Expression And Preliminary Function Analysis Of AMF Gene In Esophageal Squamous Cell Cancer Cells

Esophageal cancer is one of the most common cancers and the sixth leading cause of cancer death worldwide. China have been well recognized as the highest incidence area for esophageal cancer in the world. Especially, the Taihang mountain nearby regions of our country. Although many treatment strategies have been adopted, the prognosis of esophageal squamous cell cancer (ESCC) patients is still poor owing to early invasion and metastasis. How to reduce the accidence of ESCC metastasis, which will help to improve the prognosis of patients with ESCC.In 1986, in the culture medium of melanoma cell lines, Liotta discovered a protein which stimulated mouse 3T3 fibroblasts movement, named for the autocrine motility factor (AMF). Recent studies have suggested that after connectting its receptor, AMF can stimulate cell movement. Overexpression of AMF has been observed in many human cancers including hepatocellular cancer (HCC), astrocytoma, fibrosarcoma, etc. and the expression level is associated with lymph node metastasis, staging of tumour, blood vessel vassion etc. But the invasion and metastasis of AMF in ESCC cells has not been reported. The purpose of this study is to construct eukaryotic expression vector of AMF gene and the green fluorescence protein vector, investigating the localization of EGFP-AMF proteins and effects of the AMF gene in the cell migration in ESCC cells. The results supply theorical basis for molecular targeted therapies.The results showed that EGFP-AMF proteins localized in the cytoplasm of ESCC cell lines.While p-cofilin protein was significently promoted in the ESCC transfected with pcDNA3.1(+)-AMF by Western blotting, compared to the control groups (P<0.05).These findings indicate that AMF gene promotes ESCC metastasis, and it is possiblely that AMF gene as a potential molecular target for ESCC therapy.Methods1 Extraction and identification of total RNATotal RNA was extracted from ESCC according to manufacture's instructions and identified its concentration, purity and integrity.2 Cloning of the AMF gene of ESCCA pair of primers was designed according to the AMF gene of ESCC sequences known in GenBank, then RT-PCR was used to amplify AMF gene cDNA fragment. The PCR product was analyzed by agarose gel electrophoresis and sequencing.3 Construction and identification of the recombinant eukaryotic vectorsAfter the fragment of AMF was inserted into the Hindâ…¢-EcoRâ… restriction sites of the eukaryotic expression vectors pEGFP-C1 and pcDNA3.1 (+) respectively, the recombinant eukaryotic vectors were identified by digestion and sequencing, generating the correct vectors pEGFP-C1-AMF and pcDNA3.1(+)-AMF respectively. 4 TransfectionThe recombinant vectors were transfected into ESCC cells by LipofectamineTM 2000 according to manufacturer's instructions.5 Cellular localization of EGFP-AMF fusion proteinThe location of EGFP-AMF proteins was determined by fluorescent microscope on 24 hour after transfection.6 RT-PCR detectionRT-PCR was used to detect the expressions of AMF gene before and after ransfection.7 Western blotting detectionThe expressions of recombinant AMF were detected by Western blotting using anti-his tag antibody. The expressions of the p-cofilin proteins was detected by Western blotting.Results1 Identification of total RNAThe purity and concentration of total RNA were assessed by spectrophotometry and the absobance ratio of 260/280 is grater than 1.80. The integrity of the RNA was also verified by electrophoresis in the 1% agarose gel.2 Cloning of the AMF gene of ESCC Sequences analysis of PCR product showed that the cloned fragment was about 1700 bp in length, which was consistent with the sequences submitted to GenBank.3 Identification of the recombinant eukaryotic vectorsThe expected fragments were identified to have been correctly inserted into the eukaryotic vectors with the correct ORF by restriction enzyme digestion and sequencing, and the recombinant eukaryotic vectors were named as pEGFP-C1-AMF and pcDNA3.1 (+)-AMF respectively.4 Cellular localization of the EGFP-AMFAfter transfection with empty vector pEGFP-C1, cytoplasm and nucleus of ESCC cells appeared green fluorescent under the high-power fluorescent microscope. However, after transfection with pEGFP-C1-AMF, green fluorescent were localized in the cytoplasm of ESCC cell lines.5 RT-PCRThe results of RT-PCR showed that in all three separate experiments the expression of the AMF gene improve obviously in the ESCC cells transfected with pcDNA3.1(+)-AMF.6 Western blotting detectionThe results of western blotting showed that the fusion proteins containing six His tags were expressed in ESCC cells. The expression of p-cofilin protein was significantly increased in the ESCC cells transfected with pcDNA3.1(+)-AMF, compared to the control groups.ConclusionThe AMF gene is cloned successfully, the sequence of which is same as Genbank by BLAST analyzing. Also the two eukaryotic expression vectors are constructed successfully and than transfected in ESCC cell lines respectively. The AMF gene localizes in the cytoplasm by fluorescent microscope screening. The expression level of p-cofilin increases significently. The results show that AMF gene can promote the mobility of ESCC cells, which provide basis for the reseach of function.