Different Expression Of DOC-1 MRNA In Oral Squamous Cell Carcinoma And The Biological Influence Of Exogenous P12^DOC-1 On Tca8113 Cell

Oral cancer is the most common tumor in oral and maxillafacial surgery. It often occurs on the body surface or near the body surface, so it badly impacts the normal physiological functions and social communications of the patients. Nowadays the occurrence and development of many diseases can be explained on the genic level. The mutation, inactivation and abnormal expression of oncogene and anti-oncogene are the primary cause of cancer. Several genes related to oral cancer were identified in recent year. So it is possible to investigate the occurrence and development of oral cancer on molecular level. DOC-1 is a putative tumor suppressor gene isolated and identified from the hamster oral mucosa by Todd in 1995, in Harvard University. p12 is the coded product of this gene. It is reported that p12 has the function of anti-tumor growth. Human DOC-1 gene is located in 12q24 chromosome and its cDNA is 1627 base pairs in length and encodes 115-amino acid polypeptide (12.4kDa). Objectives: Pre-experiment of our research group showed that the amount of p12 in different differentiation of oral squamous cell carcinoma is different. We used the technology of RT-PCR to investigate the difference expression of DOC-1 mRNA in different differentiation of oral squamous cell carcinoma. And we constructed an eukaryotic expression vector contained the coding region of open reading frame of DOC-1 gene to transfect Tca8113 cell line, in order to investigate the influence of biological functions of exogenous p12DOC-1 on Tca8113 cell line.Methods: 1. We chose RT-PCR by designing specific primers to detect the expression of DOC-1mRNA in oral squamous cell carcinoma and normal oral mucosa. The gradation of every product after electrophoresis was obtained from computerized gel image system. A relative figure was obtained by comparing the gradation of the product to the gradation of internal reference ofβ-actin mRNA. These relative figures were used into statistical analyzing. 2. DOC-1 ORF was obtained from HaCaT cell line by designing specific primers which aimed directly at coding region of DOC-1 ORF. Specific enzyme restriction position was added to the terminals of ORF sequence. The eukaryotic expression vector was successfully constructed by enzyme cutting, binding, transforming and sequencing. 3. The eukaryotic expression vector contained DOC-1 ORF was transfected into the Tca8113 cell line by transfection kit. The proliferation and invasion ability of stable transfected cell line were observed in order to investigate the influence of biological functions of exogenous p12DOC-1 on Tca8113 cell line.Results: 1. The gradation of DOC-1mRNA in oral squamous cell carcinoma is 1.22±0.12, while 1.58±0.15 in oral mucosa. The gradation of DOC-1mRNA in well differentiation oral squamous cell carcinoma is 1.35±0.04, 1.20±0.05 in moderately differentiation oral squamous cell carcinoma and 1.08±0.03 in poorly differentiation oral squamous cell carcinoma. 2. The vector pT7-FLAG-4 was successfully constructed by double enzyme cutting, electrophoresis and sequencing identification. 3. Compared the selected stable transfected cell line with controlled cell line, it is significantly lowered in the ability of cell proliferation and cell invasion of selected stable transfected Tca8113 cell line.Conclusions: This study confirmed the difference in expression of DOC-1mRNA in different differentiation of oral squamous cell carcinoma. The lower differentiation of oral squamous cell carcinoma, the less expression of DOC-1 mRNA. DOC-1 gene may play an important role in the early stage of oral squamous cell carcinoma. The low expression of DOC-1mRNA may be the early event of oral squamous cell carcinoma.development. The eukaryotic expression vector pT7-FLAG-4 which contains DOC-1 ORF was successfully constructed and transfected into Tca8113 cell line. The stable transfected Tca8113 cell line was selected. Compared the selected stable transfected cell line with controlled cell line, it is significantly lowered in the ability of cell proliferation and cell invasion of selected stable transfected Tca8113 cell line. The cytomorphological and cytobiological changes of Tca8113 can be induced by transfection of the eukaryotic expression vector pT7-FLAG-4 through integrating into chromosome.