| Ovarian cancer is a disease that seriously threatens women's lives and health.It is a common tumor in women in the world.At present,a effective diagnosing and screening method for early ovarian cancer is unavailable.As a result,up to 75%of ovarian cancer patients are diagnosed at an advanced stage.The probability that patients with advanced ovarian cancer survive to 5 years is less than 30%,and the mortality is the highest in gynecology cancers.Clinically,the standard treatment for ovarian cancer is surgery combined with paclitaxel(PTX)and platinum-based adjuvant chemotherapy.However,as the treatment continues,most of the ovarian cancer cells will become resistant to chemotherapy drugs,which resulted in the recurrence of ovarian cancer.Therefore,elucidating the drug resistance mechanism of ovarian cancer and overcoming this challenge are of great significance for improving the treatment and prognosis of ovarian cancer.CC motif chemokine ligand 20(CCL20)is a ligand for CC motif chemokine receptor 6(CCR6)in lung cancer,colorectal cancer and thyroid cancer.It also has been proved that CCL20/CCR6 plays an important role in the development of drug resistance.Recent studies have shown that cisplatin can stimulate macrophages to secrete CCL20 and promote the migration of ovarian cancer cells through the CCL20/CCR6 axis.At the same time,studies have found that the increased expression levels of CD44 and CD117 in SKOV3 cells are significantly related to the enhancement of epithelial mesenchymal transition(EMT),sphere formation,metastasis and multidrug resistance.However,the role of CCL20/CCR6 in CD44+CD117+ ovarian cancer cells to chemotherapeutic drugs resistance including PTX and its underlying mechanism are still unclear,and further research is needed.This study intends to isolate CD44+CD117+ subgroup cells from SKOV3 cells and determine the stemness and paclitaxel resistance of it.And further clarify the role of CCL20/CCR6 plays in regulating paclitaxel resistance in CD44+CD117+ cells in vivo and in vitro,which will provide new insights into the mechanism and clinical treatments of PTX resistance in ovarian cancer.Part ?The analysis for stemness and paclitaxel resistance in CD44+CD117+cellsObjective:This study aims to isolate CD44+CD117+and CD44+CD 117-cell subgroups of epithelial ovarian cancer cells by magnetic bead screening method,and determine the expression of CCR6 and Notch 1 in the CD44+CD117+cell subgroups and detect their role in stemness and paclitaxel resistance.Methods:(1)Magnetic bead screening was used to obtain CD44+CD117+and CD44-CD117cell subsets in human ovarian cancer cell line SKOV-3 and the distribution of CD44+CD117+and CD44-CD117-cell subsets were detected by flow cytometry.(2)qRT-PCR was used to detect the mRNA expression of Oct4 and CCR6 in CD44+CD117+ and CD44-CD117-cell subsets;We detected the protein expression level of CCR6?Oct4?ABCG1 and Notch1 in CD44+CD117+and CD44-CD117-cell subsets by using Western blotting.(3)CCK-8 assay was used to detect the IC50 of CD44+CD117+and CD44-CD117subgroup cells to PTX.(4)Sphere formation ability of CD44+CD117+and CD44-CD117-subclone cells was detected by sphere formation analysis.(5)Dichlorofluorescein diacetate method was used to determine the level of reactive oxygen species(ROS);CCK-8;The proliferation and apoptosis were detected by flow cytometry and Hoechst assay respectively.Results:(1)The proportions of CD44 positive cells separated by flow cytometry were 0.2%and 98.9%,indicating that the CD44" and CD44+subgroups were successfully separated;The proportions of CD117-in CD44-subgroup cells and CD117+in CD44+subgroup cells were 99.5%and 98.5%respectively,indicating that CD44+CD17+and CD44-CD117-subgroup of ovarian cancer cells were successfully isolated.(2)Compared with the CD44-D117-cell subgroup,the mRNA expression of CCR6 and Oct4 in the CD44+CD117+cell subgroup were significantly increased.The expression of CCR6,Oct4,Notch1 and ABCG1 in the CD44+CD117+cell subgroups were significantly increased compared with CD44-D117-cell subgroup.(3)CD44+CD117+subgroup cells have higher IC50 values for PTX at 24 h(10.22 nM)and 48 h(5.04 nM)than CD44-D117-subgroup cells at 24 h(4.99 nM)and 48 h(2.51 nM)respectively.(4)CD44+CD117+ cells showed higher sphere formation ability than CD44-D117subgroup.(5)After PTX treatment,the ROS content in the CD44+CD117+cell subgroup was significantly reduced,compared with the CD44-D117-subgroup cells.(6)Compared with the CD44-D117-cells,the apoptosis rate of CD44+CD117+cells was significantly reduced after treatment with PTX;the proliferation rate was significantly increased.Conclusions:(1)The CD44+ CD117+and CD44-CD117-ovarian cancer subgroup cells were successfully isolated.(2)CD44+CD117+subgroup cells showed higher stemness and PTX resistance than CD44-D117-subgroup cells.Part ?Effect of CCL20/CCR6 on drug resistance of ovarian cancer CD44+CD117+cell subsets by regulating Notchl signaling pathway in vitroObjective:This study aims to explore the influence of stemness and drug resistance of Notch1 interfered CD44+CD117+ subgroup in vitro and further investigated the mechanism of CCL20/CCR6 in regulating the stemness and drug resistance of CD44+CD117+ cell subsets.Methods:(1)siRNA sequences of Notch1 were transfected into CD44+CD117+cells by using Lipofectamines 2000.(2)qRT-PCR and Western blot were used to detect the expression of Oct4 in the Notch1 interfered cells after treatment with CCL20;Western blotting determined the Notch1,CCR6 and ABCG1 protein expression of the Notchl interfered cells after treatment with CCL20.(3)CCK-8 assay was used to detect the IC50 to PTX of CD44+CD117+cells and Notch1 interfered cells after treatment with CCL20.(4)Cell sphere formation test was used to detect the sphere formation ability of CD44+CD117+ cell subset and Notch 1 interfered subsets after treatment with CCL20.(5)Dichlorofluorescein diacetate method was used to determine the levels of reactive oxygen species(ROS)in CD44+CD117+cell subsets and Notch1 interfered cells after treatment with CCL20.(6)CCK-8,flow cytometry and Hoechst were used to detect the proliferation and apoptosis induced by PTX of CD44+CD117+cell subsets and Notchl interfered subsets after treatment with CCL20 respectively.Results:(1)qRT-PCR and Western blot analysis indicated that the three Notchl siRNA sequences can successfully interfere the expression of Notch 1 in CD44+CD 117+cells at mRNA and protein levels.siNotch1-2 sequence has the best interference effect.(2)qRT-qPCR and Western blot indicated that CCL20 treatment can significantly increase the expression of Oct4 in CD44+CD117+cells.Silencing Notch 1 can partially reverse the up-regulation effect of CCL20 on Oct4 expression in CD44+CD117+cells;CCL20 significantly increased the protein expression levels of CCR6?ABCG1 and Oct4 in CD44+CD117+cells by Western blotting analysis.Silencing Notch1 notably reversed the up-regulation effect of CCL20.(3)CCL20 can increase the sphere formation capacity of CD44+CD117+cells,while silencing Notch 1 can significantly impair the promotive effect of CCL20,indicating that the CCL20/CCR6 axis increases the tumorigenicity of CD44+CD117+cells through Notch1 pathway.(4)Compared with the control group(IC50=10.98 nM,4.86 nM),CCL20 can significantly increase the IC50 of CD44+CD117+cells(IC50=17.51 nM,8.41 nM)to PTX at 24h and 48 h respectively,while siNotchl significantly reverse the the promotive effect of CCL20 on PTX IC50(IC50=7.92 nM,4.22 nM).(5)CCL20 can significantly reduce PTX-induced CD44+CD117+cell ROS level,while siNotchl could attenuate the effect of CCL20 in alleviaing ROS levels in CD44+CD117+cells.(6)CCL20 can decrease the apoptosis of CD44+CD117+cells after treatment with PTX,while siNotch1 could partially reverse the effect of CCL20 in suppressing apoptosis of CD44+CD117+cells induced by PTX;CCL20 can enhance proliferation of CD44+CD117+cells after PTX treatment,while siNotchl impaired the promotive effect of CCL20,indicating that the CCL20/CCR6 axis increases the PTX resistance of CD44+CD117+cells through the Notch1 signaling pathway.Conclusions:We demonstrated that the CCL20/CCR6 axis promotes the stemness and PTX resistance of CD44+CD117+cells via Notch1 signaling pathway in vitro.Part ?To verify the effect of CCL20/CCR6 on the drug resistance of ovarian cancer CD44+CD117+cells through Notchl signaling pathway in vivoObjective:This study aims to verify the effect of CCL20/CCR6 on the stemness and drug resistance of ovarian cancer CD44+CD117+cells via the Notch1 signaling pathway by constructing CD44+CD117+and CD44-CD117-xenograft tumor models on C57BL/6 mouses.Methods:(1)Use CD44+CD117+and CD44-CD117-cells to construct xenograft ovarian cancer tumor models on C57BL/6 mice,and perform CCL20 and/or PTX injection treatments on 1d,3d and 7d;Tumor size and weight were obtained on 3d,7d,14d,21d and 28d to draw tumor growth curve.(2)Immunohistochemical(IHE)was used for CCR6 expression in tumor tissues;Western blot was used to determine the CCR6,Notchl,Oct4 and ABCG1 protein expression levels.(3)The chlorofluorescein diacetate method was used to determine the ROS levels of tumor tissues of each group.(4)We use HE staining method to stain the tumor tissues and detected the pathological changes.(5)Expression level of Ki-67 was determined via immunohistochemistry.(6)TUNEL assay determined the cell apoptosis of tumor tissues.Results:(1)The tumor volume and weight of the CD44+CD117+nude mice group was significantly higher than that of the CD44-D117-group;further analysis showed that no matter in the nude mice bearing the tumor of CD44+CD117+ cells or the nude mice bearing the tumor of CD44-D117-cells,the volume and weight of tumors were significantly increased after PTX treatment;however,compared with the PTX treatment group,PTX and CCL20 treatment can significantly increase the tumor volume and weight in nude mice.(2)Compared with the CD44-D117-group,the expression level of CCR6 in the tumors of CD44+CD117+group was significantly increased determined via IHE.In addition,compared with the PTX group,CCL20 treatment can significantly increased the expression levels of CCR6 in the CD44+CD117+and CD44-D117-group tumors;Western blot indicated that compared with the CD44-D117-group,the expressions of Oct4,CCR6,Notchl and ABCG1 in the CD44+CD117+ group were significantly increased;compared with the PTX group,CCL20 could significantly promote the expression of Oct4,CCR6,ABCG1 and Notchl in tumors of CD44+CD117+ and CD44-D117-groups.(3)Dichlorofluorescein diacetate:Compared with the tumor tissue in the CD44-D117group,the ROS levels of tumors in CD44+CD117+ group were significantly lower after treatment with PTX or/and CCL20.(3)H?E analysis:Regardless of PTX or PTX and CCL20 treatment,compared with the CD44-D117-group,the tumors of CD44+CD117+ group showed an increased amount of small round cells with hyperchromatic nuclei and scanty cytoplasm;compared with the PTX group,regardless of the CD44-D117-or CD44+CD117+ nude mouse model groups,CCL20 could significantly decrease tumor differentiation.(4)Ki-67 assay indicated that compared with the CD44-D117 group,the CD44-D117group tumor tissue presented higher proliferation after treatment with PTX;the proliferation of tissues is further increased in both groups after treatment with CCL20.(5)TUNEL assay indicated that the apoptosis rate was greatly reduced in CD44+CD117+group compared with CD44-D117-group;compared with the PTX group,no matter in the CD44-D117-group or CD44+CD117+ group tumor tissues,the apoptosis rates were further reduced after CCL20 treatment.Conclusions:We demonstrated that CCL20/CCR6 can promote the stemness and tumorigenicity of CD44+CD117+ovarian cancer cell subgroup by regulating Notch1 signaling in vivo,thereby promoting the resistance of CD44+ CD117+ ovarian cancer cells to PTX. |
PDF Full Text Request