Endoplasmic reticulum protein 29 inhibits epithelial-mesenchymal-transformation in placental trophoblast cells and promotes the development of intrahepatic cholestasis of pregnancy in pregnant rats

doi:10.21203/rs.3.rs-1880582/v1

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Endoplasmic reticulum protein 29 inhibits epithelial-mesenchymal-transformation in placental trophoblast cells and promotes the development of intrahepatic cholestasis of pregnancy in pregnant rats

https://doi.org/10.21203/rs.3.rs-1880582/v1

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Background

Intrahepatic cholestasis of pregnancy (ICP) is a common pregnancy specific liver disease, which has little impact on the mother and mainly causes adverse fetal outcomes. Endoplasmic reticulum protein 29 (ERp29) plays a key role in protein folding, transport and secretion, and participates in biological events such as apoptosis, oxidative stress, lipid metabolism, immune regulation and so on. Epithelial mesenchymal transformation (EMT) is the process of epithelial cells losing polarity and adhesion, and transforming into mesenchymal like phenotype through migration.

Methods

Subcutaneous injection of ethinyl estradiol (E2) was used to induce ICP in pregnant rats, and serum TBA, ALT and AST were detected to test whether the animal model was successfully constructed. ERp29 and EMT related molecules were detected by immunohistochemistry and western blotting. Taurocholic acid (TCA) was used to simulate the ICP environment, and TGF-β1 was added to induce the EMT process. CCK8 and TUNEL were used to detect the level of cell apoptosis. The scratch, migration, and invasion test were used to detect the EMT process of cells. ERp29 overexpression/knockdown vector were constructed and transfected to verify the role of ERp29 in the EMT process.

Results

The ICP animal model was successfully constructed. ERp29 in the placenta tissue of the ICP pregnant rats increased significantly, and the level of apoptosis increased. Compared with the normal, the placental tissues of the ICP group had high expression of E-cadherin and low expression of N-cadherin, snail1, vimentin. After HTR-8/Svneo cells were induced by TCA, EMT was inhibited, while the ERp29 increased. Knockdown of ERp29 reduced the inhibition of cell EMT. After the ERp29 knockdown vector was transfected into pregnant rats by adenovirus, the degree of inhibiting EMT in the placenta tissue of pregnant rats was inhibited, and the ICP progress was alleviated.

Conclusion

The high level of ERp29 in placental trophoblast cells inhibits the EMT process and aggravates the occurrence and development of ICP.

ICP

ERp29

EMT

apoptosis

Intrahepatic cholestasis of pregnancy (ICP) is a common pregnancy specific liver disease, which usually occurs in the middle and late trimester of pregnancy. The clinical manifestations are maternal pruritus without rash and abnormal liver function (elevated total bile acid and transaminase). ICP is associated with an increased risk of perinatal adverse outcomes, including spontaneous preterm birth, amniotic fluid meconium staining, and stillbirth. There is increasing evidence that intrahepatic cholestasis of pregnancy has a lasting impact on the health of pregnant women and fetuses[1-3].

As an important organelle in the secretory pathway, endoplasmic reticulum (ER) participates in the regulation of lipid biosynthesis and calcium flux[4]. ER stability is essential for cell survival. When exposured to environmental toxins, energy or nutrient deficiency and Ca²⁺ imbalance, misfolded or unfolded proteins increase, which is called endoplasmic reticulum stress (ERS)[5, 6]. ER resists cell damage caused by ERS and restores cell homeostasis by activating the unfolding protein response (UPR)[7]. Molecular chaperone endoplasmic reticulum protein 29 (ERp29) plays a key role in protein folding, transportion and secretion. Despite widespread expression, ERp29 is up-regulated in response to ERS and is found at high levels in some types of cells, such as secretory epithelial cells and neurons. The extensive expression and multiple roles of ERp29, as well as the up-regulation of expression through UPR during ERS, have involved ERp29 in a series of cellular processes and diseases[8]. In the past, we found that the expression of ERp29 protein was significantly up-regulated in the placenta, and it was mainly involved in the biological events, such as oxidative stress, etc[9]. The balance between various biological events, including cell proliferation, growth arrest, apoptosis and epithelial mesenchymal transformation (EMT), is essential for embryonic development and in vivo homeostasis. It is well known that apoptosis and EMT play an important role in differentiation, morphogenesis and organogenesis, wound healing, tissue remodeling and homeostasis, and disease-related processes[10]. Susceptible genetic and environmental factors lead to placental maladjustment, resulting in placental defects, apoptosis of invasive cytotrophoblast cells, insufficient expansion and reconstruction of spiral arteries, decreased uterine placental perfusion pressure and placental ischemia, which eventually lead to pregnancy diseases (ICP, hypertension and intrauterine distress, etc.)[11].

2.1 Animal model

Female wistar rats (150±10 days old, 10-14 days of pregnancy, 300±10 g) were selected. The pregnant rats were randomly divided into different groups, namely the negative control group (Neg) and the ICP experimental group (ICP). The pregnant rats in the ICP group were injected subcutaneously with ethinyl estradiol (E2, Sigma) at 1.25 mg/(kg·d) at 10:00 am every day. The Neg group was injected subcutaneously with propylene glycol (PG, Aladdin) at 2.0 ml/(kg·d) at the same time every day. The pregnant rats were treated continuously for five days, during which the weight of the pregnant rats was recorded. On the 6th day, the rats were anesthetized by intraperitoneal injection of sodium barbital (100 mg/kg), the placenta and liver tissues were collected, washed with PBS, and then set aside. At the same time, the eyeballs of pregnant rats were taken to obtain venous blood. After centrifuging, 3000 r/min, 5 min, the serum was collected, and stored at 4°C. The serum indexes (TBA, ALT, AST) were detected by kit (Gemic).

2.2Cell culture

HTR-8/Svneo cells were purchased from Shanghai EK-Bioscience Biotechnology Co., Ltd, cultured in 5% CO₂, 37℃incubator by using 1640 medium (containing 10% fetal bovine serum, 1% penicillin/streptomycin, Meilunbio). 5x10⁵ HTR-8/Svneo cells were cultured in a well, and taurocholic acid (TCA, 100 μM, MedChemExpress) was added to simulate an ICP environment. TGF-β1 (10ng/ml, PeproTech) was added to induce the EMT environment. Cells were collected after co-cultivation for 36 h for subsequent experiments.

2.3 Transfection

5x10⁵/well HTR-8/Svneo cells were cultured, after the cells adhered to 60%, the original culture medium was discarded, and 1640 without fetal bovine serum (including transfection reagent) was added. After culturing in the dark for 6 h, the culture medium was replaced by 1640 (10% fetal bovine serum), then the cells were collected after culturing for 36 h for subsequent experiments.

2.4 H&E staining

The placenta and liver tissues were washed in PBS, fixed in 4% paraformaldehyde for at least 24 h, embedded in paraffin and made into tissue sections. After fixing, dewaxing, washing with PBS, tissue sections were stained with hematoxylin for 30 s, washed with PBS, stained with eosin for 30 s, rinsed with running water for 30 min, dehydrated, mounted with resin, scanned with a pathological slide scanner.

2.5 TUNEL

The tissue sections were fixed and dewaxed, and DNase-free proteinase K (20 μg/ml, Beyotime) was added, incubated at 37°C for 30 min, then washed with PBS. TUNEL detection solution (Beyotime) was added to the samples, and incubated at 37°C for 60 min in the dark, incubated with DAPI (Bioss) for 5 min to stain nuclei. After washing with PBS, the slides were sealed with anti-fluorescence quenching solution and observed under fluorescence microscope.

2.6 IHC

After fixation and deparaffinization, the tissue sections were washed with PBS, immersed in 30% hydrogen peroxide solution, incubated at room temperature for 30 min, washed with PBS 3 times, and immersed in 0.01M citrate buffer for 30 min, washed with PBS 3 times. Add 5% BSA solution dropwise for blocking, let stand for 30 min at room temperature, and then incubate with antibody (1:200, Abcam) at 4°C overnight. After washing with PBS, the pieces were incubated with secondary antibody at 37°C for 30 min and SABC reagent (Biorad) at 37°C for 30 min, using DAB (Biorad) chromogenic solution for color development under the microscope, scanning for photography picture after counterstaining with hematoxylin.

2.7 Western blotting

Pregnant mouse placental tissue was taken and treated with protein lysate (Vazyme Biotech) to obtain protein supernatant. Protein and loading buffer (Takara) were mixed at a ratio of 3:1, boiled at 100 °C for 8 min, cooled on ice. Proteins were separated using SDS-PAGE gel, then transferred to PVDF membrane, incubated in 5% skim milk for at least 60 min, washed with TBST, and incubated with antibody (1:1000, Abcam) at 4°C overnight. After washing with TBST, they were incubated with the secondary antibody for 1 h at room temperature. After washing with TBST, high sensitivity ECL detection kit (Vazyme) was added for photo analysis.

2.8 CCK8

The HTR-8/Svneo cells (2x10³/well) were cultured in 96-well plate. When the cells adhered to 60%, the 1640 was replaced and the factor treatment was performed. CCK8 solution (Meilunbio) was added to the culture wells at 12 h, 24 h, 36 h, 48 h respectively. Then cells were incubated in the dark for 30 min, and the absorbance was measured at 450 nm.

2.9 Scratch test

The HTR-8/Svneo cells (5x10⁵/well) were cultured in 6-well plate. After the cells adhered to 90%, the original culture medium was discarded, and the cells were streaked vertically. After washing with PBS, the 1640 (serum-free) was replaced. Pictures were taken under the microscope at 12h, 24h, 36h respectively.

2.10 Migration experiment

The HTR-8/Svneo cells (1x10⁵/well) cells were cultured in the upper chamber of the transwell, 600μl 1640 (containing 10% FBS) was added to the lower chamber, then cultured for 36 h. The transwell chamber was fixed with 4% paraformaldehyde (Meilunbio) at room temperature for 30 min, and stained with crystal violet (Solarbio) for 15 min. After washing with PBS, cells remaining in the chamber on the transwell were wiped off, pictures were taken under the microscope.

2.11 Invasion experiment

45 μl of the mixture (1640: Matrigel (Corning) = 4: 1) was spread on the upper chamber of the transwell and solidified in the incubator. The HTR-8/Svneo cells (2x10⁵/well) cells were cultured in the upper chamber of the transwell, 600 μl 1640 (containing 10% FBS) was added to the lower chamber, then cultured for 36 h. The transwell chamber was fixed with 4% paraformaldehyde for 30 min at room temperature, stained with crystal violet for 15 min, washed with PBS. Cells remaining in the chamber on the transwell were wiped off, pictures were taken under the microscope.

2.12 Statistical analysis

All data were shown as mean ± SEM. Statistical analysis was performed by using GraphPad Prism software for analysis of variance (ANOVA), with P < 0.05 considered statistically significant.

3.1 Construction of ICP pregnant rat model

ICP was induced during pregnancy by subcutaneous injection of E2 into pregnant rats. During this period, the weight of ICP pregnant rats began to decrease on the 3th day, while the weight of neg group continued to increase (Fig. 1A). The results of serum indexes of pregnant rats showed that compared with pregnant rats in neg group, TBA, ALT and AST in serum of ICP group were significantly up-regulated (P < 0.05) (Fig. 1B, 1C, 1D). HE staining showed that the liver lobule structure of pregnant rats in neg group was clear and complete, the hepatocytes were arranged radially and orderly, the hepatocytes were uniform in size, the hepatocyte structure in ICP group was deformed, the outline was unclear, the hepatic cord was arranged irregularly, the hepatic sinusoid was enlarged, and the hepatocytes were swollen and denatured (Fig. 1E). The placental villous trophoblasts of pregnant rats in neg group were monolayer distributed, with the same size and orderly arrangement, while the ICP group were swollen, uneven and disordered (Fig. 1F). TUNEL results showed that after fluorescence staining, compared with the neg group, the placental tissue of pregnant rats in ICP group showed strong red fluorescence (Fig. 1G). The apoptosis index was statistically analyzed, and there was significant difference between them (P < 0.05) (Fig. 1H). This further indicates that the placental function of ICP pregnant rats has changed.

3.2 The expression of ERp29 in placenta of ICP pregnant rats increased and the process of EMT was inhibited

Western blotting showed that the ERp29 protein was up-regulated in the placenta of ICP pregnant rats (Fig. 2A). IHC results showed that ERp29 was mainly located in the cytoplasm of placental trophoblast cells, and the expression in ICP group was significantly increased (Fig. 2B). Western blotting and IHC showed that compared with Neg group, E-cadherin in ICP was highly expressed, whlie N-cadherin, Snail 1, Vimentin, MMP2 and MMP9 were decreased (Fig. 2C, 2D).

3.3 The EMT process of HTR-8/Svneo cells was inhibited in ICP environment

The ICP environment of HTR-8/Svneo cells was induced by TCA in vitro, TGF-β1 induced EMT process. The results of CCK8 experiment showed that cells continued to proliferate within 36h and gradually apoptosised after 36h (Fig. 3A). After TCA treatment, the proliferation activity of cells did not change significantly at 12h and 24h, and was significantly inhibited at 36h. TGF-β1 showed obvious ability to promote proliferation after 12 h (Fig. 3B). Western blotting showed that ERp29 in cells treated with TCA increased, and TGF-β1 inhibited its expression (Fig. 3C). Both scratch and transwell test certificated that TCA could inhibit cell migration, TGF-β1 significantly promoted cell migration (Fig. 3D, 3E). Matrigel experiment showed that TCA inhibited cell invasion and TGF-β1 promoted cell invasion (Fig. 3F). Western blotting showed that the cells in TCA group highly expressed E-cadherin and low-expressed N-cadherin, Snail 1, Vimentin, MMP2 and MMP9 (Fig. 3G).

3.4 HTR-8/Svneo cells overexpressing ERp29 can inhibit the process of EMT

ERp29 overexpression lentivirus vector and siRNA knockdown vector were constructed and transfected into HTR-8/Svneo cells. After transfection, the expression of ERp29 was significantly lower in siRNA-ERp29 group and higher in Lenti-ERp29 group (Fig. 4A). The results of CCK8 showed that the proliferation ability decreased gradually when the cells highly expressed ERp29. After knock-downing ERp29, the cell proliferation activity increased significantly at 36h, compared with the TCA group (Fig. 4B, 4C). Scratch and transwell test showed that ERp29 inhibited cell migration (Fig. 4D, 4E), and matrigel test showed that ERp29 inhibited cell invasion (Fig. 4F). The results of western blotting showed that the siRNA-ERp29 group highly expressed N-cadherin, Snail 1, Vimentin, MMP2 and MMP9, the expression of E-cadherin decreased. The Lenti-ERp29 group showed the opposite trend (Fig. 4G).

3.5 ERp29 aggravates ICP in pregnant rats

The adenovirus vector of ERp29 overexpression and knockdown were constructed. ICP pregnant rat model was established. The body weight curve showed that the body weight of other pregnant rats began to decline in varying degrees on the 3th day except Neg group. Compared with ICP group, the weight loss trend of pregnant rats in ERp29-Rat-222 group was alleviated, and the weight of ERp29-Rat-418 Group continued to decline (Fig. 5A). Compared with ICP group, the expression of TBA, ALT and AST in ERp29-rat-222 group decreased, and TBA in ERp29-rat-418 group was still high (Fig. 5B, 5C, 5D). HE staining of liver showed that the structure of hepatocytes in ICP and ERp29-Rat-418 group were deformed, the outline were unclear, the arrangement of hepatic cord were irregular, the hepatic sinuses became larger, and the hepatocytes swelled and degenerated. The structure of hepatic lobules of ERp29-Rat-222 group was intact and recovered, and the degree of hepatocyte size disorder decreased (Fig. 5E). The placental HE staining showed that the trophoblasts in ICP group and ERp29-Rat-418 group were swollen, uneven in size and disordered in arrangement, and the trophoblasts in ERp29-Rat-222 group tended to be orderly in size and arrangement (Fig. 5F). TUNEL results represented that after fluorescence staining, the placenta of ICP group and ERp29-Rat-418 group showed strong red fluorescence, and the fluorescence intensity of ERp29-Rat-222 group decreased (Fig. 5G). Statistical analysis of apoptosis index showed that there were significant differences among ERp29-Rat-222, ERp29-Rat-418 group and ICP group (Fig. 5H).

3.6 Overexpression of ERp29 inhibited EMT process in placenta of ICP pregnant rats

Western blotting showed that after adenovirus transfection, ERp29 in the placenta of pregnant rats in ERp29-Rat-222 group decreased significantly, while ERp29-Rat-222 group was still high (Fig. 6A). It can be seen from the IHC results that ERp29 in ICP group and ERp29-Rat-418 group was highly expressed in the cytoplasm of placental cells, and the expression intensity of ERp29 in ERp29-Rat-222 group was reduced (Fig. 6B). The detection of EMT related molecules in placental tissue showed that the expression of N-cadherin, Snail 1, Vimentin, MMP2 and MMP9 in ERp29-Rat-222 group was high, and the expression of E-cadherin decreased. The expression of ERp29-Rat-418 group showed the opposite trend (Fig. 6C, 6D).

ERS signal is an important determinant of cell dormancy, survival and autophagy. ERp29 is a hypothetical chaperone protein located on the built-in network. It is structurally composed of an N-terminal domain, a flexible ring and a C-terminal domain[12]. As a novel ER protein, ERp29 plays an important role in protein formation and secretion[13]. It has been reported that overexpression of ERp29 significantly inhibited cell proliferation[14]. This study showed that the ERp29 protein was significantly increased in the placenta of ICP pregnant rats. The histochemical results showed that ERp29 was mainly expressed in the cytoplasm. TUNEL fluorescence staining proved that the level of apoptosis in placental tissue of ICP pregnant rats increased significantly. In vitro cell experiments showed that HTR-8/Svneo cells highly expressed ERp29 and inhibited cell proliferation after TCA treatment. We can speculate that ERp29, as a new protein in the ER, has a regulatory mechanism to mediate trophoblast apoptosis in the occurrence and development of ICP. Our previous in vitro experiments also explained that knockdown of ERp29 in HTR-8/ Svneo cells treated with TCA inhibited G2/M block and apoptosis[15].

The placenta is the site of gas and nutrient exchange and elimination of metabolites between the mother and fetus. As a part of the main structure of placenta, the invasion of trophoblast cells into endometrium is an important physiological process of placenta formation[16, 17]. Studies have shown that extravillous trophoblast cells invade the uterine decidua and uterine myometrial spiral artery, and gradually replace the endothelial cell layer and some smooth muscle tissue in the maternal spiral artery, resulting in vascular thickening and providing sufficient blood supply for the uterus[18, 19]. EMT is the process of epithelial cells losing polarity and adhesion, and transforming into mesenchymal like phenotype through migration[20]. Most studies found that EMT transcription factors (slug, snail and twist) were up-regulated in mesenchymal like cells, while E-cadherin decreased,N-cadherin and vimentin increased[21]. In the process of phenotypic transformation from epithelial cells to mesenchymal cells, the ability of cell migration and invasion is enhanced, and the ability of cell resistance to apoptosis is also enhanced to a certain extent[22-24]. In the process of EMT, extravillous trophoblast up-regulates matrix metalloproteinases (MMPs), such as MMP2, MMP3 and MMP9. These enzymes are the main enzymes involved in the degradation of uterine basement membrane. They make cells more prone to metastasis and invasion by degrading extracellular matrix[25-30]. EMT is a process that begins during initial conception and embryo implantation[31]. When EMT is inhibited, the invasion ability of trophoblast during pregnancy is insufficient, which often leads to the impaired development of placental vascular network and induces placental ischemia/hypoxia, which threatens the safety of pregnant women and fetus[32, 33]. Our study results showed that N-cadherin, vimentin and snail 1 decreased and E-cadherin increased in the placenta of ICP pregnant rats. At the same time, metalloproteinases such as MMP2 and MMP9 were significantly degraded in placental tissue. When cells simulated ICP environment in vitro, except for the change of EMT related protein level of HTR-8/Svneo cells, functional experiments showed that cell proliferation was significantly inhibited, cell migration ability was weakened, and invasion ability was significantly inhibited. These results showed that the EMT process of placental cells was significantly inhibited during ICP.

The relationship and synergy between apoptosis and EMT have not been deeply studied. Apoptosis and EMT are independent events that play a controlling and coordinating role in time and space during embryonic development. They are also basic events closely related to the regulation of tissue and body homeostasis, immune response, tumorigenesis and cancer progression[34]. The common characteristics of apoptosis and EMT show that they are closely related. Therefore, we have reason to believe that the interaction and balance between apoptosis and EMT are very important for various related pathophysiological processes. Snail is a well-known transcription regulator, which is highly expressed in EMT[35]. Snail has been shown to weaken the cell cycle and give cells the ability to resist cell death induced by pro-apoptotic signals, indicating that the induction process of EMT is related to anti apoptotic and growth arrest effects[36]. Further study showed that snail overexpression induced EMT and inhibited mouse hepatocyte apoptosis[37]. We added TGF-β to induce EMT. Compared with HTR-8/Svneo cells treated with TCA only, TGF-β significantly promoted cell migration and invasion. We found that during the EMT, the cell proliferation activity increased and ERp29 protein decreased. The study has shown that ERp29 mainly plays a regulatory role in ERS, MET, cancer cell growth, cell integrity regulation, tumor progression, metastasis and chemical sensitivity[38]. Silencing ERp29 can promote the invasiveness of PCa cells by down regulating E-cadherin and increasing EMT[39]. In this study, after overexpression of ERp29, the epithelial cell markers increased, the ability of cell migration and invasion decreased, and the process of EMT was inhibited. After knockdown of ERp29, the cells turned to mesenchymal like phenotype and proliferated significantly. Then, we verified that reducing the level of ERp29 in placental tissue can reduce the apoptosis of placental trophoblast cells to a certain extent, inhibit the EMT process and alleviate the disorder. The clinical symptoms of ICP in pregnant rats were relieved.

Our whole experiment proved that ERp29, as a new ER protein, alleviated ICP in pregnant rats by inhibiting the EMT process of placental trophoblast cells. When HTR-8/Svneo cells highly express ERp29, EMT is inhibited, and its invasion ability is insufficient. The change of this mechanism makes the imbalance between cell proliferation and apoptosis and the impairment of placental function to some extent. Taking ERp29 as the target molecule can provide a new possibility for the diagnosis and treatment of clinical ICP.

E2	ethinyl estradiol
EMT	epithelial mesenchymal transformation
ER	endoplasmic reticulum
ERp29	endoplasmic reticulum protein 29
ERS	endoplasmic reticulum stress
ICP	intrahepatic cholestasis of pregnancy
PG	propylene glycol
TCA	taurocholic acid
UPR	unfolding protein response

Ethics approval

Experiments involving animals and humans were approved by the Ethical Committee of Wuxi maternal and Child Health Hospital Affiliated to Nanjing Medical University (2021916).

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

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

Competing interests

All authors declare that they have no conflict of interest.

Funding

This study was supported by the National Natural Science Foundation of China (grant no.82171674 and 81671489), the Major Research Foundation of Jiangsu Science and Technology Department (grant no. BE2017628), Jiangsu Province Health Committee Scientific Research Project (grant no. M2021023), Wuxi City Key Medical Talent of the Project of Invigorating Health Care through Science, Technology and Education (grant no. Z201601). Wuxi City Health Committee top-notch talent (grant no. BJ2020077), Jiangsu Province Health Committee " Six One Project " Top Talent Project (grant no. LGY2020023), Wuxi City Health Committee Youth Project (grant no. Q202032 and Q202121). Wuxi City Health Committee Translational Medicine Project (grant no. ZH202109). Wuxi City Science and technology development Fund project (grant no. Y20212035).

Author's contribution

GW: conceptualization, data curation, analysis and interpretation, methodology and writing-original draft;RD, KW: project design and administration, Investigation, collection and/or assembly of data; NY, JW: data analysis and interpretation; SZ, JC, TW: construction of animal model; XS, TY: cell culture; TZ, LL, FG: study design, writing-review/editing, final approval of the manuscript and funding acquisition. All authors read and approved the final manuscript.

Acknowledgments

Not applicable.

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No competing interests reported.

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Endoplasmic reticulum protein 29 inhibits epithelial-mesenchymal-transformation in placental trophoblast cells and promotes the development of intrahepatic cholestasis of pregnancy in pregnant rats

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