YKL-40 promotes proliferation and invasion of HTR-8/SVneo cells by activating akt/MMP9 signalling in placenta accreta spectrum disorders

Abstract YKL-40 is a secreted glycoprotein that can promote invasion, angiogenesis and inhibit apoptosis, and was highly expressed in a variety of tumours. In this paper, we investigated the impacts of YKL-40 on proliferation and invasion in HTR-8/SVneo cells during placenta accreta spectrum disorders (PAS) development. The levels of YKL-40 protein in late-pregnant placental tissue were detected using immunohistochemistry and Western blotting, and gene expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The proliferation, migration, invasion and apoptosis abilities of HTR-8/SVneo cells were detected by cell counting kit-8 (CCK-8), Transwell, scratch assay, and flow cytometry, respectively. Our current results showed that YKL-40 was significantly increased in the PAS group compared to the normal control group (P < 0.01). Biological function experiments showed that YKL-40 significantly promoted the proliferation, migration and invasion of HTR-8/SVneo cells, and inhibited cell apoptosis. Knockdown of YKL-40 inhibited the activation of Akt/MMP9 signalling in trophoblast cells. These data suggested that YKL-40 might be involved in the progression of PAS, which may be attributed to the regulation of Akt/MMP9 signalling pathway. IMPACT STATEMENT What is already known on this subject? YKL-40 is a secretory glycoprotein that can promote invasion, angiogenesis, and inhibit apoptosis and was highly expressed in a variety of tumours. Trophoblast cells resemble tumour cells in their migration and invasion. What the results of this study add? YKL-40 expression was significantly up-regulated in PAS. CCK-8 assays showed that YKL-40 remarkably enhanced the viability of HTR-8/SVneo cells. Scratch and Transwell assays demonstrated that YKL-40 significantly promoted the migration and invasion of HTR-8/SVneo cells. Additionally, YKL-40 attenuated apoptosis in HTR-8/SVneo cells. What the implications are of these findings for clinical practice and/or further research? Akt/MMP9 was involved in the regulation of YKL-40 on trophoblast invasion, which may provide theoretical basis and new ideas for the drug blocking intervention of placenta accreta.


Introduction
Placenta accreta spectrum (PAS) is the abnormal attachment and invasion of the chorionic villi in the myometrium.Pathologically, it can be classified into three categories: placenta accreta, increta, and percreta (Jauniaux et al. 2018).A recent study showed that the overall rate of PAS in the United States was 3.7‰ delivery-related discharges (Mogos et al. 2016).The primary cause of PAS is caesarean delivery.In recent years, the incidence of placenta previa and PAS has increased dramatically with the increase in caesarean delivery rates.In addition, postpartum haemorrhage and obstetric hysterectomy have increased rapidly due to the occurrence of placenta accreta and placenta previa (Pan et al. 2015, Weiniger 2020, O� glak € Olmez and Tunc¸ 2022).The pathogenesis of PAS could be attributed to the defects in uterine metaplasm, excessive invasion of extravillous trophoblast cells, and abnormal angiogenesis (Long et al. 2020).Moreover, the proliferation, invasion and migration of trophoblast cells was similar to tumour cells (Zhang et al. 2020).Hence, further exploration of the pathogenesis of abnormal trophoblast invasion in PAS will provide more help for the diagnosis and treatment of PAS in the clinic.
Chitinase 3-like-1 (CHI3L1, also known as YKL-40 in humans and BRP-39 in mice), has a molecular weight of 40 kDa (Johansen et al. 1992).It is a glycoprotein secreted by chondroid, endothelial, tumour, and various inflammatory cells.It regulates vascular endothelial growth factor (VEGF) and plays a critical role in inflammation and angiogenesis (Chen et al. 2021).YKL-40 was overexpressed in tissues of patients with different types of tumours such as bladder cancer, colorectal cancer, glioma, ovarian cancer, and prostate cancer (Larionova et al. 2020, Hao et al. 2021, Liu et al. 2021, OhPyo and Son, 2021).It was also expressed abnormally as an inflammatory factor in asthma, rheumatoid arthritis, preeclampsia and kidney disease (Madazli et al. 2008, Yeo et al. 2019, Guvenir et al. 2021, Puthumana et al. 2021).Several studies have demonstrated that YKL-40 promoted cancer cell invasion, migration, and inhibition of apoptosis in various cancers (Hao et al. 2021). Currently, G€ oz€ ukara et al. (2017) concluded that YKL-40 is overexpressed in placental tissues with PAS.Nonetheless, its mechanism and biological function has not been clarified.Several studies have shown that PI3K/Akt was involved in multiple physiological and pathological processes, including cell proliferation, cell cycle, apoptosis, inflammation, chemical damage, and tumour progression (Wang et al. 2020).In addition, the epithelial-tomesenchymal transition (EMT), migration, and invasion of trophoblasts can be promoted by activating the PI3K/Akt/Erk1/2 signalling pathway (Ding et al. 2021).Matrix metalloproteinases (MMPs) are a set of zinc endopeptidases vital for the decomposition of extracellular matrix (ECM) components and basement membrane (BM) (Jiang and Li 2021) and play a crucial role in various physiological and pathological processes, including morphogenesis, angiogenesis, inflammation, cancer invasion, and metastasis (Raeeszadeh-Sarmazdeh et al. 2020).Nevertheless, the potential relationship between YKL-40 and the Akt/MMP9 signalling pathway has not been explored in PAS.
In the present study, we found that YKL-40 mRNA and protein expression was up-regulated in placental tissues of PAS patients compared with the normal controls.The first extravillous cytotrophoblast (evCTB) cell line was established by Graham et al. (1993) and named HTR-8/SVneo.It was generated by freshly isolated evCTB from the first-trimester placenta.HTR-8/SVneo cells are often used to detect the invasive and migratory ability of trophoblasts.To further elucidate the functional role of YKL-40 in the pathogenesis of PAS, we knocked down and overexpressed YKL-40 in vitro to investigate that YKL-40 plays a vital role in PAS by activating Akt/MMP9 pathways with human trophoblastic cell line HTR-8/SVneo.

Patients
Placental tissues were obtained at the time of caesarean section from the Department of Obstetrics, Hebei General Hospital (Shijiazhuang, China) between June 2018 and December 2021.The study was approved by the Ethics Committee of the Hebei General Hospital, and all the pregnant women or their relatives provided written informed consent.In this study, a total of 25 cases of pregnant women (age 31.61 ± 3.37 years) with PAS, including 13 cases of placenta increta, and 12 of placenta percreta; meanwhile, 22 cases healthy pregnant women (age 32.14 ± 4.36 years) due to the caesarean section scar were included as the normal control group.

Collection and preservation of placental samples
Placental tissues were collected within 3 min after caesarean sections, including the implanted portion of the uterus.It was divided into two parts: one was immediately fixed in formalin and embedded in paraffin after 24 h for immunohistochemical, while the other was flash-frozen in liquid nitrogen and stored at À 80 � C refrigerator until further analysis.

Total RNA extraction and RT-qPCR
Total RNA was extracted from the placental tissues and HTR-8/SVneo cells using the RNeasy Mini Kit (TIANGEN, Beijing, China).The concentration of RNA was measured on an Ultraviolet spectrophotometer (Thermo, USA).cDNA was transcribed from 2 lg RNA using the Fast Quant RT Kit (with gDNase) (TIANGEN, Beijing, China).RT-qPCR was carried out to evaluate the expression of YKL-40 using Super Real PreMix Plus (SYBR Green) (TIANGEN, Beijing, China) on an ABI PRISM 7500 sequence detector (Applied Biosystems, USA), according to the manufacturer's instructions.GAPDH was used as an internal control to calculate the relative expression.The 2 À DDCt method was used to calculate the relative expression levels.Each sample was replicated at least three times.

Extraction of total protein and Western blot
Total protein was extracted from the placental tissues and cells using RIPA buffer containing 50 lg/500 mL or each well of the 6-well plate/200 lL protease inhibitors (Solarbio, Beijing China) according to the manufacturer's instructions.Protein samples were resolved by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane.Then, the membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 2 h before probing with anti-YKL-40 (1:700, Abcam) antibodies at 4 � C overnight, respectively.Then, the membranes were incubated with the secondary antibody for 1 h at room temperature.Finally, the blots were imaged on a hyper signal electrochemiluminescence (ECL) substrate.The gray values of the target bands were analysed by Image J software.

Cell proliferation
Briefly, transfected cell suspension (100 lL/well, 5 � 10 3 cells) was seeded into a 96-well plate in six replicates for each group at 37 � C and 5% CO 2 .The 10 lL of Cell Counting Kit-8 (Dojindo, Japan) reagent was added to the wells at 0 h, 24 h and 48 h respectively and sequentially incubated for 4 h.Then, the absorbance was measured at OD450 on an automicroplate reader (Thermo, USA).Each sample was replicated at least three times.

Matrigel invasion and migration assay
This assay was performed as described previously (Wang et al. 2019b).For invasion assays, HTR-8/SVneo cells (1 � 10 5 /well) in 100 lL serum-free RPMI1640 from the different treated groups were added in the upper part of a Transwell chamber (precovered with Matrigel).The medium with 20% FBS (700 mL) was added to the lower chamber.For migration assays, the same number of treated cells as the invasion assays was seeded in the upper chamber of each well, and 20% FBS (700 mL) was added to the lower chambers.After culturing at 37 � C for 26 h, the cells were fixed with methanol for 10 min before staining with haematoxylin for 8 min.Finally, the cells were counted from five fields at a � 200 magnification.Each experiment was repeated in triplicate.

Cell wound scratch assay
The scratch assay is used to determine the migratory ability of YKL-40 of HTR-8/SVneo cells.The cells transfected with siRNA or pcDNA plasmid were added into 6-well plate.After 24 h, a linear vertical scratch was drawn with a sterile 200 lL pipette tip.Images were captured under a microscope at 0 h and 24 h, and the wounds were selected at the same points.Wound areas (lm 2 ) were measured using ImageJ, and the cell migration rate was calculated as follows: (area difference between 0 h and 24 h)/area of 0 h � 100%.The assay was repeated at least three times independently.

Cell apoptosis analysis
The cell apoptosis was measured by flow cytometry.The collected cells were stained with Annexin V-FITC/PI Apoptosis Detection Kit (MultiSciences, China) and incubated in the dark for 15 min.Finally, cell apoptosis was assessed on a FACS scan flow cytometer (Epics XL, USA) to calculate the percentages of necrotic and apoptotic cells in 10,000 events.The assay was performed in triplicates.

Statistical analysis
All statistical analyses were conducted using IBM SPSS 25.0 (IBM Corp, Armonk, NY, USA), and the graphs were plotted using GraphPad Prism 7.04 software (GraphPad, San Diego, CA, USA).For quantitative data, normal distribution was expressed by Student's t-test with mean ± standard deviation (SD), while non-normal distribution was expressed by nonparametric test with percentile expression, and chi-square test was used for the count data.p < 0.05 was considered statistically significant.

Clinical and demographic characteristics of the subjects
No statistically significant differences were detected between PAS and control groups according to demographic data, including age and Apgar scores, but the gestational age (p < 0.01) was significantly lower and caesarean hysterectomy was higher (p < 0.01) in the PAS group than the control group (Table 1).

Expression of YKL-40 in PAS and normal pregnant placental tissues
Immunohistochemistry showed that the YKL-40 level was significantly higher in the PAS group (0.484 ± 0.002) than in the control group (0.323 ± 0.003) (p < 0.01).This finding was consistent with the Western blot and RT-qPCR results (Figure 1).

The expression of YKL-40 mRNA and proteins by knockdown or overexpression of YKL-40 in human trophoblastic HTR-8/SVneo
Transfection efficiency was measured by RT-qPCR and Western blot (Figure 2).
After YKL-40 was knocked down in HTR-8/SVneo for 24 h, the mRNA levels (Figure 2(a)) were distinctly lower than controls with transfected blank (p < 0.01).The level of YKL-40 proteins (Figure 2(c)) in the silencing group was significantly decreased compared to the si-NC group after cells were

YKL-40 knockdown inhibited HTR-8/svneo cell proliferation, invasion, and migration and promoted cell apoptosis
The CCK-8 assay explored the proliferation of HTR-8/SVneo cells by knockdown of the YKL-40 gene, as shown in Figure 3(a).We found that HTR-8/Svneo cells growth was inhibited significantly after transfection with si-YKL-40 for 24 and 48 h compared to the negative control (p < 0.05).Wound scratch and Transwell assays showed that the migratory viability silenced YKL-40 was significantly lower than that of the negative control (Figure 3(b,f), p < 0.01).Subsequently, we used a Transwell assay and observed that the invasion ability of HTR-8/Svneo cells was weaker than that of the si-NC group after YKL-40 knockdown (Figure 3(e), p < 0.01).

Overexpression of YKL-40 increased the proliferation, invasion and migration and decreased apoptosis of HTR-8/svneo cells
Transfection efficiency was measured by RT-qPCR 24 h after transfection (Figure 2b, p < 0.01).
(a) CCK-8 assay showed that the proliferation activity of OE-YKL-40 cells at 24h and 48h was significantly higher than that of over control cells (p < 0.05).(b, e) Wound scratch and Transwell assays were used to assess trophoblast cell migration.After YKL-40 overexpression, the cell migration activity was significantly increased (p < 0.01, magnification �200).(f) In addition, Transwell assays were performed to examine cell invasion ability.The invasion ability with overexpressed YKL-40 was increased significantly than the OE-NC group (p < 0.01, magnification �200).(c, d) The apoptotic rates of HTR-8/SVneo cells were measured by flow cytometric assays (p < 0.05).

YKL-40 regulated AKT/MMP9 signalling pathway in HTR8/SVneo cells
Previous studies have shown that YKL-40 regulated cell proliferation and motility, including tumour cell migration and invasion.Hence, we verified the related proteins, MMP9 and vital proteins involved in the PI3K/AKT downstream signalling pathway, including AKT phosphorylation (PAKT), AKT, after knockdown or overexpression of YKL-40 genes.

Discussion
In normal, uncomplicated pregnancies, extravillous trophoblasts invade through the uterine interstitium (interstitial trophoblasts) into spiral arteries, then line and remodel them (endovascular trophoblasts).Therefore, they are responsible for successfully establishing maternal blood flow into the placenta (Weiss et al. 2016, Xu et al. 2019a).Some evidences indicated that overexpressed YKL-40 in the serum/plasma has a high correlation with low survival in patients who have solid tumours (Bian et al. 2019).The function of YKL-40, a factor expressed in almost all tumour tissues (B€ ockelmann et al. 2021), promoted cell proliferation and invasion in tumours, but its role in the development of PAS has not been elucidated.
PAS is mainly caused by factors such as decreased, absent or abnormal decidua and/or defects in decidualization, which lead to increased penetration depth of (normal) extravillous trophoblast (Illsley et al. 2020).In addition, trophoblast and tumour metastasis are often similar in migration and invasion because they have similar molecular mechanisms (Kohan-Ghadr et al. 2016).Accumulating evidence demonstrated that YKL-40 knockdown or overexpression inhibited or promoted tumour cell migration and invasion (Jefri et al. 2015).
In the present study, 47 maternal cases were included, and the gestational age in the PAS group was lower and caesarean hysterectomy was higher than that in normal control group.The timing of delivery should be individualised.Hysterectomy has become the cornerstone of treatment in patients with PAS and postpartum haemorrhage.As for the termination time of PAS, when the mother and foetus are stable, the planned termination time varies according to different associations, and the American College of Obstetricians and Gynaecologists (ACOG) considered the best termination time to be 34-35 þ6 weeks (Allen et al. 2018).In addition, elective surgery under the condition of adequate preparation significantly reduced the risk of bleeding than emergency surgery, and also reduced the occurrence of many related complications (Miller et al. 2020).Therefore, the gestational age at termination of pregnancy in the PAS group was shorter than that in the control group, and no normal control pregnant women matched with the gestational age of PAS were obtained, which was also the limitation of this study.However, both groups were pregnant women in late pregnancy, which had limited impact on the study results.Among the 25 PAS cases, there was no significant difference in YKL-40 expression between 13 cases of placenta increta and 12 cases of placenta percreta (P > 0.05), which may be related to insufficient sample size and individual differences.
Hence, in this study, the expression and biological function of YKL-40 in the progression of PAS were investigated.Immunohistochemistry, RT-qPCR, and Western blot analysis indicated the up-regulation of both YKL-40 mRNA and protein in placental tissues of the pregnant woman with PAS.YKL-40 was consistent with the participation of Cripto-1 in controlling angiogenesis, a recent study concluded that serum Cripto-1 levels were significantly increased levels in pregnant women suffering from PAS than in pregnant women with PP and uncomplicated healthy pregnancies (Ozkose et al. 2022).Besides, further biological functions suggested that knockdown of YKL-40 inhibited cell proliferation, migration, invasion, and promoted cell apoptosis.Conversely, overexpression of YKL-40 promoted cell proliferation, invasion, migration, and hindered cell apoptosis.These findings are in line with previous studies suggesting a role for YKL-40 in promoting invasion of malignant tumour cells (Li et al. 2016, Chen et al. 2021) and that the up-regulated expression of YKL-40 may play a crucial role in PAS progression.The PI3K/Akt signalling pathway is fundamental in mediating the growth-factor-dependent regulation of trophoblast growth, migration, invasion, placental development, and foetal growth (Xu et al. 2019a, Xu et al. 2019b).A large number of studies suggested that the invasion and migration capability of HTR-8/SVneo cells and the expression of key kinases in PI3K/AKT/mTOR pathways were decreased when PI3K was inhibited (Zhu et al. 2016, Wang et al. 2019a).In addition, PI3K/AKT regulated trophoblast invasion and migration in human extravillous trophoblasts (EVTs) via the upregulation of MMP9 expression by epidermal growth factor (Liu et al. 2020).Another study indicated YKL-40 might affect the treatment efficacy of PI3K/AKT-based pathway inhibitors in glioblastoma (Wang et al. 2018).
The present results showed that the protein expression of YKL-40 was up-regulated in placental tissues from PAS.In addition, the Akt/MMP9 signalling pathway was activated by YKL-40 overexpression but inhibited by YKL-40 knockdown.Therefore, YKL-40 may play a role in the pathogenesis of PAS through Akt/MMP9 signalling pathway.Human trophoblastic cell invasion during placentation was regarded as limited to the invasion into uterine connective tissues and uterine spiral arteries (Moser et al. 2018).Although some studies have shown that trophoblastic cells are similar to tumour cell migration and invasion, YKL-40 can promote these phenomena, which are different from those in tumours.The invasion of trophoblastic cells during implantation and placentation is strictly restricted in both space and time (Kohan-Ghadr et al. 2016).Therefore, the mechanism by which YKL-40 promotes trophoblast invasion should be under intensive focus.The strength of this study is that we have demonstrated for the first time that YKL-40 may regulate PAS invasion through AKT/MMP9 signalling.Nevertheless, the present study has some limitations.We had not used inhibitors of the PI3K/AKT signalling pathway to study the effect of YKL-40 protein on trophoblast function through this pathway.And the biological function of YKL-40 was only verified using HTR-8/SVneo cells, which could be verified further with other pregnancy-related or primary cells.

Conclusion
In this study, we demonstrated that the expression and biological function of YKL-40 in placental tissues, and the expression was up-regulated in PAS.YKL-40 promoted HTR-8/SVneo cell proliferation, invasion, migration and inhibited apoptosis via Akt/MMP9 pathway.

Figure 1 .
Figure 1.YKL-40 expression was higher in PAS than in control group (PAS: n ¼ 25, control group: n ¼ 22).Immunohistochemistry showed that YKL-40 was mainly expressed in the cell membrane and cytoplasm of placental villi.(a, b) Control group placental tissues were stained light brown, nevertheless, (c, d) PAS placental tissues were stained dark brown (magnification, �200 and �400, p < 0.01).In addition, (e, f) Western blotting showed YKL-40 was highly expressed in placental tissues in PAS patients than control groups (p < 0.05).(g) RT-qPCR was used to detect the expression of YKL-40 in placental tissues of PAS and control groups (P < 0.05).

Table 1 .
Demographic Characteristics in Two Groups.