| Objective:87 cases of human colorectal cancer tissues and normal colorectal mucosa were collceted in The First Affiliated Hospital of Kunming Medical University,and were detected the expression level of non-SMC condensin I complex,cubunit H(NCAPH)among these samples,and analyzed its relationship with age,gender,tumor differentiation,tumor stage,tumor location,survival and prognosis of colorectal cancer patient,and then explored the NCAPH function in human colorectal cancer.We then investigate the function of NCAPH on proliferation,migration and apoptosis in colon cancer cell lines HCT-116 by using RNAi method in cellular level.And we investigate the effect of NCAPH on epithelial-to-mesenchymal transition(EMT)in colon cancer cell lines HCT-116.Finally,we also investigate the effects of NCAPH on the growth of colon cancer in Balb/c nude mice.Methods1.Tissue samples were collected from Department of Oncology of the first affiliated hospital of Kunming Medical University January-December 2011.We detected NCAPH expression in 87 cases colorectal cancer tissues and normal colorectal mucosa tissues by using the immunohistochemistry(IHC)staining assay.Our data are analyzed by SPSS 20.0 solftwares,Count data adoption rate,composition than the description;Measurement datawith normal distribution using X ? S,the skewness distribution data using M±Q description;Measurement data of normal distribution is compared between groups using analysis of variance(ANOVA),disorderly classification data comparing the differences between ?2 inspection;Binary classification level data to compare the Mann-Whitney rank and inspection inspection(U),multiple classification data to compare the Kruskal-Wallis rank and inspection inspection(H),survival analysis using the method of Kaplan-Meier,cancer patients survive multiple factors analysis using Cox regression.,P<0.05 was considered statistically significant analysis.Analyze its relationship with age,gender,tumor differentiation,tumor stage,tumor location,survival and prognosis of colorectal cancer patient.2.Firstly,we design three NCAPH-siRNAs by online website of Invitrogen company.After cells are transfected with three NCAPH-siRNAs,we tested the inhibitory ratio of NCAPH-siRNA by using qPCR and western blot assay,and we choose the best NCAPH-siRNA sequence.Subsequently,groups are divided into three groups:control group(normal),negative group(transfection reagent only),and NCAPH-siRNA group(NCAPH-siRNA).We detected the proliferation of cells by using CCK-8 test,the migration by using transwell and wound healing assay,the apoptosis by using flow cytometry,qPCR and western blot.3.To explore the mechanism of NCAPH promoting proliferation of HCT-116,the epithelial-to-mesenchymal transition(EMT)pathway related proteins are chosed for further study.We analyzed the epithelial marker genes(E-cadherin and?-catenin),and the mesenchymal marker genes(Fibronectin and Vimentin)by using qPCR and western blotting;and we also performed the immunofluorescence(IF)experiments to test the expression of E-cadherin and Fibronectin for research the function of NCAPH on EMT in HCT-116 cell lines.4.Transplanted subcutaneously tumor models of human colon cancer were established in nude mice with 1 × 108 different group treated HCT-116 cells respectively for 21 days.We observed and recorded the volume of tumor at 1,5,10,14 and 21 d.We take out the tumor at 21d,and pictured and weight them.We also detected the tumor tissues by using H&E staining and immunohistochemical technology.Results1.Results of IHC showed that the NCAPH expression had specificity in human colorectal cancer tissues(P=0.006).NCAPH expression(+++)in elderly patients(P<0.05)and occur in the poorly differentiated adenocarcinoma(P=0.027).In the right-sided colon,especially with high expression trend in ascending colon tissue,but did not show statistical difference because the right-sided colon cases are less than the left-sided colon,need to further expand the sample size to verify its specificity(P=0.067);NACPH low expression(-,+)in patients with higher survival than patients with high expression of(++,+++)(P=0.001);Cox regression showed that the tumor location of patients(left-sided colon,right-sided colon)(P=0.009),tumor staging(P = 0.013)and NCAPH expression intensity(P=0.005)is the main influence factors of survival in these patients.2.Results of RNAi showed that NCAPH-siRNA-2 dramatically inhibit the NCAPH level in HCT-116 cells.Thus,we chose the NCAPH-siRNA-2 in following experiments.Results of CCK-8 showed that the NCAPH-siRNA group had the lower cell viability(P<0.001)compared with control group,and there were no significant difference between negative group and control group(P>0.05).Results of cell migration showed that the NCAPH-siRNA group markedly inhibited the migration of HCT-116(P<0.001)compared with control group,and there were no significant different between negative group and control group(P>0.05).Results of cell apoptosis shown that the apoptosis of HCT-116 was induced markedly by the NCAPH-siRNA group(P<0.001)compared with control group,and there are no significant different between negative group and control group(P>0.05).The NCAPH-siRNA significantly promoted the proapoptosis related protein Bax(P<0.001),and suppressed the anti-apoptosis protein Bcl-2(P=0.0119,P<0.05).3.The results of qPCR and western blotting showed that the interference of NCAPH induced the the mesenchymal marker genes Fibronectin and Vimentin expression,and inhibited the the mesenchymal marker genes Fibronectin and Vimentin.The immunofluorescence data was consistent with above results.There were no significant different between negative group and control group.4.The growth of control and negative group tumor were rapid and the diameter of tumor were increased to about 15 mm in 21 days,and the weight was about 180 mg.However,the growth and weight of tumor in NCAPH-siRNA group were markedly decreased.The H&E staining data shown that cell proliferation was inhibited significantly in NCAPH-siRNA group.The IHC results shown that NCAPH expression were higher in NCAPH-siRNA group than control and negative group.It indicated that inhibition of NCAPH can reduce proliferation of cancer cells.Conclusion1.Compared to normal tissue,NCAPH was specificity expressed in human colorectal cancer tissues;NCAPH in right-sided colon specially in ascending colon with high expression trend,but need to be further expanded the sample size to verify its specificity;Through the analysis of survival,NACPH expression have negatively association with the prognosis of these patients,this may be related to NCAPH have higher expression ratio(+++)in elderly patients and patients with poorly differentiated adenocarcinoma increased;Cox regression showed that the tumor location,tumor staging and NCAPH expression intensity are the main factors influencing the patients survival.2.We obtained the best NCAPH-siRNA by RNAi technology.Results showed NCAPH interference can inhibit the proliferation and migration of HCT-116,and induced apoptosis.It implied that NCAPH could promote the proliferation and migration of HCT-116 and inhibit apoptosis.3.Results about EMT related proteins found that NCAPH interference can significantly increase the expressions of E-cadherin and ?-catenin,and markedly decrease the expressions of Fibronectin and Vimentin,EMT were suppressed.It implied that NCAPH promoted colon cancer through increasing epithelial-to-mesenchymal transition(EMT)pathway.However,further research about EMT signal pathway dependent experiments are needed to identify the mechanis.4.Results on nude mice found that NCAPH interference could inhibit the proliferation and growth of colon cancer cells in vivo.It implied that NCAPH promoted the proliferation and growth of colon cancer cells,and there were closely relationships between NCAPH and colon cancer. |
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