The Relationship Between TM4SF9 And Metastasis Of Colorectal Carcinoma

Colorectal carcinoma.(CRC) is a kind of maliganat tumor which has high attack rate and death rate. Although maliganat tumor has many biological characters, including metastasis, independence, and dedifferentiation and so on, but the most ioportant one is metastasis. CRC's high death rate is mainly concerned with metastasis. Important as it is, research about the mechanism of CRC metastasis is remained backward in contrast with other research field of CRC. Because mechanism regards CRC metastasis is very complicated. Rcently, with the rapid development of molecular biology, focus of carcinomor metastasis research graduly switch from cytoplast to cytomembrane, to investigate the influence of membrane receptor's denomination and mode of action to metastasis.Tetraspanins (also referred to as tetraspans or TM4SF proteins) are a family of widely expressed four-transmembrane-domain proteins. Tetraspanins are postulated to have a topology in which there are two extracellular loops (a small loop between the first and second transmembrane (TM) domains, and a large loop between the third and forth TM domains), an interconnecting intracellular loop (between the second and third TM domains), and cytoplasmic N- and C-termini. Although the external location of the extracellular loops has been confirmed experimentally, the cytoplasmic location of the four consecutive hydrophilic residues of the interconnecting loop remains apoint of speculation. More and more study certificated that TM4SF proteins play an important role in migration of malignant tumor.TM4SF9, which is a member of the tetraspanin superfamily, is prominent expression in the brain. More detailed studies show that mouse TM4SF9, is highly expressed in cortical structures including the hippocampus, and in other brain regions. TM4SF9 is important to the maintenance of brain activity. TM4SF9 contains a putative PKC phosphorylation signal at the intracellular amino terminus, suggesting a role in intracellular signalling. In contrast with other members of TM4SF, CD9 for example, stuys about the relationship between TM4SF9 and tumor are very few, let alone the relationship between TM4SF9 and colon cancer.The present study manages to screen the differential expression genes among three cell lines which own different metatasis potency via cDNA microarray, and then select genes that may play a role in the metastis of carcinoma for further investigate. We aim no only to investigate the molecular mechanism of tumor metastasis, but also to search new effective target for predict malignancy and gene therapy. Testing difference of metastasis ability of three CRC cell lines In vitro, adhesive and motility experiment were used to assay metastasis ability of LST, SW480 and Lovo cell lines. As to adhesive experiment, collageon IV was used as adhesive mediator. CRC cell lines (lxlO8/L) were incubated with collageon IV in the condition of 37 °C, 50mL/L CO2 for 1 hour. As to mobility experiment, the mobility of CRC tumor cells was assayed in a transwell cell culture chamber. CRC cell lines (lxlO8/L) were incubated in up chamber while NIH3T3 culture supernatant in down chamber in the condition of 37°C, 50mL/L CO2 for 6 hour.. The result showed that there were difference of adhesion and mobility ability among LST, SW480 and Lovo. Lovo had the strongest ability of adhesion and mobility, indicated that Lovo had the strongest ability of metastasis.cDNA microarrayThe criterion (mean signal value of a pair of sample >2, or one <2, but the other > 10; coefficient of variance < 0.33; difference of signal value more than 2 times ) was used as to judge the differential expression of genes. Among up regulated genes, Dynamin2, ARF-1 and CAPZA-2 can affect metastasis. Among down regulated genes, TM4SF9 may affect metastasis. The expression of TM4SF9 in CRC carcinoma was first reporten by our research, so we selected TM4SF9 for further research. Validation of cDNA microarray's resultSemi-quantitive RT-PCR was used to validate TM4SF9 expression level in Mrna expression level. Immunohistochemistry, Western-Blot and flow cytometry were used to validate TM4SF9 expression level in protein expression level. The results coincidence with cDNA microarray' result. Positioning study of TM4SF9 in intestine tissueImmunohistochemistry technique was used to study TM4SF9 ' s positioning of clinic CRC carcinoma and normal colon membrana mucosa tissue beside tumor. The result showed colon membrana mucosa cell coloration, and in contrast with normal colon membrana mucosa cell, tumor cells owned higher expression level than normal membrana mucosa cellEstablishing of stably expressed TM4SF9-EGFP fusion protein LST cell line TM4SF9/pEGFP- Q recombinant plasmid was successfully established by molecular cloning technique. LST cell line was transfected TM4SF9/pEGFP- Ci recombinant plasmid and pEGFP- Q plasmid by Lipofectamine 2000?. After 3 weeks screening by G418 (400ng/mL), the transected cell could stably exist in G418 (400ng/mL) culture condition. LCSM shows that TM4SF9-EGFP fusion protein located on cellular membrane, while EGFP protein located in cytoplasm. Flow cytometry shows that cell transfected TM4SF9/pEGFP- Ci recombinant plasmid own higher expressionlevel of TM4SF9 protein than that of cell transfected pEGFP- Q plasmid TM4SF9 RNA interferenceRNA interference (RMAi) technique was used to down regulate Lovo's TM4SF9 expression level. Two siRNA fragments were synthesized by chemical method, and were transfected into Lovo cell by Lipofectamine 2000?. Successfully down regulation was confirmed by flow cytometry and Western-Blot assays. Testing difference of metastasis ability after alter TM4SF9 expression level In vitro, adhesive and motility experiment were used to assay cell metastasis ability after alter TM4SF9 protein expression level. The result showed that colon cancer cells' adhesion ability was enhanced after up regulated TM4SF9's expression level, vice versa. But alteration of TM4SF9 protein expression level didn't has significant effect on colon cell lines' mobility.From the results above, we may draw conclusions as following:1. LST, SW480 and Lovo cell lines have different metastasis potency.2. LST, SW480 and Lovo cell lines can express TM4SF9, and the one that has higher metastasis potency has higher expression level..3. Colon membrane mucosa cell can express TM4SF9, and in contrast with normal colon membrane mucosa cell, tumor cells owned higher expression level than normal membrane mucosa cell.4. Colon cancer cells' adhesion ability were enhanced after up regulated TM4SF9's expression level, vice versa.5. Alteration of TM4SF9 protein expression level didn't has significant effect on colon cell lines' mobility.