Establishment Of A Detection System For Serum Exosome MiR-940 As A Marker Of Breast Cancer

Exosomes are small vesicles with a size of 80-150 nm secreted by cells.Exosomes can be released by many kinds of normal cells and tumor cells under physiological and pathological conditions.The difference in the expression levels of circulating exosomes between healthy people and breast cancer patients can identify molecular markers that can be used to diagnose and predict the outcome of breast cancer.RT-qPCR is commonly used to detect miRNA expression levels in various types of cancer.However,there is no standard for internal reference miRNAs for RT-qPCR studies of serum exosomal miRNAs in breast cancer.Clinically,the influence of factors such as the difference in the extraction method of exosomes,the difference in the initial amount of serum samples,whether the sample is hemolyzed or not on the detection of serum exosomal RNA in breast cancer is not yet known.The study have pointed out that the expression level of miR-940 in breast cancer patients is low,but the relationship between the expression of miR-940 in exosomes and breast cancer has not been clear.In this study,five kinds of software were used to screen out reference genes suitable for normalization of serum exosomal RNA.And then the serum exosomal miR-940 was used to verify the reliability of the selected internal reference genes,and the effects of three exosomes extraction methods on exosomal miRNA levels,different initial serum levels,and serum exosomal miRNA expression levels under different serum conditions were compared.Finally,we found the relationship between serum exosomal miR-940 and patient clinical pathological data.The main research contents of this paper are as follows:1.We had screened the internal reference genes suitable for normalization of breast cancer serum exosomal miRNAs.Firstly,it was verified by RT-qPCR that the expression level of each candidate internal reference gene has no significant difference in serum exosomes between breast cancer patients and normal people.The applicability of candidate internal reference genes was tested by Ge Norm,Norm Finder,Best Keeper and △CT methods.Ge Norm shows that miR-1228 and miR-191 are the most suitable internal reference combination;Norm Finder shows that miR-191 is the most suitable internal reference gene;Best Keeper shows that miR-423 is the most suitable internal reference gene;△Ct method shows that miR-1228 is the most suitable.Finally,the four methods were synthesized by Ref Finder,and it was concluded that miR-1228 and miR-191 are the internal reference genes suitable for normalization of breast cancer serum exosomal miRNA.2.We had screened a sample processing method suitable for clinical detection of serum exosomes miR-940.First,We had used different internal reference genes to verify the stability of miR-940 expression.It was found that when miR-191 and miR-1228 were used as internal reference genes,the expression level of miR-940 was significantly different between breast cancer patients and healthy people.When other candidate genes were used as internal controls,there was no significant difference in the expression level of serum exosomal miR-940 between breast cancer patients and healthy people.The RNA content of exosomes extracted by hypervelocity centrifugation method is relatively low.If the gene content is relatively low,it is not suitable to use hypervelocity centrifugation method to extract exosomes and subsequent RNA detection operation in clinical practice.However,there were no significant differences in exosomes and subsequent RNA levels between membrane affinity and precipitation methods.When the serum volume doubled,the CT value of the serum exosome miRNA decreased for three cycles,and the PCR signal increased linearly with the serum volume.In hemolytic blood,the expression levels of miR-16,miR-940 and miR-1228 extracted exosomes were significantly higher than those of unhemolyzed serum exosomes.3.We had verified the expression significance of serum exosomes miR-940 in breast cancer.According to the established standard curve of miR-940 copy number,we had calculated the copy number of exosomal miR-940 in each sample.We found that the copy number of serum exosomes miR-940 in breast cancer patients was significantly lower than that of normal people.It was also found that the level of serum exosomal miR-940 in HER2/neu-positive patients was significantly lower than the expression of serum exosomal miR-940 in HER2/neu-negative patients.The copy number of serum exosomes miR-940 in breast cancer patients without lymph node metastasis was significantly higher than that of patients with lymph node metastasis.Finally,it was found that miR-940 can be delivered to breast cancer cells and breast epithelial cells through exosomes,changing the level of miR-940 in target cells and inhibiting the proliferation and migration of target cells.