Study On The Mechanism Of Cell Cycle Regulatory Protein 50A In Ovarian Cancer Stem Cells

BackgroundEpithelial ovarian cancer(EOC)is the leading cause of gynecological cancer mortality,in this setting,optimal primary debulking surgery followed by platinum-and taxane-based chemotherapy is the standard treatment of choice.Over the past few decades,the rapid development of molecular targeted therapy and immuno-oncological cellular therapy has facilitated the treatment strategies of ovarian cancer.However,there are still 60～80%patients relapsed and the 5-year survival rate in advanced stage is only 30%.The main reason is considered to be chemo-resistance,but the mechanism is still not clear.So it becomes the enormous barrier for ovarian cancer therapy.The CSCs are interpreted as a small proportion of "self-renew" malignant stem cells with the capacity to undergo asymmetric divisions to recreate a tumor at quiescent state.Numbers of studies have found that there existed a very small number of CSCs(cancer stem cells)in tumors such as hematological system and many solid tumors,which maintained the tumorigenicity and heterogeneity by the main two characteristics of self-renew and differentiation.The quiescent cells can escape from the intracellular toxic damage then result in the chemo-resistance and progression and metastasis of tumors.With the intensive research of CSCs,a series of separation,identification methods have been established,and the molecular mechanism researches of CSCs have been in progress.In the term of ovarian cancer stem cells research,the present studies mainly focus on the following surface markers:CD133,CD24,CD117,CD44,ALDH1A1,CD90 and so on,which maintain the stemness though various mechanisms such as the changes of tumor cells and microenvironment,epigenetic modifications or signaling pathway regulation.However,there has no consensus on which ones are the certain markers of ovarian cancer.Therefore,it is significant for targeted therapy to hint the specific surface markers and develop their mechanisms furtherly.Our previous study has screened the surface OCSCs marker-CDC50A by isotope labeling method and quantitative proteomic strategy.It was proved that CDC50A might be the surface marker of OCSCs though the general identification of the CSCs properties of CDC50A surface markers positive cells(cell-lines and primary cancer cells included)in vivo and in vitro.And cell-line chemotherapy resistance and the xenograft module setting in immune-suppressed mice have confirmed the drug resistance capacity.Simultaneously,with the shRNA to down-regulate the expression of CDC50A,the properties of CSCs have been confirmed.Furthermore,the genes of CDC50A-related P4 family of ATPase phospholipid transporting have been screened by RT-PCR.On the year of 2004,NATURE proclaimed the euchromatic sequence of the human genome had finished,which has been provided significant sequence evidences for biological function and interaction of genes.Molecular cloning technology is the basis of genic study including gene structures and functions as transcription,translation,which are analyzed according to the full-length sequence.Rapid amplification of cDNA ends(RACE)is the common procedure to yield complete sequences of cDNA,which facilitate the rapid amplification of full-length cDNA 5’-and 3’-ends after a partial cDNA sequence has been obtained.The full-length cDNAs of candidate were obtained by RACE technology,which is the key process in this study to facilitate depth research on the relationship between P4-ATPases and CDC50A.P-type ATPase family has a wide distribution.With an adenosine triphosphate-dependent multi-transmembrane structure,they intermediate substances transport and then fulfil multiple important physiological functions.Amongst,the members of the P4-ATPases are only present in eukaryotic organisms and there are 11 CDC50A-related genes that encode P4-ATPases exist in human,including ATP8A1、ATP8A2、ATP8B1、ATP8B2、ATP8B4、ATP10A、ATP10B、ATP10D、ATP11A、ATP11B、ATP11C、Based on the previous study,we make researches on CSC properties of CDC50A-overexpressed SKOV3 to certify the role of CDC50A on ovarian cancer stem cells.Combined with molecular clone technology and genomics to construct plasmids carrying targeted full-length genes,then complete construction of stable cells using lenti-virus infection technology.Then investigate the effect of ATP11A or ATP11B on CSCs.After co-expression of ATP11A or ATP11B and CDC50A to investigate the effect of ATP11A or ATP11B on CDC50A-overexpressed SKOV3,further mechanisms of the properties of OCSCs were validated.Methods1.Validation of a plvx-FLAG-GFP-based vector carrying CDC50A cDNA then construction of lentivirus expression vector and construction of stable cells using lenti-virus infection technology.Certify the role of CDC50A on ovarian cancer stem cells by sphere forming and cisplatin-resistance assays.2.Based on the whole genome sequence database of ATP11A、ATP11B and expression vectors,strategies of clone were designed.The sequence of ATP11A was spliced cloning and the ATP11B was full-length obtained.The CDS region of ATP11A、ATP11B were introduced into the pLVX-IRES-mcherry、pLVX-IRES-RFP expression vectors with a C-terminal myc tag(ATP11A-myc-mcherry)and a C-terminal HA tag(ATP11B-HA-RFP),respectively.The new constructed vectors were transformed to the engineered Escherichia coli and then targeted plasmids were extracted.After gel electrophoresis、enzyme digestion analysis and gene sequencing,the plasmids were transfected into 293FT cell-lines.The transfection efficiency was tested by fluorescence microscope and flow cytometry.The expressions of the two identified genes were analyzed using western blot.The expressions of the two identified genes were analyzed using western blot.Package lentivirus vectors then infect SKOV3 to reconstruct stable cells loaded with ATP11A or ATP11B cDNA to analysis the properties of CSCs.3.Reconstruct 4 pairs of stable cells loaded with P4-ATPase and CDC50A cDNA,including double genes groups(SKOV3-CDC50A+ATP11 A,SKOV3-CDC50A+ATP11B),single genes groups(SKOV3-ATP11A+GFP,SKOV3-ATP11B+GFP,SKOV3-CDC50A+mcherry,SKOV3-CDC50A+RFP)and negative control group(SKOV3-GFP+mcherry,SKOV3-GFP+RFP).The bifluorescence cells were sorted by Moflo fluorescent and then cultured in medium.The groups of ATP11A/CDC50A or ATP11B/CDC50A were fixed for immunofluorescence analysis of the sub-cellular localization.The sphere forming capabilities were compared in 2 pair-groups of stable cells.And cisplatin resistances to CDC50A,ATP11A,ATP11B were tested in spheres cultured in medium with different concentrations of cisplatin.To further analyze the mechanism,we applied flow cytometry analysis to detection of the early apoptosis cells in the CDC50A VS vector groups or CDC50A+ATP11A/ATP11B VS CDC50A groups induced by 5uM concentration of cisplatin.Results1.Compared to negative control,SKOV3-CDC50A was more capable of generating spheres(25.5 ±4.76 VS 9.8±4.26,P=0.000).Cisplatin treating assay showed that in the condition of 5umol/l or 10umol/l CDDP,CDC50A could increase the capacity of cisplatin-resistance(P=0.040,P=0.011).In the time curve,the relative sphere number of CDC50A group decreased much more slowly(P=0.039).2.Both of ATP11A and ATP11B failed to increase sphere forming compared to negative control(10.7±2.828 VS 8.0±2.121,P=0.185;7.0±2.121 VS 6.7±1.414,P=0.795).After treated by cisplatin,the relative sphere numbers of all groups declined,and there was no statistics significance(P=0.248,P=0.093).3.compared to the CDC50A-overexpessed groups,SKOV3-CDC50A+ATP11A、SKOV3-CDC50A+ATP11B formed much more spheres(21.7±2.3 VS 12.3±3.2,P=0.015;18.7±4.2 VS 10.3±2.1,P=0.036).Cisplatin treating assay showed that when CDDP concentration was lumol/1,the groups of single gene or vector control RSN=1,in the spheres carrying double genes RSN>1.Low dose of cisplatin can induce the sphere forming in double genes groups(P=0.004,P=0.029).When the concentration was 5umol/l,both CDC50A+ATP11A and CDC50A+ATP11B groups RSN=1,however CDC50A groups RSN<1(P =0.024,P=0.025).In the condition of 10umol/l CDDP,the RSN of all groups declined.The decrease of RSN in double genes groups and CDC50A group presented no significance.In the time curve,the relative sphere number of CDC50A+ATP11A or CDC50A+ATP11B group decreased much more slowly than CDC50A group(P=0.024,P=0.024).The cisplatin-induced early apoptosis assay exhibited the apoptosis rates of CDC50A group was much lower than negative control.Besides,when co-regulation with ATP11A or ATP11B,the early apoptosis rate was smaller than the CDC50A counterpart.Conclusions1.Upregulation of CDC50A in SKOV3 can enhance the cancer stem cells property.2.Molecular cloning technology is an important means to study genes function,which can increase the targeted genes specifically and efficiently.Neither ATP11A nor ATP11B contributes to the properties of the ovarian cancer stem cells.3.Both genes of ATP11A and ATP11B contribute to the properties of the CDC50A positive cancer stem cells.Furthermore,under certain concentration of cisplatin,ATP11A and ATP11B can enhance CDC50A subsets chemo-resistance by inhibiting early apoptosis.