Establishment Of DdPCR Detection Method For Serum Hsa＿circ＿0044235 And Its Preliminary Study In Breast Cancer Diagnosis

Breast cancer(BC)is the most common malignant tumor in women worldwide,which seriously endangers women’s health.With the rapid development of diagnosis and treatment technology,the cure rate and survival rate of breast cancer patients have been significantly improved.Due to the lack of specific diagnostic markers,the majority of breast cancer patients are diagnosed in the middle and late stages.If the optimal treatment window is missed,the patient can only receive more traumatic surgical treatment.Early screening techniques,such as mammography,ultrasound,CT and biopsy,have a certain rate of missed diagnosis.It is difficult to achieve early detection and early diagnosis of BC.The deficiency of early detection technology has led to the increasing prevalence of BC year by year,and the base of BC patients is constantly expanding.Therefore,there is an urgent need for an early screening technology with less trauma,high specificity and sensitivity.CircularRNA(circRNA)is a Non-codingRNA(ncRNA)with conserved structure,strong tissue specificity and good stability.It plays an important role in a variety of diseases such as tumors,cardiovascular diseases,nervous system diseases,and It’s a potential new serum marker.The content of circRNA in peripheral blood is low,and the ability of realtime fluorescence quantitative detection is limited.More accurate detection methods are needed to explore the relationship between circRNA content and BC.Droplet digital pcr(dd PCR)is a polymerase chain reaction technology with strong specificity,high sensitivity,little interference,and can absolute quantitative detection.It has unique advantages in measuring the trace circRNA in peripheral blood.This study mainly uses the detection method of dd PCR to study the content of hsa＿circ＿0044235 in serum,excavate new serum diagnostic markers of BC,and provide new examination ideas for early diagnosis,etiological mechanism exploration,and targeted therapy of breast tumors.Objective:1.This study aims to detect the content of hsa＿circ＿0044235 in the serum of breast cancer patients and healthy people using dd PCR,and to explore the molecular diagnostic value of hsa＿circ＿0044235 by comparing the difference in the expression of hsa＿circ＿0044235 in the serum of the two groups.2.Explore the formation source of hsa＿circ＿0044235 and verify its structural characteristics.3.Combined with clinical data,the relationship between hsa＿circ＿0044235 and the clinical manifestations of breast cancer was explored.Methods:Firstly,circRNA gene sequencing technology was used to determine the serum circRNA expression profile.By comparing the serum sequencing results of the control group and the patient group,hsa＿circ＿0044235 with significant difference in expression was selected as the research target.The source,structure and performance characteristics of hsa＿circ＿0044235 were analyzed by gene sequencing and other methods.The level of hsa＿circ＿0044235 in serum of breast cancer patients was detected by q RT-PCR and dd PCR.The serum molecular diagnostic performance of hsa＿circ＿0044235 was described by drawing the receiver operating curve(ROC).Finally,combined with clinical information,the role of hsa＿circ＿0044235 in the clinical manifestations of breast cancer was analyzed.Results:1.Serum circRNA gene sequencing results showed that the Fold change(FC)of expression was 1.5,P<When 0.05 was set as the standard,there were 109 differentially expressed circrnas in the serum circRNA expression profile,of which 58 circrnas were up-regulated and 51 circrnas were down-regulated.2.The dd PCR method for detecting serum hsa＿circ＿0044235 was successfully constructed.3.Hsa＿circ＿0044235 showed a trend of low expression in the serum of breast cancer patients.4.Hsa＿circ＿0044235 has good time stability and anti-enzyme stability,and can be used as a serum diagnostic marker for breast cancer.5.Hsa＿circ＿0044235 is correlated with clinical stage,pathological classification, surgical treatment,lymph node metastasis and other clinical information of breast cancer,suggesting that hsa＿circ＿0044235 may have a certain suggestive effect on the disease progression and efficacy evaluation of breast cancer,and play a positive guiding role in the mechanism research of breast cancer.