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Objective To investigate the effects of lentivirus-mediated RNA interference (RNAi) targeting DNA binding protein A (dbpA) on the proliferation and the biological behavior of colorectal cancer cell line SW620.Methods The experiment was divided into 3 groups:KD group (siRNA-dbpA,lentivirus interference group),CON group (non-specific sequence group) and NC group (blank control group).The lentiviral vector siRNA-dbpA was constructed and verified by PCR and DNA sequencing.SW620 cells were transfected with siRNA-dbpA plasmid,nontargeting siRNA plasmid,or empty plasmid.After 48 h the transfection,the cells were examined for dbpA expression using Western blot.After 72 hrs transfection,flow cytometry was used to detect the cell apoptosis and cell cycle changes.The cell growth inhibition rate was detected by MTT (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay,and then clone formation was detected,and the ability of SW620 cells to form tumors in vivo after dbpA was silenced was studied in nude mice.Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting dbpA gene was successfully inserted into the lentiviral vector.siRNA-dbpA transfection resulted in reduced expression of dbpA in SW620 cells.After transfection,the apoptosis rate of siRNA-dbpA-transfected cells increased to 26.60％ ± 0.38％,significantly higher than that in cells transfected with the nontargeting plasmid or the empty plasmid 12.54％ ± 0.25％ and 4.46％ ± 0.19％,respectively (F =28.159,P ＜0.01).The growth inhibition test indicate that the OD value of the fifth day in siRNA-dbpA group was 0.194 ±0.037,significantly lower than that in the other two groups 0.814 ±0.043 and 1.625 ±0.061,respectively(F =23.214,P ＜ 0.01).The colony formation number is 37 ± 3,64 ± 5and 175 ± 10 respectively,siRNA-dbpA is significantly higher than that in the other two groups(F =40.254,P ＜ 0.01).After the completion of nude mouse transplantation tumor model,through the detection of tumor volume,KD group (group siRNA-dbpA) tumor volume after 14 d and CON and NC group had obvious difference (F =38.256,P ＜ 0.05),and after 21d is more significant difference in tumor size (F =40.241,P ＜ 0.01),can be clearly observed after 35 d KD group (group siRNA-dbpA) growing tumors had differences with the control group (F =30.257,P ＜ 0.05).Conclusion Lentivirus-mediated RNAi targeting dbpA can effectively suppress the expression of dbpA in colorectal tumor in nude mice,it is proved that dbpA silencing has a significant inhibitory effect on the growth of living tumor cells and decrease the proliferation of the colorectal cells.
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Objective To investigate the expression of DNA binding protein A (dbpA) in patients with colorectal carcinoma of different stages and its significance.Methods Expression of dbpA protein and mRNA in specimens of normal tissues and colorectal cancer were detected by immunohistochemistry,expression of dbpA mRNA and protein of colorectal cancer cell lines SW480,RKO,SW620,DLD-1,HT-29,SW1463 and tissues were detected by immunohistochemistry,RT-PCR and Western blot.Results There was no positive staining dbpA protein and mRNA in normal colorectal tissues.However,dbpA was expressed in epithelial cells of colorectal mucosa [dbpA mRNA:80.0 ％ (48/60),10.0 ％ (6/60);dbpA protein:83.3 ％ (50/60),10.0 ％ (6/60),P ＜ 0.01].And there was no expression in normal colorectal cell,but its expression was high in 6 colorectal cancer cells (P ＜ 0.05).The high expression of dbpA was correlation with the infiltration depth,lymph node metastasis and type of histology (P ＜ 0.05),and had effect in prognosis of colorectal cancer (P ＜ 0.05).Conclusion Elevated dbpA may be related to the pathogenesis and development of colorectal carcinoma,and dbpA may be a prognostic factor of colorectal carcinoma.
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<p><b>OBJECTIVES</b>To investigate the regression pattern of mid and low rectal cancer treated with neoadjuvant chemoradiotherapy and then to provide the pathological proofs for reasonable resectional margin in rectal cancer surgery.</p><p><b>METHODS</b>Forty cases of mid and low rectal cancer patients received concurrent chemoradiotherapy and then underwent radical operation. The whole-mount serial sections of resected rectal cancer specimen were stained with cytokeratin antibody using immunohistochemical techniques to show the residual cancer cells under the mucosa. The microscopic measurement was performed to determine the reverse infiltration of cancer cells in the rectal wall and to describe the cancer cells scatter ways in the cancer mass. The Ki-67 immunohistochemical stain was also performed to show the proliferation activity of residual cancer cells after neoadjuvant chemoradiotherapy.</p><p><b>RESULTS</b>The length of specimen was shrinking continuously during the pathologic section production and the shrink rate was 18%. There were remanent cancer cells which showed positive Ki-67 expression and the chemoradiotherapy decreased the Ki-67 expression significantly. The lower edge of remaining ulcers or scars could be used as the reference point from which the cancer infiltration could be measured. According to our measurement, the average reverse infiltration of cancer cells in the whole-mount section was (6.1±4.7) mm, the deepest one was 11.0 mm in the section which could be converted into fresh bowel length of 12.98 mm. The pathology showed that the residual cancer cells scattered in the fibrous tissue of ulcers, scars and manifested a regression of spatial distribution.</p><p><b>CONCLUSIONS</b>The rectal cancers show regression in different degrees after neoadjuvant chemoradiotherapy. The residual cancer cells in the fiber tissues manifest proliferation activity. The distal end of resection should be at least 2 cm away from the lower edge of ulcers or scars of primary tumor in the rectal wall in patients after neoadjuvant chemoradiotherapy. The circumferential resection margin should include all the fibrous scar of the tumor area to ensure the remove of tumor cells completely.</p>
Subject(s)
Aged , Female , Humans , Male , Middle Aged , Chemoradiotherapy , Neoadjuvant Therapy , Rectal Neoplasms , Pathology , General Surgery , TherapeuticsABSTRACT
Objective To evaluate the possibility of detection mutations of multiple genes in stool for secondary screening for colorectal cancer.Methods Tumor specimens and stool samples from 40 patients with colorectal cancer and 40 normal persons were examined for mutations of p53,K-ras and APC gene by polymerase chain reaction single strand conformation polymorphism(PCR-SSCP) and silver nitrate staining.Results ①The mutation rate of p53,K-ras and APC gene in the tissues and stools of colorectal cancer respectively were 57.50%,50.00%,60.00% and 42.86%,40.00%,51.43%,and no mutations were found in normal mucosa and stool.②The mutation ratioes between multiple gene and single gene had significant difference(P