Amplification Of The MIF Gene By Using RACE Method And Identification In Early Stage Squamous Carcinoma Of The Cervix

Purpose:Screening the gene expressed sequences related to early stage cervical cancer lymph node metastasis from genes chip of SCC at stage of Ib,cloning full length of gene by rapid amplification of cDNA ends(RACE),authenticating these genes in early stage squamaceous cervical cancer from the mRNA and protein expression levels.To providing the markers and bases to discuss the mechanism of early stage cervical cancer or diagnosis and therapy in gene level.Method:Firstly,we using reverse transcription PCR(RT-PCR)to identity these was screened ESTs mRNA expression levels in early stage squamaceous cervical cancer:Then,we design gene specific primer(GSP)base on EST,and cloning full length of gene by rapid amplification of cDNA ends(RACE),forecasting sequence and analyzing sequences with bioinformatics; Eventually,these sequences mRNA expression levels was examined in early stage squamaceous cervical cancer with reverse transcription polymerase chain reaction(RT-PCR),MIF that one of these sequences protein expression in blood serum was detected with ELISA,and we using RT-PCR,ELISA and Immunohistochemistry(IHC)to identity MIF expression levels in CaSki and Siha cell strains.Results.(1)we examined the mRNA expressions of AI870661,AI140221,AI985548,ADCAPE03 and ADCAPB08 in early stage SCC,the expression of AI140221 was higher in early stage SCC without lymph node metastasis than those with metastasis(P＜0.005),other ESTs were higher in early stage SCC with lymph node metastasis than those without metastasis (P＜0.01,P＜0.005).(2)we designed gene specific primer(GSP)base on AI870661 and amplificated gene of MIF with 3'RACE in early stage SCC.We obtianed a sequence that is 485bp by gene sequencing.Through BLAST software,the sequence was compared with gene and protein of MIF in GenBank database for homology analysis,there are homology up to 95%.We also found a new EST in process of RACE,and obtained GeneBank ID:EL812774. (3)We analysis the result of RT-PCR and ELISA with statistical method,the expression of MIF is higher in early stage SCC without lymph node metastasis than normal tissue of cervix,and it is higher in early stage SCC with lymph node metastasis than those without metastasis(P＜0.01, P＜0.05).The result of RT-PCR of this EST is higher in early stage SCC without lymph node metastasis than normal tissue of cervix,and it is higher in early stage SCC with lymph node metastasis than those without metastasis(P＜0.01,P＜0.05).MIF expression in CaSki cell strain is higher than Siha cell strain.Conclusion:We start from EST and amplificated gene of MIF with 3'RACE in early stage SCC at the first time.And We found a new EST in early stage SCC.We examined expression of MIF at gene level and protein level,it's expression was higher in early stage SCC without lymph node metastasis than normal tissue of cervix,and it was higher in early stage SCC with lymph node metastasis than those without metastasis.This result indicate that MIF play the part of significant role in formation and metastasis of SCC probably.And we ulteriorly find and reseach genes that those ESTs is corresponding to can provide clues for molecular mechanism of SCC.