The Complexity And Diversity Of The TCR β-chain CDR3 Repertoire In Bladder Cancer Using High-throughput Sequencing

Background:Bladderr cancer is a common malignant tumor,which the incidence of the disease is increasing year by year,the treatment effect and prognosis is poor,which can be life-threatening and serious harm to human health.Its exact pathogenesis is not fully understood.The search for new biological targets for bladder cancer surveillance and intervention has become an urgent problem to be solved.Recent studies have shown that tumor microenvironment is closely related to the development and prognosis of bladder cancer.T cell plays a central role in the tumor microenvironment.But it is basically based on the study of the changes of T cell subsets and molecular typing,and we need to further study the sequence information and diversity variation of T cell receptor(TCR)combined with tumor antigen.However,the immune repertoire can reflect the polymorphismof B cell receptor heavy and light chain,and T cell receptor alpha,beta,gamma and delta chains,can deeply dig the relationship between immune repertoire and disease.High-throughput sequencing is a massively parallel sequencing technology,based on a lower cost by generating a large number of short sequence provides several orders of magnitude higher throughput reading.Faster high-throughput data obtained on the basis of lower cost compared to Sanger sequencing.A sequence of hundreds and thousands of DNA molecules can be used to analyze the transcriptome and genome of a species.Objective:To study the complexity and diversity of T lymphocyte immune repertoire in bladder cancer patients,objective and veritably assessment immune state of bladder cancer,elucidate the etiology and pathogenesis of bladder cancer,and provides a new theoretical basis for disease prevention,diagnosis,treatment and prognosis monitoring.Methods:Collect 5 cases of surgical removal of the fresh frozen storage of bladder cancer tissue(cancer group)and its corresponding normal tissue adjacent to cancer(control group),in which Whole genome DNA were extracted.Designing primers for the region of V family and J family,to amplify the whole CDR3 area of T cell receptor(TCR).Combined with high-throughput sequencing technology and platform,to analyze the region of CDR3 diversity,length distribution characteristics and the expression frequency of each gene family of TCR beta chain in T lymphocytes of cancer group and control group.To compare the length distribution of CDR3 amino acid sequence,the frequency distribution of V,D,J and V-J combinations,and top 20 V gene usage of cancer group and control group.To screen the highly clonal amplification DNA sequence(frequency greater than 0.5%),amino acid sequence(frequency greater than 0.5%)and V-J combination(frequency greater than 1%),the special/share DNA sequence,amino acid sequence and V-J combination from cancer group and control group.In addition,we analyze the TCR diversity and differentially expressed clones according to Shannon entropy.To reveal the immunological characteristics of T lymphocytes in bladder cancer tissues and paracancer tissues,to elucidate the pathophysiological mechanism of bladder cancer,and to find new biomarkers and therapeutic targets.Results:1.The frequency of length distribution:The 5 most frequently observed CDR3 amino acid sequences lengths in cancer group were 14,11,12,13 and 10nt.The 5 most frequently observed CDR3 amino acid sequences lengths in control group were 11,13,12,10 and 14nt.Gauss distribution fitting values of cancer group and control group respectively were 0.83 and 0.98.The results showed that the CDR3 amino acid sequence length distribution of the control group was more tend to normal distribution.2.Clonal amplification degree:Compared with the control group,the highly clonal amplification(frequency greater than 0.5%)DNA sequences and amino acid sequences increased in cancer group,and clonal amplification degree was general increased.3.TCR diversity:By calculating the Shannon entropy coefficient(0.40 VS 0.59)of cancer group and control group,we found that the diversity of the cancer group was lower than that in the control group.4.Highly expanded clones(HEC)sequences:After statistical analysis,we screened the HECs in cancer group and control group,at the resolutions of DNA sequence(32 vs 14),amino acid sequences(35 vs 14).5 Highly expanded clones DNA sequences and 7 Highly expanded clones acid sequences were found in cancer group,which could be used to develop therapeutic targets and biomarkers for prognosis monitoring.5.There were significant differences in the usage frequencies of TRBV,TRBD,TRBJ,and V-J combinations between cancer group and control group.Conclusion:1.Compared with the paracancer tissue,the degree of T cell clonal expansions in the bladder cancer tissue was higher,and the immune diversity of bladder cancer tissue was significantly lower.2.The DNA sequence,amino acid sequence and V-J combination level can be used to comprehensively understand the diversity and characteristics of TCR CDR3 in bladder cancer tissues and paracancer tissues,can be used to evaluate the immune status of bladder cancer accurately and objectively,can help us to deeply understand the pathogenesis of bladder cancer,to develop therapeutic targets and biomarkers for prognosis monitoring.