The Effect Of Notch1 Gene Silencing On The Expressions Of Hedgehog Signaling Pathway In Triple-negative Breast Cancer

PurposeBreast cancer is one of the most common malignant tumors in the world,which seriously threatens women’s health.Triple negative breast cancer(TNBC)is a highly invasive breast cancer subtype,which has a worse prognosis than other breast cancer subtypes due to the lack of effective therapeutic targets,high metastasis and recurrence rate.It is urgent to find out the exact therapeutic target in order to develop more effective drugs for TNBC.Some studies has found abnormal activation of signaling pathways in TNBC,including the Notch signaling pathway and the Hedgehog signaling pathway,which plays an important role on the occurrence of tumor development.Furthermore,there is cross-talk between the Notch signaling pathway and the Hedgehog signaling pathway in some malignant tumors.So the purpose of this study is to explore the effect of silencing the Notch1 gene on the expression of the downstream target genes Hesl,and on the expression of Hedgehog signaling pathway key molecular Smo and Gli1 in the triple negative breast cancer.MethodTriple-negative breast cancer line MDA-MB-231 cells were cultured for 3-5 generations,and the cells were divided into 3 groups,namely,the experimental group,the negative control group and the blank control group.Cells of the experimental group were transfected with Notch1-siRNA,cells of the negative control group were transfected with NC-siRNA,and the cells of the blank control group were not treated.After transfection,morphological changes and growth condition of the cells were observed under the Inverted Microscope.The transfection of cells in each group was observed under Fluorescence Microscope.The transfection effect of Notchl gene was detected by RT-PCR,then the one with the highest silencing efficiency was statistically screened out of the 3 purchased Notchl-siRNA for the subsequent transfection experiments.The expressions of Notch1 gene,downstream target gene Hes1 and Smo and Glil in Hedgehog signaling pathway were detected by RT-PCR.Western Blot was used to detect the protein expressions of Notch1 gene,downstream target gene Hes1 and Smo and Gli1 in Hedgehog signaling pathway.Results1 After cell transfection,the cells in the experimental group were observed under an Inverted Microscope.Compared with the negative control group and the blank control group,the number of MDA-MB-231 cells in the experimental group decreased,some cells changed from long spindle shape to round shape,and the cell volume decreased.2 After cell transfection,the cells were observed under fluorescence microscope observation.Compared with the blank control group,there was a small number of scattered fluorescent cells in the negative control group,and there were much more fluorescent cells in all of the 3 experimental groups transfected with the purchased Notchl-siRNA.There were the most fluorescent cells in the group transfected with Notch1-homo-5723,which was estimated to be the best one.3 To select the most effective siRNA,the mRNA expression of Notchl gene in each group was detected and statistically analyzed after the transfection of Notchl-siRNA.The transfection rate of the negative control group(i.e.,NC-siRNA transfection)was 1%.In the experimental group,the transfection rate of Notch1-homo-6950 was 44%,the transfection rate of Notch1-homo-5723 was 83%,and the transfection rate of Notch1-homo-4757 was 36%.Compared with the blank control group and the negative control group,the difference of 3 siRNA in the experimental group was statistically significant(P<0.01),while the difference between the negative control group and the blank control group was not statistically significant(P>0.05).Finally,the Notch1-homo-5723 was selected as the most effective Notchl-siRNA,and it was used in subsequent experiments.4 After 24 hours of cell transfection,the mRNA expression levels of Notchl,Hes1,Smo and Glil in the experimental group were significantly decreased by RT-PCR,and the differences were statistically significant(P<0.01)compared with the blank control group and the negative control group,respectively.After 48 hours of cell transfection,the protein expression levels of Notchl,Hes1,Smo and Glil in the experimental group were significantly decreased by Western Blot,and the differences were statistically significant compared with the blank control group and the negative control group,respectively(P<0.01).Conclusion1 After silencing the Notchl gene in vitro triple-negative breast cancer cells by siRNA,the expression of Notchl and its downstream target gene Hesl can decrease significantly,proving that Hesl is a sensitive target gene of Notch signaling pathway.2 After silencing Notch signaling pathway by using Notchl-siRNA,the expression of Smo and Glil in Hedgehog signaling pathway can decrease,proving that silencing Notch signaling pathway can inhibit the activity of the Hedgehog signaling pathway in triple-negative breast cancer.