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Diagnostic investigations of PLA2G16 and CDH11 expression levels as independent prognostic markers of human osteosarcoma

Introduction The aim of this study was to facilitate and deepen the understanding of the associations of the clinical significance of PLA2G16 and CDH11 in patients with osteosarcoma. Material and methods We collected 50 paired osteosarcoma tissues and adjacent normal bone tissues and evaluated the expression of PLA2G16 and CDH11 by quantitavise reverse transcriptase real-time polymerase chain reaction. Results PLA2G16 expression was upregulated in osteosarcoma tissues when compared with adjacent normal bone tissues, and the difference was statistically significant (4.78 ±0.70 vs. 1.31 ±0.65; p < 0.05). Our data indicated that high expression of PLA2G16 was significantly related to advanced TNM stage and metastasis or recurrence (p < 0.05). The expression level of CDH11 was lower in osteosarcoma tissues (median relative expression level ± SD: 6.29 ±1.43) than adjacent normal bone tissues (mean ± SD: 13.72 ±3.08, p < 0.05). Our findings demonstrated that decreased expression of CDH11 was strongly linked to advanced TNM stage, and metastasis or recurrence (p < 0.05). Log-rank analysis showed that patients with high expression of PLA2G16 have shorter overall survival than those with low expression. Moreover, shorter overall survival was significantly correlated with decreased expression of CDH11. Multivariate Cox proportional hazards analysis showed that PLA2G16 (p = 0.028; HR = 2.621; 95% CI) and CDH11 (p = 0.023; HR = 2.81; 95% CI) expression and also metastasis or recurrence (p = 0.03; HR = 2.531; 95% CI) were independent prognostic factors for poor overall survival of osteosarcoma patients. Conclusions These findings suggest that PLA2G16 and CDH11 expression can be independent prognostic factors for poor overall survival of patients with osteosarcoma.


Introduction
Osteosarcoma is known as a primary malignancy among children and young adults and also is linked to high morbidity. Despite treatment strategies including chemotherapy and surgery, the 5-year survival rate of primary osteosarcoma was estimated to be only 50-60% [1][2][3][4][5], and it is dissatisfactory for most patients with metastasis or recurrence. Therefore, it is very important to identify effective targets and new strategies in this field.
Pla2g16, also called H-REV-107, belongs to group XVI phospholipase A 2 , and was reported to be expressed in most normal tissues [6,7]. PLA2G16 has been shown to play a role in progression and metastasis in osteosarcoma cell lines [8]. PLA2G16 has been reported to act as a class II tumor suppressive due to its function in inhibition of H-ras-induced transformation. Moreover, decreased expression of PLA2G16 was reported in some kinds of tumor such as kidney, breast, and ovary [9][10][11]. On the other hand, upregulation of PLA2G16 expression has been found in rectum, colon, stomach and lung cancers, indicating that it can play an oncogenic role in mentioned tumors [12]. Cadherins are known as cell surface molecules for cell communication and signaling through catenin [13]. The association of cadherins with tumor progression has been reported in many kinds of cancer [14][15][16]. Over 20 types of cadherins have been identified, including CDH11, reduction of which has been reported to be associated with osteosarcoma metastasis [17]. However, the role of PLA2G16 and CDH11 in patients with osteosarcoma needs further studies. In the current study, we evaluated the clinical significance of PLA2G16 and CDH11 in patients with osteosarcoma.

Ethics statement
All clinical protocols in the present study were conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines, and procedures were reviewed and approved by the Ethics Committees. In addition, all participating patients signed the consent forms.

Patients and clinical samples
We obtained 50 paired osteosarcoma tissues and adjacent normal bone tissues from different hospitals in Tehran, Iran between 2008 and December 2013. The tissues were obtained from surgical specimens and confirmed by pathological evaluation. Tumor tissues were grouped based on the sixth edition of the TNM classification of the International Union against Cancer (UICC). The clinicopathological parameters are shown in Table I.

RNA extraction and qRT-PCR
In brief, Trizol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA. A High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) was used to carry out the reverse-transcription reactions.
Quantitavise reverse transcriptase real-time polymerase chain reaction was conducted by using TaqMan universal PCR master mix on the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems). The expression levels were quantitated using the TaqMan miRNA assay kit (Applied Biosystems) for PLA2G16 (Hs00912734_m1) and GAPDH (Hs02758991_g1) with gene-specific probes. Moreover, CDH11 expression (Hs00156438_m1) and internal control glyceraldehyde-3-phosphate dehydrogenase (Hs99999905_m1) were analyzed. Moreover, relative expression levels were evaluated with the comparative cycle threshold (CT) method.

Statistical analysis
Correlation between expression levels of genes and clinicopathological parameters were analyzed using the χ 2 test. SPSS version 16.0 for Windows (SPSS Inc, IL, USA) software was used to evaluate all variables. Analysis of overall survival was performed using the log-rank test. Univariate and multivariate Cox proportional hazards analyses were applied to evaluate the prognostic role of PLA2G16 and CDH11. Statistical analysis was considered to be statistically significant at p < 0.05.

PLA2G16 expression and its correlation with clinicopathological parameters
The expression level of PLA2G16 was upregulated in osteosarcoma tissues when compared with adjacent normal bone tissues, and the difference was statistically significant (4.78 ±0.70 vs. 1.31 ±0.65; p < 0.05; Figure 1). According to the median expression level, the expression levels of genes were categorized into low and high expression groups (Table I). Our results indicated that high expression of PLA2G16 was significantly correlated with advanced TNM stage and metastasis or recurrence (p < 0.05). No significant difference was found between PLA2G16 and other clinicopathological parameters (Table I).
We found that low expression of CDH11 was strongly linked to advanced TNM stage, and metastasis or recurrence (p < 0.05). Furthermore, no significant association was found between CDH11 and other clinicopathological parameters (Table I).
Log-rank analysis showed that patients with high expression of PLA2G16 had shorter overall survival than those with low expression. Moreover, shorter overall survival was significantly correlated with decreased expression of CDH11.

Discussion
Despite treatment strategies including chemotherapy and surgery, the 5-year survival rate of primary osteosarcoma was estimated to be only 50-60% [1][2][3][4][5]15]. Therefore, the study of osteosarcoma biomarkers is very important to identify  The expression level of PLA2G16 was upregulated in osteosarcoma tissues when compared with adjacent normal bone tissues, and the difference was statistically significant (p < 0.05). Our results indicated that high expression of PLA2G16 was significantly correlated with advanced TNM stage and metastasis or recurrence. Multivariate Cox proportional hazards model analysis showed that PLA2G16 expression and also metastasis or recurrence were independent prognostic factors for poor overall survival of patients with osteosarcoma.
PLA2G16 has been found to be involved in progression and metastasis of osteosarcoma in both mouse and human osteosarcoma cell lines [8]. The PLA2G16 marker has been accepted as a class II tumor suppressive due to its function in inhibition of H-ras-induced transformation. Low expression of the PLA2G16 gene has been found in some types of tumor such as renal, breast, and ovary [9][10][11]. On the other hand, upregulation of PLA2G16 expression has been found in rectum, colon, stomach and lung cancers, indicating that it can play an oncogenic role in the mentioned tumors [12].
A recent study demonstrated that metastatic osteosarcoma was related to PLA2G16 expression at both protein and mRNA levels when comparing osteosarcoma with and without metastasis. The mentioned study showed that negative PLA2G16 protein expression was related to higher survival rates for 3-year overall survival and metastasis-free survival than the positive expression of PLA2G16. Consistent with the results of that study, our results showed that high expression of PLA2G16 was significantly correlated with advanced TNM stage and metastasis or recurrence. Log-rank analysis showed that patients with high expression of PLA2G16 have shorter overall survival than those with low expression. Increased expression of PLA2G16 mediated by mutant p53 contributes to progression and metastasis of osteosarcoma [18]. It can be useful to evaluate whether p53 mutations are associated with expression of PLA2G16 in osteosarcoma.
Furthermore, it has been indicated that PLA2G16 produced free fatty acids, arachidonic acid, and lysophosphatidic acid from phosphatidylcholine [5]. Arachidonic acid can be converted by cyclooxygenase-2 (COX-2) into prostaglandin E2 (PGE2) and other prostaglandins [19] that have an important role in regulating the invasion of cells during cancer progression [20]. Moreover, COX-2 expression has been reported to be a prognostic factor in osteosarcoma lung metastases [21]. In addition, lysophosphatidic acid was demonstrated to induce cell proliferation, invasion, and migration and promote survival [22].
In the present study, the expression level of CDH11 was downregulated in osteosarcoma tissues compared to adjacent normal bone tissues. We found that low expression of CDH11 was strongly linked to advanced TNM stage, and metastasis or recurrence. Moreover, shorter overall survival was significantly correlated with decreased expression of CDH11. In agreement with our results, CDH11 reduction has been reported to be associated with osteosarcoma metastasis [17]. Previous studies indicated that the absence or decrease of CDH11 expression is related to metastasis in osteosarcoma and retinoblastoma [23][24][25][26][27]. It has been reported that low CDH11 expression contributes to tumor invasion in many kinds of tumor [16,24,25], and the decrease or loss of cell differentiation is related to lower expression of CDH11, which may contribute to the oncogenic capacity of osteosarcoma [26]. On the other hand, in agreement with our study, a previous study showed that the CDH11 expression level was decreased from the osteoblast to the primary cell lines and further to tumor metastases, and high expression of CDH11 was significantly associated with longer survival time than those with decreased expression of CDH11 [27]. Further studies are required to clarify the role of CDH11 and its involved mechanisms in osteosarcoma. In conclusion, PLA2G16 and CDH11 expression levels can be independent prognostic factors for poor overall survival of patients with osteosarcoma.