Expression Of 8 Biomarkers Such As Survivin In TNBC And In Vitro Inhibition Of Juglone On TNBC Cell Line

Part 2:Preliminary exploration of potential biomarkers in 49 cases of triple negative breast cancerObjective:To investigate the expression of 8 biomarkers,including AR,CD8,FOXC1,DCLK-1,PD-L1,MAG-A4,Survivin and WT1,in tumor tissues of 49 patients with triple negative breast cancer and their relationship with clinicopathological characteristics.Mthods:Forty-nine paraffin-embedded tissue specimens diagnosed as primary invasive breast cancer in Nanjing Drum Tower Hospital from April 2019 to December 2020 were retrospectively collected.The expression of AR,PD-L1,CD8,FOXC1,DCLK-1,MAGE-A4,Survivin and WT1 in tumor tissues were detected by immunohistochemistry.The correlation between immunohistochemical protein expression and clinicopathological features was analyzed.At the same time,the correlation between immunohistochemical protein expression and clinicopathological features was analyzed retrospectively.Results:1.The positive expression rate of each biomarker in tumor cells:AR was 40.82%(20/49),FOXC1 was 81.63%(40/49),Survivin was 79.60%(39/49),DCLK-1 was 71.42%(35/49),MAGE-A4 was 20.41%(10/49),CD8 was 34.70%(17/49),MAGE-A4 was 34.70%(17/49),MAGE-A4 was 34.70%(17/49).WT1 was 24.49%(12/49).The positive rate of FOXC1 expression was the highest,and there was no statistical significance compared with that of the non-expression group(P>0.05).The positive rate of PD-L1 in tumor cells and tumor interstitial immune cells was 24.49%(12/49)and 49.00%(24/49),respectively.2.The correlation between the expression of each biomarker and clinicopathological features was analyzed.The expression of PD-L1 in tumor stromal immune cells was statistically significant with tumor histological grade(P=0.012),and the expression of AR was statistically significant with the site of breast cancer(P=0.014).There was a positive correlation between PD-L1 and AR expression(r=0.427,P=0.001).In addition,the expression of CD8 and WT1(r=0.272,P=0.049),Survivin and DCLK-1(r=0.326,P=0.017)and Survivin and MAGE-A4(r=0.448,P=0.001)should be positively correlated.The expression of PD-L1 and MAGE-A4 in tumor cells(r=-0.338,P=0.013)and the expression of PD-L1 and DCLK-1 in tumor cells(r=-0.790,P=0.000)were significantly negative correlated.The expression of PD-L1 and WT1 in tumor stromal immune cells(r=-0.628,P=0.000)were significantly negative correlated.There was also a significant negative correlation between AR and Survivin expression(r=-0.566,P=0.000)Conclusion:Survivin,PD-L1,CD8,FOXC1,DCLK-1,MAGE-A4 and WT1 were expressed to different degrees in triple negative breast cancer tissues,and compared with WT1 and other markers,PD-L1 and AR grade DCLK-1 may be more closely related to the occurrence and development of triple negative breast cancer.It is worth further exploring its value in triple negative breast cancer.Part3:Effect of Juglone on Proliferation and Biomarkers of Triple Negative Breast Cancer CellsObjective:To investigate the effects of Juglone(Jug)on proliferation and biomarkers of triple negative breast cancer cell lines.Methods:CCK-8(Cell Counting Kit-8)method was used to detect the growth inhibition rate of Jug and DDP on MDA-MB-231 and MDA-MB-468 triple negative breast cancer cells,and IC50 value was calculated.DAPI and PI staining and inverted fluorescence microscopy were used to observe the cell apoptosis.The effect of Jug on the cycle and apoptosis of triple negative breast cancer cells was detected by flow cytometry.Immunohistochemical staining was used to detect the effect of Jug on the expression of Survivin and other biomarkers in triple negative breast cancer cells.Results:1.Jug significantly inhibited the proliferation of MDA-MB-231 and MDA-MB-468 triple negative breast cancer cells in a time-dependent and concentration-dependent manner.24h IC50 values of Jug treated groups were 3.44μg/mL and 3.59μg/mL,respectively.48h IC50 values of Jug treated group were 2.85μg/mL and 2.91μg/mL.2.Jug could block the cell cycle of MDA-MB-231 and MDA-MB-468 triple negative breast cancer in the G0/G1 phase by affecting the cell cycle.3.The results immunohistochemical of cell slide showed that MDA-MB-231 and MDA-MB-468 cells were treated with Jug for 24h.The results of CD8 and WT1 immunohistochemical staining of MDA-MB-231 and MDA-MB-468 cells were consistent with the results before and after JUG treatment,both of which were negative.Survivin results were changed from negative to positive after Jug treatment.After Jug treatment,AR staining results of the two cells changed from moderate positive to weak positive,and from weak positive to negative.The results of DCLK-1 changed from untreated positive to weakly positive or negative.The results of MAGE-A4 staining were negative to positive expression and consistent negative expression respectively.Conclusion:Jug showed significant antitumor effects on MDA-MB-231 and MDA-MB-468 cell lines of triple negative breast cancer,and certain regulation effects on biomarkers including AR,FOXC1,DCLK-1,MAG-A4,Survivin and WT1.Perhaps these biomarkers can be used as prognostic factors for Jug treatment of TNBC,or biologics combined with Jug can be developed in the future.