| Object: Oral squamous cell carcinoma(OSCC)is one of the most common oral and maxillofacial malignancies in the world.In recent years,a large sample of epidemiological studies have shown that OSCC incidence was increasing year by year,and the age of onset was getting younger,the incidence rate was 3.6 / 10 ~ 8.0 / 10 million in China.Due to other high degrees of malignancy,the tendancy to lymph node metastasis,poor prognosis,resulting in OSCC 5-year survival rate was about 50%.At present,there is a lack of early diagnosis and prognosis of OSCC.The current study suggests that gene changes in OSCC development include gene mutation and apparent genetic modification.DNA methylation studies allow us to better understand the mechanisms of OSCC and provide a new theoretical basis for early diagnosis and prognosis of OSCC.Studies have shown that mutations and epigenetic abnormalities are important factors affecting the occurrence of OSCC.Epigenetic modification is a kind of genetic and reversible biological behavior,including methylation of DNA nucleotide cytosine,Histone modification,non-coding RNA regulation,and the like.Among them,DNA methylation research has become a hotspot of epigenetics.PRSS8 gene,also known as human prostate protein(Prostasin)or CAP-1,located on chromosome 16p11.2,not only to maintain the normal physiological function of the body,but is also involved in the regulation of growth factors,cell migration of various inflammation,epidermal growth,Tumor occurrence,progress and other processes.In recent years,it has been found that PRSS8 gene methylation and abnormal expression of PRSS8 mRNA are found in esophageal squamous cell carcinoma and colorectal cancer.Abnormal changes of PRSS8 gene may be involved in the development of cancer.The protein tyrosine phosphatase non-receptor type 22(PTPN22)gene is located on chromosome 1p13.3-13.1 and participates in epithelial adhesion.The TCGA database showed a significant difference in PTPN22 methylation level between tumor tissue and normal tissue.However,although the PTPN22 gene is potentially important in the carcinogenesis,there are few reports about it.There have been no reports of PRSS8 and PTPN22 in epigenetic changes in OSCC.Therefore,methylation-specific PCR(MSP)and RT-PCR were used to analyze the methylation status and expression of PRSS8 and PTPN22 in OSCC and to explore its relationship with OSCC and its clinical significance.In this study,52 cases of OSCC postoperative specimens were used as the subjects,by analyzing PRSS8 and PTPN22 gene promoter region methylation status and mRNA relative in OSCC tissue and the corresponding normal mucosal tissue and the pathogenesis of the patients,to provide experimental theoretical basis for the early detection and prognosis of OSCC.Method:1 Methylation status of PRSS8 and PTPN22 gene promoter regions was detected by Methylation Specific PCR(MSP)in 52 cases of OSCC and normal mucosal tissues.2 The mRNA expression of PRSS8 and PTPN22 in cancer tissues and corresponding normal mucosal tissues were detected by reverse transcriptase-polymerase chain reaction(RT-PCR)in 52 cases of OSCC.3 All experimental data were processed by SPSS21.0.Result:1 Methylation status of PRSS8 and PTPN22 genes in OSCC and normal tissuesThe promoter methylation rates of PRSS8 and PTPN22 genes were 57.7%(30/52),51.9%(27/52)in 52 OSCC tissues,34.6%(18/52),30.8%(16/52)in 52 normal tissues.The methylation rate of PRSS8 and PTPN22 gene promoters in OSCC tissues was significantly higher than that in normal tissues(P<0.05).The differences were statistically significant(χ~2=5.571,P=0.018,χ~2 = 4.798,P = 0.029).(Table 1,2)(Fig.1,2)2 Expression of PRSS8 and PTPN22 mRNA in OSCC and normal tissuesThe expression level of PRSS8 mRNA in OSCC tissues and normal tissues were 0.44 ± 0.12,0.71 ± 0.13,and the relative expression of PRSS8 mRNA was 0.71 ± 0.13.The relative expression of PRSS8 mRNA was The expression was significantly lower in the OSCC tissues than in the normal tissues(t=-10.620,P = 0.000).(Table 3)(Fig.3)The relative expression of PTPN22 mRNA in OSCC tissues was 0.44 ± 0.20,the expression level of normal tissues was 0.63 ± 0.20,and the relative expression of PTPN22 mRNA was significantly lower in OSCC tissues than in normal tissues.The difference was statistically significant(t=-4.647,P=0.000).(Table 4)(Fig.4)3 Comparison of Relative Expression of mRNA in Methylation and Non-Methylation of PRSS8 and PTPN22 GenesIn 52 OSCC tissues,the relative expression level of PRSS8 mRNA in methylation group was 0.43±0.12 and 0.52±0.11 in unmethylated group.The results were analyzed by independent t test.The results showed that the difference of the relative expression levels of PRSS8 mRNA between methylation group and non-methylated group was statistically significant(t =-2.880,P=0.006).(Table 5)The relative expression of PTPN22 mRNA in methylation group was 0.47 ± 0.18,and in methylation group was 0.56 ± 0.08.The difference of PTPN22 mRNA expression between methylation group and non-methylated group was statistically significant(t=-2.171,P=0.035).(Table 6)4 The relationship between promoter methylation status of PRSS8 and PTPN22 gene and the clinical parameters of patients.The methylation rates of PRSS8 and PTPN22 gene promoters were 53.3%(16/30)and 46.7%(14/30)respectively in the male group,63.6%(14/22)and 59.1%(13/22)in the female group(χ~2=0.552,P=0.458),(χ~2=0.785,P=0.376).The difference was not statistically significant both.The methylation rates of the ≥60 years group were 62.5%(20/32)and 56.3% respectively,the methylation rate of patients aged <60 years was 50.0%(10/20)and 45.0%(9/20),the difference was not statistically significant both(χ~2=0.788,P=0.375)(χ~2=0.624,P=0.430).The methylation rate of drinking group was 59.3%(16/27)and 51.9%(14/27)respectively.The methylation rates of non-drinking group were 56.0%(14/25),52.0%(13/25)respectively,The difference was not statistically significant(χ~2=0.056,P=0.812),(χ~2=0.000,P=0.991).The methylation rates of smoking group were 51.7%(15/29)and 48.3%(14/29)respectively.The methylation rates of non-smoking group were 65.2%(15/23)and 56.5%(13/23)respectively.The difference was not statistically significant(χ~2=0.957,P=0.328),(χ~2=0.349,P=0.554);The methylation rates were 38.1%(8/21)and 28.6%(6/21)in clinical stage Ⅰ+Ⅱ,respectively.The methylation rates of stage Ⅲ+Ⅳ were 71.0%(22/31)67.7%(21/31),the difference was statistically significant(χ~2=5.543,P=0.019),(χ~2=66.589,P=0.000);The methylation rates of lymph node metastasis group were 73.9%(17/23)and 65.2%(13/23)respectively.The methylation rate of non-lymph node metastasis group were 44.8%(13/29),41.4%(12/29),the difference was statistically significant(χ~2=4.446,P=0.035),(χ~2=2.920,P=0.047);The methylation rates of the Well and Moderate group were 47.1%(16/34)and 41.2%(14/34)respectively.The methylation rates of the poorly differentiated group were 77.8%(14/18),72.2%(13/18),The differences were statistically significant(χ~2=4.550,P= 0.033),(χ~2=4.544,P=0.033).(Table 7,8)5 The relationship between the relative mRNA expression of PRSS8 and PTPN22 in OSCC and with clinical parametersThe relative mRNA expression levels of PRSS8 and PTPN22 in male group were 0.44±0.13 and 0.41±0.19 respectively.The female group were 0.46±0.11 and 0.49±0.21,respectively.There was no significant difference between the two groups in 2 genes respectively(t=-0.586,P=0.560),(t=-1.481,P=0.145);The relative mRNA expression levels were 0.45±0.13 and 0.44±0.20 in ≥60 years group,respectively,The <60 years group were 0.43±0.10 and 0.46±0.20,respectively.There were no significant difference between the two groups in 2 genes(t=-0.481,P=0.633),(t=-0.451,P=0.654);The relative expression levels of smoking group were 0.44±0.11,0.48±0.22,and the non-smoking group were 0.45±0.13 and 0.40±0.18,the difference were not statistically significant between the two groups in 2 genes(t=-475,P=0.637),(t=1.323,P=0.192);According to the TNM staging,the relative expression levels of I+ were 0.49±0.11 and 0.56±0.12,respectively,and the Ⅱdifference were statistically significant between the two groups in 2 genes(t=2.335,P=0.024),(t=2.076,P=0.043);lymph node metastasis group were 0.42±0.11,0.44±0.17 and non-lymph node metastasis group were 0.49±0.13,0.55±0.11,the difference were statistically significant between the two groups in 2 genes(t=-2.11,P=0.039),(t=-2.68,P=0.010).The Well and Moderate group were 0.48±0.12,0.57±0.11,and the poorly differentiated group were 0.39±0.11,0.49±0.12,the difference were statistically significant between the two groups in 2 genes.(t=2.73,P=0.009),(t=2.18,P=0.033).(Table 9,10)Conclusion:1 The relative mRNA expression of PRSS8 and PTPN22 in OSCC tissues was significantly lower than that in normal mucosal tissues,suggesting that PRSS8 and PTPN22 genes may play a major role in tumor suppressor genes in OSCC.2 The promoter methylation status of PRSS8 and PTPN22 genes in OSCC tissues was significantly higher than that in normal mucosal tissues,suggesting that methylation of PRSS8 and PTPN22 genes may be one of the mechanisms of OSCC.3 The methylation rate of PRSS8 and PTPN22 promoter region was independent of the sex and age of patients,which was related to differentiation degree,lymph node metastasis and pathological grade,suggesting that methylation of PRSS8 and PTPN22 gene promoter region may be related to the prognosis of OSCC. |
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