Study Of The Expression Level Of CDK2AP1 MRNA In Oral Squamous Cell Carcinoma With LCM And RT-PCR

Oral squamous cell cancer(OSCC) is the most common tumor in oral and maxillafacial region,and accounts 80% of all malignant tumor in this region. It badly impacts the normal physiological functions and social communications of the patients for it often occurs on the body surface or near the body surface. Activation of oncogene and deactivation of anti-oncogene may be involved in the pathogenesis of oral cancer. DOC-1/CDK2AP1 is a putative tumor suppressor gene isolated and identified in 1995. Human DOC-1/CDK2AP1 gene is located in 12q24 chromosome.Its cDNA is 1627 bp in length and encodes a 115-amino acid polypeptide named p12^CDK2AP1.The molecular weight of p12^CDK2AP1 is 12.4kDa.It is reported that p12^CDK2AP1 has the function of anti-tumor growth,and it expresses negatively in OSCC cell line.LCM is an important technology to purify cells,and it was developed in 1996.With LCM single cell even cytoarchitectonic component can be dissected from tissues without damage to molecular substance and tissue morphous of cell.LCM has been used widely combined with technology of cell,DNA,RNA and protein in oncomolecularbiology.It ensures the high precision and reliability of the finding.ObjectivesThe finished work of our research group had shown that the amount of p12^CDK2AP1 and mRNA of gene CDK2AP1 in different differentiation of OSCC is different.But with heterogeneous cell the precision and reliability might be influenced.We combined the technology of LCM and RT-PCR to investigate the difference expression of DOC-1/CDK2AP1 mRNA in cancer cells, stromal cells and normal cells from different differentiation of oral squamous cell carcinoma patients. Our purpose is to provide an evidence for molecul diagnosis treatment.Methods1.We purified cancer cell,stromal cell and normal cell with lacer capture microdissection(LCM) dividely and obtained highly purified cell groups.2. We designed specific primers of DOC-1/CDK2AP1 and the internal reference ofβ-actin.And we detected the expression of DOC-1/CDK2AP1 mRNA in OSCC cancer cell, stromal cell and normal oral mucosa cell with RT-PCR.3.After Agarose Gel Electrophoresis,the gradation of every PCR product was obtained from computerized gel image analytical system. Comparing the gradation of the product to the gradations of DOC-1/CDK2AP1 andβ-actin mRNA,we obtained the relative gradation of CDK2AP1.Then we used these relative figures into statistical analyzing.ResultsThe relative gradations of DOC-1/CDK2AP1 mRNA in cancer cell, stromal cell are 1.49±0.07 and 1.47±0.10. The relative gradations of DOC-1/CDK2AP1 mRNA in poorly, moderately and well differentiated OSCC cancer cell are 0.95±0.08, 1.04±0.06 and 1.25±0.10. Statistical analysis demonstrated: CDK2AP1 mRNA expressed in normal cell and OSCC stromal cell with no significant difference. The experession level of CDK2AP1 in OSCC cancer cell was lower than that in normal cell and stromal cell,with an significant difference(P<0.01).The experession level of CDK2AP1 in well differentiated OSCC cancer cell was higher than that in moderately differentiated OSCC cancer cell with an significant difference(P<0.01). The experession level of CDK2AP1 in well differentiated OSCC cancer cell was higher than that in poorly differentiated OSCC cancer cell with an significant difference(P<0.01). The experession level of CDK2AP1 in moderately differentiated OSCC cancer cell was higher than that in poorly differentiated OSCC cancer cell without an significant difference(P>0.05).ConclusionsThis study confirmed down-regulated expression of CDK2AP1 mRNA in OSCC,and it might be associated with the differentiation of OSCC.The experession level of CDK2AP1 in poorly and moderately differentiated OSCC was lower than that in well differentiated OSCC.There was The experession level of CDK2AP1 shows no difference between poorly and moderately differentiated OSCC,it might be caused by the too small sample size of poorly and moderately differentiated OSCC.