Introduction

Preeclampsia (PE) is primarily characterized by hypertension and proteinuria. It can be accompanied by liver dysfunction, renal insufficiency, pulmonary edema, thrombocytopenia, and other multi-system and multi-organ damage. The incidence of PE is 3–5%, making it a significant cause of both morbidity and mortality1. PE is a multi-system progressive disease, and the exact pathophysiological mechanisms remains unclear. Immune, genetic, and maternal risk factors can lead to weakened trophoblast cell invasion, resulting in reduced placental perfusion. This induces the release of placental anti-angiogenic factors into the maternal bloodstream, which antagonizes pro-angiogenic factors, leading to insufficient angiogenic factor release. Consequently, excessive inflammatory responses and vascular endothelial dysfunction occur, promoting platelet aggregation, increased activation of the coagulation system, and elevated systemic microvascular resistance2,3.

Clinical studies have shown that4,5, taking low-dose aspirin, calcium, and multivitamins before 16 weeks of pregnancy can help prevent the occurrence of PE, but this intervention have no clinical significance after this gestational week. Currently, the only effective radical treatment for PE is termination of pregnancy; however, termination does not fully alleviate the impact of preeclampsia. PE increases the long-term risk of cardiovascular diseases and other complications in both the mother and the fetus. For example, the risk of maternal cardiovascular events in pregnant women with severe preeclampsia is 4.7 times higher than in normal pregnant women6, and the risk of neurodevelopmental disorders or mental illness in their children is 18% higher than in normal children7. Moreover, expectant management is a common treatment method for preeclampsia, which has garnered extensive clinical attention for improving pregnancy outcomes and maternal and fetal safety. Expectant therapy involves methods such as spasmolysis8,9, antihypertensive treatment10, anticoagulation11, and fetal lung maturation to extend gestational age, thereby effectively improving neonatal survival and maturation rates and enhancing maternal and infant outcomes.

MicroRNAs (miRNAs) are highly conserved small RNAs, typically ranging from 16 to 25 nucleotides in length. The first small RNA ‘lin-4’ and the second ‘let-7’ were reported in N. elegans in 1993 and 2000, respectively12. Mature miRNAs bind to messenger RNAs, forming RNA-induced silencing complexes that can recognize and bind to target genes in complementary or partially complementary ways. This process affects mRNA stability and regulates target gene expression and protein translation, influencing organ growth, development, cell proliferation, and apoptosis13,14,15. Studies have identified over 70 differentially expressed miRNAs in the placental tissues of PE patients. These miRNAs regulate angiogenesis, proliferation, apoptosis, immune responses, and oxidative stress by altering the levels of associated genes, playing significant roles in PE16. For instance, miR-30a-3p17, miR-547-5p18, and miR-1619 inhibit the expression of IGF-1, MKI67, and Notch2, respectively, inducing trophoblast cell apoptosis and contributing to PE. Conversely, miR-221-3p20, miR-145-5p21, and miR-576-5p22 promote trophoblast cell proliferation and placental formation by inhibiting the expression of THBS2, FLT1, and TFAP2A, respectively, thereby preventing the occurrence and progression of PE.

The PIK3R1 gene encodes the p85α protein, the regulatory subunit of the phosphoinositide 3-kinase (PI3K) complex23. Studies have shown that miR-155 can mediate apoptosis by downregulating PIK3R1, thereby activating the PI3K/AKT signaling pathway24. Specifically, the reduced expression of PIK3R1 leads to a decrease in the p85α monomer, resulting in the activation of the PI3K/AKT signaling pathway23,24. As a negative regulator of the PI3K/AKT pathway, PIK3R1 is a direct target of miR-155. Overexpression of miR-155 inhibits the expression of PIK3R1 mRNA and, consequently, the p85α protein, leading to the overactivation of the pro-apoptotic PI3K/AKT signaling pathway. This results in changes in the levels of key apoptotic proteins such as Bad, Bcl-2, and Bax, which mediate apoptosis through the downstream AKT apoptotic pathway24,25. While miR-155 is known to regulate a variety of diseases through the negative regulation of PIK3R1, its involvement in the pathogenesis of preeclampsia (PE) remains unexplored.

MiR-155-5p located in the third exon of the B-cell non-coding cluster on human chromosome 21 and is one of the earliest discovered miRNAs in humans26. Reports showed that miR-155-5p is highly expressed in the placenta of PE patients27, may aggravate PE28. This study aims to explore the mechanism of miR-155-5p in PE, providing potential targets for the prevention and treatment of PE.

Materials and methods

Clinical sample

All placental samples were obtained from preeclamptic (PE) patients and normal controls at Jinhua People’s Hospital, following ethical approval from the hospital’s Ethics Committee (Approval No. IRB-2020063-R). The PE patient group (n = 30) included women with a diagnosis of preeclampsia based on standard clinical criteria, the mean age of the patients was 30.4 years old and the mean gestational age was 38 weeks. The normal control group (n = 30) consisted of women with uncomplicated pregnancies, The mean age of the patients was 29.23 years and the mean gestational age was 38 weeks. All participants provided written informed consent prior to sample collection.

Cell culture and transfection

The HTR-8/SVneo cell line, a human trophoblast cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured using a medium with RPMI-1640, 10% FBS, penicillin, as well as streptomycin (Gibco, USA) at 37 °C with 5% CO2.

HTR-8/SVneo cells were seeded at density of 2.0 × 105 cells/mL per well, then transfected with synthetic miR-155-5p mimic or inhibitor (Anhui GENERAL BIOL, China) via Lipofectamine 3000 (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. The miR-155-5p mimic and inhibitor were designed to specifically modulate miR-155-5p expression.

RT-qPCR

The HTR-8/SVneo cells were lysed, RNA samples were extracted via a commercially available kit (Yuduo Biotechnology Co., Ltd., Shanghai, China). The cDNA was synthesized, then PCR via Rt-PCR Detection System (Bio-Rad, USA), Prepared cDNA was applied to amplification. Conditions of one cycle: 95 °C 22s, 56 °C 22s, 73 °C 22s, as well as 40 cycles were performed. The primer sequences used in the experiments were as follows: Forward miR-155-5p: 5’-ACACTCCAG CTGGGTTAATGCTAAACGTGAT-3’, reverse: 5’-CCAGTGCAGGGTCCGAGGT-3’; Forward PIK3R1: 5’-GAATCTCTAGCTCAGTATAATCCC-3’, reverse: 5’-GACAACTTGATCCTGTTGGT-3’.

Western blot

Extracts were loaded onto SDS gel electrophoresis, then transferred to PVDF membrane (Millipore), anti-PIK3R1, anti-Bax, anti-Bcl-2 (1;1000, ab191606, ab32503, ab182858, Abcam) as well as anti-GAPDH (1:3000, ab8245, Abcam ) incubate the membranes overnight, then add Goat anti-rabbit secondary antibody (1:5000, 202700514, Zhongshan Jinqiao Biotechnology Co. LTD, China) for 2 h. In the end, captured with the ECL system (Thermo Scientific, Inc.).

Immunohistochemistry

Placental trophoblast tissues were resected and embedded in paraffin. After dewaxing, rehydrated in grade ethanol. An H2O2 solution was added and slides were incubated in 0.01 M sodium citrate. Next, wash slices as well as incubated with anti-PIK3R1 (1:200, R&D Systems, Inc.). After washing, the specimens were exposed to peroxidase reagent. Washing and an SP staining kit (Beijing Solarbio Science & Technology Co., Ltd.) were applied for visualization.

Cell viability

HTR-8/SVneo cells, after resuspending, were seeded at 100 µL/well. 10 µL CCK8 reagents (Beyotime, Jiangsu, China) were added and then maintained for four hours in the 37 °C environment. A microplate reader (Thermo MK3) was applied to detect absorbance values.

5-Ethynyl-2′-deoxyuridine assay

HTR-8/SVneo was stained with EdU and then fixed with 4% paraformaldehyde. 1X Apollo reaction cocktail stained for 30 min before incubation with Hoechst 33,342. Then HTR-8/SVneo cells were imaged via fluorescence microscope (Leica, Germany).

Transwell assay

HTR-8/SVneo cells suspension (4 × 104 cells/ml) was prepared and was transferred to the upper cavity that was pre-coated with Matrigel (356234, Millipore, U.S.A.). The lower cavity was filled with RPMI-1640 medium containing 20% FBS. After 24 h, invaded cells that passed from upper chamber to lower were fixed as well as stained with 0.5% crystal violet (Sigma Aldrich, U.S.A.) 25 °C, 20 min. Cell numbers were counted under fluorescence microscope.

For migration assay, the experimental procedures were similar except that there is no Matrigel.

TUNEL assay

The HTR-8/SVneo cells were fixed on the slide with 4% acetaldehyde for 15 min, and washed. Then incubated with 0.5% Triton X-100 solution for 10 min. Then washing by phosphate buffered saline, add 50µL TUNEL reagent containing fluorescein-12-dUTP, incubated at 37 °C for 60 min. Then washed three times, DAPI coloration, hematoxylin counterstaining, and fluorescence microscope observation after sealing.

Dual luciferase reporter assay

Designed and synthesized WT as well as MUT type 3′-UTR region of PIK3R1 luciferase reporter pmirGLO vectors. After 24 h cultivation, lysing the cells as well as measuring the activities by Luciferase Reporter Assay Kit (Promega) 48 h after co-transfection. Then measured with a luciferase assay kit (Promega).

Statistical analysis

All experiments were conducted in triplicate (n = 3) to ensure reproducibility. All data were calculated through GraphPad as well as presented as mean ± SD. The Student’s t-test was applied to contrast two groups’ differences, and contrasts among multiple groups with the analysis of variance were with the Tukey post-hoc test. P < 0.05 was a significant difference.

Results

Expression of miR-155-5p and PIK3R1 in placental trophoblastic tissue of PE patients

We found that miR-155-5p in the placental trophoblast of PE patients was markedly higher than in normal human placental trophoblast, while the level of PIK3R1 was significantly reduced (Fig. 1A). Further western blot and IHC tests revealed that the PIK3R1 protein level was notably reduced in the placental trophoblast tissue of PE patients (Fig. 1B and C).

Fig. 1
figure 1

(A) Then mRNA levels of miR-155-5p and PIK3R1. (B) Protein expression of PIK3R1. (C) Immunohistochemistry of PIK3R1. N = 3. **p < 0.01, ***p < 0.001 versus normal group.

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Expression of miR-155-5p affects the function of trophoblast cells

To verify miR-155-5p’s effect on trophoblast cells, we increased as well as decreased miR-155-5p expression by transfection (Fig. 2A). Further experiments showed that inhibit miR-155-5p markedly enhanced cell viability, cell proliferation, migration as well as invasion, while overexpression of miR-155-5p led to the opposite effect (Fig. 2B-D). Additionally, inhibition of miR-155-5p reduced the occurrence of apoptosis in HTR-8/SVneo cells, resulting in a decrease in the expression of Bax as well as an increase in the expression of Bcl2, respectively, while overexpression of miR-155-5p reverses this result (Fig. 2E and F).

Fig. 2
figure 2

(A) Expression of miR-155-5P. (B) Cell viability of HTR-8/SVneo. (C) EDU positive HTR-8/SVneo cells. (D) Migration as well as invasion of HTR-8/SVneo. (E) Cell apoptotic of HTR-8/SVneo. (F) Protein expression of Bax as well as Bcl-2. N = 3. *p < 0.05, ***p < 0.001, versus NC inhibitor; #p < 0.05, ###p < 0.001 versus NC mimics.

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miR-155-5p directly bound with PIK3R1

We used Starbase 3.0 to predict the target gene and found PIK3R1, Fig. 3A showed the binding region of miR-155-5p as well as PIK3R1, and the targeting relationship was verified (Fig. 3B). In cells with miR-155-5p inhibition, mRNA level as well as protein expression of PIK3R1 were notably increased, while miR-155-5p overexpression decrease the PIK3R1 (Fig. 3C and D).

Fig. 3
figure 3

(A) Binding site of miR-155-5p to PIK3R1. (B) Luciferase activity of HTR-8/SVneo co-transfected with luciferase reporter vector containing PIK3R1 and miR-155-5p mimics vector. ***p < 0.001, versus NC mimics. (C) mRNA level of PIK3R1 in HTR-8/SVneo with miR-155-5p inhibitor and mimics. (D) Protein expression of PIK3R1 in HTR-8/SVneo with miR-155-5p inhibitor and mimics. N = 3. *p < 0.05, ***p < 0.001, versus NC inhibitor; ###p < 0.001 versus NC mimics.

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Overexpression of miR-155-5p regulates HTR-8/SVneo cells through PIK3R1

In order to further verify the role of PIK3R1, we increased the mRNA level as well as protein expression of PIK3R1 (Fig. 4A and B). Increasing PIK3R1 can reverse the effects of miR-155-5p overexpression, increase cell viability, enhance cell proliferation, migration as well as invasion (Fig. 4C-E), reduce the occurrence of apoptosis, and change the expression of Bax and Bcl2 proteins (Fig. 4F and G).

Fig. 4
figure 4

(A) mRNA level of PIK3R1. (B) Protein expression of PIK3R1. (C) Cell viability of HTR-8/SVneo. (D) EDU positive HTR-8/SVneo cells. (E) Migration as well as invasion of HTR-8/SVneo. (F) Cell apoptotic of HTR-8/SVneo. (G) Protein expression of Bax as well as Bcl-2. N = 3. *p < 0.05, **p < 0.01, ***p < 0.001 versus NC mimics, #p < 0.05, ##p < 0.01, ###p < 0.001 versus miR-155-5p mimics + pc-NC.

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Discussion

Uterine spiral artery remodeling disorder is considered to be a very important part of the pathogenesis of preeclampsia. The insufficient ability of trophoblast cells to invade the muscular layer of the spiral artery causes spiral artery stenosis, which leads to the failure of physiological transformation of the spiral artery, blood flow disorder, insufficient perfusion of the uterus and placenta, ischemia and hypoxia, and oxidative stress, resulting in abnormal development of placenta1. Further, the placenta secretes various cytokines into the maternal circulation, causing extensive vascular endothelial cell damage. Therefore, the abnormal function of placental trophoblast cells is related to PE.

MiR-155 plays a vital role in PE. For instance, miR-155 expressed highly in placental PE patients, can inhibit cell proliferation as well as invasion by targeting FOXO3, and promote its apoptosis and inflammatory response29. Our study selected miR-155-5p, main active substance of miR-155, for verification. It was found that miR-155-5p expression was markedly increased in PE tissues, while knockdown of miR-155-5p could increase cell viability, as well as the ability of proliferation, migration as well as invasion. Overexpression of miR-155-5p significantly attenuated these cell functions. Besides, apoptosis is a pathological phenomenon that occurs widely in PE. Studies have found that magnesium sulfate inhibits the apoptosis of vascular endothelial cells in PE rat models30, while human umbilical vein endothelial cells exposed to PE maternal serum increase apoptosis31. Herein, the knockdown of miR-155-5p expression can observably reduce the apoptosis. On the contrary, overexpression of miR-155-5p can increase apoptosis.

PI3K/Akt is the most important signaling pathways that widely exist in various cells to regulate physiological processes like cell metabolism, proliferation, migration, invasion as well as apoptosis. It can regulate trophoblast function and affect PE32. We predicted that PIK3R1 is a negative regulator of the PI3K/AKT pathway. Down-regulation of PIK3R1 gene expression can activate the PI3K/AKT pathway23,33. It’s reported that multiple genes regulated by miR-155-5p are involved in the regulation of the PI3K/AKT pathway34, and miR-155 can target the PIK3R1-mediated PI3K/AKT pathway to affect osteoarthritis35, indicating that miR-155-5p can indeed regulate PI3K/AKT signaling pathway through PIK3R1, which is consistent with our results. However, the regulatory pathway of miR-155-5p-PIK3R1-PI3K/AKT has not been studied in PE. However, it has been reported that other miRNAs can affect PE via PI3K/AKT pathway. For example, miR-483 affects PE by targeting IGF1 to regulate endothelial progenitor cells36, miR-15a-5p promotes the progression of preeclampsia via regulating PI3K/AKT pathway through CDK137. This indicates that miRNA affects the development of PE via PI3K/AKT pathway is a confirmed mechanism and also provides favorable support for this study. In this study, mRNA level as well as protein expression of PIK3R1 were markedly decreased in PE tissues, and its expression was negatively correlated with miR-155-5p level. To verify its effect, we increased the expression of PIK3R1 in cells overexpressing miR-155-5p, which could reverse the effect of miR-155-5p on cells, increase cell viability, strengthening cell proliferation, migration as well as invasion, while reducing the occurrence of apoptosis.

However, we acknowledge some limitations in our study. Firstly, the HTR8 cell line used in our experiments has limitations in modeling preeclampsia. The HTR8 cell line does not syncytialize, which means it does not fully replicate the behavior of primary trophoblasts in vivo. This limits its ability to accurately mimic the complex processes involved in trophoblast invasion and placental development. While the HTR8 cell line is useful for studying trophoblast-like cells, it is not a perfect model for the primary trophoblast function that is disrupted in preeclampsia. Therefore, future studies using more representative models, such as primary trophoblasts or in vivo models, are needed to confirm these results. In addition, the potential off-target effects of miR-155-5p mimic and inhibitor were not systematically assessed in this study, which could be a limitation of our findings.

Conclusion

In summary, this study confirmed the effect of miR-155-5p on PE by changing the expression of miR-155-5p in placental trophoblast HTR-8/SVneo, obtained PIK3R1 through database screening, further confirming that miR-155-5p affects PE by regulating PIK3R1. In the future, we will verify miR-155-5p’s role in PE through animal models, and provide possible drug therapeutic targets for clinical treatment and prevention of PE. Studies have confirmed that one of the mechanisms for the therapeutic effect of aspirin, a drug for the treatment of PE, is to further regulate the function of trophoblast cells by affecting miRNA38. In the future, we will also explore the interaction between miR-155-5p and drugs for the treatment of PE based on miR-155-5p, so as to provide stronger support for clinical drug selection for the treatment of PE.