MiRNA Profiling Reveals A Potential Role Of Milk Stasis As Microenvironment In Breast Neoplasia

Objection: The incidence of breast cancer is rising,。Breast cancer gradually become thefirst malignant tumor in women. Breast cancer cells mostly change from epithelial cells. Inthe clinic, we find that many patients with milk remains in their breast instead of dischargingduring feeding, resulting in ‘milk stasis’. The duration required for milk stasis for milk stasisranges from several years to more than30years with nomal PRL. Tumor microenvironmentplays an important role in breast carcinogenesis. Milk acts as an important microenvironmentof breast cancer, but its role in breast carcinogenesis is largely unknown. Recently peoplefound miRNAs in human body fluid such as serum, milk,saliva and so on. These miRNAs aredirectly related to the cell activity. So, studying the miRNA profiling between milk stasis onlyand milk stasis plus tumor group can help us to find the changes of microenvironment forepithelial cell, and also find the function of special miRNAs in neoplasia.Methods: Samples were collected by lavaging the milk ducts from the two groups. Afterisolating total RNAs using miRNA Isolation kit, we did high-throughput sequencing andselected miRNAs from full lengh small RNAs. Then we got miRNAs profiles with differentexpression in the two groups, also got novel miRNAs. For the candidate of miRNAs, we didtarget genes prediction and metabolic pathway analysis of possible biological processes. Realtime RT-PCR was used to verify the expression of miRNAs in20samples. Finally wecompared the expression between milk stasis samples and the same patients’ serum in7pairsof samples.Results: We successfully extracted miRNAs from the two groups. We identified266known miRNAs and271novel miRNAs in10milk stasis only samples,271known miRNAsand140novel miRNAs in10milk stasis plus breast tumor samples by high-throughput sequencing. MiRNA profiles were different between the two groups. Furthermore,we foundten significantly down-regulated miRNAs such as let-7,miR-29a, miR-146and miR-223,while seven significantly up-regulated miRNAs such as miR-451,miR-185,miR-107,miR-92,miR-10were in the milk of milk stasis plus tumor patients. We analyzed the results of targetgenes prediction such as molecular function, cellular component and biological process, alsogot metabolic pathway analysis of possible biological processes,3in milk stasis only groupand9in milk stasis plus tumor group. We identified13miRNAs which high expressed in bothgroups with no signiftant difference, especially miR-140. Three kinds of the identifiedmiRNAs were selected using real time RT-PCR, confirming that these miRNAs were highlyexpressed. The results also showed that those three kinds of miRNAs were more abundant inthe milk than in the blood.Conclusion: We make the first miRNA profiling in milk stasis and we found thehigh-expression miRNAs all took part in the “regulation of cancer inordinately”. Significantlydifferent expression miRNAs could regulate the “adhesion junction” pathway. We found10miRNAs significantly down-regulated in milk stasis plus tumor group,6tumor suppressor,1oncogene and3dual character.There might be a close relationship between them andinformation transmission by ‘MAPK signaling’ pathways. Suggesting that they mightcontribute the microenvironment of epithelial cells as a role of suppressor.7miRNAs(5oncogene,1tumor suppressor and1dual character), most target genes are on themembrane,significantly up-regulated in milk stasis plus tumor group might act as oncogenein the microenvironment joined nine pathways about neoplasia and information transmissionamong epitheliar cells. miR-140might be internal reference.MiRNAs in the milk could be abetter biomarker for breast tumor than in the blood.