Research And Application On The Expression Of Clusterin In Esophageal Squamous Cell Carcinoma

Background The carcinoma of esophagus, mostly is esophageal squamous cell carcinoma (ESCC), is one of the most frequently diagnosed malignancy in the world , and is the second killer of malignant tumors in China. Esophageal carcinoma has endangered people's health and life greatly. There is high mortality of esophageal carcinoma in our country. The exact reason caused ESCC has remained unknown yet, many kinds of complication and genes may involved in the development of this illness. The traditional methods used to diagnose ESCC are mainly by X-ray or endoscopy and biopsy. It has been an instancy to find a more useful and easier method for the diagnosis of ESCC. It may provide a simpler and more useful tool to diagnose and screen carcinoma of esophagus, via identifying tumor-related biomarker especially those serum proteins, but no specific cancer-related biomarker of esophageal squamous cell carcinoma is found still.Clusterin is a widely distributed protein in many kinds of body fluids, and is involved in many physiological processes, such as its functions in the interaction of cell-cell, membrane lipid recycling, in apoptotic cell death, and as a stress-induced secreted chaperone protein, and others. The importance of clusterin in the appearance and development of tumor hasbeen hotspot recently. Many scholars have found the abnormal expression of clusterin in many kinds of disease.but the expression of clusterin in ESCC has not been detected still.With the support of the Special Funds for Major State Basic Research Project (973 project), we have found that clusterin deleted in the tissue and serum of ESCC, by 2-diamensional electrophoresis and mass spectrometry. This task is to explore the change of its gene in ESCC, and the change of this protein in the serum of ESCC, in order to find a biomarker to screen and diagnose ESCC.Objective To investigate the change of clusterin gene in ESCC tissue and mechanism of this abnormal expression; and find the deletion or miss splicing of ESCC gene by multi-region RT-PCR , in order to discover a new target used to the diagnosis and treatment of ESCC. By comparing the level of sera clusterin via statistic analysis, we find the standard of serum clusterin level used to diagnose ESCC. We also analyze the relationship of serum clusterin and clinical guidelines of ESCC, demonstrating the feasibility of clusterin to be a biomarker of ESCC.Materials and Methods Both ESCC and normal esophageal tissues, also the sera, are from the patients of Cancer Hospital, Chinese Academy of Medical Sciences. All the cases are diagnosed by pathologist. Semi-quantitative RT-PCR for multi-region alteration analysis, and ELISA for serum conceration of clusterin level, are processed. Total RNAs were isolated from ESCC and so the consulting specimens. Reverse transcription reactions were performed on 5.0/xg of total RNAs using SuperScriptTM First-Strand synthesis for RT-PCR. The PCR steps were performed using TaqDNA polymerase. The amplified multiproducts were analyzed on 12-20g/L agarose gels. Each PCR reaction was done triplicate. Quantitative analysis of the serum clusterin of ESCC and matched pairs is performed by human clusterin EIA kit, according to the manufacture's instruction. Constitute standard of serum clusterin for the diagnosis of ESCC via ROC curve. Quantitative data are analysed via SPSS 10.0, p<0.05 is regarded tobe meaning.Results This study compared the difference of clusterin gene of esophageal squamous cell carcinoma and matched normal tissues by semi-quantitative RT-PCR for analysis, data showed that clusterin gene is significantly downregulated in cancer tissues. Also there is an N-terminal deletion or miss splicing located on lbp-437bp, the N-terminal deletion of clusterin may be essential for its alterations of biogenesis in carcinoma of esophagus. We found that the conceration of clusterin of "healthy" serum is 76.0U66.60w g/ml, while 4.40±4.80 u g/ml in ESCC sera [p<0.00\). And the concentration of clusterin is a relatively independent index regardless the level of albumin, bilirubbin, or cholsterone, sugar etc in sera.Conclusions Clusterin, a 75-80kDa heterodimeric, disulfide-linked glycoprotein is expressed in a wide variety of tissues and secreted in all human fluids. Clusterin has been found highly conserved and implicated in a variety of biological processes. Abnormal expression of clusterin, both protein and mRNA, has been established in diseases where either abnormal cell death or proliferation is occurring. For the first time, we find that there was gene down-regualtion or deletion in ESCC tissue, and suggest that there was an N-terminal deletion or miss splicing located on lbp-437bp that induced a wrong transcription form of the clusterin in ESCC, and may have close relation with the tumorgenesis of ESCC. We also create a referenced standard of sera clusterin conceration through ELISA, in order to find an easier way used for the screen and diagnosis of ESCC. We admit this view that the significance of alterations of clusterin gene expression during tumorigenesis remains a mystery. Its possible roles in cell survival, cell death and neoplastic transformation remain an additional debate.