| IntroductionRenal cell carcinoma is the most common malignancy of the urinary system, which is the heavy threat for people’s lives and health. According to statistics, the incidence of renal cell carcinoma was more younger, and about 37 male and 16 female patients in every 100,000 population were diagnosed with kidney cancer. Because kidney cancer patients don’t have obvious early symptoms,25%-30% of patients have until been in late stage to hospital for therapy. Meanwhile, renal cancer patients are not sensitive to radiotherapy and chemotherapy, therefore, surgery section was the main therapy method for patients with kidney cancer. However, more than 20% of patients may be relapsed. Therefore, the current treatment for kidney cancer is still not satisfactory.Clear cell carcinoma is the most common histological type in renal cell carcinoma, accounting for 70%-90% patients. However, the mechanism about renal clear cell carcinoma formation and development is not clear, which has brought great difficulties for its treatment. Therefore, further fundamental research about renal cells is still great significant. In recent years, with the rapid development of molecular biology, more and more protein correlated with renal cell carcinoma was discovered, which not only helps assess the prognosis of patients with kidney cancer, but also helps the clinical diagnosis of kidney cancer, local recurrence or distant metastasis prediction. Furthermore, those protein is expected to become molecular targeted drug development targets and used for clinical treatment. Therefore, to find new genes associated with renal cell carcinoma will help to elaborate mechanisms of renal cell carcinoma and provide valuable reference for diagnosis and treatment.PGAM1 belongs to PGAM family members, is located on human chromosome 10q25.3 region. Present study showed PGAM1 is involved in the regulation of glucose metabolism, catalyzes glyceraldehyde 3-phosphate acid to 2-phosphoglycerate. Meanwhile, PGAM1 is involved in the regulation of a variety of life activities. Recent studies have found that PGAM1 expression is closely related to tumorigenesis and development of tumor. High PGAM1 expression was found in colorectal cancer, liver cancer, lung cancer and breast cancer tissues, but is hardly detected in the brain tumor. Nevertheless, whether PGAM1 expression is associated with the occurrence and development of renal clear cell carcinoma is unclear. At the same time, the biological function of PGAM1 and the relationship between PGAM1 expression and patients’s prognosis in renal cell carcinoma is unknown. Therefore, we are aimed to explore the relationship between PGAM1 with renal cell carcinoma in this study.Object1. To investigate the expression and its clinical significance of PGAM1 in renal clear cell carcinoma and normal kidney tissues.2. To investigate the relationship between PGAM1 expression and patients’ prognosis in renal clear cell carcinoma.3. To investigate the biological function and its mechanism of PGAM1 in renal clear cell carcinoma.Methods1.192 cases of renal cell carcinoma and 80 cases of normal renal tissue distal from renal cell carcinoma were collected, fixed, dehydrated into paraffin tissue blocks, PGAM1 expression was detected by using immunohistochemical staining. The relationship between PGAM1 expression and clinicopathological parameters and patient prognosis was analyzed in renal clear cell carcinoma.2. PGAM1mRNA was detected in three pairs of clear cell renal cell carcinoma and paired normal renal tissues using qRT-PCR. Meanwhile, PGAM1 protein expression was detected in 8 cases of renal clear cell carcinoma and paired normal renal tissues by Western blot.3. PGAM1 expression was detected in RLC-310 and Caki-2 clear cell carcinoma cell line using qRT-PCR. Meanwhile, the siRNA fragments were transiently transfected into RLC-310 and Caki-2 renal cell carcinoma cell lines to knockdown PGAM1 expression, which would be validated by qRT-PCR and Western blot.4. The effect of PGAM1 downregulation on the apoptosis of RLC-310 and Caki-2 renal cell carcinoma cell lines was measured by flow cytometry; Meanwhile, MTT assay was used to evaluate the effect of PGAM1 downregulation on the proliferation of RLC-310 and Caki-2 renal clear cell carcinoma cell lines.5. Using qRT-PCR and Western blot, the influence of PGAM1 downregulation on P53, Caspase 3 and Caspase 9 expression in renal clear cell carcinoma was investigated to further explore the biological function mechanism.Results1. PGAM1 expression in clear cell renal cell carcinomaFirstly, we examined the expression PGAM1 in 192 cases of renal cell carcinoma and 80 cases of distal normal renal tissues by immunohistochemistry. Results showed that PGAM1 expression was mainly located in the cytoplasm of normal kidney and renal clear cell carcinoma tissues. In normal kidney tissues,20 cases with negative expression (25.0%),34 cases with weak positive expression (42.5%),20 cases with moderate positive expression (25.0%) and 6 cases with strong positive expression (7.5%) were showed.59 cases with negative expression (30.7%),45 cases with weak positive expression (23.4%),71 cases with moderate positive expression (37.0%) and 17 patients with strong positive expression (8.9%) were recorded in renal clear cell carcinoma. Statistical analysis showed that there was significant differences in the two group (P=0.017). Subsequently, we conducted a semi-quantitative analysis, and found low PGAM1 expression was shown in 104 cases (54.2%) in renal clear cell carcinoma, and 88 cases (45.8%) showed high expression. In normal renal tissues,54 cases (67.5%) were low expression and 26 patients (32.5%) was showed high expression. Significant difference was shown in the two groups (P=0.044).Then, we detected PGAM1 mRNA expression in three pairs of clear cell renal cell carcinoma and paired normal distal renal tissues by qRT-PCR. Results showed that PGAM1 expression in renal clear cell carcinoma was higher with 4.2 times than normal renal tissue expression with significant statistical analysis (P=0.004). At the same time, we also quantitatively detected PGAM1 protein expression in eight renal clear cell carcinoma and matched normal kidney tissues by Western blot. Results showed that PGAM1 expression in renal clear cell carcinoma was higher with 3.2 times compared with normal renal tissues, with significant statistical differences (P=0.001).Meanwhile, we also evaluated the correlation between PGMA1 expression and clinicopathological features in renal clear cell carcinoma. Results showed that PGMA1 expression correlated with age, tumor size and T stage, but no significant correlation with sex, tumor location, and smoking history. Among 92 patients aged> 57 years,12 patients showed high PGMA1 expression, which was significantly lower than those in patients≤57 years (43/100), the difference was statistically significant (P=0.005). In 58 patients with tumor diameter≥5 cm,34 patients showed high expression, which was significantly higher than those with tumor diameter<5 cm (54/134), the difference was statistically significant (P=0.0027). In 80 patients with T2-T3 stage,46 cases were showed with PGAM1 expression, which was significantly higher than those with T1, obvious differences were statistically significant (P=0.008).2. The relationship between PGAM1 expression and patients’ prognosis in renal cell carcinomaBased on patients’s prognosis in renal cell carcinoma, we analyzed the correlation between PGAM2 expression and clinicalpathological features in renal clear cell carcinoma by using Kaplan-Meier method. The results showed that patients’ prognosis was associated with PGAM1 expression, tumor size and T stage in renal cell carcinoma. The mean survival time of patients with high PGAM1 expression was 48 months, which was significantly lower than those with low expression PGAM1 (56 months), the difference was statistically significant (P=0.000). Meanwhile, the results showed that the mean survival time of patients with tumor diameter<5 cm was 55 months which was significantly higher than those with tumor diameter>5 cm (47 months), the difference was statistically significant (P=0.000). The average survival time of patients with T1 stage was 55 months, which was significantly higher than those with T2-T3 stage (49 months), the difference was statistically significant (P=0.000). However, no significant correlation was found between patients’prognosis with age, sex, tumor location and smoking history (P>0.05).Subsequently, we discuss the impact of the correlation between PGAM1 expression and clinicopathological features on patients’ prognosis. We found that in patients aged <57 years old,, PGAM1 low expression showed worse prognosis (P=0.000). In patients with aged >57 years old, no significant correlation between PGAM1 expression and patients’prognosis (P=0.203). In male patients, PGAM1 low-expression showed significantly worse prognosis (P=0.000). Similarly, in female patients, PGAM1 high expression showed significantly worse prognosis (.P=0.013). In the left location of renal cell carcinoma, PGAM1 expression showed worse prognosis (P=0.000). In the right location of renal clear cell carcinoma, PGAM1 expression wasn’t correlated with prognosis (P=0.579). In patients with a smoking history or non-smoking, PGAM1 high expression were significantly related to worse prognosis (.P=0.000, P=0.006). In patients with tumors size with smaller than 5cm, PGAM1 expression was significantly correlated with worse prognosis (.P=0.000). In patients with tumor>5cm, no significant correlation between PGAM1 expression and patients’prognosis (P=0.47). In patients with T1 stage, PGAM1 expression significantly correlated with worse prognosis (P=0.000), while no significant correlation between PGAM1 expression and patients’prognosis was observed in T2-T3 stage patients (P=0.945). Multivariate analysis indicated that PGAM1 and tumor size can be used as an independent prognostic factor in patients with renal cell carcinoma, but T stage was not as an independent prognostic factor in patients with renal cell carcinoma.3. The effect of PGAM1 downregulation on the biological functions of renal cell carcinoma cellsWe firstly examined the expression PGAM1 in clear cell renal cell carcinoma cell line RLC-310 and in Caki-2 by qRT-PCR. Results showed that PGAM1 was expressed in RLC-310 and Caki-2 renal cell carcinoma cell lines, while higher expression was showed in the RLC-310 cells. Subsequently, we interfered PGAM1 expression by siRNA, which was validated by using qRT-PCR and Western blot. After transfection, qRT-PCR and Western blot showed that PGAM1 expression in RLC-310 and Caki-2 cells were significantly reduced, and the difference was statistically significant (P=0.001, P=0.008), indicating PGAM1 expression was successfully knocked down in RLC-310 and Caki-2 cells.Subsequently, the effects of PGM1 downregulation on cell proliferation was evaluated in clear cell carcinoma by MTT assay. The results showed that in the RLC-310 cells, the OD value was significantly lower in cells with PGAM1 downregulation compared with control group (P=0.000), indicating cell proliferation was inhibited. Similarly, in the Caki-2 cells, the OD value was significantly lower in cells with PGAM1 downregulation compared with control group (P=0.000), indicating cell proliferation was inhibited. At the same time, flow cytometry was used to detect the influence of PGAM1 downregulation on cell apoptosis in renal clear cell carcinoma. Results showed that PGAM1 down-regulated in RLC-310 cells, the apoptosis rate was 15.67%±0.52%, while the control group apoptosis was 5.21%±1.04%, and the difference was significant (P=0.000). After PGAM1 expression down-regulation in Caki-2 cells, the apoptosis rate was 11.83%±1.09%, while the control group apoptosis was 4.81%±1.33%, the difference was significant (P=0.002).4. The mechanisms of PGAM1 in Clear cell carcinomaFirstly, we examined the expression of P53, Caspase 3 and Caspase 9 in RLC-310 renal carcinoma cell lines by qRT-PCR. Results showed that the expression of P53, Caspase 3 and Caspase 9 was significantly up-regulated compared with those in control group after PGAM1 downregulation in RLC-310, and the difference was statistically significant (P<0.05). Subsequently, we also examined the expression of P53, Caspase 3 and Caspase 9 in Caki-2 renal carcinoma cell lines. Results also showed P53, Caspase 3 and Caspase 9 expression were also significantly increased compared with those in control group after PGAM1 downregulation with a significant difference (P<0.05).Subsequently, in order to further validate that the influence of PGAM1 downregulation on the expression of P53, Caspase 3 and Caspase 9, we performed Western blot assay. In RLC-310 renal cancer cell lines, results showed PGAM1 down-regulation induced the up-regulation of P53, Caspase 3 and Caspase 9 with statistical significance (P<0.05). Similarly, in Caki-2 renal carcinoma cell lines, PGAM1 down-regulation increased the expression of P53, Caspase 3 and Caspase 9. Gradation value detection analysis showed that the OD values of P53, Caspase 3 and Caspase 9 were significantly decreased when PGAM1 was downregulated (P<0.05), indicating that PGAM1 increased the expression of P53, Caspase 3 and Caspase 9.Conclusions:1. PGAM1 expression in renal clear cell carcinoma was significantly higher than those in surrounding normal tissue, which was related to the occurrence and development of renal clear cell carcinoma.2. PGAM1 expression is correlated with clinicopathological features in renal cell carcinoma, and affects the patient’s prognosis, which could be served as an independent prognostic factor in renal clear cell carcinoma.3. PGAM1 downregulation can promote renal cell apoptosis and inhibit the proliferation in renal clear cell carcinoma, which is correlated with P53/Caspase 3/Caspase 9 signaling pathway. |
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