Effect of umbilical cord mesenchymal stem cells in human ovarian cancer SKOV3 cells

Objective :To investigate the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on apoptosis and proliferation of human ovarian cancer SKOV3 cells and to explore mechanism. Methods :hUC-MSCs were isolated and cultured by tissue block adherent culture method. The hUC-MSCs phenotype were identified by flow cytometry. The hUC-MSCs lysate and conditioned medium,directly combine were used to treat SKOV3 cells.The effects on the proliferationapoptosismechanism of SKOV3 cells were examined by cell counting kit-8(CCK-8)Annexin V-FITC/PIquantitative real time polymerase chain reaction(RT-qPCR) and spheroid formation assays.Establish ovarian cancer xenograft models1X 10 6 hUC-MSCs and 2 X 10 6 hUC-MSCs were administrated into the mice t rear back tumor tissue. After three injections of hUC-MSCs, the nude mice were sacrificed after 1 week of observation.Remove tumor tissue. Observed tumor volume changes every 3 days after the start of the experiment. The expression of CD34 and VEGF were detected by immunohistochemistry. Results :Human umbilical cord mesenchymal stem cells were cultured and isolated from tissue block. Flow cytometry results revealed that the hUC-MSCs marks CD44 and CD29, but not CD45 and CD34 were expressed on obtained cells. The apoptosis of SKOV3 cells was induced by hUC-MSCs lysate, conditioned medium and Transwell co-culture method in SKOV3 cells, and the apoptosis rate was higher with increasing concentration. hUC-MSCs conditioned medium and Transwell co-culture method can inhibit cell proliferation. After adding experimental factors, the conditioned medium and Transwell co-culture method can down-regulate the transcription of PI3KCA, AKT and BCL-2 genes in SKOV3 cells, and up-regulate the Caspase-3 gene.The tumor volume of the experimental group was smaller than that of the control group during the observation period. The expression levels of CD34 and VEGF in the experimental group were significantly lower than those in the control group(P<0.05). Conclusion : The conditioned medium of hUC-MSCs and the co-culture method of hUC-MSCs and SKOV3 can significantly inhibit the proliferation of SKOV3 cells, which is mainly achieved by inhibiting PI3K/AKT signaling pathway. hUC-MSCs can inhibit the growth of subcutaneous subcutaneous transplantation and the expression of CD34 and VEGF in ovarian cancer. It provides a new idea for the treatment of ovarian cancer.


Introduction
Ovarian cancer (OvCa), one of the most common gynecological maligancies in women worldwide, which the incidence is second only to cervical and uterine cancer [1− 2] .According to statistics, there were nearly 300,000 new cases of OvCa in 2018 [3] . Because the anatomical part of the ovary is located deep in the pelvis and the lack of specific symptoms in patients with OvCa makes early diagnosis difficult, initial diagnosis typically occurs when the tumor has reached an advanced stage.
About 70% -75% of patients are in advanced stage at the time of onset; their 5-year survival rate is only about 20% [4] . Although application of treatments such as surgical resection, chemotherapy, radiotherapy and targeted therapy significantly made substantial progress, many OvCa patients still have no improve overall survival (OS) time [5] .Therefore, in order to improve the survival rate of OvCa patients, new methods need to be explored.MSCs were originally found in bone marrow and are pluripotent stem cells derived from mesoderm. MSCs have low immunogenicity, tumor orientation and targeted migration ability [6− 7] . Many studies have shown that when MSCs aggregate or contact tumor tissues, they can directly inhibit tumor growth or play a dual role by directly acting or secreting certain cytokines; Therefore, there is no unified understanding of the relationship between hUC-MSCs and tumorigenesis and tumor progression. In this study, we applied hUC-MSCs lysate and conditioned medium to ovarian cancer SKOV3 cells, and co-cultured hUC-MSCs with human ovarian cancer SKOV3 cancer cells, and observed the effects on the proliferation and apoptosis of human ovarian cancer SKOV3 and its occurrence Preliminary investigation of the mechanism, establishment of a model of OvCa xenograft, the application of hUC-MSCs to the xenograft model, and observation of its effect on the xenograft model.

hUC-MSC isolation,culture and treatment
We isolated hUC-MSCs using an adherent tissue method. Brieflfly, a 10 cm umbilical cord from a full-term healthy newborn(The umbilical cord was obtained from a healthy full-term Caesarean section in the Department of Obstetrics and Gynecology, Henan Provincial People's Hospital.)was washed with PBS (containing 1% penicillin-streptomycin double-resistant solution, Beijing Solarbio Science & Technology Co.,Ltd) 3 times. Then, we cut the cord into small pieces, dislodged the umbilical vein and umbilical artery, and left Wharton's jelly at last. Wharton's jelly was then cut into 1 mm × 1 mm × 1 mm and cultured in DMEM/F12 medium (Gibco, Grand Island, NY) containing 10% FBS (Gibco, Grand Island, NY) ; then cultured in humidified atmosphere, the medium was changed every 72 hours, and the cell fusion degree reached 80% before 1: 3 passage.3-6 passages (P3-6) were selected for the following experiments.
Cell lines culture and treatment SKOV3 cell lines of human origin were purchased from Cyagen Biosciences Inc.
(Guangdong,China)and cultured in DMEM/F 12 medium containing 10% FBS. then cultured in 25 mm cell culture flask The umbilical cordexplants were cultured in 37℃, 5% CO 2 humidified atmosphere, the medium was changed every 72 hours, and the cell fusion degree reached 80%.
Flow cytometry and hUC-MSCs identifification hUC-MSCs were trypsinized, and the cell concentration was adjusted to 1 × 10 6 /mL in PBS. Then, 200 µl of the suspension was incubated with 5 µl of antibodies against CD29 CD45 CD44 and CD34 without light for 30 min. Primary antibodies were directly conjugated with FITC and phycoerythrin. For isotype control, non-specifific FITC-conjugated IgG was substituted for the primary antibodies. Lastly, the samples were analyzed using flflow cytometry.

Lysate preparation
Select 3-6 passages hUC-MSCs with cell fusion to 80% and trypsinize them. After washing 3 times with PBS, resuspend them in DMEM medium without FBS, Count on the cell counting plate and adjust the cell concentration to 1 × 10 6 cells / ml. The cells were lysed by repeated freeze-thaw methods, and the collected cells were quickly frozen under liquid nitrogen for 10 minutes, taken out and placed at room temperature to slowly thaw, repeatedly frozen and thawed for more than 10 times, and stored in a refrigerator at -20 ° C for later use.Add 0.5 × 10 6 SKOV3 cells to four group, 1 / 2 times group, 1times Group, 2 times group, and then add 1 ml DMEM / F12, 0.25 ml lysate + 0.75DMEM / F12, 0.5 ml lysate + 0.5 ml DMEM / F12, 1 ml lysate, and incubate for 48 h. Each experiment was repeated 3 times.
Add 0.5 × 10 6 SKOV3 cells to four 25 cm 2 culture flask, numbered as CM control group, 50% group, 75% group, and 100% group. The control group was 5 ml of SKOV3 complete medium (CM concentration was 0%) and cultured in an incubator for 48 h. Each experiment was repeated 3 times.  Table 1.
Total RNA was extracted from treated SKOV3 cells using TRIzol reagent (TaKaRa, Japan). 1 µg RNA was reverse transcribed using the MMLV reverse transcriptase kit according to the manufacturer's instructions (TaKaRa, Japan). The GAPDH,PI3KCA AKT,bcl-2 and caspase-3 primers were provided directly and the sequences were kept secreted by Sangon Biotech (Shanghai) Co.,Ltd. All data were normalized using the internal control GAPDH.

Genes
Primers when the tumors have a diameter of about 0.5 cm. OvCa cells nude mice were randomly divided into 3 groups of 5 mice each, which were respectively the normal control group, the 1 × 10 6 hUCMSCs group, and the 2 × 10 6 hUCMSCs group. 1 × 10 6 and 2 × 10 6 hUCMSCs were injected into the dorsal tumors of ovarian cancer-bearing nude mice, respectively, for 3 consecutive weeks and once a week. The control group was injected with physiological saline, and hUCMSCs were injected 3 times.
The nude mice were sacrificed and the tumor tissues were removed after 1 week.

Immunohistochemistry
Formalin-fixed paraffin-embedded mouse tumor tissue sections were first deparaffinized in xylene and rehydrated through graded ethanol. Subsequently, the sections were boiled for 10 min in citrate buffer (pH 6.0, 10 mM) for antigen retrieval. Endogenous peroxidase activity was then inhibited by exposure to 3% hydrogen peroxide for 10 min. The sections were then blocked with 5% BSA and incubated with properly diluted VEGF and CD34 primary antibody at 37˚C for 1 h.
After the sections were washed with PBS, they were then incubated with diluted secondary antibody for 20 min. Finally, the sections were visualized with 3,3'diaminobenzidine (DAB) and then counterstained with hematoxylin for examination under a light microscope (x100, SN:9G15626; Olympus). Immunohistochemistry experiments were performed in strict accordance with the kit instructions, and images were collected under a microscope.

Statistical analysis
All experiments were conducted at least in triplicate. Data were presented as the means ± standard error. Statistical analysis was performed using SPSS 25.0 software. Potential differences between groups with different treatments were determined using one-way ANOVA or an independentsample t-test. A value of P < 0.05 was considered to indicate a statistically significant difference.

Characterization of hUC-MSCs
Adherent spindle-shaped cells and classical MSCs colonies were observed under inverted microscope. We chose P6 hUC-MSCs to identify the cell phenotype by flflow cy [1]tometry. Inverted phase contrast microscopy showed that P3 hUC-MSCs and P6 hUC-MSCs, as shown in Fig. 1A

hUC-MSCs CM promotes SKOV3 cells apoptosis and inhibits proliferation
Flow cytometry was conducted to identify changes in the apoptosis rate of SKOV3  (Fig.3 A-F) P 0.05.

Co-culture of hUC-MSC and SKOV3 cells promotes SKOV3 cells apoptosis and inhibits proliferation
Flow cytometry was conducted to identify changes in the apoptosis rate of SKOV3 cells treat with Co-culture of hUC-MSC according to Annexin V staining. The results indicated that the apoptosis rates of SKOV3 cells did not differ markedly between the control group and 0.5 × 10 6 group, 1 × 10 6 group, and 2 × 10 6 group at either 60 h (31.60±0.53%, 40.63±0.74%, 45.53±0.67%, 56.53±0.57%). P 0.05).

Caspase-3 in SKOV3 cells.
To verify the underlying pathway, we detected PI3K/AKT signaling pathway related genes and related apoptotic genes in SKOV3 cells treat with hUC-MSCs CM or in the hUC-MSCs co-culture system. The RT-PCR results showed that the expression of PI3KCA in SKOV3 cells treat with hUC-MSCs CM relative to the control group in the 50% group, 75% group, and 100% group was 0.74 ± 0.07, 0.60 ± 0.07, 0.40 ± 0.08, respectively; the relative expression levels of AKT in each group were 0.80 ± 0.03, 0.60 ± 0.04, 0.44 ± 0.05, respectively; the relative expression levels of BCL-2 in each group were 0.63 ± 0.02, 0.40 ± 0.04, 0.25 ± 0.09, respectively; the relative expression levels of Caspase-3 in each group were 1.52 ± 0.11, 2.02 ± 0.08, 2.58 ± 0.29, respectivel(P 0.05) (Fig.5 A);The RT-PCR results showed that the expression of seen subcutaneously on the back of the nude mice. The nodules were hard and had good mobility, and the surrounding skin was free of ulceration and swelling. The tumor formation rate was 100%. The tumor-bearing nude mice had no significant changes in their activities, diet, and mental state before tumor implantation.

Umbilical cord mesenchymal stem cells inhibit the volume of ovarian cancer xenografts
During the entire observation period, the quality of the tumor-bearing nude mice in the control group and the experiment was group generally stable; the tumor volume of the tumor-bearing nude mice in the control group and the 1 × 10 6 hUCMSCs group and the 2 × 10 6 hUC-MSCs group showed an overall increase. At the same times, as the number of hUC-MSCs injected increases, the volume of the transplanted tumor decreases (Fig.6).

Umbilical cord mesenchymal stem cells inhibit VEGF and CD34 expression in ovarian cancer xenografts
Observation of the light microscope after HE staining in the control group and the experimental group showed that tumor cells were abundant, densely arranged, obvious cell atypia, large nuclear deep staining, significantly increased nuclear and cytoplasmic ratios, and more abnormal nuclear and mitotic phases. can prevent cancer cell proliferation by up-regulating PTEN levels in aggressive cancer cells [10] .The results of Wang Y et al. Showed that the artificial fusion of human mesenchymal stem cells and esophageal cancer resulted in reduced hybrid cell growth, apoptosis and proliferation, and inhibition of tumorigenicity [11] . Then there are some studies that show that umbilical cord mesenchymal stem cells can promote tumor development. Karnoub et al. Demonstrated that mesenchymal stem cells promote cell metastasis by secreting cell-derived factor-1 (SDF-1), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF) [12] . However, there are relatively few reports on the effects of umbilical cord mesenchymal stem cells on ovarian cancer cells, and there are different opinions. Some studies have shown that co-culture of mesenchymal stem cells and human SKOV3 ovarian cancer cells can promote apoptosis, and The fusion of SKOV3 cells can also significantly reduce tumorigenicity [13] .
The PI3K / AKT signaling pathway is one of the important signal transduction pathways in cells, and plays an important role in inhibiting apoptosis, regulating the cell cycle, and promoting cell invasion and metastasis [14] . The P13K / AKT signaling pathway plays an important role in the proliferation, invasion, cell cycle progression, angiogenesis, and drug resistance of ovarian cancer, so P13K / AKT / mTOR signaling pathway inhibitors have become a new direction in the treatment of ovarian cancer [15] .
In this study, an isolation method of hUC − MSCs tissue block adherent culture was cells and the microenvironment. The process is complicated, and the current mechanism is still unclear [16] .VEGF has the ability to increase capillary permeability, promote tumor cells to enter the vascular wall, and metastasize. Its biological activity is regulated by oncogenes, hypoxia, cytokines, and inflammatory factors [17] . MVD assay is to study the expression of vascular endothelial cells in solid tumor stroma, but not in parenchymal cells. CD34 can efficiently and specifically label vascular endothelial cells [18] . Zhao assist in the experience, Qiuyun Yang contribute in experience design and interpretation of the results, All authors read and approved the final manuscript.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The animals were maintained in accordance with institutional policies, and all experiments were performed with approval of the Committee on the Use and Care of Animals of the Henan Province People's Hospital.and all patients were asked to read and approve/sign informed consent forms prior to any participation.All experiments involving the handling of human tissues were performed in accordance with Tenets of the Declaration of Helsinki.

Consent for publication
Written informed consent for publication was obtained from the participants before the collection of any samples.   Effect of SKOV3 cells treat with Co-culture of hUC-MSC A:The apoptosis of the control group The Volume of xenograft tumors in nude mice bearing each group Figure 7 Role of hUC-MSCs in SKOV3 Ovarian cancer cell-derived tumor xenograft growth in vivo. A:H&