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Introduction

The incidence of spontaneous abortion (SA) is 10-15% in clinical pregnancies globally (1). The definition of SA is clinically confirmed pregnancy loss within 24 weeks of pregnancy (2,3). The etiology of pregnancy loss is complicated, and genetic factors such as chromosomal abnormalities and single gene disorders, endocrinological factors such as diabetes mellitus and thyroid disease, environmental factors such as alcohol and tobacco and infectious factors such as viral and bacterial factors are all the risk factors of SA (4,5). However, there may still be other unknown factors contributing to SA, therefore further investigation is needed.

The cellular mechanisms underlying recurrent abortion are the proliferation and apoptosis of cytotrophoblasts and human decidual cells (6). Zhang et al (7) reported that microRNA (miR)-184 is highly expressed in decidual stromal cells, decidual immune cells and peripheral blood of patients who experience recurrent spontaneous abortion (RSA). Mechanically, miR-184 promotes apoptosis and inhibits the proliferation of trophoblast cells by targeting and regulating WIG1(7). miR-520 also enhances the apoptosis of trophoblast cells by targeting poly (ADP-ribose) polymerase (PARP)1 in human-trophoblast-derived HTR-8/SVneo cells (8). Ferroptosis is form of programed cell death that is different from apoptosis both in morphology and biochemical levels such as caspase-3 activation, and is characterized by the accumulation of reactive oxygen species (ROS) as a result of iron accumulation and lipid peroxidation (9). Ferroptosis is involved in several diseases, such as ischemia/reperfusion-induced organ injury, stroke and cancer, and ferroptosis inhibition is effective in treating ischemia/reperfusion-induced organ injury and stroke in a number of experimental models in vivo (10-12). However, whether ferroptosis is involved in the process of SA is still unknown.

Long non-coding RNAs (lncRNAs), which are >200 nucleotides in length, have important roles in a number of cellular processes, including posttranslational regulation and carcinogenesis (13,14). H19 is a lncRNA that is 2.3 kb long and is primarily located in cytoplasm. During embryonic development, H19 is highly expressed; however, after birth, the expression of H19 is decreased (15). Most studies investigating H19 have focused on its role in carcinogenesis, including tumor growth and metastasis (16,17), and although several studies have analyzed the expression and methylation status of H19 in SA (18,19), whether H19 regulates apoptosis and ferroptosis in SA is unknown.

The present study analyzed the expression levels of H19 and the apoptosis- and ferroptosis-associated genes Bcl-2, Bax and phospholipid hydroperoxide glutathione peroxidase (GPX4) in SA tissues. Importantly, the current study also further evaluated the regulatory correlations between H19 and Bcl/GPX4/Bax both at the mRNA and protein levels.

Materials and methods

Patient sample collection

The placental villi tissues from patients with SA (SA group) and clinically normal pregnancies terminated for non-medical reasons (control group) were collected. The SA group consisted of 30 women aged 21-38 years. The criteria for inclusion in SA group were as follows: i) Normal chromosome number and structure of both the mother and father, ii) no thyroid dysfunction, diabetes and other systemic diseases, iii) no reproductive endocrine disease, iv) no deformity of genital tract and uterus and v) the activity and quality of sperm of the father were normal. The control group included 30 women aged 21-38 years who requested termination due to an unplanned pregnancy. The criteria for control group were as follows: i) No vaginal bleeding, ii) no abdominal pain. All women in these two groups were between gestational weeks six to nine. The present study was approved by The Medical Ethics Committee of the First People's Hospital of Yunnan Province (approval no. 2018-012, Kunming, China) and written informed consent was provided by each patient.

Cell culture

HTR-8/SVneo cells (henceforth referred to as HTR-8) were purchased from the American Type Culture Collection and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml in CO2 at 37˚C.

Cell transfection

A H19 knockdown lentiviral vector (pGLVU6-puro-H19-shRNA) and negative vector (non-targeting control, pGLVU6-puro-NC-shRNA) were constructed by Shanghai GenePharma Co., Ltd. 3 µg H19 knockdown lentiviral vector/negative vector were co-transfected with 2.5 µg pMD2.G and 7.5 µg psPAX2 plasmids into 293T cells (75 cm2 flasks) for lentivirus packaging with Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.). The sequences are as follows: H19 short hairpin (sh)RNA, 5'-CCGGCAGCCTTCAAGCATTCCATTACTCGAGTTTTTG-3'; NC shRNA, 5'-CCGGTTCTCCGAACGTGTCACGTTTTTTG-3'. Lentivirus infection was applied to HTR-8 cells with 50-60% confluence. Reverse transcription-quantitative (RT-q)PCR was used to examine the efficiency of H19 knockdown after 72 h of infection.

RT-qPCR

TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA of tissues and cells according to the protocol of the manufacturer. A QuantiTect Reverse Transcription kit (Qiagen China Co., Ltd.) was used to synthesize cDNA by incubating at 42˚C for 15 min. H19, Bcl-2, Bax and GPX4 levels were detected by using a SYBR Green RT-qPCR assay kit (Takara Biotechnology Co., Ltd.), and GAPDH was used as the endogenous control. The PCR program was as follows: Stage 1, 95˚C for 30 sec; stage 2, 95˚C for 5 sec and 60˚C for 34 sec, for 40 cycles. The primer sequences were as follows: Bcl-2, forward, 5'-GGTGGGGTCATGTGTGTGG-3' and reverse, 5'-CGGTTCAGGTACTCAGTCATCC-3'; Bax, forward, 5'-CCCGAGAGGTCTTTTTCCGAG-3' and reverse, 5'-CCAGCCCATGATGGTTCTGAT-3'; GPX4, forward, 5'-GAGGCAAGACCGAAGTAAACTAC-3' and reverse, 5'-CCGAACTGGTTACACGGGAA-3'; H19, forward, 5'-CGTGACAAGCAGGACATGACA-3' and reverse, 5'-CCATAGTGTGCCGACTCCG-3'; GAPDH, forward, 5'-GGAGCGAGATCCCTCCAAAAT-3' and reverse, 5'-GGCTGTTGTCATACTTCTCATGG-3'. Quantitative measurements were evaluated using the 2-ΔΔCq method (20). The mRNA levels of H19, Bcl-2, Bax and GPX4 in shCON group were normalized to 1.

Western blotting

HTR-8 cells were collected and lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology) for 25 min on ice. Protein concentrations were evaluated using the BCA method. Equal amounts of proteins (7-10 µg per sample) were loaded per lane on a 12% gel, resolved using SDS-PAGE and subsequently transferred to PVDF membranes. Then the membranes were blocked with 5% non-fat milk for 1 h at the room temperature. The membranes were treated with primary antibodies overnight at 4˚C. Next, the membranes were treated with the secondary antibodies for 1 h at room temperature. The protein signals were detected using ECL Detection Reagent (Beijing Solarbio Science & Technology Co., Ltd.). The primary antibodies including anti-Bcl-2 (1:1,000; cat. no. 15071), anti-Bax (1:1,000; cat. no. 5023) and anti-β-actin (1:5,000; cat. no. 3700) antibodies were purchased from Cell Signaling Technology, Inc., and anti-GPX4 (1:1,000; cat. no. ab125066) was purchased from Abcam. The secondary antibodies including anti-mouse IgG, HRP-linked antibody (1:3,000; cat. no. 7076) and anti-rabbit IgG, HRP-linked antibody (1:3,000; cat. no. 7074) were purchased from Cell Signaling Technology, Inc. β-actin was used as the internal control.

Cell viability analysis

After H19 shRNA infection for 48 h, HTR-8 cells were seeded in 96-well plates. The cells were then divided into six groups, the shCON, shH19, shCON+Z-VAD-FMK, shH19+Z-VAD-FMK, shCON+Ferrostatin-1 and the shH19+Ferrostatin-1 group. After treatment for 48 h, cell viability was examined by using the Cell Counting Kit-8 method as previously described (21). Z-VAD-FMK (Selleck Chemicals) was a strong apoptosis inhibitor, and ferrostatin-1 (Selleck Chemicals) was a ferroptosis inhibitor which could depress the cellular lipid peroxidation (22).

Statistical analysis

Data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS version 19.0 software (IBM, Corp.). Data were analyzed using a two-tailed unpaired Student's t-test (two groups) or analysis of variance (ANOVA) with Tukey's post hoc test for multiple comparisons. The correlation between Bax, Bcl-2 and GPX4 mRNA expression with H19 levels was analyzed using Pearson's correlation. P<0.05 was considered to indicate a statistically significant difference. All in vitro experiments were repeated in triplicate.

Results

Expression levels of H19 and apoptosis- and ferroptosis-associated genes in the SA group

Sixty patients were included in the present study, including 30 cases in the SA group and 30 cases in the control group. RT-qPCR was used to detect the RNA expression levels of H19, Bcl-2, Bax and GPX4. The results showed that the RNA expression levels of H19 were significantly lower in SA group compared with those in the control group (Fig. 1A). It was further found that the mRNA expression of anti-apoptosis gene Bcl-2 was significantly lower, and the mRNA expression of proapoptotic gene Bax was significantly higher in SA group compared with those in control group (Fig. 1B and C). Notably, the ferroptosis-associated gene GPX4 was also significantly downregulated in SA group compared with the control group (Fig. 1D).

Figure 1 mRNA expression of H19, Bcl-2, Bax and GPX4 in SA. (A-D) Reverse transcription-quantitative PCR was used to detect the expression of H19, Bcl-2, Bax and GPX4 in the SA group (n=30) and control group (n=30). **P<0.01 and ***P<0.001 vs. control. SA, spontaneous abortion; lncRNA, long non-coding RNA; GPX4, phospholipid hydroperoxide glutathione peroxidase. Circle, expression data of each control sample; square, expression data of each SA sample.

Correlation analysis was applied to evaluate relationship between the RNA expression levels of H19 and apoptosis- and ferroptosis-associated genes. lncRNA H19 expression was significantly positively correlated with Bcl-2 expression (R=0.4730, P=0.0083), and significantly negatively correlated with Bax expression (R=-0.3714, P=0.0433; Fig. 2A and B). Notably, it was also demonstrated that H19 expression was significantly positively correlated with GPX4 expression (R=0.6450, P=0.0001; Fig. 2C).

Figure 2 Correlation between the expression of H19 and (A) Bcl-2, (B) Bax and (C) GPX4 in the SA group. lncRNA, long non-coding RNA; GPX4, phospholipid hydroperoxide glutathione peroxidase. Circle, expression data of each SA sample. SA, spontaneous abortion.

Western blotting was used to evaluate the expression of apoptosis- and ferroptosis-associated proteins. The protein levels of Bax were higher in SA group, and Bcl-2 levels were lower in SA group compared those in the control group. In addition, GPX4 expression was downregulated in SA group compared with the control group (Fig. 3).

Figure 3 Protein expression of Bcl-2, Bax and GPX4 in SA group compared with the control group as analyzed using western blotting. SA, spontaneous abortion; GPX4, phospholipid hydroperoxide glutathione peroxidase.

Silencing H19 downregulates Bcl-2 and GPX4 and upregulates Bax in HTR-8 cells

HTR-8 cells were transfected with the shH19 lentiviral construct, and the results showed that the expression of H19 was significantly decreased by ~80% (Fig. 4A). Then, the expressions of Bcl-2, Bax and GPX4 were evaluated using RT-qPCR and western blotting. The results showed that silencing H19 significantly reduced expression of Bcl-2 and GPX4 and increased Bax expression at both the mRNA and protein levels (Fig. 4B-E).

Figure 4 Silencing H19 downregulates Bcl-2 and GPX4 expression and upregulates Bax expression at both the mRNA and protein levels. (A-D) Reverse transcription-quantitative PCR was used to detect the expression of H19, Bcl-2, Bax and GPX4 in H19 silenced HTR-8 cells. (E) Western blotting was used to detect the protein expression of Bcl-2, Bax and GPX4 in the H19 silenced HTR-8 cells. The experiments were performed three times. **P<0.01 and ***P<0.001 vs. control. sh, short hairpin; shNC, negative control group; shH19, H19 knockdown group; GPX4, phospholipid hydroperoxide glutathione peroxidase; lncRNA, long non-coding RNA. Circle, expression data of shCON group; square, expression data of shH19 group.

The viability of HTR-8 cells following H19 knockdown and apoptosis or ferroptosis inhibitor treatment was further examined. The results showed that H19 silencing significantly reduced the viability of HTR-8 cells, and Z-VAD-FMK (an apoptosis inhibitor) and Ferrostatin-1 (a ferroptosis inhibitor) (22) could partially rescue the decreased viability of HTR-8 cells induced by H19 knockdown (Fig. 5).

Figure 5 Apoptosis and ferroptosis inhibitors partially rescue decreased cell viability induced by H19 knockdown in HTR-8 cells. Cell viability was detected by using a Cell Counting Kit-8 assay. The experiments were performed three times. ***P<0.001 vs. shH19. shNC, negative control group; shH19, H19 knockdown group. Filled circle, cell viability of shCON group; square, cell viability of shH19 group; upturned triangle, cell viability of shCON+Z-VAD-FMK group; downturned triangle, cell viability of shH19+ Z-VAD-FMK group; diamond, cell viability of shCON+Ferrostatin-1 group; unfilled circle, cell viability of shH19+Ferrostatin-1 group.

Discussion

SA is the most frequently occurring pregnancy disorder and is a serious threat to women's health (23). SA has been reported to be associated with several risk factors, including endocrine disorders, anatomic deformation, immunodeficiency and chromosomal abnormalities (24-27). However, other risk factors and underlying molecular mechanisms of SA still need further investigation.

H19, a lncRNA, is involved in the pathogenesis of several diseases, including lung cancer and pre-eclampsia (28,29). Although a previous study reported that the methylation of H19 differentially methylated regions is increased in the placental samples of SA cases (23), the role and underlying mechanism of H19 in SA are still largely unclear. The present study revealed that the expression of H19 was lower in SA group compared with that in control group.

Trophoblast cells participate in the formation of placenta and have important functions in embryo implantation (30). Apoptosis of trophoblast cells is involved in SA (31). The apoptotic rate and mRNA and protein expression of Fas and FasL were significantly higher in recurrent spontaneous abortion compared with the abortion group (32). Li et al (33) reported that knockdown of Storkhead-box protein 1 induced apoptosis and decreased the migration and proliferation of HTR-8/SVneo cells via mediating the PI3K/AKT signaling pathway (33). A previous study also reported that miR-184, which is highly expressed in RSA, repressed the proliferation and promoted the apoptosis of trophoblast cells by targeting WIG1 and upregulating its downstream molecule Fas (7). Dong et al (8) also found that miR-520, which is highly expressed in villi in RSA, enhanced the apoptosis of human trophoblast cells by targeting and regulating PARP1 expression. Increased cyclin-dependent kinase inhibitor 1 and Bax expression was associated with increased apoptosis of cytotrophoblasts and human decidual cells in the RSA group (34). However, the regulatory mechanisms underlying trophoblast apoptosis are still largely unknown. The present results demonstrated that the mRNA and protein expression of Bcl-2 was lower and the expression of Bax was higher in SA compared with control tissues. Notably, the expression of Bcl-2 was positively correlated with H19 expression, and Bax expression was negatively correlated with H19 levels. In vitro analysis revealed that silencing H19 decreased Bcl-2 and upregulated Bax expression at both the mRNA and protein levels.

Ferroptosis is a caspase-independent, non-apoptotic and newly defined type of cell death characterized by high levels of lipid peroxidation (35). GPX4 is the key regulator of ferroptosis by controlling the activity of cyclooxygenase and lipid-modulating lipoxygenase enzymes (36). A recent study has reported that an inactivating mutation of GPX4 has a dominant-negative effect on male fertility (36). However, the roles of ferroptosis and GPX4 in SA are still unknown. The present findings reported that GPX4 expression was lower in SA group compared with that in control group and was positively correlated with H19 expression. In addition, silencing H19 downregulated GPX4 expression levels. Overall, these results indicated that ferroptosis was involved in SA.

The mechanisms by which lncRNA H19 regulates Bcl-2, Bax and GPX4 expression is still unclear. A previous study reported that H19 could directly binds to the miR-17-92 cluster and suppress STAT3 activity, decreasing the expression of its target genes Bcl-2 and Bcl-2L1 (37). H19 is also reported to positively regulate Bcl-2 expression by sponging miR-877-3p in myocardial ischemia/reperfusion injury (I/RI) (38). Whether H19 regulates Bcl-2 by these two pathways or other pathways in SA still needs to be explored.

There are some limitations to the present study. Firstly, the regulatory association between H19 and Bcl-2, Bax and GPX4 was confirmed in only one cell line. In the future, more cell lines should be used to verify the present findings. Secondly, an Annexin V/PI and lipid peroxidation assays should be used in the figure to investigate the regulatory association between H19 and apoptosis and ferroptosis.

Overall, the present study demonstrated that H19 was downregulated in SA cases, and H19 expression was positively correlated with Bcl-2 and GPX4 levels, and negatively correlated with Bax expression. In addition, silencing H19 downregulated Bcl-2 and GPX4, and upregulated Bax at both the mRNA and protein levels. The results of the current study may provide a novel insight for the pathogenesis for SA.

Acknowledgements

Not applicable.

Funding

The present study was funded by The Open Project Program of Yunnan Provincial Key Laboratory for Birth Defects and Genetic Diseases (grant no. 2017ZDKFKT001).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

RXB and ZYT designed the study. RXB and ZYT performed the experiments. ZYT wrote the paper. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by The Medical Ethics Committee of the First People's Hospital of Yunnan Province (approval no. 2018-012, Kunming, China) and written informed consent was provided by each patient.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References