion and invasion ability of BTCs(P<0.05).In conclusion,immortalized BTCs was established through exogenous TERT gene transduction.The immortalized BTCs had similar biological characteristics to the primary cells,and have stronger VEGFA secretion and invasion ability under hypoxia condition,and were associated with HIF-1α up-regulation.

Key words: Dairy cows, Trophoblasts, Hypoxia, Migration, Invasion, HIF-1α

SU Yue, GUO Yong, GAO Yuping, XIAO Longfei, LIAO Chenxing, QI Xiaolong, SHENG Xihui, XING Kai, WANG Xiangguo, NI Hemin. The Mechanism of HIF-1α in Migration and Invasion of Dairy Cow Placental Trophoblast Cells[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 202-209. doi: 10.7668/hbnxb.20193290.

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Figures/Tables 5

Tab.1 Target gene amplification primer sequences

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	产物长度/bp Product size	用途 Application
TERT	F: TATGCCGTGGTCCAGAAGG	384	qPCR
	R: CAAGAAATCATCCACCAAACG
HIF-1α	F: AGCTTGCTCATCAGTTGCCACTC	110	qPCR
	R: AGCATCCAGAAGTTTCCTCACACG
Glut1	F: TGTCTGGCATCAACGCTGTCTTC	144	qPCR
	R: CCTGCTCGCTCCACCACAAAC
VEGFA	F: CCTTGCCTTGCTGCTCTACCTTC	152	qPCR
	R: AGATGTCCACCAGGGTCTCGATG
GAPDH	F: CGGCACAGTCAAGGCAGAGAAC	116	qPCR
	R: CCACATACTCAGCACCAGCATCAC

Fig.1 Isolation,purification and identification of dairy cows BTCs A and B indicated morphological characteristics and giemsa stain results under a phase-contrast inverted microscope(A.bars=1 mm;B.bars=100 μmol/L),respectively;C and D indicated the expression of CK7 and CD90 in BTCs(bars=100 μmol/L).

Fig.2 Biological characteristics of immortalized BTCs The relative protein expression level of TERT in cells;B.The relative mRNA expression level of TERT in cells;C.The growth curve of cells;D.The endocrine ability of PL in cells.Different lowercase letters indicate significant difference. The same as Fig.3—4.

Fig.3 Establishment of hypoxia models of BTCs A.Cell viability;B,C,D.The relative protein and mRNA expression level of HIF-1α in cells;E.The relative mRNA expression level of Glut1 and VEGFA in cells.

Fig.4 The effects of hypoxic states on the expression of HIF-1α and VEGFA and migration and invasion ability of BTCs A.The relative protein expression of HIF-1α in cells;B.The relative mRNA expression of HIF-1α in cells;C.The endocrine ability of VEGFA in cells;D,E.The migration and invasion ability of cells(D. bars= 1 mm;E. bars= 100 μm).

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