Research On The Role And Mechanism Of MiR-140-3p In Breast Phyllodes Tumor

Study objectivesPhyllodes Tumor(PT)and Fibroadenoma(FA)belong to fibroepithelial tumors,which are a heterogeneous group of bidirectionally differentiated tumors with proliferation of both epithelial and stromal components.PT is divided into benign,borderline and malignant according to histological features,of which the most common is benign phyllodes tumor(BPT),accounting for approximately 60%-75%of all phyllodes tumors.The clinical manifestations of FA and BPT are similar.There are many studies on clinical data between the two groups,but the results are different due to different sample sizes.Collecting and analyzing the clinical data of FA and BPT has certain clinical significance for understanding the two diseases.At the same time,BPT and FA are difficult in clinical practice in differential diagnosis,and both have similar clinical,imaging and cytopathological findings.Existing diagnostic methods such as ultrasound,molybdenum target,magnetic resonance imaging and needle biopsy are difficult to provide accurate differential diagnostic results,so it is of great clinical significance to find new and reliable tumor markers for differentiating BPT from FA.Benign phyllodes tumor,borderline phyllodes tumor and malignant phyllodes tumor are different stages of breast phyllodes tumor.For the treatment of PT,surgery is the main treatment.For malignant PT,the role of radiotherapy and chemotherapy is not clear,and the mechanism of its occurrence and development is also unclear.In this experiment,the molecular mechanism exploration study in BPT primary cells as well as HT1080 cell lines derived from the same mesenchymal tissue is important to study the development of PT.Study methodIn this study,we collected the data of 981 typical FA cases from 2014 to 2020 and 156 BPT cases from 2009 to 2020 in Qilu Hospital for retrospective analysis to understand the similarities and differences in clinical data between the two.The DEmiRNAs(differential expression microRNAs)between BPT and FA were selected by miRNA-Seq technology,and the stability and difference of their expression were further verified by qRT-PCR(Quantitative Real-time PCR)in primary cells and fresh tissues,so as to find miRNAs that can be used as differential markers of FA and BPT.The downstream targets of miRNAs were predicted by multiple Shengxin websites,and the results were intersected to speculate the downstream target genes of the correlation.The correlation between miRNAs and their downstream targets was then verified by westernblot,immunohistochemistry with dual-luciferase reporter assay.The expression of miR-140-3p in HT1080 cell line was up-regulated or inhibited by transfection technique to observe the effect of miR-140-3p on HT1080 cell line,so as to speculate the possible role and mechanism of miR-140-3p in PT,especially malignant PT.The pathway and target of miR-140-3p were further explored by cell counting,scratch assay,cell cycle detection,apoptosis detection and other techniques to deepen the study and exploration of PT.ResultsIn terms of clinical data,compared with FA patients,BPT patients were on average 4.22 years older,with an average long diameter of about 1.48 cm longer,the probability of mass occurrence and the rate of mass occurrence were lower,and the proportion of patients with reproductive history and lactation history was higher;there was no significant difference between the two groups in terms of disease duration,menopausal status and family history of cancer.Three statistically significant DEmiRNAs with fold changes greater than 1.2 and high expression were selected by miRNA-Seq technology,which were miR-140-3p,miR-126-5p and miR-10b-5p.After further validation by qRT-PCR in fresh specimens it was found that the expression difference of miR-140-3p was statistically significant between the two groups,and the miR-140-3p expression level in the BPT group was down-regulated by about 70%compared with the FA group.The same differential trend was also reflected in primary cells.The Shengxin website predicted and took the intersection to obtain 46 downstream target genes of miR-140-3p.The results of Western blot showed that the contents of PD-L1 and BCL2 in fresh specimens of BPT group were higher than those of FA group,but they were not statistically significant.Immunohistochemical results showed that the expression levels of PD-L1 and BCL2 in paraffin specimens of malignant PT were much higher than those in benign PT,borderline PT and FA specimens.In HT1080 cell line,the results of westernblot assay showed that the expression levels of PD-L1 and BCL2 were decreased in the high miR-140-3p expression group,and vice versa in the low expression group.The results of the scratch assay showed that the migration ability of the high miR-140-3p expression group was lower than that of the low expression group.The results of dual-luciferase reporter assay showed that when miR-140-3p was highly expressed,the fluorescence intensity of the WT plasmid group was significantly decreased compared with the MUT plasmid group,and the same trend appeared in the PD-L1 and BCL2 plasmids,which was statistically significant.The results of flow cytometry showed that there was no significant difference in the cell cycle of cells in the high miR-140-3p expression group,but the proportion of apoptosis in cells in the high miR-140-3p expression group was significantly higher than that in the low expression group.Conclusion1.BPT is better in older and obese people.BPT masses are more common than FA masses.BPT masses with a long diameter of more than 5 cm are more common.At the same time,BPT occurs more frequently and simultaneously than FA.2.miR-140-3p expression differed significantly between FA and BPT and has the potential to be used as a differential marker between the two.3.PD-L1 and BCL2 are downstream targets of miR-140-3p.4.In the HT1080 cell line,highly expressed miR-140-3p significantly inhibited the growth rate and migration ability of the cells and promoted apoptosis,but had no significant effect on the cell cycle.