Bioinformatics Analysis Of CST Gene Family And Expression Of CST2 In Breast Invasive Ductal Carcinoma

Background: The incidence and mortality of breast cancer in women are increasing year by year,and the molecular mechanism leading to the development of breast cancer remains unclear.The changes in the expression levels of many genes and proteins play an important role in the occurrence and development of breast cancer.Therefore,it is particularly important to explore the changes of expression levels of related genes and proteins during the development and progression of breast cancer.Objective: This study found that CST family members have different expression variations in different types of breast cancer databases by querying the cBioProtal database and are associated with prognosis in breast cancer patients.The evolutionary relationship analysis of CST family was carried out to determine the structural characteristics and related information of the family members.The expression of CST2 in breast invasive ductal carcinoma tissues and control tissues was examined,and the differences in expression of CST2 in tumor tissues and control tissues were discussed.Relationship with clinicopathological features.Methods: 1.Analysis of evolutionary relationship of CST family:(1)download all known human genes,identify eligible CST gene family members and annotate.(2)Building a phylogenetic tree from the protein sequences of CST gene family members.(3)Domain,conserved component analysis and functional annotation of family member protein sequences.(4)Analysis of intron and exon information of CST family members.(5)Determining the location of CST family members on the chromosome.2.Total RNA was extracted from 10 pairs of breast invasive ductal carcinoma and benign breast lesions.Six pairs of cancer and normal control tissue RNA samples were selected for whole transcriptome sequencing.Eight pairs of RNA samples into cDNA were selected for real-time quantitative PCR to analyze the expression changes of CST2 mRNA transcription level.3.Immunohistochemistry was used to detect the expression of CST2 protein in 74 cases of invasive ductal carcinoma of the breast and 35 cases of benign breast lesions.The relationship between CST2 protein expression and clinicopathological parameters was analyzed.Results: 1.Cystatin family analysis results:(1)Annotation gives information on the gene ID of 42 CST family members,the location of each gene on thechromosome and the length of the gene sequence.(2)According to the evolution tree,all members are divided into Group I to Group III,all members contain the Cystatin domain.(3)Family members each contain 1-6 motifs,each of which varies in length from 11 aa to 50 aa.(4)Family members contain 1-11 exons,ranging in length from 500 bp to 26 Kbp.(5)Family members are scattered on five chromosomes,and the distribution is not obvious.2.Transcriptome sequencing results showed that CST2 mRNA was significantly elevated in 6pairs of breast invasive ductal carcinoma tissues compared with adjacent normal tissues.3.qRT-PCR showed that CST2 mRNA was significantly higher in 8 pairs of breast invasive ductal carcinoma than in adjacent normal tissues(P<0.05).4.Immunohistochemistry results:(1)CST2 protein is only expressed in small amounts in breast invasive ductal carcinoma cells,and most of the expression in myoepithelial or basal cells of benign breast lesions.The positive rate of CST2 in breast invasive ductal carcinoma was significantly lower than that in benign breast lesions(P<0.05).(2)The expression of CST2 was correlated with age(P<0.05),but not with tumor size,ER,PR,Her-2 status,lymph node metastasis and TNM stage(P>0.05).Conclusions: Bioinformatics analysis clarifies the evolutionary relationship of CST2 family members and provides valuable bioinformatics information for further study of the CST family.CST2 mRNA expression is elevated in invasive ductal carcinoma of the breast.CST2 protein is expressed in cancer cells of breast invasive ductal carcinoma,and most of the muscle epithelial or basal cells of benign breast lesions are expressed in breast cancer.Protein expression is related to age of breast cancer patients.