| Background:Osteosarcoma(OST) is a primary malignant bone tumor. Thanks to the improvement of chemotherapy and operational techniques, patients have a 65-70% chance of 5-year survival when the tumor was still localized. However, most of the remaining patients suffered from lung metastasis during the first 5 years, which thereby reduced survival rates. Therefore, to understand the pathogenesis of osteosarcoma and to identify a novel prognostic or predictive biomarker is of a significant importance. The initiation and progression of osteosarcoma is a multiple process that includes genetic and epigenetic changes amongst others.Recently, more and more researches have showed that epigenetic alterations lead to the malignant transformation and development of osteosarcoma, and provide a better understanding of the molecular mechanisms of this tumor. mGluR4 is predominantly a presynaptic glutamate receptor, which is expressed in several key locations in the basal ganglia circuits that control movement. GRM4 was detected in various human cancers, and although the mRNA and protein expression of GRM4 were also over-expressed in osteosarcoma and related to progression and malignancy. The expression of mGluR4 in osteoblast and osteoclast indicates that glutamate signals are involved in cell differentiation and regulation during bone formation and reabsorption.Purpose:To detect the methylation status and expression of metabotropic glutamate receptor 4(GRM4), as well as its potential clinical impact in osteosarcoma patients.Methods:Subjects and specimens:A total of 63 osteosarcoma patients were diagnosed and treated in the First Affiliated Hospital of Guangxi Medical University, with the diagnosis verified histopathologically. The clinical and histological information such as age, gender, Enneking stage, metastasis, tumor location and histologic type were collected. Osteosarcoma tissues were obtained during biopsies or surgeries, while tissues obtained from patients who underwent chemotherapy or other anti-neoplasm therapy were excluded. The normal bone tissues were obtained from another 105 patients not suffering from any tumor. The osteosarcoma samples included 35 males and 28 females with the average age being 24.6 years(range 6 to 72 years). A follow-up was conducted once 2 month in the first 2 years, then every 6 months until the final follow-up, the average follow-up period was 20.0 months(range 1 to 36 months). The study was approved by the Institutional Ethics Review Board at the First Affiliated Hospital of Guangxi Medical University; the written informed consent was obtained from all patients or guardians.GRM4 gene promoter methylation detection:The status of methylation of the GRM4 gene was detected by the pyrosequencing method in the osteosarcoma tissues and the normal bone tissues.GRM4 gene mRNA expression detection:The mRNA expression of metabotropic glutamate receptor 4(GRM4) was investigated using Reverse transcriptase polymerase chain reaction(RT-PCR).GRM4 gene protein expression detection:The protein expression of metabotropic glutamate receptor 4(GRM4) was investigated using Western Blot(WB).Statistical analysis:SPSS 16.0(IBM, Armonk, NY, USA) for Windows was used for all statistical analysis. Independent t-test was conducted to compare the methylation levels, the mRNA and protein expression of the two groups. Spearman correlation was calculated between methylation and gene expression of GRM4. Then, Chi-square test or Fisher’s exact test were performed to detected the relationships between GRM4 methylation levels and clinicopathological characteristics(including age, gender, Enneking stages, histologic type, location and metastasis). The overall survival(OS), recurrence-free survival(RFS) and progression-free survival(PFS) were analyzed by Kaplan-Meier analysis and log-rank test, and the patients who were still alive at the last follow-up were censored. Multiple Cox proportional hazard regression model was performed to assess the value of GRM4 methylation in predicting the recurrence and progression of osteosarcoma. A two-sided P value<0.05 was considered as statistically significant.Results:Aberrant GRM4 DNA hypomethylation:The average methylation was 3.79±0.83% and 4.08±0.74% in the osteosarcoma tissues and normal bone tissues(P<0.05), which indicated that the osteosarcoma tissues was significantly more hypomethylated than that in the normal bone tissues.GRM4 mRNA expression:GRM4 mRNA expression were detected in osteosarcoma tissues and normal bone tissues by RT-PCR. The results demonstrated that there is a significant increase in mRNA expression in osteosarcoma tissues when compared to normal bone tissues (1.71±0.15 vs. 0.56±0.13,P<0.05).GRM4 protein expression:GRM4 protein expression were detected in osteosarcoma tissues and normal bone tissues by Western Blot(WB), a markedly higher expression was observed in osteosarcoma tissues as compared with normal bone tissues(1.28±0.08 vs.0.35±0.05, P<0.05). Importantly, a significant inverse correlation between methylation and expression of GRM4(r=-0.211, P=0.016).Relationship between GRM4 methylation levels and clinicopathological parameters:According to GRM4 methylation status, the methylation levels will be divided into quartiles:Q1 (≥4.51, n=16), Q2 (3.34-4.51, n=16), Q3 (3.04-3.34, n=15) and Q4 (≤3.04, n=16). The relationship between the methylation and clinicopathological characteristics was then investigated. The results showed that the GRM4 methylation levels was associated with tumor metastasis(P<0.05) and recurrences(P<0.05), but not associated with age, gender, Enneking stages, histologic type or tumor locations(all P> 0.05).Survival Analysis:For further analyses, we stipulated Q4 as the "hypomethylation group" and Q1, Q2 and Q3 as the "hypermethylation group", the overall survival(OS), recurrence-free survival(RFS) and progression-free survival(PFS) were analyzed. The result suggested that there was a significantly prolonged median survival time for the GRM4 hypermethylation group when compared with the hypomethylation group(P<0.05). A similar trend was observed for recurrence-free survival(P<0.05) and progression-free survival (P<0.05).Conclusion:(1)The pyrosequencing results indicated that the osteosarcoma tissues were significantly more hypomethylated than the normal bone tissues. (2)The RT-PCR results demonstrated that there is a significant increase in mRNA expression in osteosarcoma tissues when compared to normal bone tissues. (3) The WB results demonstrated that a markedly higher expression was observed in osteosarcoma tissues as compared with normal bone tissues. (4) The relationship between the methylation and clinicopathological characteristics was then investigated. The results showed that the GRM4 methylation levels was associated with tumor metastasis(P<0.05) and recurrences(P<0.05). (5)The survival analysis result suggested that there was a significantly prolonged median survival time for the GRM4 hypermethylation group when compared with the hypomethylation group(P<0.05). A similar trend was observed for recurrence-free survival(P<0.05) and progression-free survival (P<0.05). |
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