SOX9 stands for a bio-marker for prostate cancer detection


 Background

Prostate cancer is one of common cancers around the world, and in our country the incidence and mortality of PCa are both increasing. More and more reports have revealed that SOX9 is involved in various human cancers. In this study, we aimed to explore the relationship between SOX9 expression and diagnostic value of PCa patients.
Methods

In this study, quantitative real-time PCR (qRT-PCR) was performed to determine the expression of SOX9 of the 131 PCa patients and 74 healthy volunteers. And receiver operating characteristic (ROC) curve was used to determine the diagnostic value of SOX9 for PCa patients.
Results

The results of qRT-PCR showed that the expression of serum SOX9 in PCa patients was higher than that in healthy controls (P < 0.05). And the expression of SOX9 was significantly associated with PSA (P = 0.001), differentiation (P = 0.000), and lymph node metastasis (P = 0.000). Besides, the area under the ROC curve (AUC) was 0.966 with the sensitivity of 93.2% and specificity of 87.8% respectively. The optimal cutoff value of SOX9 was 2.34.
Conclusions

Our results found that SOX9 is a novel oncogene for PCa, and may be a novel and effective biomarker for the diagnosis of patients with PCa.


Results
The results of qRT-PCR showed that the expression of serum SOX9 in PCa patients was higher than that in healthy controls (P < 0.05). And the expression of SOX9 was signi cantly associated with PSA (P = 0.001), differentiation (P = 0.000), and lymph node metastasis (P = 0.000). Besides, the area under the ROC curve (AUC) was 0.966 with the sensitivity of 93.2% and speci city of 87.8% respectively. The optimal cutoff value of SOX9 was 2.34.

Conclusions
Our results found that SOX9 is a novel oncogene for PCa, and may be a novel and effective biomarker for the diagnosis of patients with PCa.

Background
Prostate cancer (PCa) is one of the most common cancers in male genitourinary system. And in the worldwide, the incidence of PCa is at the second of all the malignant tumors only after lung cancer in incidence of elderly men [1]. PCa is a clinically heterogeneous multifocal disease, and the incidence is continuously rising. The carcinogenesis and mechanisms of PCa is multistep process. Because there are no obvious characteristics at the early stages, most PCa patients have already been at advanced stages or accompanied by metastasis when they are diagnosed. Moreover, although prostate-speci c antigen (PSA) has been used for the earlier detection of clinically localized PCa, the PSA testing has low speci city for PCa [2][3][4][5]. Thus there are no reliable predictors of PCa behavior and aggressiveness. So nding effective biomarkers to achieve early diagnosis and treatment has become very important for PCa patients.
SOX9 belongs to the SRY-related HMG box (SOX) family of developmental transcription factors [6]. As DNA binding proteins, SOXs widely exist in multiple tissues in the process of embryonic development [7,8]. In mammals, SOX9 is been widely studied. SOX9 has a decisive role for sex determination and the development and maturation of bone, testicular, kidney, heart, brain and pancreas [9][10][11][12][13][14]. The regulation of SOX9 can occur in multiple links, including transcription, translation, posttranslational modi cation, nuclear transport and the interaction of coenzyme.And In addition, it was reported that SOX9 was associated with several human cancers. For example, gastric cancer, non-small cell lung cancer and pancreatic cancer [15][16][17] What's more, SOX9 has also been reported in prostate cancer, that it was required for cancer initiation and contribute to PCa tumor growth and invasion [18]. However, SOX9there are few reports about the correlation of SOX9 expression with the diagnosis of PCa patients.
In the current study, we aimed to detect the serum expression level of SOX9 in PCa patients with qRT-PCR method and to investigate the association between SOX9 expression and diagnosis of PCa patients.

Methods
Patients and specimens collection All of 131 PCa cancer patients con rmed by pathologists were nally recruited in the study from Huaihe Hospital of Henan University. 74 healthy volunteers were collected as the control group, and no one had been diagnosed with any malignancy in the group. Our study was approved by the Ethics Committee of Huaihe Hospital of Henan University, and the written consents were also obtained from all participants.
The clinicopathological features of the PCa patients were listed in Table 1, including age, tumor size, PSA, differentiation, TNM stage, and lymph node metastasis. The serum samples were taken from all the 131 PCa patients and 74 healthy volunteers, and the samples were collection before patients accepting any surgery and therapy. Then all the collected serum samples were put into blood collection tube of EDTA and stored at -80℃ until use. Primescript™ RT reagent Kit (Takara, Japan). In this study the expression of SOX9 was determined using the SYBR Green® Realtime PCR Master Mix (QPK-201, Toyobo Co, Ltd, Osaka, Japan). The primer sequences of SOX9 and β-actin were as follows: SOX9 forward: 5'-AGCGAACGCACATCAAGAC-3' and reverse: 5'-GCTGTAGTGTGGGAGGTTGAA-3', β-actin forward: 5'-GGTGGCTTTTAGGATGGCAAG-3', reverse: 5'-ACTGGAACGGTGAAGGTGACAG-3'. β-actin was used as an internal control. The relative expression level of SOX9 was normalized to β-actin and calculated using the 2 −ΔΔCt method.

Statistical analysis
All statistical analyses were performed with software SPSS 19.0 and GraphPad Prism 5. The SOX9 expression level was presented as mean ± SD. Student's t-test was used to analyze the expression differences between two groups. The associations between serum SOX9 expression and various clinicopathological characteristics were assessed using Chi-square tests. To determine the diagnostic performance of serum SOX9 expression in distinguishing PCa patients from healthy controls, we performed receiver operating characteristic (ROC) analysis. P < 0.05 was considered as statistically signi cant.

Increased expression of SOX9 in PCa patients
To examine the SOX9 expression in 131 PCa patients and 74 healthy controls, qRT-PCR was performed.
The results in Fig. 1 showed that SOX9 expression was signi cantly higher in PCa tissues than that in healthy volunteers (P < 0.05).
The correlation of SOX9 expression with clinicopathologic features of PCa patients In this study, Chi-square test was used to detect the association between SOX9 expression level and the clinicopathological data of PCa patients. The results were shown in Table 1. SOX9 expression was signi cantly associated with PSA (P = 0.001), differentiation (P = 0.000), and lymph node metastasis (P = 0.000). However, there was no signi cant association with the age, tumor size and TNM stage of PCa patients (all P > 0.05).

Diagnostic value of SOX9 expression in PCa patients
ROC curves in our study showed that SOX9 could be used to distinguisht PCa patients from the healthy volunteers with the area under the curve (AUC) value of 0.966 (Fig. 2) and sensitivity of 93.2%, speci city of 87.8% respectively. And the optimal cutoff value for SOX9 expression level is 2.34.

Discussion
PCa is the most prevalent cancer in men and as one of the malignant tumors of male genitourinary system, the biological behaviour of PCa is the most complex and the incidence is the higher in developed countries [19]. In our country, the PCa incidence of upward trend is obvious in recent years. Based on the data of center for disease control and prevention of Shanghai for recent 20 years, the incidence of PCa sharp increased and the advanced metastasis PCa is currently incurable [20]. For PCa, carcinogenesis and the mechanisms affecting the progression and prognosis are multistep processes [21]. Although the previous reports have shown that many factors including lncRNA HCG11, miR-129, as well as TMPRSS2-ERG fusion gene have affected the development and prognosis of PCa [22][23][24], the prediction and diagnosis of PCa patients was still poor. Thus it is important to nd a novel biomarker to improve the diagnostic e ciency of PCa.
Human SOX family has several members, and the functions of the gene family are various. For example, SOX genes regulate development and differentiation of many cell types. SOX genes within the same subgroup can counteract the function of the genes in another subgroup and the same SOX gene could regulate different stages of development in one cell type [25,26]. SOX9 is a member of SOX family, which is a developmental transcription factor playing a key role in the regulation of sex determination, cartilage development, intestinal differentiation [27]. At the 7.5 weeks of pregnancy, the expression of SOX9 can be found in embryonal tissue [28]. In the existed studies, SOX9 acts as an important role in many tissues and organs [9]. In recent years, researchers have found that as a transcription factor [29,30], Abnormal expression of SOX9 is associated with the occurrence of many diseases, including many human cancers. For example, Guo et al. found that the expression of SOX9 was higher in HCC tissues than adjacent tissues and the overexpression of SOX9 in HCC tissues is of great predictive value on tumor progression and poor prognosis [31]. Shao et al. found that the expression of SOX9 in gactric cancer was also higher than that in adjacent non-cancerous tissues. The expression of SOX9 was associated with the growth, invasion, and metastasis of gastric cancer, as well as the prognosis [32].
Moreover, SOX9 was also identi ed as one of the earliest molecular expressed in the primordial prostate.
The conditional deletion of SOX9 demonstrated a requirement for SOX9 in ventral prostate differentiation and development. The study of Huang et al. reported that SOX9 expression played an important role in prostate development and the initiation of PCa [18]. The study of Qin et al. showed that the combination of HIVEP3 and SOX9 help to predict the tumor progression and prognosis of PCa patients [33]. However, the diagnostic value of SOX9 in PCa remains still unclear and is an active focus of our current research.
In our study, we found that the expression level of SOX9 was higher in PCa patients than that in healthy group. The similar result was in agreement with the previous studies. In addition, we also focused on the relationship between the expression level of SOX9 and the clinicalpathological features of PCa patients. The results showed that the expression level of SOX9 was signi cantly correlated with PSA, differentiation, and lymph node metastasis. And the result of ROC curves analysis indicated that the expression of SOX9 could be used as a diagnostic biomarker for PCa patients.

Conclusions
In conclusion, our current data may provide a novel biomarker for the diagnosis of PCa, but the further investigation should be implement about the SOX9 acting on PCa patients. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.   ROC analysis for evaluation of the accuracy of serum SOX9 to distinguish PCa patients from healthy volunteers.