The Construction Of Yeast Two-hybrid System Of FasL In The Protein-interactions And Its Significance In Hepatic Metastasis Of Colorectal Carcinoma

Colorectal Carcinoma (CRC) is one of the commonest carcinoma in our country.Its morbidity and mortality rates are all the second top diseases in carcinoma of the digestive tract. The morbidity of CRC is increasingly higher year by year in recent years, but the total mortality descends a little, which is due to the increased diagnosis and treatment techniques. The key point of determining the prognosis of CRC is how to find, diagnose and treat earlier, and to prevent postoperated CRCs from recurring and metastasizing. So it is very important to investigate the mechanisms of its occurrencing, recurring and metastasizing. In nowadays, searching some new, more specific and hypersensitive makers to diagnose its recurrence and metastasis, and finding some molecular treating targets is the hot spot.Aims To construct the yeast two-hybrid system for the study of proteomics in the field of the protein-interactions, and to screen the proteins which interact with FasL, and to investigate the mechanisms of hepatic metastasis of CRC with FasL and its interacting proteins. To provide theoretical evidence for screening some earlier diagnosing makers and some molecular treating targets in the hepatic metastasis of CRC.Methods We cloned the FasL gene into the pGBKT? vector as the bait, then screened the fetal liver cDNA library, and have got a series of specific proteins that interact with FasL protein. By transferring their genes into human HR-8348 cells, we study their influences on the drug-resistance of CRC cells. We examinedthe expressions of these proteins in clinic postoperated CRC tissues to analyze the expression diffences between "different CRC TNMs. Using the bioinformatics, we analyzed the interacting proteins in the roles of the mechanism of hepatic metastasis of CRC.Results We screened several proteins that interaction with FasL protein, including metallothionein 1K-. 1G> 2A, cathepsin B, fatty acid synthase, interferonalpha-inducible protein 27, phospholipid scramblase, Ser/Thr-like kinase, anchor attachment protein, fibulin-5. In the experiments of drug-resistance, we verified that metallothionein and cathepsin could enhance HR-8348 cells with higher drug-resistance. By the means of immunohistochemistry, we have examined the expressions of FasL, ;.metallothionei.n and cathepsin in CRC tissues. The results showed that they expressed much higher in CRC tissues than in normal intestinal mucosa (p<0. 05), but there were no significance difference in various CRC TNMs p/0. 05) . Their coexpressions were higher in CRC tissues than in normal intestinal mucos, and the coexpressions of various CRC TNM tissues had no significance difference (p>0. 05) .Conclusions We had successfully constructed the yeast two-hybrid system, and preliminarily identified that the interaction between FasL, metallothionein, cathepsin, Ser/ Thr-like kinase, and anchor attachment protein were related to the drug-resistance and the hepatic metastasis of CRC cells. And the interaction between FasL, fatty acid synthase and phospholipid scramblase were related to the carcinogenesis and proliferation of CRC. Perhaps, FasL, metallothionein and cathepsin may become the makers of diagnosing CRC, and FasL may become the molecular treating targets of CRC and its hepatic metastasis.