Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells

Glioblastoma multiforme (GBM) is the most aggressive form of Background malignant glioma and is also known as grade IV astrocytoma. This might be due to the presence of cancer stem cells with high pluripotency and ability of self-renewal. Recently, it has been reported that tumor stroma cells, including mesencyhmal stem cells (MSCs), secrete factors that affect cancer cell growth. Until now, the role of MSC secretomes in cancer stem cell pluripotency remains unclear. The aim of this study was to analyze the effect of MSC secretomes in conditioned medium (CM) on the expression of pluripotency markers of GBM cells. : Umbilical cord-derived MSCs (UCSCs) were grown on serum-free Methods alphaMEM for 24 hours to prepare the UCSC-CM. Human GBM T98G cells were treated with UCSC-CM for 24 hours. Following this treatment, expression of pluripotency markers SOX2, OCT4 and NANOG genes was analyzed using quantitative RT-PCR. : SOX2 and OCT mRNA expression was 4.7-fold (p=0.02) and 1.3-fold Results (p=0.03) higher in CM-treated cells compared to the control. However, there was no change in NANOG mRNA expression. This might be due to there being others factors regulating NANOG mRNA expression. : UCSC-CM could affect the expression of SOX2 and OCT4 in Conclusions human glioblastoma multiforme T98G cells. Further research is needed to elucidate the mechanism by which pluripotency markers are expressed when induced by the UCSC secretome.


Introduction
Glioblastoma multiforme (GBM) is a primary brain tumor that arises from glial cells (glioma), and is the most aggressive form of malignant glioma.According to the WHO, GBM is also known as grade IV astrocytoma 1 .The life expectancy of patients with GBM is very low, usually less than 1 year despite available options such as surgery and chemoradiation.Failure of therapy might be due to the presence of cancer stem cells with high pluripotency and ability of self-renewal 2 .Cancer stem cells have an improved the ability to repair their own DNA 3 , and can be identified by the analysis of transcription factors found in embryonic stem cells such as OCT-4 (octamer-binding transcription factor 4), SOX2 [SRY (sex determining region Y)-box 2] and NANOG.Those transcription factors play an essential role in sustaining the pluripotency and self-renewal ability of embryonic stem cells 4,5 .
Recently, it has been reported that tumor stroma cells, including mesencyhmal stem cells (MSCs), secrete factors that affect cancer cell growth.Previous studies have demonstrated that MSCs were recruited from bone marrow and that they home around the cancer cells to support tumor growth and metastasis 6 .
Other studies have shown that MSCs can trigger cancer growth by inducing angiogenesis, suppressing the immune system, forming cancer-associated fibroblasts (CAF) that contributing to the tumor growth, epithelial mesenchymal transition (EMT) and metastasis 7 .The role of MSCs in cancer growth has been widely investigated.Nevertheless, the role of MSC secretomes in cancer stem cell pluripotency remains unclear.The aim of this study was to analyze the effect of MSC secretomes in conditioned medium (CM) on the expression of pluripotency markers of GBM cells.

Cell culture
The Human Glioblastoma Multiforme T98G UCSC-CM treatment of T98G cells 400,000 T98G cells were seeded in triplicate on a 12-well-plate in high glucose DMEM/10% FBS/1% streptomycin-penicillin/1% amphotericin and allowed to adhere overnight.The following day, medium of T98G cells was replaced by 50% (v/v) UCSC-CM and incubated for 24 hours.
Reaction protocol was as follows: cDNA synthesis for 10 minutes at 42°C, inactivation with iScript Reverse transcriptase for 5 minutes at 95°C, then 40 PCR cycles for 10 seconds at 95°C, followed by 30 seconds at 57°C for OCT4, 59°C for SOX2, 60°C for NANOG; then 30 seconds at 72°C.Following the PCR cycles, the protocol was subsequently continued with the melting curve analysis, i.e. 1 minute on 95°C; 1 minute on 55°C; 10 seconds on 55°C (80 cycles, increase 0.5°C every cycles).

Statistical analysis
All data were presented as means ± SD from triplicate experiments.Statistical analysis was performed using Student's t-test using PASW 18 software, with p < 0.05 as a cut-off for determining a significant difference.

Cell morphology
Cell morphology was observed under an inverted microscope (100X magnification) after 24 hours of the CM treatment.
There was no difference in morphology between control and CM-treated T98G cells, as shown in Figure 1.Both control T98G cells and CM-treated T98G cells appear with fibroblast morphology, adhering to the plate.There was no change in cell shape and the cell membrane was still intact.

Pluripotency marker expression
Pluripotency marker expression was analyzed by measuring SOX2, OCT4 and NANOG mRNA expression.Relative mRNA expression was calculated using the Livak method (2 -ΔΔCT ) with 18S rRNA as the reference gene.SOX2 mRNA expression was significantly higher in CM-treated T98G cells compared to control (Figure 2).SOX2 mRNA expression was up-regulated 4.7-fold in CMtreated T98G cells.Moreover, OCT4 mRNA expression was also significantly increased in CM-treated T98G cells compared to the control (Figure 3).OCT4 mRNA expression increased 1.2 fold in CM-treated T98G cells.Nevertheless, there was no significant change in NANOG mRNA expression (Figure 4).

Discussion
Similar to normal stem cells, stem cell-like properties such as pluripotency in glioma CSCs are maintained by a core set of transcription factors, including SOX2, OCT4, and NANOG.Up-regulation of this set of genes is associated with poor outcome in terms of tumor malignancy, recurrence and metastasis 11 .
Here, we demonstrated that the pluripotency markers SOX2 and OCT4 were up-regulated in UCSC-CM-treated cells.This indicates that UCSCs secrete certain factors that support the self-renewal capacity of GBM cells.Liu et al showed that bone marrow-derived MSCs produce a cytokine meshwork that stimulates CSCs 12 .
There is accumulating evidence suggesting that the regulation of stem cell-like properties requires a two-way interaction between CSCs and their microenvironment, particularly the MSCs.For instance, secretion of interleukin-1 (IL-1) in colon cancer cells could stimulate MSCs to produce prostaglandin E2 (PGE2).Then, PGE2 collaborated with IL-1 to produce other cytokines and chemokines such as IL-6, CXCL1 & CXCL8 by MSCs, leading to enhancement of the cancer stem cell population 13 .Wu et al proved that cytokines (IL-6, CXCL-8) were detected in conditioned media from MSCs.Those cytokines stimulated the expression of pluripotency markers (SOX2, OCT4, cMyc), as well as NF-κB &AMPK/mTOR signaling pathways in colon cancer cells 14 .In another study by Luo et al., CCL5 secreted by recruited BM-MSCs were found to induce prostate CSCs via androgen receptor signaling 15 .Investigating the secreted components of our UCSC-CM could allow us to determine more targeted CSC therapy in GBM.
Unlike SOX2 and OCT4 expressions, NANOG mRNA expression in T98G cells was not affected by UCSC-CM in our study.This is might be due to the abundance of NANOG pseudogenes 16,17 and also due to the way NANOG mRNA undergoes unique N 6 -methyladenosine (m 6 A) posttranscriptional modification as part of its regulation 18 .Furthermore, microRNA-134 has been reported to suppress proliferation and invasion of T98G cells by reducing NANOG expression 19 .Further studies are required to elucidate the involvement of UCSC secretomes in inducing the differential expression of pluripotency markers.

Conclusions
The conditioned medium of umbilical cord-derived mesenchymal stem cells could affect the expression of SOX2 and OCT4 as pluripotency markers in human glioblastoma multiforme T98G cells.

Data availability
Dataset 1: 18S rRNA Cq values.18S rRNA Cq was used to calculate SOX2, OCT4 and NANOG mRNA expression using the  The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com

Figure 2 .
Figure2.SOX2 mRNA expression. 100 ng of total RNA was amplified using quantitative reverse transcriptase polymerase chain reaction to detect SOX2 mRNA expression.The expression was relatively calculated using Livak formula with 18S rRNA gene as a reference gene.All values are means ± SE, n = 9.Significant differences at *(p<0.05).SOX2 expression was 4.7-fold (p=0.02)higher in the conditioned medium-treated T98G cells compared to the control.

Figure 3 .
Figure3.OCT4 mRNA expression. 100 ng of total RNA was amplified using quantitative reverse transcriptase polymerase chain reaction to detect OCT4 mRNA expression.The expression was relatively calculated using Livak formula with 18S rRNA gene as a reference gene.All values are means ± SE, n = 9.Significant differences at *(p<0.05).OCT4 expression was 1.3-fold (p=0.03)higher in the conditioned medium-treated cells compared to the control.

Figure 4 .
Figure 4. NANOG mRNA expression. 100 ng of total RNA was amplified using quantitative reverse transcriptase polymerase chain reaction to detect NANOG mRNA expression.The expression was relatively calculated using Livak formula with 18S rRNA gene as a reference gene.All values are means ± SE, n = 9.Significant differences at *(p<0.05).There was no significant difference in NANOG expression between control and conditioned mediumtreated T98G cells.

3 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells.
Hardiany NS, Huang P, Dewi S, et al.:

Dataset 4 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells.
F1000Research. 2018.Data Source 24.Hardiany NS, Huang P, Dewi S, et al.: