ObjectiveTo determine the expression change of activating transcription factor 5 (ATF5) in human rectal cancer tissue, and analyze the correlation between ATF5 expression and the clinicopathologic parameters of rectal cancer. MethodsNinetytwo paired samples of rectal cancer tissue and more than 5 cm distant normal rectal tissue were obtained from inpatients between March 2009 and October 2009 in this hospital. ATF5 mRNA and protein expressions were detected by quantitative real-time RT-PCR and immunohistochemical staining. ResultsThirty-three (35.9%) cases of rectal cancer showed ATF5 mRNA overexpression; however, the expression level of ATF5 mRNA in the rectal cancer tissue was not statistically different from that in the normal rectal tissue (P=0.363). There was no evidence for the relationship between the ATF5 mRNA expression and the patients’ age, gender, histological type, tumor differentiation degree, invasive depth, lymph node metastasis, distance metastasis, or TNM stage. In contrast, the positive expression rate of ATF5 protein in the rectal cancer tissue was significantly higher than that in the normal rectal tissue (P=0.000). Moreover, the ATF5 protein expression was correlated with the tumor differentiation degree (P=0.013), but not with other clinicopathologic features (Pgt;0.05). ConclusionsThe results suggest that ATF5 protein may be related to the carcinogenesis and differentiation of human rectal cancer. However, further researches are required to prove the correlation.
Objective To detect expression of Ras association domain family 1A （RASSF1A） gene in the colonic carcinoma tissue and to analyze the relationship of this expression to its clinical features. Methods Immunohistochemistry and Western blot methods were employed for detecting the RASSF1A protein expressions in 34 colonic carcinoma tissues and corresponding normal colon tissues. RT-PCR was employed for detecting RASSF1A mRNA expression. Results ①The RASSF1A protein expression in the colonic carcinoma tissues was significantly lower than that in the normal colontissues by using immunohistochemistry〔35.3% （12/34） versus 97.1% （33/34）， P＜0.05〕.There were significant relati-onships of RASSF1A protein expressions to the tumor differentiation and TNM stage （P＜0.05）， in other words， the positive rates of RASSF1A protein in the moderately and well differentiated andⅠ＋Ⅱof TNM colonic carcinoma tissues were all higher （P＜0.05）. ② The RASSF1A protein expression in the colonic carcinoma tissues was significantly lower than that in the normal colon tissues by using Western blot 〔0.316 8±0.019 6 versus 0.914 4±0.177 6， P＜0.05〕， which was close to the result of RT-PCR〔0.158 9±0.223 7 versus 0.572 3±0.193 9， P＜0.05〕. Conclusions Absentexpre-ssion of RASSF1A gene in the colonic carcinoma tissue might play an important role in tumor genesis and tumor progre-ssion， and it might become useful early detection of the colonic carcinoma.
ObjectiveTo clone full-length cDNA of rat galectin-9 and construct recombinant adenovirus granule containing rat galectin-9 gene. MethodsThe galectin-9 gene was amplified by RT-PCR from rat liver tissue and inserted orientationally into plasmid pDC316-GFP digested by restriction endonucleases NotⅠ and HindⅢ. The recombinant pDC316-GFP-galectin-9 shuttle plasmid was identified by PCR, restriction endonuclease digestion and sequencing, and then co-transfected with rescue plasmid pBHGlox△E1.3Cre into HEK-293 cells by liposome reagent. Recombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) was generated by sitespecific recombination and confirmed by PCR, and then Ad5-galectin-9 was propagated in HEK-293 cells and purified. The infectious titer of viral stock was determined by TCID50 assay. ResultsConstruction of pDC316-GFP-galectin-9 shuttle plasmid was confirmed to be correct by PCR, restriction endonuclease digestion and sequencing. Construction of recombinant adenovirus Ad5-galectin-9 was confirmed to be correct by PCR. The infective titer of Ad5-galectin-9 was 1.4×109 U/ml. ConclusionRecombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) is successfully constructed, which provides the foundation of further research on the function of galectin-9 gene.
ObjectiveTo detect expressions of PTEN and Ki-67 in primary thyroid cancer tissues and explore its clinical significances. MethodsThe expressions of PTEN protein and Ki-67 protein in 40 cases of paraffin-embedded tissues of primary thyroid cancer and the corresponding paracancerous tissues were detected by immunohistochemical method. The expressions of PTEN mRNA and Ki-67 mRNA in 14 cases of resected fresh tissues of primary thyroid cancer and the corresponding paracancerous tissues were detected by RT-PCR method. The relations between clinicopathologic characteristics and expression of PTEN protein or Ki-67 protein in the primary thyroid cancer tissues were analyzed. Results① The PTEN protein positive expression rate and the PTEN mRNA in the primary thyroid cancer tissues were significantly lower than those in the corresponding paracancerous tissues[35.0% (14/40) versus 60.0% (24/40), P<0.05; 0.225 7±0.036 3 versus 0.503 6±0.037 5, P<0.05], the Ki-67 protein positive expression rate and Ki-67 mRNA in the primary thyroid cancer tissues were significantly higher than those in the corresponding paracancerous tissues [72.5% (29/40) versus 42.5% (17/40), P<0.05; 1.212 1±0.042 1 versus 0.293 6±0.027 4, P<0.05]. ② The expressions of PTEN protein and Ki-67 protein were associated with the histological grading, pathological type, tumor stage, and presence of regional lymph node metastasis (P<0.05), which not associated with the patient's gender, age and integrity of tumor capsule or not (P>0.05). ③ The PTEN and Ki-67 protein expressions in the primary thyroid cancer tissues had a significantly negative correlation (rs=-0.605, P=0.000), which in the corresponding paracancerous tissues had no correlation (rs=-0.021, P=0.899). ConclusionPTEN and Ki-67 genes abnormally express in thyroid cancer tissue, which might be related with occurrence and development and its mechanism of primary thyroid cancer. Combination of two genes might contribute to identification of pathologic type, judge of biological behavior, and tumor stage of primary thyroid cancer, which might serve as a new target for diagnosis and treatment of it.
ObjectiveTo explore the influence of patients who accepted chemotherapy of Folfox4 scheme before operation for the expression of miR-196 and HoxB8 in colorectal cancer, and illustrating the differences between the miR-196 and HoxB8 expressions in colorectal cancer tissues and sensitivity to chemotherapy with Folfox4 scheme and its corre-lation and significance. MethodsFluorescence quantitative PCR (RT-PCR) and immunohistochemistry were used to determine the expressions of miR-196 and HoxB8 in 50 specimens of neoadjuvant chemotherapy group (chemotherapy sensitive group and chemotherapy insensitive group) and 30 specimens which received the surgery directly (no-chemo-therapy group), and analyzing the relationship and discrepancy between miR-196 and HoxB8 in these groups. ResultsThe RT-PCR examination showed that the relative expression levels of miR-196 and HoxB8 in the neoadjuvant chemo-therapy group were lower than the no-chemotherapy group (0.646 8±0.683 9 vs.1.000 0±0.000 0, P < 0.01;0.607 6±0.418 9 vs.1.000 0±0.000 0, P < 0.01).Expression of miR-196 in the chemotherapy sensitive group was higher than the chemotherapy insensitive group (0.948 9±0.691 0 vs.0.344 7±0.536 1, P < 0.01), however, the expression of HoxB8 mRNA in the chemotherapy sensitive group was lower than the chemotherapy insensitive group (0.489 9±0.371 5 vs.0.725 3±0.437 5, P < 0.05).Expression positive rate of HoxB8 protein in chemotherapy sensitive group was lower than the chemotherapy insensitive group (Z=-2.396, P=0.017).The expressions of miR-196 and HoxB8 in the neoadjuvant chemotherapy group had negative relationship (r=-0.595, P < 0.01), which was also exist in the no-chemo-therapy group (r=-0.435, P < 0.01). ConclusionsThe neoadjuvant chemotherapy with Folfox4 scheme before oper-ation can reduce the expression levels of miR-196 and HoxB8 in colorectal cancer tisssues.The different expression levels of miR-196 and HoxB8 could influence the sensitivity of neoadjuvant chemotherapy with Folfox4 scheme in colorectal cancer.The high level expression of miR-196 could restrain the expression of HoxB8, and then increase the sensitivity of chemotherapy with Folfox4 scheme in colorectal cancer.
Objective To discuss the changes of c-kit/scf mRNA and protein in guinea pig gallbladder fed on high cholesterol diet. Methods Twenty guinea pigs were divided into two equal groups of 10 each：the control group and lithogenic group. Normal diet and high cholesterol diet was given to each group respectively. The period of stone permeation was six weeks. RT-PCR and Western blot were used to determin the expressions of c-kit and scf mRNA and protein. Results RT-PCR results showed that the expressions of c-kit mRNA（t=6.985，P＜0.01） and scf mRNA （t=6.028， P＜0.01）decreased significantly in lithogenic group compared with the control group. Western blot results showed that the expressions of c-kit protein （t=10.256， P＜0.01） and scf protein （t=9.586， P＜0.01）decreased significantly in lithogenic group compared with the control group. Conclusions The expressions of c-kit/scf mRNA and protein decrease during the formation of cholesterol gallstones in guinea pigs fed on high cholesterol diet. Inhibition of c-kit/scf pathway may play a role in the formation of cholesterol gallstones.
Objective To investigate the difference of minichromosome maintenance protein 2 (MCM2) mRNA expression among colonic normal mucosa, colonic adenoma and carcinoma. Methods The expressions of MCM2 mRNA were determined by real-time RT-PCR in 12 colonic normal mucosa, 33 colonic adenomas and 12 colonic carcinomas. Data were evaluated by relative expression software tool (REST-XL). Results The expressions of MCM2 mRNA elevated in turn by colonic normal mucosa, adenoma and carcinoma. The expression of MCM2 mRNA in colonic carcinomas was significantly higher than that in adenomas， as well as in normal mucosa (P=0.001), while the difference of MCM2 mRNA expressions between adenoma and normal mucosa was insignificant (P=0.184). Conclusion The difference of MCM2 mRNA expressions between adenoma and carcinoma indicated the potential value on early diagnosis for colonic carcinoma.
Objective To investigate the different influence of the expression levels of the epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) after the unilateral phrenectomy in piglets. Methods Thirty-six piglets were divided into 3 groups according to their ages during the operation (10 d,30 d,50 d). In each group, 6 piglets underwent the left cervical phrenectomy and 6 piglets were used as the shamoperation controls. The expression levels of EGF and KGF were determined by the real time quantitative RT-PCR at 2 weeks after operation.Results The melting curves of RTPCR showed that there was a single peark at the temperature of 80.0, 84.5 and 89.0℃ of EGF,KGF and GAPDH, respectively. In the experimental group, the expression levels of EGF were 3.53±0.36 and 1.73±0.29, and the expression levels of KGF were 4.71±0.42 and 2.77±0.29 in thepiglets undergiong the operation at their ages of 10 d and 30 d.Compared with the control group,the expression levels of EGF (4.60±0.41，2.18±0.24) and KGF(6.05±0.42，3.58±0.31) showed that there was a significant decrease postoperatively in the piglets undergoing the operation at their ages of 10 d and 30 d（P＜0.05）. However, there was no significant change in the piglets undergoing the operation at their ages of 50 d（P＞0.05）. The expression levels of EGF and KGF were significantly decreased with the lung development of the piglets（P＜0.05）. Conclusion The unilateral phrenectomy performed in the piglets younger than 30 d may cause abnormity of the EGF and KGF expression levels. The piglets older than 50 d may not cause a significant influence.
Objective To study the expression and significance of multidrug resistance-associated protein (MRP) gene in hepatocellular carcinoma (HCC). Methods Reverse transcription polymerase chain reaction (RT-PCR) assay was used to detect the expression of MRP mRNA in 25 fresh specimens of the primary HCC and its surrounding liver tissues. Immunohistochemistry LSAB technique was adopted to test MRP in 60 HCC specimens. The drug sensitivity was also tested by flow cytometry.Results The positive expression rates of MRP mRNA and MRP protein in primary HCC were 44.00%(11/25) and 45.00%(27/60) respectively. All the intensity of expression was low, but significant higer than its surrouding liver tissues (P＜0.05). The intensity and expression rate of MRP protein in 5 recurrent HCC had a tendency to increase. There was a correlation between the expression of MRP mRNA and MRP protein in 25 patients using RT-PCR and immunohistochemistry assay (Plt;0.05). Detected by flow cytometry, the average sensitivity of drugs in vitro of 60 HCC sp-cimens were 5-FU （15.80±7.63）%，DDP（18.45±9.59）%，ADM（17.95±7.99）%，MMC（16.60±8.69）% and CTX（17.40±10.14）%. Only 5FU and ADM were significantly affected by the expression of MRP protein (Plt;0.05).Conclusion The expression of MRP in primary HCC may be one of the important mechanisms of the intrinsic and acquired drug resistance in HCC. To study the expression of MRP could give a predictive value in HCC chemotherapy.
【Abstract】ObjectiveTo investigate the relationship between the expression of urokinase-type plasminogen activator (uPA) mRNA and breast cancer, lymph node metastasis. MethodsSixty patients with breast tumor were selected randomly and the expression of uPA mRNA was detected with RT-PCR. The patients were divided into benign group (18 cases) and malignant group (42 cases) which included 22 cases with lymph node metastasis and 20 cases without lymph node metastasis. The relationship between uPA mRNA expression and breast cancer, lymph node metastasis was analyzed. ResultsAmong these 18 benign tumors, low expression of uPA mRNA was found in 2 cases and the others were negative. While in 42 cases of malignant tumor, uPA mRNA were positive in 22 cases of lymph node metastasis, 16 of which were high expression, 5 of which were moderate expression, and 1 was low expression. uPA mRNA were positive in 18 of 20 cases of nonmetastatic lymph node, 1 of which was high expression, 5 of which were moderate expression and 12 of which were low expression, the other 2 were negative expression. The expression of uPA mRNA had significant difference between benign and malignant tumors (P＜0.05). The expression in lymph node metastasis was much higher than no lymph node metastasis (P＜0.05). ConclusionThe expression of uPA mRNA in malignant breast cancer is obviously higher than that in benign breast tumor. The expression tensity of uPA is highly relevant to lymph node metastasis in malignant breast cancer, which can provide evidence for clinical staging and therapy.