| BackgroundColorectal cancer(CRC)is one of the deadliest gastrointestinal malignancies with a high incidence and mortality rate worldwide.In recent years’ colorectal cancer has been placed as the third most common cancer among all cancers.Although adjuvant progress has been made in the prevention,diagnosis and therapy of colorectal cancer,metastasis and recurrence are considered the major problem for successful treatment.Several factors are involved in the heterogeneity and recurrence of colorectal cancer,though cancer stem cells are recently believed to play major role in the etiology of colorectal cancer.Cancer stem cells(CSCs)have been settled as a cellular mechanism that contributes to phenotypic and functional heterogeneity in diverse cancers.Although cancer stem cells account for a very minor population of cancer,they are considered a major threat for prognosis and treatment response of various cancers including colorectal cancer.Colorectal cancer stem cells(CR-CSCs)share the major biological characteristics of stem cells from other solid tumors.Several studies indicate the existence of colorectal cancer stem cells in human colorectal cancer and their contribution to clinical tumor progression,chemoresistance,and therapeutic collapse.Over the past years,many surface markers haven been proposed for the identification and characterization of colorectal cancer stem cells including CD44,CD133,CD166,Lgr5,ALDH,Nanog,Sox2,and Oct-4.However,identifying cancer stem cells in CRC remains a difficult challenge.Lysine-specific demethylase 2B(KDM2B)is a member of the Jmj C domain-containing histone demethylase(JHDM)family which is known to function either as a tumor suppressor gene or oncogene.KDM2 B participates in several aspects of normal cellular processes such as cell differentiation,cell senescence and stem cell self-renewal.KDM2B gene expression is also associated with several abnormalities including Genitourinary Anomalies,Mental Retardation Syndrome and Tumor.Increased KDM2 B gene expression is reported in breast cancer,ovarian cancer and glioma.Meanwhile,KDM2 B was reported to decrease the expression of several factors in cancer cells.These factors are reported to have an important function in tumor cell survival,tumor growth,invasion,and metastasis.Apart from that,KDM2 B has been reported to control cancer stem cell self-renewal in some cancers.However,the impacts and underlying mechanisms of KDM2 B on the CR-CSCs have not been explored.Enhancer of zeste homolog 2(EZH2)is a component of PRC2 that mediates methylation of H3K27 and functions in the maintenance of embryonic stem cell pluripotency and plasticity.It is an important regulator of cancer development and progression.Suppressed EZH2 expression is found to affect tumor cell proliferation and invasion.Thus,EZH2 is considered a tumor suppressor.The expression of EZH2 is regulated by various oncogenic transcription factors and cancer-associated non-coding RNA that are critical for cell proliferation,tumorigenesis,and stem cell maintenance.Furthermore,studies have reported that EZH2 inhibition and knockdown dramatically decrease CSCs tumorigenicity.EZH2 gene expression has been reported to be regulated by KDM2 B in some abnormalities.However,in colorectal cancer,their relationships are not fully eluted.KDM2 B and EZH2 play important role in the maintenance of CSC self-renewal capacity and tumorigenic ability,however,the biological functions of those genes in colorectal cancer remain unclear.Several pathways have been proposed in the regulation of colorectal cancer stem cells,including,ERK pathway,Egfr/Akt/Nf-κB pathway.The impact of the PI3K/AKT signaling pathway in cancer development and progression is well documented.This signaling is crucial in cancer because it advances cell growth and survival.Apart from its role in solid tumors,PI3K/Akt pathway plays an important role in cancer stem cell.A previous study reported that the PI3K/Akt pathway plays an important role in colon CSC sphere formation and growth,which represents CSC properties of stemness and proliferation.Emphasizing the interest in this particular signaling pathway remains a chance and a challenge for cancer therapy.Objectives1.The first objective of this study is to determine the functional role of KDM2 B gene expression in colorectal cancer cell progression and colorectal cancer stem cell features.2.To confirm the regulatory correlation between KDM2 B and EZH2 and determine their influence on colorectal cancer stem cell characteristic regarding cells self-renewal ability of colorectal cancer cells in vitro via activating PI3K/AKT signaling pathway.3.To evaluate the functional role and expression of KDM2 B in vivo.Methods1.Immunohistochemistry(IHC)was used to determine protein expression level in human tissues both adjacent normal tissue and tumor tissue.2.In colorectal cancer HT-29 and DLD-1 cells,the transient downregulation of KDM2 B was achieved by small interfering RNA(si RNA)against KDM2 B.The cells were divided into 3 groups:(1)si KDM2B#1,(2)si KDM2B#2 and(3)non-specific si RNA(NC)using lipofectamine 2000.3.Achievement of stable downregulation of KDM2 B and EZH2 was done by using Lentivirus vectors in both cells line HT-29 and DLD-1.The cells were divided into4 groups:(1)sh RNA-KDM2 B,(2)sh RNA-EZH2,(3)negative control(NC)and the(4)non-transfected group was labeled as control.4.RT-q PCR was applied to measure the m RNA level of KDM2 B expression after transient and stable transfection.The western blot and was performed to determine protein level of KDM2 B,EZH2,CD133,CD44,ALDH1,P21,Cyclin D,P27,PI3 K,p PI3 K,AKT and p AKT.5.In order to determine the effect of KDM2 B on proliferative cell ability we performed CCK-8,colony formation,and comet assays in both HT-29 and DLD-1 cells line.6.To determine whether KDM2 B gene expression is associated with CRC stem cells features,we measured sphere formation assay and the expression of CRC stem cells markers(CD133,CD44,ALDH-1)in HT-29 and DLD-1 cells line.7.To determine the regulating roles of KDM2 B on EZH2 transcription,we analyzed the impact of KDM2 B on the cytoplasm and nuclear protein levels of EZH2 by Cytoplasmic and Nuclear protein fractionation assay.8.Magnetic Sorting-Based Separation was used to isolate the stem cells CD133+/CD44+ and CD133-/CD44-subpopulations form HT-29 cell.9.Immunofluorescence Staining(IF)was used to determine the effects of KDM2 B and EZH2 in colorectal cancer HT-29 and DLD-1 cells and cancer stem cell CD133+/CD44+ and CD133-/CD44-subpopulations.10.The scratch wound healing assay and Transwell migration and invasion assays were performed to evaluate the effect of KDM2 B and EZH2 in migratory and invasive potentials of HT-29 and DLD-1 cells.11.In order to measure the role of KDM2 B tumorigenicity of CRC in vivo,BALB/c nude mice were inoculated with transfected DLD-1 cells and non-transfected DLD-1 cells.Results(1)The IHC score showed that KDM2 B gene expression was highly expressed in colorectal tumor tissues compared with adjacent normal tissues(P < 0.001)and the expression level of KDM2 B in stage I-II and stage II was higher than stage II-III+III(P < 0.05).The correlation between clinical features and KDM2 B gene expression showed that overexpression of KDM2 B positively correlated with tumor stages(P = 0.012)and TNM(tumor/nude/metastases)classification(N: P = 0.033,T: P = 0.029,< 0.05),however there was no significance in M(P > 0.05).(2)The Western blot results showed that KDM2 B protein expression was higher in colorectal cancer cells(HT-29,ROK,LOVO,and DLD-1)compared with normal epithelial cells(CCD841Co N).The transfection efficiency of KDM2 B was further confirmed by western blot and q PCR.The result showed significantly decreased KDM2 B protein and m RNA levels in si KDM2 B #1 and si KDM2 B #2 groups compared with the NC group(P<0.01).The quantified collected data,were normalized by using GAPDH and beta-tubulin.(3)CCK-8 assay showed a significant decrease in HT-29 and DLD-1 cell proliferative rate of si KDM2 B #1 and si KDM2 B #2 groups compared to the NC group(P<0.01).Also in both cell lines HT-29 and DLD-1,the average colony count in the si KDM2 B #1 group(334 ± 11.31,672 ± 5.65)and si KDM2 B #2(267 ± 18.38,616 ± 55.86)was significantly decreased than in the NC group(470 ± 11.31,789 ± 25.46)(P<0.01).In addition,the comet assay showed that the knockdown of KDM2 B induced cell DNA damage in HT-29 and DLD-1 cells.The result showed increased comet tail in si KDM2 B #1 group(69 ± 8.41,68 ± 6.84),si KDM2 B #2(78.42 ± 3.32,73.11 ± 9.03)transient KDM2 B knockdown group compared to negative control(2.13 ± 0.52,5.09 ± 9.03)(p < 0.001).The proteins studies showed that the expressions of P21 and P27 were increased by KDM2 B knockdown,whereas the cyclin D expression was decreased KDM2 B knockdown compared with NC.(4)To determine whether KDM2 B downregulation in CRC is associated with stem-like properties,a sphere formation assay was performed.Western blot results show that KDM2 B protein expression level was higher in sphere cells compared to adherent cells.Moreover,after successful downregulation of KDM2 B in HT-29 and DLD-1 cells,the results showed reduced sphere size in si KDM2 B #1 group(23± 2.82,30 ± 2.12),si KDM2 B #2(27 ± 2.83,37 ± 2.12)compared with NC(57 ± 4.25,28 ± 3.54)(P < 0.05).Similar results were obtained in the average sphere number;si KDM2 B #1 group(10 ± 1.41,12 ± 2.83),si KDM2 B #2(13.5 ± 2.12,12 ± 1.41)and NC(23 ± 3.54,28.5 ± 3.54).Furthermore,western blot showed that the downregulation of KDM2 B was capable of decreasing the CRC cell stemness markers including CD44,CD133 and ALDH-1.(5)The stable downregulation of KDM2 B was confirmed by a fluorescent microscope and by performing q RT-PCR.The result showed that more than 90% of infected cells had green fluorescence under the fluorescence microscope and the m RNA expression level of KDM2 B was lower in both cells(p < 0.001).After stable downregulation of KDM2 B,we run western blot,immunofluorescence and cell fraction to confirm the relationship between EZH2 and KDM2 B.The result showed that EZH2 expression was markedly reduced by downregulation of KDM2 B at protein level(p < 0.05),and knockdown of KDM2 B decreased the protein levels of EZH2 in the cytoplasm and increased the levels in the nucleus.Furthermore,to investigate whether the KDM2 B and EZH2 could regulate PI3K/Akt signaling pathway,we examined the effect of KDM2 B and EZH2 on the downstream proteins of the PI3K/Akt pathway in CRC HT-29 and DLD-1 cells.The result showed that downregulation of KDM2 B and EZH2 in HT-29 and DLD-1 cells decreased the expression of p PI3 K and p AKT and increased the expression of PI3 K and AKT(P<0.01,P<0.05).(6)Moreover,the colorectal cancer stem cell study showed that after stem cell isolation,the protein levels of KDM2 B and EZH2 were significantly higher in the CD133+/CD44+ cells population than in CD133-/CD44-cells.Then,after a successful downregulation of KDM2 B and EZH2 in CD133+/CD44+ cells,we observed that,the results for the spheres formation analysis showed a significant decrease in the number of spheres in sh KDM2 B and sh EZH2 groups compared with control and NC groups.In addition to the sphere formation study,the proteins studies showed that the expressions of AKT,PI3 K expression were increased after KDM2 B and EZH2 knockdown in CD133+/CD44+ cells.The surface markers CD133,CD44,and ALDH-1 showed decreased expression in sh KDM2 B and sh EZH2 as compared with control and NC as well.(7)Scratch wound healing assay showed that there was significant retardation of cells to wound area of sh KDM2 B and sh EZH2 groups compared to Control and NC groups in both cells HT-29 and DLD-1 cells,(P<0.01).The numbers of migrated cells penetrating through Transwell chamber without Matrigel in the sh KDM2B-HT-29(42.0 ± 5.66),sh EZH2-HT-29(40.0 ± 7.07)and sh KDM2B-DLD-1(52.0 ± 5.66),sh EZH2-DLD-1(48.0 ± 1.41)groups were significantly decreased than in the control groups(91.0 ± 4.94,110.5 ± 3.54)(P < 0.01).The Transwell invasive assay showed similar results,where the invading cells in the sh KDM2B-HT-29(33.5 ± 7.78),sh EZH2-HT29(30.5 ± 2.12)and sh KDM2B-DLD-1(37.0 ± 2.82),sh EZH2-DLD-1(33.0 ± 2.83)groups were reduced compared with control groups(65.0 ± 5.65,70.0 ± 4.24)(P<0.01).(8)KDM2B transcriptionally decreased the expression of EZH2 in CRC cells,we analyzed the protein levels of EZH2 in the same cohorts of TMA sections for KDM2 B and immunostaining with a specific anti-EZH2 antibody.The results showed that EZH2 was highly expressed in the nucleus of tumor tissue and the Pearson correlation showed a positive correlation between EZH2 and KDM2 B expression in CRC Human Tissues(P <0.001).(9)KDM2B knockdown was shown to retard tumor growth from the inoculated mice.The in vivo study showed that there was significant increase in tumor growth in mice inoculated with untreated DLD-1 cells(control)and NC while we observed a reduction in tumor growth in mice inoculated with sh KDM2 B respectively.The measurement of tumor volume after 4 weeks in sh KDM2 B groups was 68.80 ±17.18 while the control and negative control were 278.05 ±42.70;251.07 ±22.42(P < 0.0001).Furthermore,the western blot results of the tissue samples showed that EZH2 was markedly downregulated at protein levels in the sh KDM2 B group compared with the control and negative control groups.The immunohistochemistry result showed that the tumor inoculated with sh KDM2 B cells to have lower intensity of KDM2 B and EZH2 expression,compared to the tissues samples from the mice inoculated with the control(DLD-1 untreated cells)and NC groups.This finding was consistent with in vitro results.Conclusion(1)KDM2B is highly expressed in CRC tissue as compared with normal tissue and was strongly related to the clinical stage and TNM stage(2)Downregulation of KDM2 B decreased colorectal cancer cell viability,induced DNA damage,concluding that KDM2 B might act as an oncogene.(3)Downregulation of KDM2 B decreased CRC cancer cell spheroids,decreased the expression of colorectal cancer stem cell surface markers CD133,CD44,ALDH-1 concluding that KDM2 B regulates CRC stemness.(4)Downregulation of endogenous KDM2 B transcriptionaly decreased the expression of EZH2 and PI3K/AKT pathway.Knockdown of KDM2 B and EZH2 reduced the migration of colorectal cancer cells.KDM2 B and EZH2 were indispensable for colorectal cancer stem cells maintenance in vitro by regulating PI3K/AKT signaling pathway.EZH2 expression is positively correlated to KDM2 B in CRC tissues,and both play important role in CRC progression.(5)The in vivo study confirms the oncogenic function of KDM2 B in CRC cells and the relationship between KDM2 B and EZH2.Repression of KDM2 B expression shown to inhibit tumor growth from the mice inoculated with sh KDM2 B.Moreover,KDM2 B seems to regulate EZH2 expression in vitro and in vivo.Altogether,we conclude that our findings show that KDM2 B is an important oncogenic gene in colorectal cancer cells and colorectal cancer stem cells,and this functional relation between KDM2 B and EZH2 could be exploited for the development of novel colorectal cancer and colorectal cancer stem cells therapy. |
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