Objective:To investigate the expression and methylation status of the SALL4 gene in placental tissues of fetal growth restriction (FGR) and its effects on trophoblast cell proliferation, migration, and invasion. Methods:Placental tissues were collected from 20 full-term FGR patients and 20 healthy term controls who underwent regular prenatal examination and cesarean section at the Third Affiliated Hospital, Zhengzhou University between July 2023 and February 2024. SALL4 mRNA and protein expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Methylation specific polymerase china reaction (MSP) assessed promoter methylation levels. HTR8/SVneo cells were transfected with SALL4-targeting small interfering RNA (si-SALL4) or negative control small interfering RNA (si-NC). HTR8/SVneo cells were treated with the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dC) to inhibit gene methylation (5-Aza-dC group) or with 10% RPMI-1640 medium as a vehicle control. Transfection efficiency (for siRNA) and the efficacy of 5-Aza-dC-induced demethylation were assessed by qRT-PCR and Western blot. The functional effects of SALL4 knockdown and methylation inhibition on trophoblast cells were evaluated using proliferation assays, scratch wound healing assays, and Transwell invasion assays. Statistical analyses included independent t-tests and Chi-square test. Results:(1) Human tissues: FGR placentas showed lower SALL4 mRNA (0.802±0.194 vs. 1.015±0.186, t=3.55) and protein expression (0.445±0.114 vs. 0.701±0.113, t=3.19), alongside higher methylation rates of SALL4 [80% (16/20) vs. 15% (3/20), χ2=14.44] compared to controls (all P<0.05). (2) In vitro: si-SALL4 transfection reduced HTR8/SVneo proliferation (OD450 at 48 h: 0.653±0.021 vs. 0.827±0.040, t=6.60), migration [healing rate at 48 h: (24.317±2.637)% vs. (49.327±1.961)%, t=13.18], and invasion [counted invaded cells: (133.000±6.557) vs. (272.667±18.009) cells, t=12.62] versus si-NC (all P<0.05). Conversely, 5-Aza-dC treatment increased HTR8/SVneo proliferation (0.917±0.042 vs. 0.783±0.031, t=－4.47), migration [(71.097±3.354)% vs. (51.632±2.877)%, t=－7.63], and invasion [(384.000±12.166) vs. (202.833±7.095) cells, t=－13.69] versus vehicle control (all P<0.05). Conclusions:Hypermethylation of the SALL4 promoter in FGR placentas suppresses its expression, impairing trophoblast cell function. Demethylation restores SALL4 expression and enhances cellular proliferation, migration, and invasion, involving in the occurrence and development of FGR disease.

Objective:To investigate the expression and methylation status of the SALL4 gene in placental tissues of fetal growth restriction (FGR) and its effects on trophoblast cell proliferation, migration, and invasion. Methods:Placental tissues were collected from 20 full-term FGR patients and 20 healthy term controls who underwent regular prenatal examination and cesarean section at the Third Affiliated Hospital, Zhengzhou University between July 2023 and February 2024. SALL4 mRNA and protein expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Methylation specific polymerase china reaction (MSP) assessed promoter methylation levels. HTR8/SVneo cells were transfected with SALL4-targeting small interfering RNA (si-SALL4) or negative control small interfering RNA (si-NC). HTR8/SVneo cells were treated with the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dC) to inhibit gene methylation (5-Aza-dC group) or with 10% RPMI-1640 medium as a vehicle control. Transfection efficiency (for siRNA) and the efficacy of 5-Aza-dC-induced demethylation were assessed by qRT-PCR and Western blot. The functional effects of SALL4 knockdown and methylation inhibition on trophoblast cells were evaluated using proliferation assays, scratch wound healing assays, and Transwell invasion assays. Statistical analyses included independent t-tests and Chi-square test. Results:(1) Human tissues: FGR placentas showed lower SALL4 mRNA (0.802±0.194 vs. 1.015±0.186, t=3.55) and protein expression (0.445±0.114 vs. 0.701±0.113, t=3.19), alongside higher methylation rates of SALL4 [80% (16/20) vs. 15% (3/20), χ2=14.44] compared to controls (all P<0.05). (2) In vitro: si-SALL4 transfection reduced HTR8/SVneo proliferation (OD450 at 48 h: 0.653±0.021 vs. 0.827±0.040, t=6.60), migration [healing rate at 48 h: (24.317±2.637)% vs. (49.327±1.961)%, t=13.18], and invasion [counted invaded cells: (133.000±6.557) vs. (272.667±18.009) cells, t=12.62] versus si-NC (all P<0.05). Conversely, 5-Aza-dC treatment increased HTR8/SVneo proliferation (0.917±0.042 vs. 0.783±0.031, t=－4.47), migration [(71.097±3.354)% vs. (51.632±2.877)%, t=－7.63], and invasion [(384.000±12.166) vs. (202.833±7.095) cells, t=－13.69] versus vehicle control (all P<0.05). Conclusions:Hypermethylation of the SALL4 promoter in FGR placentas suppresses its expression, impairing trophoblast cell function. Demethylation restores SALL4 expression and enhances cellular proliferation, migration, and invasion, involving in the occurrence and development of FGR disease.