Expression Of HINT1 And FAK In Lesions Of Malignant Melanoma And Their Significance

Acral lentiginous melanoma(ALM) is the major subtype melanoma and is a relatively rare disease in China.The biologic behavior and evolution and prognosis of ALM were distinct from other subtypes of cutaneous melanomas.However the genetics and molecular feature of ALM were not well-recognized. Gene expression of HINT1/PKCI-1 and FAK were analyzed in ALM and common nevocellular nevus. We aimed to test whether they could be as the new diagnositic markers or prognostic tool or new therapeutic target for ALM.Studies on the genes expression were hampered by the lack of fresh tissues. We therefore sought to utilize the formalin fixed and paraffin embedded (FFPE) cuteanous tissue specimens as the source of material to retrospective study mRNA expression of more and more new recognized genes during the progress of a disease.The method of extraction of RNA from FFPE cuteanous tissue for reverse transcription and polymerase chain reaction (RT-PCR) were established.The feasibility and stability of this method were investigated. The results of RT-PCR assays in FFPE tissue were compared with the result of in situ hybridization and immunohistochemistry assay.RNA were extracted from FFPE sections by utilizing a proteinase K digestion and guanidine isothiocyante-phenol extraction, RT-PCR reaction were performed subsequently.The effects of different digest protocol were compared and the impact of different primers and different amplicon size were studied.The protocol for mRNA isolation and RT-PCR reaction from different years archival tissue were optimized. The result was stability under the amplicon size was less than 120bp and the reverse transcription step was primed with the specific antisense primer.There were no difference among different years archival tissue.The results of RT-PCR from the same five tissues that either frozen or FFPE show very similar. On this base the expression level of HINT1/PKCI-1 mRNA and FAK mRNA were compared in ALM and NN by semi-quantitive RT-PCR.