The Promoter Methylation And MRNA Expression Of Dickkopf-3 Gene In Oral Squamous Cell Cancer

Objective: Oral cancer,which is one of the common malignant tumors of head and neck,has high morbidity in our country.The epidemiological studies have shown that it is related with many pathogenic factors such as smoking,drinking,oral hygiene and so on,and onset age tend to be younger.Although multidisciplinary sequence treatment has greatly promoted the oral cancer treatment in a certain extent,the five-year survival rate is still low.DKK-3(Dickkopf-3),as one of the important inhibitors in the Wnt signaling pathway,has been shown low mRNA expression and high promoter methylation status in abundance of malignancies such as lung cancer,esophageal cancer,thyroid carcinomas and so on.All the researches have shown that DKK-3 can be acted as a candidate tumor suppressor gene involved in tumorigenesis and progression.At present,there is few reports about DKK-3 gene in oral and maxillofacial malignant tumor.The object of this experiment is to explore the relationship between the DKK-3 gene and cancer accurance by detecting DKK-3 gene mRNA expression levels and its promoter region methylation status in oral squamous cell carcinoma tissue and corresponding normal oral mucosa tissues,and to provide theory basis for DKK-3 as a new biological indicator in the target gene therapy of oral cancer.Methods:The 51 patients with primary oral squamous cell carcinoma who were treated with excision surgery during Sep.2014 to Dem.2015 in the Fourth Hospital of Hebei Medical University were elected,and were compared with the corresponding adjacent normal oral mucosa tissues.The cancer tissues were proved to be squamous cell carcinoma and adjacent tissues were proved to be non-tumor tissue by pathological detection.All the tissues were used immediately after surgery and cryopreserved in-80° refrigerator.The experiment was applied with RT-PCR and the methylation-specific polymerase chain reaction(Methylation-Specific PCR,MSP)respectively to investigate the DKK-3 gene m RNA expression levels and its promoter region methylation status in oral squamous cell carcinoma tissues and corresponding normal oral mucosa tissues.At the same time,processing the results by statistical software SPSS13.0 and analysing the relationship between the results and clinical data,such as age,gender,smoking,clinical stage and lymph node metastasis.Results:1 The relative expression quantity of DKK-3 mRNA in oral squamous cell carcinoma tissues was 0.41±0.27,significantly lower than that in corresponding oral mucosa tissues(0.50±0.29).The difference had statistical significance(t=-12.580,P<0.05).In the meanwhile,the relative expression quantity of DKK-3 mRNA in smoking group was 0.22±0.16,significantly lower than that in no smoking group(0.63±0.21).The difference had statistical significance(t=-8.018,P<0.05);The relative expression quantity of DKK-3 mRNA in clinical stage ?+? was 0.46±0.29,significantly higher than that in clinical stage ?+?(0.28±0.19).The difference had statistical significance(t=2.214,P<0.05);The relative expression quantity of DKK-3 mRNA in lymph node metastasis group was 0.24±0.17,significantly lower than that in no lymph node metastasis group(0.50±0.28).The difference had statistical significance(t=-4.114,P<0.05).However,there was no relationship between the relative expression quantity of DKK-3 mRNA and clinical data,such as gender and age(P>0.05);2 In the oral squamous cell carcinoma tissues,the frequency of DKK-3 gene promoter region methylation was 49.0%,significantly higher than that in corresponding normal oral mucosa tissues(7.8%).The difference had statistical significance(?~2=19.273,P<0.05).In the meanwhile,the frequency of DKK-3 gene promoter region methylation in smoking group was 64.3%,significantly higher than that in no smoking group(30.4%).The difference had statistical significance(?~2=5.790,P<0.05);The frequency of DKK-3 gene promoter region methylation in clinical stage ?+? was 38.9%,significantly lower than that in clinical stage ?+?(73.3 %).The difference had statistical significance(?~2=5.027,P<0.05).However,there was no relationship between the frequency of DKK-3 gene promoter region methylation and clinical data,such as gender,age and lymph node metastasis(P>0.05);3 In the oral squamous cell carcinoma tissues,the relative expression quantity of DKK-3 mRNA in methylated tissues was 0.30±0.21,which was lower than that in unmethylated tissues(0.51±0.29).The difference had statistical significance(t=-2.817,P<0.05).Conclusion:1 The expression of DKK-3 gene mRNA in oral squamous cell carcinoma was significantly lower than that in corresponding normal oral mucosa tissues.The result suggests that DKK-3 gene may be a tumor suppressor gene in oral squamous cell carcinomas,and its low expression or silence may leads to the occurrence and progression of the oral cancer.The relative expression quantity is associated with smoking,clinical stage and lymph node metastasis,but has nothing with gender or age.2 The frequency of DKK-3 gene promoter region methylation in oral squamous cell carcinoma was significantly higher than that in corresponding normal oral mucosa tissues.The result indicates that the hypermethylation of the DKK-3 gene promoter may be one of the important mechanism in the occuring of oral cancer.At the same time,DKK-3 gene promoter region methylation status is associated with smoking and clinical stage,but has nothing to do with gender,age or lymph node metastasis.3 In the oral squamous cell carcinoma tissues,the relative expression quantity of DKK-3 mRNA in methylation group was lower than that in unmethylation group.The result suggests that DKK-3 gene promoter region hypermethylation could be the main reason for its transcriptional silencing.