Construction And Application Of Methods For Screening And Validating Breast Cancer Biomarkers

Breast cancer is the main malignant tumor affecting women’s health all over the world.The incidence of breast cancer is the highest in female malignant tumor.The research of breast cancer has great social and medical significance.Currently,the number of breast cancer biomarkers for diagnosis and treatment is limited,so more meaningful biomarkers should be screened out.In tradition,tumor tissue samples are used in biomarkers screening,but many tissue samples can not be got from patients who are not able to be operated.Due to the rapid growth of tumor cells,the adhesion force between cells is low.A large number of tumor cells will be off and into the circulating blood to become circulating tumor cells(CTCs).Compared with solid tumor tissues,the single-CTC has the complete genetic information of tumor cells without interference from other cells or nucleic acids.Therefore,it’s necessary to establish a screening method based on a single-CTC for screening tumor markers.However,single-CTC screening platform need to overcome the following key technologies:(1)efficient CTC capture method.CTCs can high sensitivity obtained by using the capture method;(2)CTC identification method without damnification.The captured cells can be identified without affecting the detection of single cells RNA;(3)determination method of single cell transcriptomes profiles,which can bu used to analyze the gene expression in single cells,in order to find out the molecular markers.First of all,to capture CTCs,we used immunomagnetic enrichment technique combined with fluorescent staining.The detection rate of the health people was 0%,and the detection rate of patients was 44%.Moreover,we proved that there is significant correlation between the quantity of CTCs,age,ER,PR and pathological type.And the quantity of CTCs can be used to predict the efficacy of neoadjuvant chemotherapy.Furthur more,axillary lymph node dissection was verified to cause the spread of CTCs.Secondly,the quantity of CTCs can not reflect the molecular characteristics of the tumor.The abnormal expression is the important reason for the loss of tissue function.The tranditional tumor tissue detection is the average value of tens of thousands of cells actually.We hope to study the single-CTC level gene expression.CTC capture,stain and storage are the essential protocols for single-CTC mRNA quantification.However,the effects of these operations on mRNA levels in a single-cell have not be reported in detail.Here we investigated the effects of capture process,stain reagents and storage conditions on mRNA quantification of a single-cell by using the solid phase-reverse transcription assay coupled with qPCR.The results showed that CTC capture by antibody coated nanospheres did not affect the mRNA levels in a single-cell.While,washing step would cause mRNA loss unless the RNase inhibitor added in the washing buffer.Moreover,the fixing and permeabilization solutions caused the dramatical loss of mRNA in a single-cell,indicating that we should use small molecular dye,which could permeate through cell membrane by a diffusion process,or choose surface targets for CTC identification to avoid the use of the fixing and permeabilization solutions.Furthermore,the isolated single-CTC should be frozen at-80℃ to avoid the distortion of mRNA in a single-cell if it could not be analyzed immediately.Our study comprehensively analyzed the effects of these operations on mRNA levels in a single-cell.Then,we need to amplification mRNA from pg to ng level in order to screen biomarkers by high throughput sequencing method on the basis of the above research.Therefore we established single CTC cDNA library construction technology.The introduction of ddATP to control the tail length of polyA has greatly improved the efficiency of cDNA generation.In addition,according to the RNA quantity of single CTC in actual clinical samples is less than tumor cell lines,we increased the PCR amplification to 3 times.cDNA library established can meet the requirements of high throughput sequencing.Finally,we collected a group patients of the lymph node metastasis and a group patients of lymph node without metastasis with first-episode.We preliminary found 198 gene which expression is differences between the two groups by screening mark on the transcriptome of organization level.Specific markers of lymph node metastasis with first-episode were riched in the ECM-receptor interaction pathway.The markers selected by scRNA-seq need further verification.Different expression may be derived from copy number variation on the DNA level and may also represent functional deficiency on the protein level.We established a high sensitive and non-invasive method based on digital PCR to verify the copy number variation from the DNA level;Further,we constructed a versatility and low-cost knockout tool enzyme that can knockout the potential markers of target gene to validate the loss of function on protein levels in vivo.In the respect of methodology,mainly according to clinical demand the paper constructed molecular markers screening method based on single CTC transcriptome analysis,digital PCR which can be used to verifing copy number variation and knockout enzyme which can be used to verify function of markers.We detect the clinical samples by using these methods.