A Study On The Screening Of Calreticulin As A Marker For The Diagnosis And Treatment Of Bladder Cancer By Lectin Chip

Objective:Until now,the gold standard for diagnosing bladder cancer was cystoscopy and biopsy.At present,there are no reliable noninvasive diagnostic markers for the diagnosis of bladder cancer and its pathological classification.In this study,lectin chip technology was used.Detection of non-muscle-invasive bladder cancer(NMIBC)and muscle-invasive bladder cancer(NMIBC)cancer,MIBC)and urine glycoprotein glycochains in healthy adults,to search for glycoprotein markers associated with the occurrence and development of bladder cancer,so as to serve as a new marker for the diagnosis of bladder cancer.Method:(1)Lectin chips were constructed.Five groups of urine protein samples from NMIBC patients before and after surgery,MIBC patients before and after surgery and healthy people were incubated with the lectin chips,and then fluorescence intensity was measured by scanning.(2)The results of lectin microarray were analyzed,and the three lectins with the largest difference in fluorescence intensity in NMIBC patients before and after surgery were selected to enrich glycoproteins,and then the differentially expressed glycoproteins were verified by SDS-PAGE gel electrophoresis,Coomassie bright blue staining and WB experiment.A high abundance and high weight coefficient glycoprotein was selected by mass spectrometry.(3)Three groups of aptamers with the highest binding force were selected by SELEX assay.The optimal aptamer was constructed by aptamer sandwich method.The expression level of the target protein in the five groups of samples was detected by ELONA sandwich method.Result:(1)After lectin microarray scanning,it was found that the expression levels of four lectin recognized sugar chains,LTL,WFA,AAL and PHA-E+L,were significantly increased in the group before NMIBC compared with those after NMIBC.The comparison between preoperative and postoperative MIBC showed that the expression of sugar chain recognized by Con A、PSA and Jacalin was significantly increased.Compared with healthy people before and after NMIBC,there were significant differences in the expression levels of sugar chains recognized by various lectins,such as DSA,ACA,PWM,PHA-E+L,and LTL.(2)LTL,WFA and AAL were selected to enrich glycoproteins.Coomash bright blue staining showed that LTL and WFA mainly enriched glycoproteins with molecular weight slightly lower than35 k D,while AAL mainly enriched glycoproteins with molecular weight less than 15 k D.WB experiment also found three distinct protein bands.Calcium reticulin(CRT)with high abundance and high weight coefficient was screened by mass spectrometry.(3)Three groups of aptamers with the highest binding ability to the target protein were screened by SELEX technology.The expression level of the protein was detected by ELONA sandwich method,and the results were consistent with the screening results of the lectin chip.Conclusion:(1)There were differentially expressed glycoprotein chains in the five groups of samples,and the protein was calreticulin.(2)calretin has the potential to be used as a diagnostic and/or prognostic marker for bladder cancer.(3)Aptamers screened by calretin can be used as a powerful tool for urine detection and have a good application prospect.