Effects of epigallocatechin-3-gallate on the proliferation of colonic cancer cell line SW620 and PAK1 gene expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of epigallocatechin-3-gallate (EGCG) on the proliferation of SW620 cells and the expression of PAK1 gene.</p><p><b>METHODS</b>Human colonic cancer cell line SW620 was treated with EGCG at 40, 60 and 80 micromol/L and cultured in RPMI 1640 medium for 0, 24, 48 and 72 h. The proliferation of SW620 cells was observed by MTT assay before and after EGCG treatment, and the expression of PAK1 protein was observed by Western blotting.</p><p><b>RESULTS</b>SW620 cells treated with EGCG displayed a slowed growth in comparison with the control cells, and the growth rate decreased with the increase of EGCG concentration. PAK1 protein expression was lowered in SW620 cells after EGCG treatment for 48 h.</p><p><b>CONCLUSION</b>EGCG can inhibit the proliferation and partially reduce the expression of PAK1 protein in SW620 cells.</p>

Effect of p21-activated kinase-1 on the invasiveness of colorectal carcinoma cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of p21-activated kinase-1 (PAK1) gene transfection on the invasiveness of human colorectal carcinoma SW480 cells in vitro.</p><p><b>METHODS</b>SW480 cells in routine culture were transfected with the recombinant plasmid EGFP-C1/PAK1 via Lipofectamine(TM) 2000. The expression of PAK1 protein in SW480 cells was detected using Western blotting, and the changes of the invasiveness of SW480 cells were evaluated using Boyden chamber invasion assay.</p><p><b>RESULTS</b>Forty-eight hours after transfection with pEGFP-C1/ PAK1, the PAK1 protein expression increased significantly in comparison with those in negative and vector control groups. The invasiveness of the SW480 cells was significantly enhanced after the transfection.</p><p><b>CONCLUSION</b>The PAK1 gene transfection can increase the expression of PAK1 in SW480 cells and enhance the invasiveness of the cells. PAK1 can be associated with the invasiveness and metastasis of colorectal carcinoma cells.</p>

PCR-based assembly of the DNA sequence coding for tetanus toxin C fragment / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos).</p><p><b>METHODS</b>To allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing.</p><p><b>RESULTS</b>Three 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene.</p><p><b>CONCLUSION</b>PCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.</p>

Construction and identification of recombinant Lactococcus lactis highly expressing human granulocyte-macrophage colony stimulating factor / 南方医科大学学报

<p><b>OBJECTIVE</b>To transfer human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene into Actococcus lactis and obtain recombinant Lactococcus lactis highly expressing hGM-CSF (LL-CSF).</p><p><b>METHODS</b>The optimized hGM-CSF gene sequence capable of expression in Lactococcus lactis was cloned into the vector pNBC1000, which contained P59 promoter, RBS, MCS, USP45 signal peptide and USP45 stop codon, to generate the recombinant plasmid pNCSF. pNCSF was subcloned into a shuttle vector pTR1001c to acquire the plasmid pTRCSF, which was transferred into Lactococcus lactis to obtain LL-CSF by means of electroporation. SDS-PAGE was used to verify the expression of hGM-CSF protein by the constructed LL-CSF.</p><p><b>RESULTS</b>DNA sequencing and restriction enzyme digestion indicated the successful construction of the recombinant plasmid pNCSF, pTRCSF and the recombinant bacterium LL-CSF that was capable of steady and efficient expression of hGM-CSF as shown by SDS-PAGE.</p><p><b>CONCLUSION</b>The recombinant Lactococcus lactis LL-CSF has been successfully constructed, which can be valuable for studying the biological activity of recombinant hGM-CSF and for evaluating the potential clinical application of the protein.</p>

Short hairpin RNA-mediated survivin gene silencing inhibits invasion and metastasis of human colon carcinoma cell line SW480 in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of short hairpin RNA (shRNA) targeting survivin on adhesion and invasion of human colon carcinoma cell line SW480 in vitro.</p><p><b>METHODS</b>According to the sequence of the coding region of survivin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to prepare the hairpin construct as the DNA templates for the target shRNA. The shRNA templates were cloned into shRNA expression vector pRNAT-U6.1/Neo, and the resulted vector pRNAT-U6.1/Neo-survivin was transfected into SW480 cells using Lipofectamine 2000. Western blotting was performed to evaluate survivin gene silencing induced by shRNA transfection at the protein level, and the biological behaviors of the SW480 cells were investigated by cell-matrix adhesion, invasion and gelatin-zymography assays.</p><p><b>RESULTS</b>Western blotting revealed significantly lowered survivin protein expression in transfected SW480 cells, and survivin gene silencing induced by shRNA significantly suppressed the metastatic potential of SW480 cells in association with suppressed MMPs activity.</p><p><b>CONCLUSIONS</b>Survivin may play an important role in modulating human colorectal carcinoma cell invasion and metastasis, and survivin gene silencing can inhibit human colorectal cancer cell invasion and the production of MMP-2 and MMP-9. Survivin may affect invasion and metastasis of human colorectal carcinoma cells via regulating the production of MMPs.</p>

Screening and preliminary analysis of colorectal carcinoma-associated antigen genes / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen and identify the genes coding for colorectal carcinoma-associated antigen and analyze the bioinformation of their cDNA sequences.</p><p><b>METHODS</b>Immunoscreening of the cDNA phage-display library derived from human colorectal carcinoma was performed with autologous or allogeneic serum antibody from patients with colorectal cancer through SEREX approach. After amplification of the positive phage clones, the phage DNA was extracted and purified with Qiagen kit, and the fragment sizes of the cDNA of positive clones were identified by PCR and EcoR I and Hind III restriction endonucleases. The cDNAs of the positive clones were ligated into pUCm-T vector and sequenced. The bioinformation of cDNA sequences were analyzed against GenBank+EMBL+DDBJ+PDB Sequences Database.</p><p><b>RESULTS AND CONCLUSION</b>Eleven positive clones were obtained after immunoscreening, and the sizes of the cDNA fragments were 1100, 1300, 1000, 2000, 1200, 1200, 700, 900, 600, 1200 and 1000 bp, respectively, representing 9 antigen genes, including 7 with homology with the known genes. Among the 11 obtained positive clones, 3 were the same cDNA having homology with interferon-induced transmembrance protein-1 and possessing anti-proliferation effect; another 6 represented different genes, namely human BAC clone RP11-453E17 whose function have not been cleared, human cartilage-hair hypoplasia region gene responsible for cartilage-hair hypoplasia, human chromosome 5 clone CTD-2030B15 with insertion mutation, human gene similar to anti tumor necrosis factor-alpha antibody light-chain Fab fragment associated with tumor growth, mRNA of human beta-2-microglobulin in relation to tumor cell proliferation, and human aldolase A gene promoting tumor cell proliferation. The other two cDNA sequences were not identified for homology with currently known genes in GenBank, and their functions awaits further investigation.</p>