| Background:Colorectal cancer(CRC)is one of the common gastrointestinal tumors,Surgical resection of primary or metastatic tumors is the best treatment for patients,but most patients with colorectal cancer can not achieve complete cure.Therefore,radiotherapy and chemotherapy in the treatment of colorectal cancer is important.Cancer stem cells(CSCs)that are present in various solid tumors(such as breast cancer,pancreatic cancer,colon cancer,etc.)resist to existing radiotherapy and chemotherapy regimens.Traditional chemotherapy drugs can rapidly break down tumor cells,but not tumor stem cells.The presence of CSCs may be the underlying cause of cancer malignant biologic behavior.Cell mitosis is the basis for eukaryotic cell differentiation and proliferation.non-SMC condensing I complex subunit D2(NCAPD2)and non-SMC condensingII complex subunit D3(NCAPD3)play a key role in eukaryotic cell mitosis.Previously,we found that the expression of NCAPD2 and NCAPD3 were anomaly high in colorectal cancer by DNA High-throughput sequencing.Baicalin is a flavonoid compound extracted from baicalensis.The chemical formula is C21H18O11.Previous study shows that baicalin has an significantly inhibitory effection on the growth of colon cancer HCT116 cells in nude mice.The mechanism was related to G2/M arrest and induction of tumor cell apoptosis.Aim:To observe the expression of NCAPD2 and NCPAD3 in colorectal cancer tissues and cells,trying to explore the possible relationship between NCAPD2 and NCAPD3 and the occurrence and development of colorectal cancer.To observe the effects of baicalin on the stemness and the expression of NCAPD2/NCAPD3 of human colorectal cancer RKO cells,HCT116 cells,SW480 cells and HCT116 stem cells in vitro,To explore the mechanism of anti-tumor effect of baicalin,and to provide theoretical basis and research direction for clinical targeted therapy of colorectal cancer.Methods:1.Colorectal normal tissue and colorectal cancer tissue frozen samples were selected,High throughput DNA sequencing was used to check the expression of NCAPD2 and NCAPD3.Choose the same sample,Western bolt detects the expression of NCAPD2 and NCAPD3.2.Extracting Total RNA from Selected colorectal paraffin block samples of colorectal normal tissue,ulcerative colitis tissue and colorectal cancer tissue,qRT-PCR detects the expression of NCAPD2 and NCAPD3.Combined with clinical data of patients,conducting correlational analyses.3.cell culture and proliferation.The colorectal cell FHC and colorectal cancer cell line RKO,HCT116 and SW480 was purchased from cell bank of American Type Culture Collection.Cells were kept in incubator with 37℃,5%CO2 and saturated humidity.Cells were trypsinized and passaged at the ratio of 1:5 as the cells grow prior to completely cover the bottom of the bottle.4.Exponential phase cells from subjects,with different concentration of baicalin in dealing with 48hours later,Western bolt detect the marker of stemness and the expression of NCAPD2 and NCAPD3.5.Stem cell sorted,culture and proliferation.Isolation of HCT116 stem cells in serum-free medium,and seeded into Ultra-low adhesion in a density of 200 cells/ml with Stem cell conditioned medium.Cells were kept in incubator with 37℃,5%CO2 and saturated humidity.It is passaged When the diameter of the sphere forming cell reaches 100μm.6.Identification of stem cell stemness.the self-renewal capacity of stem cell was Identified by tumor sphere serial passage culture.The expression level of CD 133,CD44,Nanog,Oct4,SOX2 and NCAPD2,NCAPD3 in HCT116 sphere forming cell and normal cell was analyzed by western blot and RT-PCR.7.HCT116 sphere forming cell,with different concentration of baicalin in dealing with 48hours later,seeded into Ultra-low adhesion in a density of 200 cells/ml with Stem cell conditioned medium.To observe the capacity of sphere forming.8.HCT116 sphere forming cell,with different concentration of baicalin in dealing with 48hours later,western blot detect the expression level of CD 133,CD44,Nanog,Oct4,SOX2 and NCAPD2,NCAPD3.9.Statistics analysis.Using software SPSS21.0,the indicators are expressed in M ± SD,Two sample for means were compared by t test,all groups were compared by one-way ANOVA.If P<0.05 considered statistically significant differences.Results:1.High throughput DNA sequencing shows the expression level of NCAPD2 and NCAPD3 in colorectal cancer tissue is higher than that in colorectal normal tissue.Western blot assay shows the expression level of NCAPD2 and NCAPD3 in colorectal cancer tissue is higher than that in colorectal normal tissue(P<0.01).In cells,the expression level of NCAPD2 and NCAPD3 in HCT116 and RKO is significantly higher than that in FHC(P<0.01).There is not significant difference among SW480,HT29 and FHC.2.QRT-PCR assay shows that the expression level of NCAPD2,NCAPD3 in ulcerative colitis tissue is higher than that in colorectal normal tissue(P<0.01).In colorectal cancer tissue,the expression level of NCAPD2 is higher than that in colorectal normal tissue,but it has no significance(P=0.0878),the expression level of NCAPD3 is significantly higher than that in colorectal normal tissue(P<0.01).3.Combined with clinical data of patients,Female patients with CRC have a higher expression of NCAPD3 than female patients without CRC(P<0.05),but it has no significant difference of the expression of NCAPD2 and NCAPD3 between female and male patients with CRC.Combined with patient’s age,number of metastasis lympho node,T stage of CRC,tumor differentiation and the tumer histologic type,it has no significant difference of the expression of NCAPD2 and NCAPD3.4.In cells,the expression level of NCAPD2 and NCAPD3 in HCT116 and RKO is significantly higher than that in FHC(P<0.01).There is not significant difference among SW480,HT29 and FHC.NCAPD2 and NCAPD3 in HCT116 stem cell show a lower expression than that in HCT116 cell(P<0.01).5.After 48 hours treated in different concenteations(50,100,200,)μg/ml of RKO,HCT1 16 and SW480 cells,the expression level of CD133,CD44,Nanog,OCT4,SOX2,NCAPD2 and NCAPD3 is significantly higher than that in the control group.Moreover,with the increase in the concentration of drugs the expression level showed a downward trend.6.After 48 hours treated in different concenteations(50,100,200,)μg/ml of HCT116 stem cell,the expression level of CD133,CD44,Nanog,OCT4,SOX2,NCAPD2 and NCAPD3 is significantly higher than that in the control group.Moreover,with the increase in the concentration of drugs the expression level showed a downward trend.Conclusion:1.NCAPD2 and NCAPD3 are highly expressed in ulcerative colitis tissue and colorectal cancer tissue,and NCAPD2/NCAPD3 may be involved in the development and progression of colorectal cancer.2.Baicalin inhibits the stemness and the expression of NCAPD2/NCAPD3 of colorectal cancer cells in a certain concentration-dependent.3.Baicalin inhibits the sternness and the expression of NCAPD2/NCAPD3of colorectal cancer HCT116 stem cells in a certain concentration-dependent.4.The inhibitory effection of baicalin on the stemness of colorectal cancer HCT116 stem cells may be related to the inhibition of NCAPD2 and NCAPD3 expression. |
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