| Part 1 The Clinical Significance of HER4 Expression in ColorectalCancerObjective:To investigate the HER4 expression in human colorectal cancer,and study the clinical significance of HER4 in colorectal cancer.Methods:1 we tested the expression of HER4 in 90 patients by immunohisto chemistry.2 we examined the association between HER4 and various clinicopa-thological characteristics,as well as the relationship between HER4 and survival.Result:1.Clinicopathological featuresA total of 90 patients were interviewed by telephone,of which 73samples received survival information successfully,and the follow-up rate was81.1%.Among these respondents,28 patients were colon cancer and 45 were rectal cancer.36 were males and 37 were females;38 patients were?60 years old and 35 patients were>60.41 patients were in stage I and stage II,and 32patients in stage III.2 cases were in stage T1,14 cases in T2,22 cases in T3 and35 cases in T4.There were 41 cases with negative lymph node metastasis and32 cases with positive.Of the patients followed,19 cases died,and the reasons of death were postoperative metastasis,11 cases of which were lung metastasis,4 cases bone metastasis and 5 cases of multiple metastasis.The overall survival rate was 73.97%.2.The HER4 expression in colorectal tissues and colorectal cancer tissues.HER4 was negative in normal colorectal tissues.The patients with HER4positive expression were 25 cases?34.2%?and negative were 48 cases?65.8%?.HER4 was mainly localized in cytoplasm and membrane of colorectal cancer tissues was pale yellow,brown and tan by staining.3.The relationship between HER4 expression and the clinicopathological parameters of colorectal cancerThe HER4 expression was not associated with genders,ages,tumor sites and T stages?P>0.05?.The HER4 presence was associated with lymph node metastasis?P<0.05?.Patients with positive lymph nodes were more likely to show HER4 positive than patients with negative lymph nodes?60.0%vs40.0%,P=0.039?.4.The relationship between HER4 and the 5-year survival rate of colorectal cancer patients.There was significant difference in patients with HER4 negative and positive for 5 years survival.The prognosis of HER4 positive patients was inferior to that of HER4 negative?P=0.020?.Summary:The expression of HER4 in colorectal cancer was associated with lymph node metastasis.The prognosis of HER4 positive patients was inferior to that of negative patients.Part 2 Construction Colorectal Cancer Cells Lines with HER4 ExpressionSilencedObjective:To construct HER4-targeted short hairpin RNA?shRNA?and its expression vector,then to determine the silencing effect in hunman colorectal cancer cells.Methods:1 Synthetized four lentiviral shRNA vectors,carrying human HER4 gene,ligased the HER4 interference sequences to lentiviral vector.2 Packaged the lentivirus,which was used to transfect 293T cells,and then obtained high titer lentiviral solution measured by condensing the supernatants.3 Evaluated the inhibitory effect on target gene by Western blot and RT-PCR,and constructed colorectal cancer cells lines with HER4 expression silenced.Result:1.Lentivirus vector constructionHER4-shRNA1-4 interference vector were sequenced,and lentiviral shRNA vector carrying human HER4 was successfully constructed by RNAi.2.The titer of lentivirus determinationThe titers of 4 lentiviruse groups were measured by fluorescence microscope and serial dilution.The titer of lentivirus was measured as follows:The infection titers of HER4-shRNA1,2,3,4 were 3×108,1×108,1×108,2×108TU/mL,respectively.3.Lentivirus infected cell linesThe infection efficiency of shHER4-1,HER4-2,shHER4-3 and shHER4-4 could be above 70%when the titer of lentivirus reached 1×108TU/mL and dilution ratio was 1:5.4.The effect of shRNA Lentivirus on the HER4 mRNA expression in HCT116.The HER4 mRNA expression levels in shHER4-1,shHER4-2,shHER4-3and shHER4-4 were 1.18±0.22,3.77±0.55,1.17±0.15,0.54±0.01,respectively.Compared to the negative?1.13±0.13?and blank control?1.0±0.13?,the HER4 mRNA expression level in shHER4-4 was significantly lower?P<0.05.?5.The effect of shRNA Lentivirus on the HER4 protein expression in HCT116.The HER4 gray values in HCT116,shHER4-4 and negative control were2070±50,1446±48,769±37,respectively.The HER4 protein expression levels in shHER4-4 cells significantly decreased than that of HCT116 and negative controls?P<0.05?.Summary:We construct colorectal cancer cells lines with HER4expression silenced,provid materials for the next experiment.Part 3 Effects of HER4 on Growth,Apoptosis,Adhesion and EpithelialMesenchymal Transition in Colon Cancer CellsObjective:To investigate the effect of HER4 on tumor growth,adhesion and cell apoptosis in colorectal cancer.To explore the role of HER4 in epithelial mesenchymal transition?EMT?in colorectal cancer.Methods:1 We examined the proliferation,apoptosis and adhesion of all three cell lines HCT116,negative control and shRNA-HER4 cell lines by employing MTT assay,flow cytometry,cell matrix adhesion assay.2 Used Western blot to measure the expression levels of E-cadherin,N-cadherin and Vimentin in HCT116 of the there cell lines.3 Used double staining immunofluorescence to test the expression levels of E-cadherin and Vimentin in HCT116 of the there cell lines.Results:1.The proliferation of HER4 expression silencingCompared with two control groups,the OD values of shRNA-HER4group decreased in 24h,48h,and 72h,respectively.There were significant differences between shRNA-HER4 group and control groups.2.The cell apoptosis of HER4 expression silencingThe apoptosis rates of HCT116 cells in shRNA-HER4,blank and negative control groups were?3.43±0.67?%,?0.76±0.33?%and?0.9±0.35?%,respectively.There were significant differences between shRNA-HER4 group and control group.3.The effects of HER4 expression silencing on cell adhesionThe number of cells adhered in the shRNA-HER4 was significantly higher than that in the blank and negative controls?P<0.05?.There were significant differences between shRNA-HER4 group and controls.4.The effect of HER4 expression silencing on the epithelial mesenchymal transition.The expression level of epithelial marker E-cadherin significantly increased in shRNA-HER4 cells group compared to the blank and negative controls?P<0.05?,while the expression level of mesenchymal markers N-cadherin and Vimentin significantly decreased compared to the blank control and negative controls?P<0.05?5.The expression levels of E-cadherin and Vimentin detected by double staining immunofluorescenceThe expression of E-cadherin was in the cell membrane with red fluorescence.Vimentin was green fluorescence.There were significantly increased in E-cadherin with red fluorescence of shRNA-HER4 group compared to blank and negative control cells?P<0.05?.While there were significantly decreased in Vimentin with green fluorescence of shRNA-HER4compared to blank and negative control cells?P<0.05?.Summary:HER4 could promote the proliferation,inhibit the apoptosis,and decrease the colorectal tumor cells adhesion through epithelial mesenchymal transformation to increase the tumor metastasis.Conclusion:1.The expression of HER4 in colorectal cancer was associated with lymph node metastasis.The prognosis of HER4 positive patients was inferior to that of negative patients.2.We construct colorectal cancer cells lines with HER4 expression silenced,provid materials for the next experiment.3.HER4 could promote the proliferation,inhibit the apoptosis,and decrease the colorectal tumor cells adhesion through epithelial mesenchymal transformation to increase the tumor metastasis. |
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