Establishment Of Real-time Quantitative PCR Assays For Detecting Il-17 Of Triple Negative Breast Cancer

Background:Breast cancer is the most common malignant tumors in females worldwide.The occurrence of breast cancer is the highest among all the malignant tumors in women and it's death rate is the second cause of fatality of female malignant tumors.According to the gene expression characteristics of the specimens,breast cancer is divided into four subtypes:Luminal A,Luminal B,HER2 over-expressing and basal-like or triple negative type.Different breast cancer subtypes have different cell surface receptors,the treatment and prognosis of different subtypes were distinctly different.Triple negative breast cancer?TNBC?means that breast cancer cells express none of estrogen receptor,progesterone receptor and human epidermal growth factor receptor 2.Triple negative breast cancer has attracted widespread attention because of its poor prognosis,high recurrence and metastasis rate.Interleukin-17?IL-17?is mainly secreted by T cells.It is a central member of the mammalian immune system and has the property of promoting the development of inflammation.It can act on a broad of inflammatory cells to induce the secretion of cytokines,chemokines,metalloproteinases and the like.In recent years,IL-17 has been found to play an important role in tumor immunity and treatment prevention.However,the expression of IL-17 protein in many tissues is rather limited,whereas the IL-17 mRNA expresses in many tissues.Therefore,the purpose of this study is to establish a method to stably detect the IL-17 mRNA level,and detect the expression of IL-17 in TNBC,study the relationship between IL-17 and TNBC to provide new ideas for exploring the pathogenesis,diagnosis and treatment of TNBC.Objective:To establish a real-time quantitative PCR method for the detection of IL-17,and use this method to detect the level of IL-17 mRNA in triple negative breast cancer tissues.Furthermore,the relationship between the expression of IL-17 mRNA and clinicopathological feature in TNBC tissues was analyzed.Methods:1.Acquisition of fresh breast tissues Fresh human breast tissues were obtained from Department of Thyroid/Breast and Vascular Surgery of XiJing Hospital from January 2016to March 2017,snap frozen and stored in a-80?refrigerator,and part of each tissue sample was isolated to be fixed in formalin for pathology detection to determine the pathogenic type of breast cancer tissues.2.Pathological examinationThe well-fixed specimens were embedded in paraffin and stained with hematoxylin-eosin?HE?.The histopathological changes of breast tissues were observed with optical microscope to determine the tissue samples as breast cancer tissue or normal breast tissue.Then immunohistochemical staining was used to determine the breast cancer tissue as triple negative breast cancer so that they can be used in this study.3.Primer design and synthesisThe sequences of human IL-17 and?-actin in Genebankwere selected as references,?-actin as internal reference,and Primer 5software was used for analysis and the region with high homology was used to design primers in this study and these two primers were both synthesized by Takara?China?Biotech.4.Total RNA extraction and reverse transcription Trizol method was used to extract total RNA from triple negative breast cancer tissues and normal breast tissues,then the total concentration of extracted RNA was quantified by NanoDrop nucleic acid analyzer.Reverse transcription was performed to synthesize cDNA which was stored at-20?refrigerator for spare.5.Determination of annealing temperature of IL-17 and?-actinAccording to the Tm values of IL-17 and?-actin primers synthesized by Takara?China?Biotech,the temperature gradient of 43?⁵8?and 50?⁶0?were set respectively for PCR amplification and the optimal annealing temperature was selected.6.Real-time quantitative PCR reactionReverse transcribed cDNA was used as a template for PCR amplification,and then the amplified products were examined used agarose gel electrophoresis assay.7.Expression of IL-17 mRNA in breast tissuesThe initial template quantity of IL-17 and?-actin mRNA in triple negative breast cancer tissues and normal breast tissues were detected by Real-time quantitative PCR method.The value of IL-17/?-actin was regarded as the relative expression of IL-17 mRNA in these two groups.8.Immunohistochemistry was used to detect the expression of Ki-67 and E-cadherin in TNBC groupImage-Pro@Plus?IPP?advanced image analysis software was used for image information analysis,and the IOD value of immunohistochemical sections of TNBC tissue specimens was quantitatively determined.9.Correlation analysis of IL-17 mRNA with Ki-67 and E-cadherinCorrelation analysis was made between IL-17 mRNA expression in TNBC tissue specimens and Ki-67,E-cadherin immunohistochemical quantitative index IOD10.The relationship between IL-17mRNA and clinicopathological features in TNBC patient specimensThe expression of IL-17 may be different in tumor tissues of TNBC patients.In order to further clarify,different clinical and pathological indexes are stratified Results:1.The general situation of tissue specimensThe experimental group and control group specimens were confirmed byimmunohistochemical staining,and the final inclusion of the experimental study included 16 cases triple negative breast cancer and 18cases normal breast tissue.2.Determination of IL-17 and?-actin annealing temperatureAnalyzed the PCR melting curve and selected 49.3?and 53.9?as the optimal annealing temperature of IL-17 and?-actin respectively according to the principle that the smaller the Ct value was and the higher the peak height was,then the higher the detection sensitivity was.3.The specificity of Real-time quantitative PCR of IL-17 and?-actinThe melting curves of the two gene fragments and the results of 2%agarose gel electrophoresis assay were analyzed.These outcomes indicated that there was no nonspecific amplification reaction,no production of primer dimmer,and the specificity of designed primer was high.4.The sensitivity of Real-time quantitative PCR of IL-17 and?-actin The results of fluorescence quantitative PCR showed that PCR amplification efficiency was similar with each other,and there was strict linear relationship between the Ct value of quantitative PCR and the initial copy number.5.The reliability of Real-time quantitative PCR of IL-17 and?-actinThe standard curves of IL-17 and?-actin were analyzed,and the values of E,R²and Slope of IL-17and?-actin were 100.8%,0.997,-3.612 and 97.9%,0.997,3.579 respectively.6.The stability and repeatability of Real-time quantitative PCR of IL-17 and?-actin All of the Ct values were collected to calculate the mean value,standard deviation and the coefficient of variation?CV?.The coefficient of variation of?-actin was the highest?2.04%?when diluted 10³-fold,the coefficient of variation of IL-17 was the highest?1.95%?when it was diluted 10³-fold with all the coefficient of variation less than 2.5%,indicating that the established RT-qPCR method in this study had good stability and repeatability.7.Detection of IL-17 mRNA using Real-time quantitative PCRStatistical analysis of RT-qPCR results in two groups of specimens was performed,and the relative expression level of IL-17 mRNA was calculated as the ratio of the initial amount of template IL-17 gene to?-actin gene,the results showed that the relative expression of IL-17 gene in TNBC tissue was[6.98±0.54)×10^-3],and that of normal breast tissue was[?5.05±0.51?×10^-3],and the difference in these two group was statistically significant?t=7.873,P=0.021,P<0.001?.8.Expression of Ki-67 and E-cadherin in group TNBC and their correlation with IL-17The IOD value of Ki-67 in group TNBC[19.33±2.61)×10²];IOD value of E-cadherin[16.71±2.46)×10²].The relative expression of Ki-67 and IL-17 mRNA is obviously positive correlation?P<0.001?,E-cadherin is obviously negative correlated with the relative expression of IL-17?P<0.001??9.The relationship between IL-17mRNA and clinicopathological featureIn the tumor tissues of TNBC patients,the expression of IL-17 was related to the axillary lymph node metastasis and clinical staging.The positive axillary lymph node metastasis and the high clinical stage were both high expression of IL-17?all P<0.05?.There was no correlation between the expression of IL-17 and age,tumor diameter and histological type?all P>0.05?.Conclusions:The real-time quantitative PCR assay for detecting human IL-17 was successfully established.The expression of IL-17 in TNBC tissue was significantly higher than that in normal breast tissue,The relative expression of IL-17 mRNA in group TNBC patients was positively correlated with Ki-67,and was negatively correlated with E-cadherin,suggesting that IL-17 may be associated with poor prognosis in TNBC patients.There was high expression of IL-17 in the positive axillary lymph node metastasis and high clinical stage in TNBC patients,but no correlation between the expression of IL-17 with the age of the patients,tumor diameter and the histological type.