Effects of MFG-E8 expression on the biological characteristics of ovarian cancer cells via the AKT/mTOR/S6K signalling pathway

Abstract In this study, we assessed the effects of MFG-E8 on the biological characteristics of ovarian cancer cells and explored the underlying mechanisms. Human ovarian cancer SKOV3 cells were transfected with MFG-E8 siRNA or NC siRNA. CCK-8, cell adhesion, scratch-wound, and Transwell assays were used to detect changes in cell metastatic processes. Effects of MFG-E8 silencing on the proteins involved in AKT/mTOR/S6K signalling pathway were assessed using qRT-PCR and Western blotting. Transient silencing of MFG-E8 in SKOV3 cells decreased cell proliferation and downregulated the expression of CDK4, cyclin D1, and caspase-3 proteins. Cell adhesion, migration, and invasion were also suppressed. p-AKT, p-mTORC1, and p-p70S6K levels decreased following MFG-E8 knockdown. Hence, MFG-E8 enhances carcinogenesis and affects the AKT/mTOR/S6K signalling pathway in ovarian cancer cells. In conclusion, our results suggested that MFG-E8 could promote ovarian cancer via AKT/mTOR/S6K signalling pathway which improved our understanding of the molecular mechanisms involved in ovarian cancer. IMPACT STATEMENT What is already known on this subject? Milk fat globule-epidermal growth factor 8 (MFG-E8) is expressed in several types of cancers such as oesophageal, breast, and liver. However, the mechanism of MFG-E8 involving in EOC remains unknown. We previously found that MFG-E8 expression was related to pathological staging, tissue differentiation, platinum sensitivity, ascites state, and other clinicopathological characteristics. What the results of this study add? Due to a series of in vitro studies, we confirmed that MFG-E8 is involved in the process of proliferation, invasion and metastasis. Our results show that silencing MFG-E8 can significantly inhibit the expression of cyclin D1 and CDK4 in EOC SKOV3 cells. MFG-E8 enhances carcinogenesis and affects the AKT/mTOR/S6K signaling pathway in ovarian cancer. What the implications are of these findings for clinical practice and/or further research? Taken together, our findings suggest that MFG-E8 may be an oncogene in EOC and provide new insights into the mechanism of MFG-E8 in the progression of EOC.


Introduction
Ovarian cancer is a common gynaecological malignancy.Although ovarian cancer accounts for only 3% of cancer cases, 6% of cancer-related deaths are due to ovarian cancer, making it the fifth leading cause of cancer-related mortality in females (Siegel et al. 2015).Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer, accounting for approximately 90% of ovarian malignancies, with an overall survival rate of <50% mainly resulting from late diagnosis due to vague clinical symptoms.EOC is generally diagnosed only when the disease has progressed to an advanced stage; it has limited treatment options and exhibits a high degree of resistance to chemotherapy (Lheureux et al. 2019).Currently, treatments such as surgery and combined radiotherapy and chemotherapy are widely used for patients with EOC.However, the mortality of patients in the mid-and late-stages of EOC has not substantially improved, presumably because the common characteristics of ovarian cancer are silent metastasis and aggressiveness, including the spread of tumour cells into the abdominal cavity, rapid formation of large amounts of ascites, and formation of tumour blood vessels (Matulonis 2011).Therefore, it is necessary to understand the basis of the high invasiveness and heterogeneity of ovarian cancer at the molecular level.
We previously found that MFG-E8 expression was related to pathological staging, tissue differentiation, platinum sensitivity, ascites state, and other clinicopathological characteristics (Li et al. 2020).To better understand the role of MFG-E8 in EOC, we investigated the effects of MFG-E8 silencing on the biological characteristics of EOC cells in vitro and examined the related mechanisms.Our results will provide new insights into the pathogenesis of EOC and strategies for its treatment.

Cell lines
The human ovarian cancer cell line SKOV3 was obtained from the Cell Resource Centre of the Chinese Academy of Sciences (Shanghai).The cells were cultured in RPMI1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% foetal calf serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin (Gibco) at 37 � C in a 5% CO 2 incubator.

Cell counting kit-8 (CCK-8) assay
Cell proliferation was evaluated using a CCK-8 (Dojindo, Japan).First, the SKOV3 cells were seeded in a 96-well plate with the density of 3000-5000 cells per well.After 24 h, the cells were transfected with Msi or Csi.CCK-8 reagent(10 mL) was added to each well at 24, 48, 72, 96, and 120 h after transfection.The culture plate was gently tapped to mix the fluid, and the cells were then incubated for 2 h at 37 � C. The optical density (OD) at 450 nm of each well was measured using a multifunction microplate reader.

Cell migration and invasion assays
A Transwell system (8 mm pore size; Corning Inc., Corning, NY, USA) was used for the cell migration and invasion assays.First, 1 � 10 5 cells were plated in the upper Transwell chamber (24well size) with RPMI1640 containing 0.1% foetal bovine serum (FBS), and the lower chamber was filled with 600 lL of complete medium (RPMI1640 containing 10% FBS).After incubation for 24 h, the cells that remained in the insert chamber were gently scraped off, and the cells that had migrated to the bottom membrane were fixed with formalin for 20 min, stained with 0.1% crystal violet, and imaged under a microscope.
For cell invasion assays, the chambers were pre-coated with 100 lL of Matrigel (BD Biosciences, San Jose, CA, USA) diluted to 1:8 with RPMI1640 and then air-dried.After irradiation with ultraviolet light, 2 � 10 5 cells were plated in the insert chamber.The cells were cultured at 37 � C in a humidified incubator containing 5% CO 2 for 48 h.The subsequent steps were conducted as performed for the migration assay.The experiments were repeated at least three times.

Adhesion assay
Briefly, 96-well flat-bottom culture plates were pre-coated with 100 lL of Matrigel (BD Biosciences, San Jose, CA, USA; diluted to 1:8 with RPMI1640), 10 mg/L human fibronectin (Proteintech, Rosemont, IL, USA), or 10 g/L bovine serum albumin (BSA), and then incubated at 4 � C overnight.SKOV3 cells were transfected with Msi or Csi for 48 h.The cells were seeded in 96-well plates at a density of 2 � 10 4 cells/well and incubated for 2 h at 37 � C in a CO 2 incubator as described above.The cells were then rinsed three times with phosphate-buffered saline (PBS) to remove unadhered cells.After adding 10 lL of the CCK-8 reagent to each well, the OD of the sample was measured at 450 nm.Adhesion ratio ¼ [(treatment group OD value/BSA group OD value) À 1] � 100.

Scratch-wound assay
A cell monolayer wound was created in Msi and Csi cells in six-well culture plates using a 20 lL pipette tip.The cells were washed with PBS and incubated in 1% FBS medium.
The process of cell migration to fill wound areas was monitored at 200� magnification using a microscope (Olympus CX43, JPN) with a digital camera at 0, 12, 24, and 48 h after plating.

Statistical analysis
GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for all statistical analyses.Data are presented as mean ± standard error of the mean (SEM) from at least three independent experiments.Comparisons were made using a t-test or one-way ANOVA.Statistical significance was set at p < .05.

Efficiency of siRNA interference
At 48 h after transfection of SKOV3 cells with MFG-E8 siRNA or NC siRNA, MFG-E8 expression was determined at the mRNA and protein levels using qRT-PCR and Western blotting, respectively.MFG-E8 mRNA expression was considerably decreased by the three siRNAs (Msi#1, Msi#2, and Msi#3) compared with that in the vehicle or control siRNA group (Figure 1(A)).Consistent with the qRT-PCR results, Western blotting showed that MFG-E8 expression was significantly lower in the Msi#3 group than in the other two groups (Figure 1(B)).Thus, as Msi#3 could effectively interfere with MFG-E8 expression, subsequent experiments were performed using Msi#3 in conjunction with Csi.

Effects of MFG-E8 siRNA transfection on cell proliferation
We used the CCK-8 assay to analyse the functional role of MFG-E8 in SKOV3 cell proliferation.SKOV3 cell proliferation was significantly inhibited following transfection with MFG-E8 siRNA compared with that in cells transfected with Csi at 48, 72, 96, and 120 h after transfection (Figure 1

(C)).
To explore the mechanism underlying the effect of MFG-E8 on SKOV3 cell proliferation, we analysed CDK4, cyclin D1, and caspase-3 protein levels using Western blotting and found that their expression was downregulated after MFG-E8 silencing in SKOV3 cells (Figure 1(D)).Thus, MFG-E8 may be involved in cell cycle regulation.

Effects of MFG-E8 on cell adhesion, migration, and invasion
We measured the effect of MFG-E8 silencing on cell adhesion.After transfecting SKOV3 cells with MFG-E8 siRNA, the rates of adhesion to both Matrigel and fibronectin were significantly different between Csi-and Msi-transfected cells (Figure 2(A)), indicating that MFG-E8 silencing inhibited SKOV3 cell adhesion to artificial basement membranes.
To explore whether MFG-E8 is required for tumour cell migration and invasion, we used scratch wound-healing and Matrigel invasion/migration assays, respectively.The scratch area of Msi-transfected cells was significantly lesser than that of Csi-transfected cells at 24 and 48 h (Figure 2(B)).The results of both the invasion and migration experiments showed that the number of MFG-E8 siRNA-transfected cells passing through the chamber was significantly lower than that of the Csi-transfected cells (Figure 2(C)).Thus, MFG-E8 silencing could inhibit SKOV3 tumour cell migration and invasion.

Discussion
In the present study, we knockdown the expression of MFG-E8 in human ovarian cancer SKOV3 cells using small interfering RNA.We found that (1) MFG-E8 could promote the proliferation of SKOV3 cells via cell cycle regulation.( 2) MFG-E8 could stimulate the migration and invasion as well as the adhesion of SKOV3 cells.(3) MFG-E8 increased cell proliferation and tumour growth via activation of the Akt/mTOR/ p70 S6K signalling pathway.
Despite these findings, the role of MFG-E8 in ovarian cancer remains unclear.Here, CCK-8 assay was used to investigate the effect of MFG-E8 siRNA transfection on the proliferation of human ovarian cancer SKOV3 cells.The result revealed that MFG-E8 silencing significantly inhibited cell proliferation.Consistent with the study on oral squamous cell carcinoma, MFG-E8 promoted tumour cell proliferation and growth (Yamazaki et al. 2014).
The G1/S phase transition of the cell cycle is controlled by cyclin-dependent protein kinases.The kinase-cyclin complex, which modulates the kinase activity, regulates cell cycle progression by targeted phosphorylation.The activity of these complexes peaks during the G1/S transition of the cell cycle and promotes cell mitosis (Lee et al. 2019).Cyclin D mainly regulates the G1/S cell cycle transition, and its expression is closely related to the abnormal proliferation of tumours (Blain 2008).Besides, MFG-E8 could promote the proliferation of human pulmonary artery smooth muscle cells via p-Akt/ cyclin D1 pathway (Wang et al. 2021).Our present study showed consistent changes in cell proliferation and expression of cyclin D1 and CDK4 after MFG-E8 silence.Previous studies have focussed on the role of MFG-E8 in artery smooth muscle cells (VSMC) (Wang et al. 2012(Wang et al. , 2021)), however, our study confirmed that MFG-E8 is involved in cell cycle regulation, which could help us better understand the mechanism of MFG-E8 in tumours.
In addition to traditional cyclin-dependent protein kinases, cell checkpoint genes, such as ataxia-telangiectasia gene (ATM), cell cycle checkpoint kinase 2 (Chk2), p53 gene, Rb gene and P21cip1 are others cell cycle regulators that play an unneglectable role in inhibiting cell division (Jerzak et al. 2018, Manu et al. 2019, Mustofa et al. 2020, Liebl and Hofmann 2021, Engeland 2022).In our previous study, we evaluated the expression of MFG-E8 in ovarian cancer tissues (Li et al. 2020).In this study, we found that the high expression of MFG-E8 in ovarian cancer tissues could promote cell proliferation.Thus, we evaluated the role of MFG-E8 silence in the expression of CDK4 and cyclin D1 proteins.However, the relationship between MFG-E8 expression and cell checkpoint genes above needs to be confirmed in further investigation.
The occurrence and development of tumours involve several aspects such as excessive cell proliferation, abnormal apoptosis, adhesion to the extracellular matrix (ECM), invasion, and migration ability (Liotta and Stetler-Stevenson 1991).The ECM provides structural support for cells and important regulatory signals to control cell growth, metabolism, and differentiation.Cell adhesion is involved in regulating gene expression and cell growth, differentiation, and apoptosis.Ovarian cancer has a unique growth and metastasis pattern, which differs from other solid tumours that can metastasise to distant sites through the vascular or lymphatic systems.Ovarian cancer cells can adhere to peritoneal mesothelial cells and form metastases. Finally, extensive abdominal lesions involving the network membrane, gastrointestinal tract, liver, spleen, and other organs can lead to the death of patients.Therefore, reducing the ability of cells to adhere to the ECM can inhibit tumour cell invasion and migration to a certain extent.Tibaldi coated xCELLigence micropores with rhMFG-E8 and performed adhesion tests with ovarian cancer cells and found that rhMFG-E8 could promote cell adhesion (Tibaldi et al. 2013).In our study, RNA interference technology was used in cell-ECM adhesion experiments using two different biological materials to simulate the ECM.The results revealed that after MFG-E8 silencing, the cell adhesion rate was significantly lower, confirming that MFG-E8 can regulate the adhesion of tumour cells to the ECM.
Tumour cells generally migrate into other tissues.Indeed, invasion and metastasis are the most fundamental manifestations of the malignant phenotype of tumours and are the main indicators used to assess the degree of malignancy of tumour cells (Makale 2007).To further study the influence of MFG-E8 on the invasion and migration abilities of SKOV3 cells, we conducted invasion experiments on an artificial basement membrane.The invasion ability of SKOV3 cells decreased after MFG-E8 silencing.We also used a Transwell chamber and cell scratch test to determine the effect of MFG-E8 silencing on the migration ability of SKOV3 cells.The results show that MFG-E8 silencing inhibited the migration and motility of tumour cells.
Studies have found that higher levels of apoptosis may be related to a poor prognosis in patients with cancer (Haitel et al. 2001, Gurova andGudkov 2003).Caspase-3 expression in tumour cells leads to increased levels of vascular endothelial growth factor in tumours, thereby promoting angiogenesis and tumour growth (Feng et al. 2017).Furthermore, in radiation-treated glioma cells, caspase-3 mediates the proangiogenic response through the NF-jB-PGE2 pathway (Feng et al. 2015).In human head and neck cancer and advanced breast cancer cases, high caspase-3 expression and high relapse rates have been associated with low survival rates (Huang et al. 2011).We found that after MFG-E8 silencing, caspase-3 was downregulated, which further supports the above view.
Phosphorylated proteins are the main mediators of intracellular signal transduction.Phosphorylation not only activates proteins but also amplifies the transmitted signals and induces biological responses both inside and outside the cell.The AKT/mTOR/S6K signalling pathway, which is an important signal transduction pathway in cells, is involved in various aspects of cell growth, proliferation, and metabolic regulation.Abnormal functioning of this pathway can cause developmental defects and metabolic diseases, in addition to promoting tumour formation (Khan et al. 2013, Li et al. 2019).In another study, MFG-E8 was reported to be an important activator of the AKT pathway (Zhao et al. 2017).Here, we found that MFG-E8 silencing in SKOV3 cells inhibited not only AKT phosphorylation but also the phosphorylation of mTOR and its downstream S6K, suggesting that MFG-E8 may participate in the biological functions of tumour cells by regulating the mTOR/S6K signalling pathway.
At present, some studies indicated that MFG-E8 may play an important role in tumour progression and inducing an immunosuppressive tumour microenvironment (Kanemura et al. 2018, Okamoto et al. 2020).However, the effect of MFG-E8 on the immucell infiltration in ovarian cancer and the involved mechanism are not determined in this study and require further research.
In conclusion, this study showed that MFG-E8 silencing can inhibit cell proliferation, adhesion, migration, and invasion, among other processes, as well as reduce cyclin D1, CDK4, and caspase-3 protein levels and AKT, mTOR, and S6K phosphorylation levels.This suggests that MFG-E8 activates the AKT/mTOR/S6K signalling pathway, ultimately leading to enhanced tumour cell proliferation, invasion, and migration capabilities.Our findings provide new insights into the mechanism of MFG-E8 in EOC progression, opening the way for future study of the MFG-E8 molecular and its role during the tumour progression.

Figure 2 .
Figure 2. MFG-E8 functional assays for adhesion, migration, and invasion following RNAi silencing in SKOV3 cells.(A) Comparison of the adhesion ability of SKOV3 cells between groups using a cell-matrix adhesion assay ( �� p < .01).(B) Wound-scratch assays were performed in Csi and Msi cell cultures at 48 h.C. Effects on migration and invasion of SKOV3 cells with MFG-E8 siRNA or NC siRNA using Transwell assay ( ��� p < .001).

Figure 3 .
Figure 3. Levels of Akt/mTOR/S6k signal pathway-related proteins in SKOV3 cells after transfection with MFG-E8 siRNA or NC siRNA detected using Western blotting.