Construction And Selection Of Subtracted CDNA Library Of Mouse Hepatocarcinoma Cell Lines With Different Lymphatic Metastasis Potential

Background:Metastasis is the most lethal attribute of a cancer. The 90 percent of cancer is carcinoma. And lymph nodes are often the first organ to develop metastasis. Lymph node metastasis forms a bridgehead for further spread. It is very important to make the molecular mechanism of lymphatic metastasis clearly. But the molecular mechanism of lymphatic metastasis remains poorly understood. A mouse hepatocarcinoma cell line (Hca-F) with high lymphogenous metastatic potential and its syngeneic cell line (Hca-P) with low lymphogenous metastatic potential were separated from hepatocarcinoma (HCC) in mice. There are many different techique to detect the difference of gene expression between the cells with different biological phenotypes, such as Expressed sequencing tag(EST), Serial analysis of gene expression(SAGE), subtractive hybridization, mRNA differential display (DD-RTPCR), cDNA representative difference analysis (RDA), suppressive subtractive hybridization(SSH), and cDNA microarry. Suppressive subtractive hybridization has been described recetently. SSH involves a process called normalization, which equalizaes the relative amount of each cDNA species in the hybridization step. Thus , differentially expressed genes with low abundance can be detected effectively, and this procedure is more simple and effective than other techniques such as traditional subtraction. Object: In order to make the molecular mechanism of lymphatic metastasis clearly, we try to detect the difference of gene expression between mouse hepatocarcinoma cell lines with different lymphatic metastasis potential using SSH method. Method: we used mouse hepatocarcinoma cell lines with different lymphatic metastasis potential which are named as Hca-F(high) and Hca-P(low) in this study. Total RNA was extracted by Trizol Reagent. And mRNA was isolated from total RNA using Oligotex. cDNA was synthesized frommRNA of Hca-F and Hca-P cells. We used cDNA of Hca-F as tester and cDNA of Hca-P as driver. Purified tester cDNA was digested with Rsa I ,and then subdivided into two equal groups. The two tester part was ligated to adptorl and adptor2R in separate ligation reactions. Subtractive hybridization was performed by annealing an excess of driver cDNA with each sample of adaptor ligated tester cDNA. The cDNAs were heat denatured and incubated in 68 for 8 hours. Two samples were mixed together and hybridization with freshly denatured driver cDNA for 16 hours in 68. After filling in the ends, two rounds of PCR amplification were performed to enrich desired cDNAs containing both adaptors. Secondary PCR products were used as templates for PCR amplification of G3PDH at 20,25,30,35 cycles to assure subtraction efficiency. PCR Products were cloned into T/A cloning vector. The ligation products were transformed into DH5 a competent cells. Randomly selected individual 160 clones and then used for clone PCR amplification. Isolated the vector DNA from positive clones for sequencing. The results of sequencing was used to analyse sequence homologies in the genebank database. Results: The A260/A280 was 1.9, which showed extracted RNA and mRNA were pure. And agarose gel electrophoresis showed that RNA was not destroyed by Ribonucleases. We founded that Tester cDNA and driver cDNA were completely digested by agarose gel electrophoresis. The subtracted cDNA libraries after secondary PCR amplification looked like smears. Subtraction efficiency showed the effectively reduced abundance of non-differentially expressed genes. In nonsubtracted cDNA library, housekeeping gene G3PDH PCR products were visible after 25 cycles. However, subtracted cDNA library required 35 cycles for G3PDH to be detected. There were 800 positive bacreria clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95 percent clones contained 100-700bp inserts. Analysis of 15 sequenced cDNA clones randomly picked fom the SSH library revealed 4 known genes (mouse heat shock protein 84KD, DNA helicase, ribosomal protein S13 ethanol induced 6) and 3 expressed...