The expression levels of HSPB8 in placental tissues of patients with preterm PE and normal preterm pregnant women were analyzed using Gene Expression Omnibus (GEO) datasets (GSE102897 and GSE147776) downloaded from the national center of biotechnology information website (https://www.ncbi.nlm.nih.gov/) (28-30). GSE102897 dataset included the placenta tissues from normal human (n=3) and patients with severe preeclampsia (n=3). GSE147776 dataset included placenta tissues from normal human (n=8) and patients with severe preeclampsia (n=7). The analysis platform for GSE102897 and GSE147776 datasets were GPL22120 and GPL20844, respectively. The Biogrid database (https://thebiogrid.org) was used to predict the proteins that could interact with HSPB.

The human trophoblast cell line HTR-8/SVneo was acquired from American Type Culture Collection. The cells were routinely maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (MACGENE Biotechnology) at 37˚C in the presence of 5% CO₂. For the H/R treatment, when HTR-8/SVneo cells reached 50-60% confluence, they were exposed to 2% O₂, 5% CO₂ and 93% N₂ to achieve hypoxic conditions for 8 h. Subsequently, the cells were transferred to an atmosphere containing 20% O₂ and incubated for 16 h. A PE cell model was established by two cycles of H/R (31). The cells in the control group were cultured under standard culture conditions with 5% CO₂ for 48 h.

To overexpress HSPB8, HTR-8/SVneo cells were transfected with a HSPB8 plasmid (Ov-HSPB8) or the empty vector plasmid (Ov-NC). c-Myc was silenced by transfection of the cells with short interference (si) RNA sequences specific to c-Myc (siRNA-c-Myc-1, 5'-GAGCTAAAACGGAGCTTTTTTGC-3'; siRNA-c-Myc-2, 5'-GAGGAAGAAATCGATGTTGTTTC-3'). The cells transfected with a scrambled sequence [siRNA-negative control (NC)] (5'-AAGACAUUGUGUGUCCGCCTT-3') were considered to be the negative control. The aforementioned plasmids were all provided by Shanghai GenePharma Co., Ltd. The siRNA sequences were provided by OBiO Technology (Shanghai) Corp., Ltd. Subsequently, 4 µg Ov-HSPB8, Ov-NC, 100 nM siRNA-c-Myc-1/2 and siRNA-NC were transfected into HTR-8/SVneo cells with the application of Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. At 48 h post transfection, the cells were collected for subsequent experiments.

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) obtained from Beyotime Institute of Biotechnology. Following transfection and H/R exposure, HTR-8/SVneo cells were seeded into 96-well plates (1x10⁴/well). Following incubation at 37˚C for 24, 48 and 72 h, a CCK-8 solution was added to each well for an additional 2 h incubation at 37˚C. The optical density was recorded at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

The wound healing assay was conducted to evaluate the migratory ability of HTR-8/SVneo cells following the indicated treatment. Briefly, HTR-8/SVneo cells were seeded into six-well plates at a density of 1x10⁵ cells/well. When HTR-8/SVneo cells reached 90% confluence, a straight wound was created on the bottom of the plates using a sterile 100 µl pipette tip. Subsequently, the cells were incubated in serum-free RPMI-1640 medium for 24 h. The gap area was imaged using an inverted light microscope (Olympus Corporation) and the width of the wound was analyzed by ImageJ software (version 1.48v; National Institutes of Health).

Invasion of HTR-8/SVneo cells was assessed with the application of Transwell assay. A total of 5x10⁴ HTR-8/SVneo cells were suspended in 200 µl serum-free RPMI-1640 medium and were plated into the upper compartments of Transwell chambers (8 µm pore size; Corning, Inc.) coated with Matrigel (BD Biosciences) at 37˚C for 1 h. The lower chambers were subsequently filled with the normal RPMI-1640 medium containing 10% FBS. Following culture of the cells for 48 h at 37˚C, they were fixed with 4% paraformaldehyde for 20 min at 37˚C and stained with 0.1% crystal violet for 10 min at room temperature. The images were obtained with an inverted light microscope (Olympus Corporation) and the invading cells were counted using ImageJ software.

The content of ROS in HTR-8/SVneo cells was determined utilizing a ROS Assay kit (Beyotime Institute of Biotechnology) and the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) according to the instructions provided by the manufacturer. DCFH-DA (10 µmol/l) was applied for the staining of the cells for 20 min at 37˚C in the dark. The content of cellular ROS was evaluated by detecting the fluorescence intensity of the cells in each group using a confocal microscope (Olympus Corporation) with 488 nm excitation and 525 nm emission filters.

The mitochondrial membrane potential was quantified by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbo-cyanine iodide (JC-1) staining (Thermo Fisher Scientific, Inc.). HTR-8/SVneo cells were subjected to 10 mg/ml JC-1 staining for 10 min at 37˚C away from light. The normal mitochondrial potential exhibited red fluorescence (JC-1 aggregates) and the damaged mitochondrial potential displayed green fluorescence (JC-1 monomers). The images (five fields of view) were obtained using a confocal microscope (Olympus Corporation).

After H/R exposure and/or transfection, HTR-8/SVneo cells were washed three times with cold PBS, and then cell lysis buffer (Beyotime Institute of Biotechnology) was added for cell lysis. Following centrifugation at 1,000 x g for 10 min at 4˚C, the supernatant was collected for detection. The content of malondialdehyde (MDA; cat. no. A003-4-1) and the activity of superoxide dismutase (SOD; cat. no. A001-3-2) in the supernatant were detected using the corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute) according to the instructions provided by the manufacturer. The optical density was measured using a microplate reader (Bio-Rad Laboratories, Inc.).

The induction of apoptosis of the transfected cells was assessed by the TUNEL assay (Beyotime Institute of Biotechnology). HTR-8/SVneo cells were cultured on glass coverslips overnight. Following fixation with 4% paraformaldehyde at room temperature for 1 h, the cells were blocked with 3% H₂O₂ (dissolved) for 20 min at room temperature. Following permeation with 0.1% Triton X-100 at 4˚C for 2 min, the slides were incubated with 50 µl TUNEL reaction mixture for 1 h at 37˚C, mounted with Vectashield^® mounting medium. Following addition of DAPI solution at 37˚C for 10 min away from light, the cell samples were analyzed using an inverted fluorescence microscope (Olympus Corporation) in five random fields.

HTR-8/SVneo cells were lysed on ice for 30 min in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitors. The lysed protein samples (500 µg cell lysate) were incubated with antibodies against 2 µg anti-IgG (cat. no. ab172730; Abcam), anti-HSPB8 (cat no. 15287-1-AP; ProteinTech Group, Inc.) or anti-c-Myc (cat no. 10828-1-AP; ProteinTech Group, Inc.) antibodies and incubated overnight at 4˚C. Then, 50 µg protein A magnetic beads (cat. no. #sc-2003; Santa Cruz Biotechnology, Inc.) were added for capturing the complexes of HSPB8 and c-Myc. After the IP reaction, 50 µg protein G/A agarose beads were centrifuged at 1,000 x g for 3 min at 4˚C to the bottom of the tube. The supernatant was then carefully absorbed, and the agarose beads were washed three times with PBS. A total of 15 µl 2X SDS sample buffer was finally added for boiling at 100˚C for 5 min. Western blotting was used to analyze the expression levels of the target proteins.

TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used to extract the total RNA from HTR-8/SVneo cells. The RNA was reverse transcribed to complementary DNA (cDNA) using the PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. Quantitative PCR for the associated genes was conducted by RT-qPCR using SYBR Green master mix (Vazyme Biotech Co., Ltd.) on an ABI Prism 7500 Sequence Detector (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation for 2 min at 94˚C; followed by 35 cycles for 30 sec at 94˚C and 45 sec at 55˚C. β-actin was selected as the endogenous control. The calculation of the expression values was performed using the 2^-ΔΔCq method (32). Specific primers were shown below: HSPB8 forward, 5'-TTCCCAGACGACTTGACAGC-3', and reverse, 5'-GCCAATTGCGCTATCCTGTG-3'; c-Myc forward, 5'-GCAATGCGTTGCTGGGTTAT-3', and reverse, 5'-TCCCTCCGTTCTTTTTCCCG-3'; β-actin forward, 5'-GCCTCGCCTTTGCCGAT-3', and reverse, 5'-AGGTAGTCAGTCAGGTCCCG-3'.

Total proteins were extracted from HTR-8/SVneo cells using a RIPA lysis buffer (Beyotime Institute of Biotechnology). The concentration levels of the proteins were determined with the application of a bicinchoninic acid assay (BCA) kit (Beyotime Institute of Biotechnology). Equal amounts of proteins (40 µg proteins per lane) were separated using 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, followed by probing with primary antibodies at 4˚C overnight. Following addition of the HRP-conjugated goat anti-rabbit secondary antibody (1:2,000; cat. no. 7074S; Cell Signaling Technology, Inc.) for 1 h at room temperature, the bands were visualized using an ECL Plus Chemiluminescence Reagent kit (Pierce; Thermo Fisher Scientific, Inc.). The gray values of the protein bands were determined by ImageJ software (version 1.48v). The intensity of β-actin was used as a loading control. Anti-HSPB8 (1:2,000; cat. no. ab151552) and anti-c-Myc (1:2,000; cat. no. ab185656) antibodies were acquired from Abcam. Anti-Bcl-2 (1:1,000; cat. no. 4223T), anti-Bax (1:1,000; cat. no. 41162S), anti-cleaved caspase3 (1:1,000; cat. no. 9664T), anti-Caspase3 (1:1,000; cat. no. 9662S) and anti-β-actin (1:1,000; cat. no. 4970T) antibodies were provided by Cell Signaling Technology, Inc.

Data from three independent replicates were presented as the mean ± standard deviation and analyzed using GraphPad 8.0 statistical software (GraphPad Software, Inc.; Dotmatics). The differences between the two groups were calculated by the unpaired Student's t-test. One-way analysis of variance (ANOVA) followed by Tukey's test was appointed for data comparison among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

In order to identify the role of HSPB8 in PE, the GEO datasets (GSE102897 and GSE147776) were used for analysis and HSPB8 expression was found to be significantly reduced in placental tissues of preterm patients with PE compared with that noted in normal preterm pregnant women (Fig. 1A and B). Following exposure of HTR-8/SVneo cells to H/R conditions to mimic the PE pathology, HSPB8 expression was also significantly decreased compared with that of the control group (Fig. 1C). These data revealed the abnormal low expression of HSPB8 in placental tissues of patients with preterm PE and H/R-induced HTR-8/SVneo cells.

Subsequently, HSPB8 was overexpressed by transfection with HSPB8 plasmid and the transfected efficiency was assessed by RT-qPCR and western blotting. Significantly elevated HSPB8 expression was observed in the Ov-HSPB8 group compared with that noted in the empty vector control group (Fig. 2A and B). The results of the CCK-8 assay indicated that H/R stimulation decreased the viability of HTR-8/SVneo cells compared with control cells, which was significantly increased following transfection of the cells with the HSPB8 plasmid compared with the H/R + Ov-NC group (Fig. 2C). Moreover, overexpression of HSPB8 significantly restored H/R-induced limitations on the migration and invasion of HTR-8/SVneo cells compared with those noted in the H/R + Ov-NC group (Fig. 2D and E). The aforementioned results demonstrated that under H/R conditions, upregulation of HSPB8 expression accelerated the proliferation, migration and invasion of HTR-8/SVneo cells.

DCFH-DA staining indicated notably enhanced fluorescence intensity in HTR-8/SVneo cells in response to H/R stimulation, suggesting the elevated ROS levels in the H/R group (Fig. 3A). By contrast, HSPB8 overexpression markedly reduced the fluorescence intensity induced by H/R conditions compared with the H/R + Ov-NC group. In addition, in comparison with the control group, H/R stimulation increased JC-1 aggregate formation and decreased JC-1 monomer levels in HTR-8/SVneo cells, indicating the damaged mitochondrial potential in H/R-induced HTR-8/SVneo cells (Fig. 3B). Subsequent overexpression of HSPB8 caused elevated levels of JC-1 aggregates and reduced the levels of JC-1 monomers (Fig. 3B). Concomitantly, H/R caused a significant increase in the MDA content and a decrease in SOD activity, which were significantly reversed following transfection of the cells with the HSPB8 plasmid (Fig. 3C and D). Moreover, in contrast with the control group, significantly enhanced TUNEL staining was noted in the H/R group in HTR-8/SVneo cells compared with the control group, as determined by downregulation of Bcl-2 expression and upregulation of Bax and cleaved caspase 3 expression (Fig. 4A and B). However, overexpression of HSPB8 attenuated the effect of H/R on TUNEL staining and the expression levels of the aforementioned apoptotic proteins compared with the H/R + Ov-NC group (Fig. 4A and B). These results implied that HSPB8 overexpression alleviated oxidative stress and induction of apoptosis of HTR-8/SVneo cells exposed to H/R conditions.

To explore the potential mechanism of HSPB8 in the regulation of H/R-induced HTR-8/SVneo cells, the Biogrid database was used to predict the proteins that could interact with HSPB8; c-Myc was found to interact with HSPB8 (Fig. 5A). It was also shown that c-Myc expression was significantly downregulated in HTR-8/SVneo cells under H/R conditions compared with the control cell, and that HSPB8 overexpression significantly upregulated c-Myc expression compared with the Ov-NC group (Fig. 5B). The subsequent Co-IP data demonstrated the binding between HSPB8 and c-Myc (Fig. 5C). Subsequently, c-Myc expression was silenced. HTR-8/SVneo cells transfected with siRNA-c-Myc-1/2 displayed significantly decreased c-Myc expression levels compared with that of the siRNA-NC group (Fig. 5D and E). Due to the lower c-Myc expression in the siRNA-c-Myc-1 group, HTR-8/SVneo cells transfected with siRNA-c-Myc-1 were selected to perform the following experiments. It was observed in Fig. 5F-H that knockdown of c-Myc expression partially attenuated the elevated effects of HSPB8 overexpression on the proliferation, migration and invasion of H/R-induced HTR-8/SVneo cells. Collectively, these findings confirmed that HSPB8 binds to c-Myc to regulate the proliferation, migration and invasion of HTR-8/SVneo cells exposed to H/R conditions.

In contrast to the H/R + Ov-HSPB8 + siRNA-NC group, HTR-8/SVneo cells in the H/R + Ov-HSPB8 + siRNA-c-Myc group exhibited the highest ROS fluorescence intensity, the lowest level of JC-1 aggregate formation and the highest level of JC-1 monomers (Fig. 6A and B). In addition, c-Myc-silencing elevated MDA levels and reduced SOD activity compared with that noted in the negative control group (Fig. 6C and D). Consistent with these observations it was found that the decreased TUNEL levels, the upregulated Bcl-2 expression levels and the downregulated Bax and cleaved caspase3 expression levels in the H/R + Ov-HSPB8 group compared with the H/R group were all reversed by knockdown of c-Myc expression in HTR-8/SVneo cells exposed to H/R conditions compared with the siRNA-NC group (Fig. 7A and B). These data provide evidence that HSPB8 binding to c-Myc inhibited the induction of oxidative stress and apoptosis of HTR-8/SVneo cells exposed to H/R.