Claudin-3 expression in colorectal carcinoma and its significance / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of claudin-3 in colorectal carcinoma and its association with the occurrence, progression and prognosis of colorectal cancer.</p><p><b>METHODS</b>Forty surgical specimens of colorectal carcinoma and 22 adjacent normal tissues resected between October, 2010 and January, 2013 at Nanfang Hospital were examined for claudin-3 expression using immunohistochemistry, which was analyzed in association with the clinicopathological parameters and the survival of the patients.</p><p><b>RESULTS</b>Claudin-3 was expressed mainly on the cell membrane, and its positivity rate was significantly higher in cancer tissues than in normal tissues (92.50% vs 59.09%, P<0.05). In 13 cases claudin-3 expression was detected in both the cancer tissues and adjacent normal tissues with average expression scores of 4.538 and 3.269, respectively (P<0.05). In the cancer tissues, the strongly positive expression rate was significantly higher in poorly differentiated tissues (85.71%) than in well (21.43%) and moderately (36.48%) differentiated tissues (P<0.05), and was higher in cases with lymph node metastasis than in those without (61.11% vs 22.72%, P<0.05). The strongly positive expression rate of claudin-3 was not correlated with the patients'age, gender, tumor location or tumor size (P>0.05). Of the 33 cancer patients followed up, 14 had a postoperative survival time no longer than 3 years and 19 had longer survival time, and their average expression scores differed significantly (4.50 vs 3.526, P<0.05).</p><p><b>CONCLUSION</b>Claudin-3 is over-expressed in colorectal cancer tissues, and its high expression may promote the occurrence and progression of colorectal cancer. Claudin-3 may serve as a molecular biomarker for early diagnosis and prognostic evaluation.</p>

Effect of small interfering RNA targeting Rac1 gene on colony formation of SW480 cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a vector expressing small interfering RNA (siRNA) against Rac1 gene and observe its effect on soft agar colony formation of SW480 cells in vitro.</p><p><b>METHODS</b>Oligos of 64 base pairs for hairpin RNA targeting Rac1 were chemically synthesized and annealed. The siRNA constructs for Rac1, produced by inserting the annealed oligos into the downstream of H1 promoter of linearized pSUPER, were confirmed by restriction digestion and DNA sequencing. The constructed Rac1-siRNA was transfected into SW480 cells and Western blotting was performed to assess the expression and interference efficiency of siRNAs against Rac1.The soft agar colony formation assay was used to study the effect of Rac1 gene silencing on SW480 cells.</p><p><b>RESULTS</b>Restriction digestion and DNA sequencing showed that the siRNA targeting Rac1 gene was successfully constructed. The siRNA could effectively down-regulate the expression of Rac1 in SW480 cells. Soft agar colony formation assay showed that the colony number and diameter of SW480 cells was reduced after siRNA transfection.</p><p><b>CONCLUSION</b>A vector expressing hairpin RNA against Rac1 gene are successfully produced, which significantly reduces the colony numbers and size of SW480 cells in vitro, suggesting that Rac1 plays an important role in the growth of colorectal cancer in vitro.</p>

Effect of X-ray exposure on soluble tumor necrosis factor receptor-p75 release in hepatocellular carcinoma HepG2 cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of X-ray exposure on the release of soluble tumor necrosis factor receptor-p75 (sTNFR-p75) in hepatocellular carcinoma HepG2 cells in vitro.</p><p><b>METHODS</b>Enzyme-linked immunosorbent assay (ELISA) was used to examine the levels of sTNFR-p75 in the supernatants of HepG2 cells before and after X-ray exposure. The cell apoptosis was analyzed by flow cytometry and transmission electron microscope(TEM), and the morphological changes of the cells were examined under optical microscope and transmission electron microscope(TEM).</p><p><b>RESULTS</b>X-ray exposure of the cells resulted in a strong increase of cell apoptosis (P<0.05) and sTNFR-p75 production in the cells as compared with the those before the exposure (P<0.01). Optical microscopy revealed apoptotic changes of HepG2 cell after the exposure, shown as cell shrinkage, spherical cell morphology, cytoplasmic and nuclear condensation. Apoptotic bodies were detected by TEM.</p><p><b>CONCLUSION</b>X-ray exposure induces HepG2 cells apoptosis by inhibiting the release of sTNFR-p75 into the supernatant.</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>

Cloning of human migfilin N-terminal domain and preparation of anti-migfilin polyclonal antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone migfilin-N terminal sequence into E.coli and obtain a fusion protein for preparing rabbit polyclonal antibody against migfilin, thereby facilitating the study of the role of migfilin in the biological behavior of colon cancer.</p><p><b>METHODS</b>Based on human migfilin cDNA sequence, a pair of primers was designed to amplify migfilin-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-1 with EcoRI/XhoI sites, and the target recombinant plasmids were identified with enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL(21) cells, the GST-migfilin-N fusion protein was expressed with IPTG induction. Glutathione-sepharose beads were used to purify the fusion protein, and anti-migfilin polyclonal antibody was produced by immunization of rabbits with the purified GST-migfilin N-terminal fusion protein. The resultant anti-migfilin polyclonal antibody was purified by protein A beads and used for Western blotting for detecting migfilin expression in different cell lines.</p><p><b>RESULTS AND CONCLUSION</b>The migfilin-N terminal gene fragment was cloned successfully, and purified GST-migfilin N-terminal fusion protein and anti-rabbit migfilin polyclonal antibodies were obtained. Western blot analysis demonstrates that the antibodies specifically detected migfilin expression in the cell lines, which may facilitate further investigation of the role of migfilin in the biology of colon cancer.</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>

X-ray irradiation enhances tumor necrosis factor receptor p55 expression in human colorectal cancer cells and inhibits the release of its soluble form in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of X ray on human colorectal cancer cells for their tumor necrosis factor receptor-p55 (TNFR-p55) expression and release of soluble soluble TNFR-p55 (sTNFR-p55) in vitro.</p><p><b>METHODS</b>The protein expression of TNFR-p55 in Lovo cells exposed to X-ray was detected using immunohistochemistry, and enzyme-linked immunosorbent assay was used to examine the levels of sTNFR-p55 in the supernatants of the cell culture. The cell apoptosis of the exposed cells was analyzed with flow cytometry, and the changes in cell morphology were observed microscopically.</p><p><b>RESULTS</b>X-ray exposure of cells resulted in a strong increase in TNFR-p55 expression of (P<0.01) and LoVo cell apoptosis (P<0.05). The levels of sTNFR-p55 in the supernatant of cells with X-ray exposure was significantly lowered in comparison with the levels before exposure (P<0.01). Optical microscopy showed that the exposed LoVo cells shrank and became spherical with cytoplasmic condensation and nuclear pyknosis.</p><p><b>CONCLUSION</b>X-ray exposure can induce LoVo cell apoptosis by increasing TNFR-p55 expression on the cell membrane and inhibiting the release of sTNFR-p55 in the supernatants.</p>

Role of Rac1 activation in migration and invasion of colorectal cancer cell line SW480 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the role of activation of Rac1 in colorectal cancer cell migration and invasion.</p><p><b>METHODS</b>Rac1 L61 plasmid and control plasmid were transfected into colorectal cancer cell line SW480 cells. Pull down assay by Western blotting was carried out to measure the amount of activited Rac1, and transwell permeable supports were used to assess the migration and invasion of SW480 cells with different activitivity of Rac1.</p><p><b>RESULTS</b>The transfection ratio of SW480 cells was more than 80%. Pull down assay showed that the activited Rac1 was significantly higher in the SW480 cells transfected with Rac1 L61 plasmid than that in the control, and the amount of migrating and invasing SW480 cells transfected with Rac1 L61 plasmid in the Transwell permeable supports were significantly more than those in controls (migrating cell numbers: 43 +/- 9 vs. 22 +/- 5, P < 0.01; invasing cell numbers: 73 +/- 13 vs. 38 +/- 1, P < 0.01).</p><p><b>CONCLUSION</b>The activation of Rac1 plays an important role in the migration and invasion of colorectal cancer cells.</p>