| Background:Colorectal cancer(CRC) is one of the most common malignant tumor in the world,the CRC patients are always in late stage when diagnosed,five year survival rate is low.While colorectal advanced adenoma is the most important precancerous lesions of CRC.A large amount of data shows,the patients of TNM stage I could be cured by surgery,the 5 years of survival rate of stage II has been close to 78%,while the 5 years of survival rate of stage III and IV has been less than 40%.So the research of exploring the best methods to screen CRC of TNM I II stage and CRA has high clinical significance.Recent study showed,mi R-135 b in human fecal exfoliated cells expressed higher in CRC and CRA than controls,the difference had statistical significance,however,its diagnostic significance was low,which may be related with the low efficiency of fecal exfoliated cells’ enriching methods.We used h NOK/h Sef monoclonal antibody-folic acid beads to enrich fecal exfoliated cells and evaluate its efficiency.We evaluate the significance of mi R-135 b in early CRC,CRA and normal controls’ fecal exfoliated cells for the diagnosis of CRC of TNM I II stage and CRA and its relation with the clinical characteristics of CRC patients.Objective:(1) To verify the expression difference of mi R-135 b in CRC tumor tissue and adjacent mucosa, and its relation with TNM stage.(2) To estimate the efficiency of h NOK/h Sef monoclonal antibody-folic acid beads.(3) To estimate the expression difference of mi R-135 b in fecal exfoliated cells in CRC of TNM I II stage and CRA,and its relation with the clinical characters of CRC patients.Methods:1. From September 2014 to December 2015,we collect 18 CRC patients who admitted to GI Endoscopy Center of Beijing Military General Hospital,all the patients above were confirmed pathological diagnosed,they had no other tumors or any treatment,such as surgery,radiation therapy or chemotherapy.We Collect the tumor tissue and adjacent mucosa of the patients when they were enduring the colonoscopy,the tissues should be put into the freezing tube immediately and stored in-80℃ refrigerator.Then we extract the total RNA of the tissues,examine its integrity by electrophoresis and the relative expression quantity of mi R-135 b by real-time PCR to verify the expression difference in CRC tumor tissue and adjacent mucosa, and its relation with TNM stage.2. From September 2014 to December 2015,we collect 102 patients who admitted to GI Endoscopy Center of Beijing Military General Hospital,including 52 cases of CRC of TNM I II stage,34 cases of CRA(diameter>1cm) and 16 cases of normal controls.ALL the patients above were confirmed pathological diagnosed,they had no other tumors or any treatment, such as surgery,radiation therapy or chemotherapy.Feces samples were collected when the patients were enduring the 4th diarrhea after they took catharsis drugs,then the samples were sent to library immediately.We used h NOK/h Sef monoclonal antibody-folic acid beads to enrich fecal exfoliated cells,then the samples were made into cell suspension and divided into parts,one part was used for cell counting and extracting DNA to examineβ-actin of the cells by real-time PCR; another part was used for extracting the RNA and examining the relative expression quantity of mi R-135 b by real-time PCR.We analyses the relationship of mi R-135 b in fecal exfoliated cells and the CRC patients’ clinical characteristics.Results:(1) The OD value of total RNA was 1.8-2.0,which meant the samples were not polluted,the electrophoresis graph showed 28s/18s/5s clearly,which meant the RNA was complete.(2) mi R-135 b in CRC patients with carcinoma tissue expression is significantly higher than in the adjacent tissues(P=0.021),while its expression level raised with the development of the TNM stages.(3) The cell counting was 1.0×106/ml before the reaction while the counting was 0.97×106/ml after the reaction;the β-actin was positive in all samples.(4) miR-135 b of fecal exfoliated cells in advanced adenomas expressed higher than that of normal controls,the difference was statistically significant(P=0.029);mi R-135 b in the early days of feces off colorectal cancer cells expressed higher level than that of normal controls,with statistically significant difference(P=0.038).mi R-135 b in colorectal cancer of TNMI,II of fecal exfoliated cells expressed no statistical difference(P=0.290).According to the result of ROC curve analysis,when the cut-off point >1.63,mi R-135 b of fecal exfoliated cells in advanced adenomas and CRC of stage I and II,the sensitivity were 76.92% and 78.24%,while specificity was 87.50% in normal controls,AUC were 0.70(P=0.02) and 0.80(P < 0.001).(5) The expression of mi R-135 b showed no relationship with the patient’s age,sex,tumor differentiation,TNM stage and their positions,the p values were all greater than0.05,which showed no statistical significance.Conclusions:(1) The expression difference of mi R-135 b in CRC tumor tissue was higher than adjacent mucosa, and the level raised with the development of the TNM stages.(2) The efficiency of h NOK/h Sef monoclonal antibody-folic acid beads was significant.(3) mi R-135 b of the fecal exfoliated cells has clinical significance in diagnosing CRA of TNM I and II and CRA,while the expression level showed no relationship with the patient’s age,sex,tumor differentiation,TNM stage and their positions. |
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