| Background: In 2020,the IARC has released its latest figures on the cancer burden around the worldwide.From this report,we can learn that female breast cancer surpassed lung cancer for the first time in this year.Breast cancer in women now has the highest number of new cases of the cancer around the worldwide.There are five types of cancer that cause cancer deaths worldwide,and one of them is breast cancer what we now talking about.Nowadays,breast cancer is not only the tumor with the highest incidence,but also the tumor that causes the most female deaths all over the world.In2020,the number of female deaths due to breast cancer reached 68,0000 in just one year.In the face of the increasing incidence,we still lack a particularly effective means of targeted prevention and treatment.Cancer stem cells(CSCs)are the "seeds" of tumors,accounting for only a small part of countless tumor cells.They not only have the ability to initiate tumorigenesis,but also have similar characteristics to normal stem cells.Due to the existence of cancer stem cells,even after treatment of tumors,metastasis and recurrence will occur,which is a considerable degree of harm to cancer patients.miRNA(micro RNA)is an endogenous short-stranded RNA,that cannot encode protein production by themselves,but it can affect gene expression by binding to the 3’UTR of m RNA,resulting in inhibition of m RNA translation or direct degradation m RNA it targets.Abnormally expressed miRNAs are not only participate in regulating of the origin and progression of tumors,but also of many other diseases,more and more studies have provided clear evidence for this.In breast cancer,the expression levels of many miRNAs are reduced,including miR-4500.miR-4500 needs and deserves more and more extensive research on the occurrence and development of breast cancer.Members of the DVL gene family,including DVL1,DVL2 and DVL3,can be used as key signal regulator molecule in both classical and non-classical signal pathways of Wnt.Existing studies have suggested that DVL3 is highly expressed in a variety of tumor tissues,including breast cancer,but the mechanism of DVL3 in breast cancer has not been clearly explained,and it is not clear what role it plays in the proliferation of breast cancer stem cells.Objective: Based on the previous findings,this study focuses on solving the following problems:(1)Verify the expression levels of miR-4500 and DVL3 in breast cancer cells as shown in the literature;(2)Verify that DVL3 is the direct target of miR-4500 and down-regulates its expression under its effect;(3)To observe the changes in the biological behavior of breast cancer cells and the tumor stem cell-related markers when the level of miR-4500 was increased or the expression level of DVL3 was decreased;(4)To observe whether the abnormal expression of miR-4500 affects the activation of Wnt/ β-catenin signal pathway through DVL3.Methods:(1)the expression levels of RNA and proteins of miR-4500 and DVL3 in MCF-10 A,MCF-7 and MDA-MB-231 cell lines were detected by RT-qPCR and Western blot techniques.(2)miR-4500 was transfected into MCF-7 MDA-MB-231,the transfection efficiency was first confirmed by RT-qPCR,and then the changes of DVL3 in RNA and protein levels were detected to proved that miR-4500 could target and regulate DVL3 and reduce its expression preliminally.At the same time,target relationship between miR-4500 and DVL3 is verified by the dual luciferase reporter gene experiment.(3)miR-4500 and sh DVL3 were transfected into MCF-7,MDA-MB-231,RT-qPCR and Western blot to confirm the transfection efficiency.CCK8,scratch,transwell and flow cytometry were used to observe the differences of cell proliferation,scratch healing,metastasis and invasion and apoptosis before and after transfection.In addition,the expression changes of tumor stem cell-related markers ALDH1,SOX2,OCT4 and β-catenin in the Wnt/β-catenin pathway were detected by Western blot.Results:(1)Compared with the expression of miR-4500 and DVL3 in MCF-10 A at RNA and protein level,the expression of miR-4500 was significantly decreased in RNA level of both two breast cancer cell lines,while the expression of DVL3 was significantly increased at RNA and protein level in both two breast cancer cell lines.(2)comparing the experimental results shown by cells before and after transfection,after increasing the RNA level of miR-4500,the cell proliferation level,scratch healing rate,metastasis and invasion ability of MCF-7 and MDA-MB-231 were all inhibited,but significantly promoted the degree of apoptosis;After the expression of DVL3 was suppressed,the breast cancer cell lines showed the same results as the increased expression of miR-4500.(3)In MCF-7 and MDA-MB-231,the increase of miR-4500 expression was the same as the inhibition of DVL3 expression,which could reduce the expression of ALDH1,SOX2 and OCT4.(4)The protein expression of β-catenin,an important molecule in Wnt/ β-catenin pathway,was inhibited after sucssful transfection of miR-4500 and DVL3. |
