High PLA2 level is correlated with glioblastoma progression via regulating DNA replication

Abstract Phospholipases A2 (PLA2) are a superfamily of enzymes, playing a critical role in the development of various human cancers. However, the mechanism of PLA2 as an oncogene in glioblastoma remains largely unknown. In this study, we explored the effects of PLA2 on glioblastoma and investigated the underlying mechanism. The results showed that PLA2 was highly expressed in glioblastoma. Patients with a high PLA2 level have low overall survival than those with low PLA2 expression. PLA2 overexpression promoted glioblastoma cell proliferation and viability and inhibited cell apoptosis by inducing cell cycle transition from G1 to S stage. Knockdown of PLA2 inhibited tumor growth in the xenograft mice model. In addition, PLA2 knockdown decreased the protein level of MCM2 and MCM5. These findings identify PLA2 as an oncogene in glioblastoma progression and provide a promising strategy to treat glioblastoma in the future.

associated with the initiation and progression, including prostate carcinomas, gastrointestinal and colorectal carcinomas. [9][10][11][12] Therefore, PLA2 has also been identified as a potential target of cancer therapy.
However, the function of PLA2 as an oncogene in GBM remains largely unknown. In this study, we mainly investigated the functions of PLA2 in GBM progression and explored the molecular mechanism.
This study showed that high expression of PLA2 was correlated with low overall survival and poor prognosis. PLA2 knockdown induced the loss of MCM function by inhibiting MCM2 and MCM5 expression, leading to DNA damage, further resulting in GBM cell apoptosis increase and cell growth suppression.

| Tissue samples from patients with GBM
PLA2 expression between tumour and normal tissues of GBM was analyzed from the Sixth People's Hospital of Nantong. A total number of 11 GBM tissues and adjacent normal tissues were obtained from the Sixth People's Hospital of Nantong. Informed consent was signed by all the patients. Tissues were frozen in liquid nitrogen and stored at −80°C before experiments. All samples were collected in accordance with ethical guidelines, and written informed consent was received. All patients were approached based on the approved ethical guidelines, and patients who agreed to participate in this study were required to sign consent forms before being included in the study. All experimental protocols and methods were approved by the Medical Ethical committee of the Sixth People's Hospital of Nantong No 20210622.

| Cell lines and culture
Glioblastoma cell lines (SHG-44, U251 and SNB-19) and one normal glial cell (HEB) were purchased from ATCC (American Type Culture Collection) and cultured in RPMI1640 (Gibco; Invitrogen) supplemented with 10% FBS (fetal bovine serum; GIBCO, USA). All these were maintained in an atmosphere incubator at 37°C and 5% CO 2 .

| Western blotting assay
Total protein was extracted from glioblastoma cells and tissues using lysis RIPA buffer containing phosphatase and protease inhibitors.
After that, 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels were used to separate the protein. Then, these proteins were transferred to PVDF membranes at 250 mA for 2 h. The membranes were blocked with 5% skim milk at room temperature for 1 h. Subsequently, these membranes were incubated at 4°C for 16 h with antibodies targeted against PLA2, GAPDH, MCM2, MCM5 and γ-H2AX. Then, they were washed with TBST and incubated with secondary antibodies. Protein signals were visualized using an ECL kit (Millipore).

| Colony formation assay
Cells were seeded in 6-well culture plates for 2 weeks. Individual colonies were fixed and stained with a solution containing 0.2% crystal violet in 10% ethanol for 30 min. Colony images were taken after washing and air-drying.

| MTT assay
Cells were seeded in 6-well plates and incubated overnight. Then, cell viability was analyzed by the MTT assay according to the manufacturer's instructions. The absorbance at a wavelength of 450 nm was measured for the supernatant of each well using the plate reader Multiskan EX (Thermo Fisher Scientific Inc.,).

| Cell cycle analysis
Cells were fixed with 70% ethanol overnight at 4°C. After being washed with phosphate-buffered saline, the cells were stained with RNase A/PI for 30 min at 37°C. After that, the cell cycle phase was detected by using a flow cytometer.

| Caspase 3/7 activity analysis
After transfection, the cells were seeded in 96-well plates and then collected and lysed by lysis buffer. The supernatant fluid was obtained after centrifugation. Reaction buffer and caspase 3/7 substrate was mixed with the supernatant fluid. After that, the 405 nm absorbance value was measured by the SpectraMax M3 microplate reader.

| Animal experiments
Ten female Balb/c nude mice were obtained. All animal experiments followed the guidelines of animal research. StableSHG-44 cells with PLA2 knockdown were injected into the right oxters of five mice individually. The remaining were injected with SHG-44cells as the control group. Tumour volume was detected every 3 days by using a digital caliper. After 24 days, the tumour weight was measured after mice were sacrificed.

| Statistical analysis
Data are presented as mean ± standard error of the mean (SEM).
Statistical analysis was performed with SPSS software. Data analysis was performed by using two-tailed Student's-tests. p < 0.05 was regarded statistically significant.

| PLA2 was highly expressed in GBM
To explore the functions of PLA2 in glioblastoma, we used multiple Oncomine analyses to detect the expression of PLA2 based on published datasets. Interestingly, an increased expression of PLA2 was defined in glioblastoma tissue relative to normal tissue ( Figure 1A).
In accordance with this bioinformatics result, PLA2 was highly expressed in tumour tissues compared with adjacent normal samples ( Figure 1B). Next, three glioblastoma cell lines, namely, SHG-44, U251 and SNB-19 and one human normal glial cell line, HEB, were used.
Increased PLA2 productions were observed in glioblastoma cells compared with normal cells ( Figure 1C,1D), suggesting that PLA2 expression might play an oncogenic role in glioblastoma development.

| PLA2 promoted cell proliferation and viability
To explore the effect of PLA2 on the proliferation of glioblastoma cells, SHG-44 and SNB-19 cells were transfected with si PLA2 or pcDNA3.1-PLA2 to establish PLA2 knockdown or overexpression cells. The protein levels of PLA2 in these established cells were verified using Western blotting assay (Figure 2A). MTT assay and colony formation assay were further used to detect cell proliferation and viability. The OD value of cells with PLA2 overexpression was significantly higher than that of control cells, whereas the value was decreased in cells with PLA2 knockdown compared with that of control cells ( Figure 2B). In addition, PLA2 overexpression caused the increase of glioblastoma cell viability, and PLA2 silencing suppressed this process ( Figure 2C and 2D). Therefore, these findings indicate that PLA2 promoted cell proliferation and viability.

| PLA2 increased MCM2 and MCM5 expression, and decreased γ-H2AX protein level
This study found that PLA2 induced G1/S cell cycle transition. It is well-known that G1 and S stage are involved in DNA replication.
Recent evidence supports PLA2 participation in the cell replication machinery by associations at DNA replication origins. 13,14 Thus, this study further investigated the role of PLA2 in the regulation of  Figure 4C). Furthermore, the colony formation assay also showed a rescue phenomenon after knockdown of MCM5 ( Figure 4D). These findings suggest that blocking PLA2 inhibits DNA replication initiation-related genes, MCM2 and MCM5, thus, inducing DNA damage.

| PLA2 knockdown decreased glioblastoma growth in the xenograft mouse model
To further investigate the effect of PLA2 on glioblastoma growth in vivo, SHG-44cells transfected with sh PLA2 were injected into the flank region of nude mice subcutaneously. Tumour volume was measured every three days. After 24 days, mice were sacrificed, and tumour tissues were obtained. The result in vivo was consistent with that in vitro. PLA2 knockdown significantly inhibited tumour volume and weight compared the control group ( Figure 5A-5C). In addition, PLA2 knockdown suppressed MCM2 expression and increased the γ-H2AX protein level in tumour tissues ( Figure 5D).

| DISCUSS ION
The phospholipase A 2 (PLA 2 ) superfamily contains more than 50 enzymes in mammals that are subdivided into several distinct families on a structural and biochemical basis. It plays an important role in cancer cell proliferation, invasion and metastasis, and also is used as a biomarker for cancer diagnosis or progression evaluation. An increasing body of evidence supports the excessive activation of PLA2 in many cancers, such as lung cancer, hepatocellular carcinoma, osteosarcoma and glioma as well. 17 However, the function of PLA2 as an oncogene in GBM remains largely unknown. Thus, this study explored the effects of PLA2 on glioblastoma development and the underlying mechanism. The results proved that PLA2 played an oncogenic role in glioblastoma.
In detail, this study found that PLA2 was highly expressed in Thus, we further investigate the protein level of γ-H2AX, a marker of DNA damage. PLA2 knockdown decreased the γ-H2A protein level in glioblastoma, suggesting that PLA2 blocking caused DNA damage.
It was found that high levels of PLA2 repaired DNA damage more efficiently and resumed transcription and growth in breast cancer; while their low-PLA2 expressing counterparts eventually committed to apoptosis. 21 PLA2 is reported to be involved in driving checkpoint recovery after DNA damage, a key pathway that allows cancer cells to overcome damage response arrest. 14

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no potential competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article. F I G U R E 5 PLA2 silencing inhibited tumour growth in vivo. A. A stable PLA2knockdown (shPLA2) SHG-44 cell line was subcutaneously injected into 6-week-old immunocompromised mice. At the end of the assay, tumours were removed and photographed. B. Tumour volume was measured every three days. PLA2 knockdown inhibited tumour volume. C. Knockdown of PLA2 inhibited tumour weight. D. PLA2 silencing caused the decrease of the MCM2 protein level and the increase of γ-H2AX in tumor tissues. Data represent mean ± S.E.M. *, p < 0.05. ***, p < 0.001