The Mechanism Of CD44v16 On Breast Cancer Cells Migration And Invasion Ability

ObjectiveA new CD44 variant(CD44v16)was found in MCF-7/ADR cells through previous research in our laboratory.It was confirmed by experiments that it is closely related to the resistance of breast cancer cells and the enhancement of breast cancer cell stemness.In order to further understand the role of CD44v16 in breast cancer cells and its specific molecular biological mechanism that enhance breast cancer invasion and metastasis,it is expected that it will provide more theoretical basis for breast cancer recurrence and metastasis.Methods1.According to the sequence of CD44v16,designed and synthesized lentiviral particles by Gima Company,transfected into breast cancer cells MCF-7 and MDA-MB-231,and select cells by puromycin to obtain stable overexpressing CD44v16.we divided cells into three groups: CD44v16 overexpression group,negative control group(NC group),and blank group(N group).The real-time quantitative qRT-PCR to verify the relative mRNA expression of CD44v16 in MCF-7and MDA-MB-231 with infected by lentiviral particles.2.CCK8 and flow cytometry methods to assess the cell proliferation rate and apoptosis in MCF-7/CD44v16 and MDA-MB-231/CD44v16.3.The change of cell invasion and migration ability in MCF-7/CD44v16 and MDA-MB-231/CD44v16 by Wound healing and Transwell invasive cell test.4.In order to figure out the mechanism of CD44v16 enhanced migration and invasion.We used the real-time quantitative qRT-PCR and western blotting to detected the expression of MMP7 and total JNK and ERK1/2 protein and phospho JNK and ERK1/2 protein.5.To verified the expression of MMP7,JNK and ERK1/2 total protein andphospho JNK/ERK protein after pretreatment with JNK inhibitor(SP600125)and ERK1/2 inhibitor(U0126)for 24 hours by Western blotting in each group of cells.Results1.MCF-7 and MDA-MB-231 cells were infected by lentiviral particles.MCF-7/CD44v16,MDA-MB-231/CD44v16 and MCF-7/NC,MDA-MB-231/NC were successfully established.Puromycin was used to select transfected cells when the green fluorescent protein(GFP)expression rate is more than 90% in a fluorescence microscope.The mRNA expression of CD44v16 in MCF-7/CD44v16 was more higher than MCF-7/NC and MCF-7(P<0.001),and the mRNA expression of CD44v16 in MDA-MB-231/CD44v16 was significantly higher(P <0.01).2.The proliferation of CD44v16 overexpress in MCF-7 and MDA-MB-231 cells was more higher than other cells after 24 hours(P<0.001)by CCK8 assay;We also found that MDA-MB-231/CD44v16 and MCF-7/CD44v16 had lower apoptosis rates than other cells by flow cytometry(P<0.001).3.The wound-healing assay showed that the cell migration ability of MCF-7/CD44v16 and MDA-MB-231/CD44v16 after serum-free culture for 48 hours was significantly higher(P<0.001);Transwell invasive cells test showed that MCF-7/CD44v16 and MDA-MB-231/CD44v16 had a significantly higher invasion ability(P<0.001).4.The relative mRNA and protein expressions of MMP7 in MCF-7/CD44v16 were significantly higher than MCF-7/NC and MCF-7(P<0.001)by PCR and Western blot assays;The results showed that the expression of phospho-JNK protein and phospho-ERK1/2 protein in MCF-7/CD44v16 were significantly higher than MCF-7/NC and MCF-7 groups(P<0.001).5.The cells of each group were pretreated with JNK inhibitor(SP600125)and ERK1/2 inhibitor(U0126)for 24 hours.The JNK and ERK1/2 signaling pathways of MCF-7/CD44v16 were succssfully inhibited.The cells pretreated was successfully inhibited the overexpression of MMP7 by CD44v16 after pathway inhibition(P<0.001).Conclusion1.CD44v16 overexpressed in MCF-7 and MDA-MB-231 cells.The results show that CD44v16 overexpressed can enhance cell proliferation,inhibit apoptosis and promote cell migration and invasion.2.We found CD44v16 induced MMP7 overexpression through JNK and ERK1/2signaling pathways.We could inhibit CD44v16 induced MMP7 overexpression by cells after pretreament of medicine which can inhibited JNK and ERK1/2 signaling pathway.