A Study On The Role And Mechanism Of STON2 In Regulating Stemness In Ovarian Cancer Stem-like Cells

Ovarian carcinoma is the most lethal gynecological neoplasm.American Cancer Society predicts that 22 240 women will be diagnosed as ovarian cancer and 14 070 women will die from the disease in 2018.As we know,the overall 5-year survival rate of ovarian cancer patients is 47%,but the advanced stage patients only 29%from 2006 to 2012.Noteworthy,such survival situation has little changed since platinum-based treatment was introduced over 30 years ago.Even though about 70%of ovarian cancer patients respond to initial chemotherapy,most of them ultimately develop to recurrence and metastasis.Emerging evidences have demonstrated there is a small population of tumor cells with stem cell characters,so-called cancer stem cells(CSCs)or cancer stem-like cells(CSLCs),in ovarian cancer tissues.These cells possess the ability of self-renew,differentiation,and oncogenic potential,which are regarded to be the source of cancer recurrence.Thus,the strategy of CSC-targeting therapy is being expected to improve thoroughly the prognosis of ovarian cancer patients.Unfortunately,the identification and eradication of CSCs are not ideal as we initially hope.Persistent exploration of CSC characteristics may be significative and helpful for the development of novel CSC-targeting drugs.Our previous study have successfully enriched and identified ovarian CSLCs.Using LC-MS/MS label-free quantitative proteomics and bioinformatics analysis,we identified Stonin2(STON2)as one of the differentially expressed proteins in ovarian cancer stem-like cells.However,so far little is known about STON2 roles in cancer cells,especially in the sternness-regulated performance.According to STON2 function on endocytosis reported by previous studies,we speculated that the lower expression of STON2 in ovarian cancer stem-like cells may contribute to the maintenance of cell sternness.In this study,we validated the down-regulation of STON2 filtered by proteomic screening and confirmed that STON2 knockdown promotes sternness in ovarian cancer cells,for example,the proportion of CD44+CD24-subpopulation,sternness-related proteins Nanog and C-myc,and sphere-forming ability increased,EMT-related markers E-cadherin decreased,while N-cadherin and Fibronectin increased,vice versa.Using whole transcriptome shotgun sequencing and gene function experiment,we found that MUCI was the most significant difference.Using RT-PCR and Western blot,we confirmed the expression of MUC1 increased following STON2 knockdown.We verified that MUC1 positively regulated sternness characteristics in ovarian cancer stem cells.For example,the proportion of CD44+CD24-and sternness-related proteins Nanog and C-myc increased following MUC1 exaltation.In addition,Co-overexpressed STON2 and MUC1,the sphere-forming ability increased again.Togather,we verified MUC1 as a downstream target participating in STON2 mediating stem-like properties.By methylated pyrosequencing,we confirmed the level of MUC1 promoter methylation x and DNMT1 expression decreased,while MUC1 increased,following STON2 knockdown.Finally,we verified that DNMT1 negetively regualated MUC1.Taken together,we confirmed STON2 modulated MUC1 expression via DNMT1-mediated promoter methylation.The aim of our study is to uncover the mechanism of the modulation of cell stemness in ovarian CSCs,for promoting the development of C SC-targeting therapy for ovarian cancer.Part I The effect of STON2 on stemness in ovarian cancer stem-like cellsObjective:To observe the different expression of STON2 in ovarian cancer stem cell-like cells,which is cultured in serum-free medium.To inverstigate the effect of STON2 on the sternness in ovarian cancer stem cell-like cells,through detecting the proportion of CD44+CD24" subpopulation,sternness-related proteins Nanog and C-myc,EMT-related markers and sphere-forming ability.Methods:In this part,we first confirmed that STON2 was differentially expressed in ovarian cancer stem cell-like cells by Western blot.Then we regulated STON2 expression using shRNA and overexpression plasmid,and observed the sternness characteristics in ovarian cancer stem-like cells.For example,the proportion of CD44+CD24-subpopulation were detected by FCM.sternness-related proteins Nanog and C-myc were detected by Western blot,EMT related markers E-cadherin,N-cadherin and Fibronectin were detected by RT-PCR,sphere-forming ability,and tumorigenicity in NOD/SCID mice were observed.In an aim to elucidate the role of STON2 in regulating the sternness of ovarian cancer stem cell-like cells.Results:1.We confirmed that STON2 expression was down-regulated in ovarian cancer stem cell-like cells using RT-PCR and Western blot(p<0.05).2.STON2 knockdown promoted stem-like properties in ovarian cancer cells,the proportion of CD44+CD24-subpopulation,sternness-related proteins Nanog and C-myc,and sphere-forming ability increased.3.STON2 knockdown promoted EMT in ovarian cancer cells,E-cadherin decreased,while N-cadherin and Fibronectin increased.4.STON2 knockdown promoted tumorigenicity in NOD/SCID mice5.STON2 over-expression inhibited stem-like properties in ovarian cancer cells,the proportion of CD44+CD24-subpopulation,sternness-related proteins Nanog and C-myc,and sphere-forming ability decreased.6.STON2 over-expression inhibited EMT in ovarian cancer cells,E-cadherin increased,while N-cadherin and Fibronectin decreased.Conclusions:1.STON2 expression is down-regulated in ovarian cancer stem cell-like cells.2.STON2 knockdown promotes stem-like properties and EMT in ovarian cancer cells..3.STON2 over-expression inhibites stem-like properties and EMT in ovarian cancer cells.Part ? MUC1 acted as a downstream target participating in the process of STON2 regulating stemness in ovarian cancer stem cell-like cellsObjective:To screen and verify the downstream signaling molecules involved in STON2 regulating stemness in ovarian cancer stem cell-like cells via RNA sequencing and bioinformatics analysis.Methods:In this part,we first collected shNC and shSton2 cancer stem cell-like cells,extracted total RNA,and performed RNA-Seq assay on the Illumina Hiseq platform(three independent samples in eac group).The data obtained from the sequencing was analyzed by bioinformatics,and differentially expressed genes were clustering analyzed by KEGG and GO analysis.Futhermore,MUC1 was the most significant difference.Then we confirmed the expression of MUC1 following STON2 knockdown using RT-PCR and Western blot.We regulated the expression of MUC1 via shRNA and overexpression plasmid,and observed stemness characteristics in ovarian cancer stem cells.For example,the proportion of CD44+CD24-subpopulation were detected by FCM.stemness-related proteins Nanog and C-myc were detected by Western blot.In addition,to investigate the effect of MUC1 overexpression on the sphere-forming ability of STON2 overexpression,we overexpressed STON2 and MUC1 at the same time.Results:1.RNA sequencing was carried out and differentially expressed 824 genes was discovered,including 299 down-regulated genes and 525 up-regulated genes.Furthermore,MUC1 was the top 1.2.GO and KEGG cluster analysis showed that binding and enzyme inhibitory activity were the main signal pathways.3.RT-PCR and Western blot analysis confirmed that MUC1 was the downstream target of STON2(p<0.05).4.MUC1 konckdown inhibited stem-like properties in ovarian cancer cells,the proportion of CD44+CD24-subpopulation and sternness-related proteins Nanog and C-myc decreased.5.MUC1 over-expression promoted stem-like properties in ovarian cancer cells,the proportion of CD44+CD24-subpopulation and sternness-related proteins Nanog and C-myc increased.6.MUC1 acted as a downstream target participating in the process of STON2 regulating sternness in ovarian cancer stem cell-like cellsConclusions:1.MUC1 is the downstream target of STON22.MUC1 positively regulates stem-like properties in ovarian cancer cells.3.MUC1 acts as a downstream target participating in the process of STON2 regulating sternness in ovarian cancer stem cell-like cellsPart ? STON2 regulated DNA promoter methylation and expression of MUC1 via DNMT1Objective:To investigate the regulation mechanism between STON2 and MUC1.Methods:First,we transfected cells with siNC and siRNA-STON2 24h,then treated with DNA methylation inhibitors,and observed the mRNA level of MUC1 by RT-PCR.secondly,we knockdown STON2 with siRNA,and detected the level of MUC1 promoter methylation in the region which is enriched with CpG using pyrosequencing analysis.Then observed the expression of DNMT1 and MUC1 by Western blot follwing STON2 knockdown using siRNA.Finally,we regulated the expression of DNMT1 by siRNA,and investigated the expression of MUC1 by RT-PCR and Western blot.Results:1.We confirmed that the level of MUC1 promoter methylation and DNMT1 expression decreased,while MUC1 increased,following STON2 knockdown,2.Western blot verified that MUC1 increased following DNMT1 konckdown.Conclusions:1.STON2 regulates MUC1 expression by altering its DNA promoter methylation.2.DNMT1 mediates the process of STON2 in regulating MUC1 promoter methylation.