| BackgroundCancer is a public health threat in humanity.Increasing studies reported that a cluster of cancer stem cells(CSCs)play an essential role in the development of tumor,which have the ability of self-renewal,indefinite proliferation and replication.The actual origin of CSCs is currently hypothesised that they are from normal tissue-specific stem cells in their origin tissues.Based on the oncogenic mutations or the change of microenvironment,the normal stem cells are endowed with some properties typical of malignant cells such as the activation of survival pathways and the ability of indefinite proliferation and then turn into the uncontrolled growth of cancer cells.Normal cells evolving progressively to a neoplastic state is a multistep process,including alterations in different genes,signal pathways and cytokines,which result in the cells to acquire the traits that enable them to become tumorigenic and ultimately malignant.Briefly,the hallmarks of cancer cells study by Hanahan Duglas are as followings: 1)sustainning proliferative signaling;2)evading growth suppressors;3)avoiding immune destruction;4)enabling replicative immortality;5)tumor-promoting inflammation;6)activating invasion metastasis;7)inducing angiogenesis;8)genome instabilty mutation;9)resisting cell death;10)deregulating cellular energetic.Recently,studies on cancers indicate that CSC strongly influences tumor recurrence and metastasis.Furthermore,it has been recognized as the main reason of resistance to conventional radiotherapies and chemotherapies.However,the mechnism of this is still not fully clearly yet.Cancer cells have been the most popular,widely and depth studied object on cancer research.In addition,cancer cells do not manifest cancer disease alone,and the microenvironment plays another essential role in the development of cancer.When the neoplastic cancer cells infiltrate or metastasize to another place in vivo,the microenvironment of the cells are going to meet is already changed,especially in the hematopoietic system.It is well konwn that the prostate tumor cells often metastasise to bone marrow,which strongly indicates that the bone marrow microenvironment is suitable for prostate CSCs to survive or in some cases to become dormancy in bone marrow.However,the specific molecular biological mechanism remains unclear.In this study,we examined the effect of different interleukins(IL-3,6,10,11 and 24)on the prostate cancer(PCa)cells,and specifially explored their influence on the stemness of the prostate cancer cells,aiming to shed new light on why prostate cancer cells are prone to metasise into the bone marrow and some times can be dormant there.The development of cancer is closely related with activation of oncogenes and inactivation of cancer suppressor genes,which eventually result in impart aberrant properties on normal cells and the transformation into cancer cells.CSCs have been recognized as the main cause of treatment resistance towards radiotherapy and chemotherapy.CD44 and CD133 were reported as molecular biomarkers for CSC,however,the study of these markers related with the clinical behaviour need further explored.In addition,for the development of tumor is complexity,and there is remarkable variability even within a given tumor type,typically called tumor heterogeneicity.Therefore,the optimal treatment strategies should be different,so-called “personalized cancer medicine”.In view of therapy selection based on experiences from clinical trials,it is essential to explore the correlation between CSC biomarkers and the clinical behaviour.In this study,we focused on one gene screened by transcriptome deep sequencing,Cacna2d1 which was highly expressed in the cancer stem-like cells in our lab.To supply the optimal therapy strategies and to improve patients' survival,immunohistochemistry(IHC)was used to investigate the relationship of the Cacna2d1 gene expression with the clinical behaviour in 238 cases ovarian cancer patient samples.In fact,the development of cancer is a combinational result inviolving muti-signal pathways and multi-factors.It is well known that the mitochondria are protected by double integrity membranes,but they are still easily damaged by intrinsic or extrinsic noxious stimulus,including oxidative stress and toxic drugs injury.It has been indicated that intensive mitochondrial dysfunction will induce cell apoptosis or necrosis,while persistent mitochondrial dysfunction,especially inherited by genetic alterations usually result in metabolic disorder-Warburg effect.In 1956,Dr.Otto Warburg first reported that cancer cells exhibited high rates of glucose uptake and lactic acid production,even in the presence of oxygen.It has been demonstrated that cancer cells preferred aerobic glycolysis to oxidative phosphorylation(Ox Phos).Increasing studies reported that Warburg effect will confer growth advantages on tumor cells,including the provision of faster production of ATP,amino acids for protein synthesis,nucleic acids for DNA duplication,and lipids for cell bio-membrane synthesis that might be needed in cancer cells proliferation,as well as for generating an acidic environment,which is harmful to normal cells but has no effect on cancer cells.In addition,the metabolic reprogramming in tumor cells may produce less reactive oxygen species(ROS),so that the genome of cancer cells may elude damage by a high concentration of ROS,resulting in apoptosis resistance in cancer cells.Furthermore,the genetic mutations of mitochondrial TCA cycle rate-limiting enzymes were strongly related with tumorigenesis.Studies has revealed that the mutation of fumarate hydratase(FH)is associated with hereditary leiomyomatosis and renal cell cancer(HLRCC).Based on the above theoretical knowledge,exploring the functional correlation to metabolic reprogramming of the Krebs cycle genes in the mitochondria may provide novel knowledge in developing therapeutic strategy to target CSCs.To do so in our study,the TALEN gene editing technology was used to establish ?-oxoglutarate dehydrogenase(OGDH)and fumarate hydratase(FH)gene knock out rats models,hoping to study why the cells in the gene knockout rats were forced to perform aerobic glycolysis,and then to explore the potential informce on cell stemness and related molecular pathological alteratiobs in vivo.In summary,in terms of cell biology,we focused on the tumor microenvironment,and studied the cytokines interleukins-3,6,10,11 and 24,which are derived from bone marrow,in order to know how these cytokines influence the cell stemness of prostate cancer cells.In a clinical pathological study,the cancer stem cell marker Cacna2d1 was explored in terms of its clinicopathological correlation and survival association by IHC in a series of 238 cases of ovarian cancer tissue samples.In vivo studies,the key enzymes genes OGDH and FH of TCA were separately knocked out in SD rats by TALEN gene editing technology,in order to explore the potential mechanism of metabolic dysregulation in cancinogenesis.In this study,we focused on the cells,clinical and animal models to explore how the stemness of cells and cancer cells are influenced by cytokines,cancer stem cell factor and key TCA cycle-related genes,aiming to better understand the mechanism how CSCs are accumulated,so that novel therapeutical options can be developed there after.Part ?:ILs-3,6 and 11 increase,but ILs-10 and 24 decrease stemness of human prostate cancer cells in vitroMethods1.Sulforhodamine B assay to examine the optimal effect dose of ILs-3,6,10,11 and 24 on the growth in LNCa P and PC-3 cells2.Wound healing assay to detect the cell mobility.3.Transwell assay to detect the effect of ILs on the migration and invasion ability of LNCa P and PC-3 cells.4.Flow cytometry analysis to evalue the effect of ILs on the apoptosis and chemosensitivity.5.RT-PCR,Western Blot and immunofluorescence assay were used to detect the expression of cancer stem marker-SOX2 in cells.6.Colony formation assay to explore the effect of ILs on the prostate cells stemness.7.CD44 and ABCG2 expression were detected by flow cytometry on cells.8.All data are analysed by using SPSS 22.0 software and expressed as mean ± the standard deviation(SD).Differences between two groups were examined by Student t test,three or more groups were subjected to one-way analysis of variance(ANOVA).Statistical significance was accepted at the level of P-value less than 0.05.Results1.IL-3,IL-6 and IL-11 were significantly increased the ability of growth in LNCaP and PC-3 cells lines,IL-10 and IL-24 were significantly decreased the growth ability in both cell lines.The optimal effect concentration of ILs is 5ng/ml.2.IL-3,IL-6 and IL-11 were significantly increased the mobility,migration and invasion ability in both cell lines,while IL-10 and IL-24 were significantly decreased the mobility,migration and invasion ability in both cell lines.3.IL-3,IL-6 and IL-11 were significantly reduced the number of apoptosis cells and increased the chemoresistance in both cell lines.Meantime,they also upregulate the expression of CD44 and ABCG2.While IL-10 and IL-24 were significantly increased the number of apoptosis cells and increased the chemosensitivity in both cell lines,and downregulate the expression of CD44 and ABCG2.4.IL-3,IL-6 and IL-11 were significantly increased the expression of SOX2 in gene level and protein level and increase the clonogenicity in both cell lines.IL-10 and IL-24 were significantly decreased the expression of SOX2 in gene level and protein level and decreased the clonogenicity in both cell lines.Part ?: Prognostic and clinicopathological significance of Cacna2d1 expression in epithelial ovarian cancers: a retrospective studyMethods1.Immunohistochemistry(IHC)was performed in a series of 238 cases epithelial ovarian cancer samples(EOCs),including 158 serous ovarian cancer samples(SOCs).2.Analysis the correlation between the expression of the potential CSC maker Cacna2d1 and clinicopathological parameters in statistically.3.SPSS 22.0(SPSS Inc,USA)was used for all the data analyses.The Chi-square test was used to evaluate the association between categorical variables.Medians and survival analyses were investigated using the Kaplan-Meier method,and groups were compared with log-rank tests.In addition,multivariate analyses were performed using Cox Regression method.P-values less than 0.05 were considered to indicate statistical significance.Results1.In the 238 epithelial ovarian cancer(EOC)samples,positive Cacna2d1 protein expression was observed in 83.6%(199/238)of the EOC tumors,among which 92(38.7%)tumors were weakly positive and 107 tumors(44.9%)were highly positive.Among the 158 serous carcinomas,the Cacna2d1 positivity was 148(93.7%),in which 60(38.0%)were weakly positive,and 88(55.7%)were highly positive.Most strikingly,the Cacna2d1 was specifically expressed in the infiltration front areas of the EOC tumors.2.Cacna2d1 was significantly associated with advanced FIGO stage(P<0.001),histological subtype(P=0.017)and tumor differentiation(P=0.015).The Cacna2d1 protein expression in the serous ovarian carcinomas(SOCs)showed that Cacna2d1 expression was significantly associated with FIGO stage(P<0.001)and the tumor differentiation(P=0.007).3.Positive Cacna2d1 protein expression was significantly associated with shorter progression free survival(PFS)and poor overall survival(OS)in both total EOCs and serous carcinomas,although multivariate analyses did not reach statistical significance.Part ?:Generation of TALEN-mediated OGDH and FH knockout rat modelMethods1.Transcription activator-like effector nucleases(TALENs)genome editing technology was used to establish ?-oxoglutarate dehydrogenase(OGDH)and famarate hydratase(FH)gene knockout rat models.The most effective TALEN plasmids were selected and then injected it into the embryos cytoplasm.DNA sequencing was applied to verify the mutation.2.The SD rats were followed by observing food taking,running activity and fur color,measuring the body weight at regular time points.3.The protein expression of OGDH was detected by Western Blot.RT-PCR and Western Blot were also used to examine the expression of FH on gene level and protein level in the tissues of the SD rats' heart,liver,lung,kidney,brain,spleen,stomach and testis,respectively.4.Expression of FH protein was also examined with an immunofluorescence microscope in the primary fibroblasts isolated from SD rats.5.Embryos sequencing was performed to examine the homozygous FH KO rats in the E8.0 and E15.0 gestation rats.6.Clinical haematology and biochemical examinations were applied to explore the pathological changes,blood smears to detect the morphology of the cells.7.Histological and immunohistochemistry(IHC)evaluation were used to evalue the pathological changes of kidney.8.All data are expressed as mean ± SD,and the student t-test was used to evaluate the significance differences between two groups.P<0.05 was regarded as significance difference in statisticlly.Results1.OGDH KO rat model and FH KO rat model were constructed successfully,in which there were a 8 base-pair deletion and a 11 base-pair deletion in the first exon of the OGDH and FH gene,respectively.The result of DNA sequencing showed that all of them were heterozygous OGDH KO(OGDH+/-)rats or heterozygous FH KO(FH+/-)rats.2.Compared with the WT rats,the litter size was decreased when the WT rats were mated with the FH+/-KO rats,and significantly reduced when OGDH+/-KO rats or FH+/-KO rats were mated.There was no behaviour difference except that OGDH+/-KO or FH+/-KO male rats showed significantly higher body weight within the 16-week observation period,especially in the 10-week and 14-week for OGDH+/-KO or FH+/-KO male rats,seperately.3.Compared with the WT rats,the OGDH protein expression was decreased,the rates of decreased up to 50.6%(P<0.01).In additon,the expression of FH protein in the FH+/-KO rats were also variably reduced in the tissues of the heart,liver,lung,kidney,brain,spleen,stomach and testis.4.The result of immunofluorescence microscope showed that FH protein expression was detected in the cytoplasmic of fibroblasts.Additionally,compared with the WT rats,FH protein expression was reduced in the fibroblasts of the FH+/-KO rats.5.There were no homozygous FH KO rats observed within the six-month period mating experiments including FH FH+/-KO rats mating with FH+/-KO rats and FH+/-KO rats mating with WT rats.The embryos of some pregnant rats in the,FH FH+/-KO rats mating with FH+/-KO rats group were further examined by genomic study,and no homozygous FH KO embryo was revealed,which strongly implys a possibility of embryo lethality of homozygous FH KO.6.Clinical haematology analysis showed that,compared with the WT rats,the values of white blood cell(WBC),platelet hematocrit(PCT),%mononucleosis(%MONO),#lymphocyte(#LYMPH)and #eosimophil(#EOS)in the FH+/-KO rats were observed decreased with 49.0%(P=0.004),14.7%(P=0.039),24.5%(P=0.025),51.6%(P=0.007)and 58.0%(P=0.023),respectively.Furthermore,biochemical examinations were found that blood urea nitrogen(BUN)and creatinine(CRE)in the FH+/-KO rats were increased with 20.1%(P=0.012)and 12.7%(P=0.010),while the blood uric acid(UA)was decreased about 25.0%(P=0.024).All above strongly suggested hematopoietic and kidney dysfunction in the FH+/-KO rats.7.Histologically,small foci in the medullary part of the kidney in the FH+/-KO rats revealed anaplastic alterations with prominent pleomorphic larger nuclei and slightly coarse chromatin in the tubular epithelial cells around glomeruli.Those anaplastic cells were also strongly positive for Ki67,p53 and SOX9,and such findings are most probably related to the kidney dysfunction.Conclusions1.IL-3,IL-6 and IL-11 may promote the ability of proliferation,migration,invasion and anti-apoptosis in LNCa P and PC-3 cells by upregulating the expression of cancer stem cell markers-SOX2,CD44 and ABCG2,which in turn may enhance the chemoresistance and clonogenicity in both cell lines.All above experiment results indicate that IL-3,IL-6 and IL-11 treated prostate cancer cells have high degree of stemness,and these tumor cells are more CSC-like.However,the IL-10 and IL-24 has the opposite effect on the prostate cancer cells,and significantly inhibit the ability of proliferation,migration and invasion in both cell lines by downregulating the expression of cancer stem cell markers-SOX2,CD44 and ABCG2,which might be recognized as tumor suppressor factors in prostate cancer.2.Cacna2d1 positive expression was closely related with the clinicopathological parameters and poor prognosis in the EOCs.It is strongly indicated that the Cacna2d1 may play a crucial role in promoting aggressive EOC behavior and progression.Cacna2d1 may serve as a novel predictive prognostic marker and a potential target for therapeutic intervention in EOCs.3.We have successfully established OGDH and FH gene knock out rat models.We did find some clue that the gene products in the cells in the rat models were reduced.In the FH+/-KO rats,the expression of Ki67,p53 and SOX9 were significantly strongly positive in the kidney epithelial cells.A long period of KO rats mating experiments were performed,and no homozygous FH KO rat was revealed,which strongly implyed a possibility of embryo lethality of homozygous FH KO.Collectively,our results demonstrate the potential value of this OGDH+/-KO rat and FH+/-KO rat models in the studies of metabolic disorders and tumorigenesis in vivo,which might be regarded as promising and novel insight strategy for the study of stemness regulation and related carcinogenesis and CSCs. |
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