ABSTRACT
Objective Recently, the incidence of breast cancer has been the highest among female malignant tumors. Therefore, potential biomarkers are urgently needed to predict and prevent breast cancer. This study was aimed to explore the expression and clinical significance of differential miRNAs and their target genes in breast cancer by establishing miRNA expression profile in breast cancer tissues. Methods From January 2015 to December 2018, a total of 137 cases of breast cancer tissues with paired paracancerous tissues and 20 cases of breast fibroadenoma tissues were collected from the department of breast surgery, affiliated hospital of Guizhou Medical University. The tissues were divided into breast cancer group, paracancerous group, breast fibroadenoma group and lymph node metastasis group. High-throughput sequencing technique was used to detect the miRNAs in breast cancer tissues and paired paracancerous tissues. Real-time PCR verified the expressions of the three miRNAs with the most significant expression differences in different groups. Finally, bioinformatics analysis was used to predict the target genes and investigate the expression of miRNAs and proteins of target genes in different breast diseases. Results A total of 157 upregulated and 162 downregulated miRNAs were screened by high-throughput sequencing. Mir-hsa-miR-532-3p and hsa-miR-1260b were the most significant in upregulated miRNAs while has-let-7c-5p was the most significant in downregulated miRNAs. Meanwhile, bioinformatics analysis showed that their target gene was EZH2. Compared with para-cancerous group, expressions of hsa-miR-532-3p and hsa-miR-1260b were significantly upregulated while hsa-let-7c-5p was significantly down-regulated in breast cancer group and lymph node metastasis group (all P<0.05). The miRNA expressions of target gene EZH2 in breast cancer group, breast fibroadenoma group and lymph node metastasis group (1.24±0.01, 4.02±0.01, 15.97±0.01, respectively) were upregulated when compared with the para-cancerous group (1.00±0.00), and the similar conclusion could be drawn in EZH2 protein expression. Conclusion Hsa-miR-532-3p, hsa-miR-1260b and hsa-let-7c-5p were closely related to breast cancer, which may promote the occurrence and development of breast cancer by inducing the transcriptional expression of EZH2. HsamiR-532-3p, hsa-miR-1260b, hsa-let-7c-5p and EZH2 may be potential tumor biomarkers of breast cancer.
ABSTRACT
To synthesize salbutamol immunogen and develop an enzyme immunoassay (ELISA), a new salbutamol immunogen was synthesized using 4-aminobenzoic acid as a linker to connect hapten with carrier protein. An enzyme immunoassay based on the antibody prepared was developed and applied to detect salbutamol residue spiked in swine liver. An unusual coating antigen, clenbuterol-ovalbumin (OVA) conjugate instead of salbutamol-OVA conjugate, was used in the immunoassay and the results were discussed based on the structures of related compounds. The antibodies showed high sensitivity in the heterologous assay when using clenbuterol-OVA as a coating antigen, with an IC50 value of 8.97 ng mL(-1) toward salbutamol. The antibodies prepared showed high cross-reactivity with clenbuterol (107%) and were promising for the simultaneous determination of salbutamol and clenbuterol residues in food and food products. Recovery rates from the salbutamol-spiked swine liver samples were in the range of 70%-99%, while the intra-assay and inter-assay coefficients of variation were <13.3% and <14.3%, respectively. In summary, the antibodies of salbutamol have been successfully prepared. Sensitive and stable analysis for the detection of salbutamol residues in swine liver was obtained based on the competitive ELISA methods developed in this study.