Discovery Characteristics Of CD44,CD24-expressing Cervical Cancer Cells And Investigate The Potentially Mechanism Of Its Radioresistant

Objective:Cervical cancer is the most commom malignancy that endanger the health of women.Surgery,radiotherapy and chemotherapy are main treatment routes for cervical cancer.The failure to cure advanced cervical cancer is associated with metastatic and recurrence.A highly recurrence in five years,the fail treatment of cervical cancer after recurrence largely due to its high degree of resistance to chemotherapy and radiotherapy.Achievements of CSC povide new promise for the treatment and prognosis of the disease.CCSC become the hot point for Gynecologic Oncologists.Theory of CSC may promote us to change the traditional concept of cervical cancer treatment.This would be important for addressing and understanding the possible implication of CCSC in cervical cancer treatment.There have been a number of publications showing that stem-like cells were existed in cervical cancer tissues and cell lines.However,up to date,no specific tumor markers has been used to successfully isolate or enrich stem cells from cervical cancer cell lines.Siha cells that repeatedly exposed to 2 Gy dose of irradiation 25 times were named radioresisitant cancer cells,we found that radioresisitant cancer cells have a higher expreesion of CD44+/CD24+after radiation.In this study,we firstly acquired CD44+/CD24+Siha cells by flow cytometry.We further designed to prove whether the CD44+/CD24+Siha cells have the characteristics of stem cell by Suspension sphere-culture,Tumor Xenografts,Trans well Assay,morphological changes after irradiation by transmission electron microscopy and Colony Formation assay.To explore the feasibility of CD44+/CD24+ in high purification of CSCs in Siha cell line.With further research,it is possible for us to investigate the radio-resistance whether associated with cell cycle.Methods:1 Cell cultureSiha cells were maintained in RPMI 1640 medium supplemented with 10%fetal serum and cultured in 5%C02,37? condition.2 Siha cells were trypsinized into single cell suspension and stained with CD44-PE,CD24-FITC antibodies,Then analysis by flow cytometry(subculture or cryopreservation).3 CD44+/CD24+Siha cells and parent Siha cells were suspended in serum-free Dulbecco's modified Eagle's medium to observe its capacity of cell's spherical clusters.4 Tumorigenecity in nude miceA moderate concentration of CD44+/CD24+Siha cells and parent Siha cells were resuspended in 1:1 media and matrigel.Extracting 0.1ml injected in the right flank region of 4-week-old female NOD/SCID mice.Then observated the tumor-forming time and tumor-forming rate in nude mice and analysis the transplanted tumor 'volumes.5 Cells irradiatingA dose rate of 300 cGy/min,a linear accelerator at 6MV,The source skin distance was 100cm.6 Electron microscopeThe intracellular ultrastructure of CD44+/CD24+Siha cells and parent Siha cells were observed after 4gy irradiation by transmission electron microscopy.7 The invasion and migration of two groups by Matrigel-Transwell invasion assayThe CD44+/CD24+Siha cells and parent Siha cells were irradiated with different dose.The cells were added to the up transwell chamber that coated by Matrigel.After cultured in 5%CO2,37? condition 24 hours.The cell were fixed with4%paraformaldehyde and stained with Giemsa.Counting the number of cells that through Matrigel biological glue holes with microscope.8 Clonogenic AssayThe cells of two groups that irradiated with increasing X-ray treatment(up to 8Gy)were plated into six-well dishes.When appearing visible clones at the bottom of plates ended its culture.The cell were fixed with 4%paraformaldehyde,stained with Giemsa and counted,colonies containing>50 cells were counted with a light microscope.Then calculated Plating Efficiency(PE)and Surviving Fraction(SF).Three independent experiments were performed.9 Assays of Cell Cycle response to irradiateCells were harvested and trypsinized at 24 hour after 4Gy irradiation.The cell concentration is 1×106).The cells were fixed with75%ethanol for 24 hours at 4 ?,washed with PBS,stained with cell cycle agent for 30min.The cells were evaluated by flow cytometry.10 Data analysisThe SPSS 21.0 and GraphPad Prism 5 program software was employed to analyze the data.Data collected from all experiment were expressed as mean±s.d or media±q.r.One-way analysis of variance,rank sum test and t'test were performed to analyze the date between groups.P<0.05 was chosen for statistical significance.Results:1 The proportion of CD44+/CD24+ in Siha cells was detected by flow cytometryThe proportion of CD44+/CD24+ cells in Siha cells accounted for 1.12%.The proportion of CD44+/CD24+ cells in radioresistant cells was 4.68%.2 The mammosphere forming rate in two groups by suspension sphere-cultureThe mammosphere of CD44+/CD24+ cells were bigger and tighter.The mammosphere forming rate was(36-.0±4.52)%VS(6.1±2.85)%.The difference was statistically significance in two group.3 The state of growth and volume of transplantation tumor in parallel with time3.1 The result displayed a significant difference in Tumor-forming time(4.50±3.25day)VS(12.88±2.69day).The result indicate that CD44+/CD24+Siha cell are more malignant than parent Siha cell.3.2 There was a significant difference in transplantation tumor's volume of nude mice at each time.4 The intracellular ultrastructure of two group observed by transmission electron microscopy.The CD44++/CD24+Siha cells have integrated cell membrane,complete nuclear membrane and nuclear,there was no significant changes in cytoplasmic contents.However the parent cell displayed vesicle,cells contraction,nuclear fragments and nuclear body formation.5 As the cell survival curve showed,CD44+/CD24+Siha cells and parent cells show a progressive decrease in parallel with an increasing dose of radiation.The CD44+/CD24+Siha cells decresed lightly.The SF2Gy,DO and Dq of CD44+/CD24+Siha cells was higher than parental group.SF2GyCD44+/CD24+group=0.894±0.027 VS SF2Gyparent siha group=0.745±0.056,(P<0.05);DOCD44+/CD24+group=4.588±1.037 VS Doparent Siha group=3.195±0.519(P<0.05);DqCD44+/CD24+group=4.114±0.854 VS Dqparent Siha group=2.154±0.337(P<0.05).6 Effects of two groups on invasion capability were detected by Transwell Assay.At the dose of OGy,2Gy,4Gy,6Gy,8Gy,the invasion ability of CD44+/CD24+Siha cell was significantly higher than parent Siha cell(P<0.05).For CD44+/CD24+Siha cell group,there was no statistically significant differences at 2Gy and 4Gy.But there was statistically significant differences at 6 Gy and 8 Gy.For parent Siha group,there was no statistically.significant differences at 2Gy,but statistically significant differences at 4 Gy,6 Gy and 8 Gy.7 Effects of two groups in Cell cycle proportion Changes before radiation and after radiation measured by FCM.The proportion of CD44++/CD24+Siha cells in G2 Phase before radiation and after 4Gy radiation was statistically significantly difference(P<0.05).However,there was no statistically significanily difference for parent Siha cell(P>0.05).Conclusion:1 In this report,the CD44+/CD24+Siha cells showed a highly mammosphere forming,tumorigenic,extensive proliferation capability.The CD44+/CD24+Siha cells exhibit the characteristics of stem cells.It is feasible to purify CSCs in Siha cell line by CD44+/CD24+.2 CD44+/CD24+Siha cells posses a higher degree of resistance to irradiation than parent Siha cells.3 The Stronger radio-resistance of CD44+/CD24+Siha cell may have a relation with G2 phase arrest.