ObjectiveTo detect the expression of Prox1 (prospero-related homeobox 1) gene in primary hepatocellular carcinoma (HCC), and to analyze the correlation of Prox1 gene expression with pathological grade and clinical stage of HCC. MethodsThe expressions of Prox1 gene in carcinoma tissues and adjacent cancerous tissues in HCC as well as normal liver tissues were detected by semi-quantitative RT-PCR, then the correlation of Prox1 gene expression with HCC pathological grade and clinical stage were analyzed. ResultsThe expression of Prox1 gene in carcinoma tissues (0.243±0.102) and adjacent cancerous liver tissues (0.537±0.235) was significantly lower than that in normal liver tissue (0.812±0.372), respectively （ Plt;0.01 or Plt;0.05）. Furthermore, the expression of Prox1 gene in carcinoma tissues was significantly lower than that adjacent cancerous liver tissues (Plt;0.05). The expressions of Prox1 gene in different pathological grade (F=97.950, Plt;0.001) and clinical stage were significantly different (F=228.300, Plt;0.001), and when compared with each other, the differences of pathological grade and clinical stage were also significant (Plt;0.001 or Plt;0.01). The expressions of Prox1 gene in HCC carcinoma tissue were negatively correlated with pathological grade (r=-0.930, Plt;0.01) and clinical stage (r=-0.980, Plt;0.01) of HCC. ConclusionsExpression of Prox1 gene may be related to the initiation and development of HCC, however, that whether Prox1 gene functions as tumor suppressor in HCC needs further investigation.
Objective To analyze the variation of intestinal microflora in patients with colorectal cancer by SYBR GreenⅠreal-time fluorescence quantitative PCR and reveal the role and significance of intestinal microflora in the colorectal cancer-associated molecular pathogenesis. Methods A set of 16S rRNA gene group of species-specific primers for Bifidobacterium spp., Lactobacillus group, Escherichia coli, and ddl gene-targeted species-specific primers for Enterococcus faecalis and feces Enterococcus were designed. Patients with colorectal cancer (colorectal cancer group, n=30) and healthy volunteers (normal control group, n=30) were included and whose feces were collected to extract bacterial genome DNA. SYBR GreenⅠ real-time fluorescence quantitative PCR was used to analyze the five mentioned bacterial amounts. Results Level of Bifidobacterium spp. (4.52±0.49) and Lactobacillus group (5.46±0.12) in colorectal cancer group were significantly lower than those （9.25±0.83 and 7.45±0.37） of normal control group (Plt;0.05), whereas levels of Escherichia coli (5.82±0.47), Enterococcus faecalis (10.6±0.30) and feces Enterococcus (5.74±0.16) in colorectal cancer group were significantly higher than those （4.68±0.32, 4.95±0.24, and 5.03±0.43） of normal control group (Plt;0.05). Conclusions The fecal microflora composition of patients with colorectal cancer is significantly decreased in Bifidobacterium spp. and Lactobacillus group, whereas increased in Escherichia coli, Enterococcus faecalis, and feces Enterococcus. These data underline that the occurrence and progress of colorectal cancer may be related to intestinal microflora.
Objective To research the expressions of miR-196b and HoxB8 mRNA in colorectal cancer and theircorrelation with clinicopathologic features，and to explore the relationship between miR-196b and HoxB8 in vivo. Methods Expressions of RNA （including miR-196b and HoxB8 mRNA） and HoxB8 protein were detected respectively by using quantitative real-time reverse transcriptase PCR and Western blot in 30 cases of colorectal cancer and corresponding normalmucous membrane tissues. Results In colorectal cancer tissues，expressions of miR-196b and HoxB8 mRNA were higher than those of the corresponding normal mucous membrane tissues （P＜0.05）. Expression of miR196b mRNA was assoc-iated with lymph node metastasis，neoplasm stages （Ⅰ＋ⅡandⅢ＋Ⅳ），and distant metastasis （P＜0.05），on the otherhand，no significant differences were observed regarding tumor site，size，gross type，depth of invasion，tissue differentiation，age，and sex （P＞0.05）. Expression of HoxB8 mRNA was no significant differences concerning lymph node metastasis，tumor stages （Ⅰ＋Ⅱ，Ⅲ＋Ⅳ），distant metastasis，tumor site，size，gross type，depth of invasion，tissue differentiation，age，and sex （P＞0.05）. The expression of miR-196b mRNA was negatively correlated with HoxB8 mRNA expression （r=-0.458，P＜0.05），and HoxB8 protein expression with no obvious correlation （r=-0.236，P＞0.05） in colorectal cancer tissues. Conclusions The expressions of miR-196b and HoxB8 mRNA in colorectal cancer tissues are higher，the high expression of miR-196b mRNA is related to the tumorigenesis and progression of colorectal cancer as well as correlated with prognosis in colorectal cancer. The miR-196b inhibits the expression of HoxB8 mRNA by binding to the3′-UTR of target HoxB8 mRNA.
Objective To explore the expression and function of NDRG2 gene in human primary hepatocellular carcinoma and normal hepatic tissues. Methods The immunohistochemical ABC method， Western blot， and Real-time PCR were used to investigate the expression and content of NDRG2 in human hepatocellular carcinoma and hepatic normal biopsies. Results The NDRG2 protein located in cytoplasm. The positive rate was 16.67%（5/30） and 100%（30/30） in hepatocellular carcinoma and normal hepatic tissues, respectively. The relative content of NDRG2 protein in hepatocellular carcinoma and normal hepatic tissues were 0.029 0±0.005 9 and 0.109 2±0.002 8. There were significant differences between human hepatocellular carcinoma and hepatic normal biopsies both in staining positive rates and relative content（P＜0.05）. The Western blot also agreed with the result，the expression level of NDRG2 protein in hepatocellular carcinoma and normal hepatic tissues was 1.13±0.15 and 1.57±0.18， respectively, there was significant difference（P＜0.05）. Also, compared with normal hepatic tissues, the expression level of NDRG2 mRNA in carcinoma tissues was reduced significantly (0.89±0.15 vs. 1.48±0.17, P＜0.05). However, there were no significant differences in NDRG2mRNA expression between Edmondson-Steiner grades. Conclusions There possibly have difference in NDRG2 expression between human primary hepatocellular carcinoma and normal hepatic tissue. NDRG2 gene may take part in the pathogenesis of human primary hepatocellular carcinoma. Futher study will be needed to study its mechanism and function.
Objective To evaluate the clinical significance of epidermal growth factor receptor EGFR) mutations in the treatment of non-small cell lung cancer ( NSCLC) . Methods Plasma DNAs solated fromblood specimens of 170 NSCLC patients, who were admitted in the First Affiliated Hospital of uangzhou Medical College from December 2005 to December 2007, were subjected to the test of EGFR utant-enriched PCR. The correlation of mutant detection with clinical characteristics was analyzed as well.Results Out of the total 170 patients, EGFR mutations were identified in 77 cases ( 77 /170, 45. 3% ) .EGFR mutations were more frequent in the patients with adenocarcinoma ( P lt; 0. 001) and in the nonsmokers P =0. 001) . In the 33 patients treated with gefitinib, those with mutations ( + ) showed a higher esponse rate and prolonged progression-free survival after the treatment compared with those with mutations( - ) ( P =0. 001 and 0. 001, respectively) . Conclusions EGFR active mutations can be specifically and ensitively detected by EGFR mutant enriched PCR assay. Plasma EGFR mutants detection is valuable in uiding clinical decision.
Objective To construct the responsive plasmid PTRE-HIF-1αof Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the cDNA of HIF-1α, which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid PTRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing. The responsive plasmid PTRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid PTRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2Tet-on cells.
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 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.
Objective To identify micrometastasis in regional lymph nodes of gastric cancer by quantitative real-time reverse transcription-PCR (qRT-PCR) assay and to evaluate the clinical significance of micrometastasis. Methods To study 320 lymph nodes collected from January 2010 to June 2010， 281 of which were from 40 patients with gastric cancer who had undergone a standard gastrectomy with lymphadenectomy， and other 39 of which were from 10 patients with gastroduodenal ulcer. Made CEA， CK-19， and CK-20 as primers， and used qRT-PCR assay in addition to hematoxylin and eosin staining to detect the micrometastasis， and to analyze the clinicopathologic characteristics.Results Totally， micrometastasis were detected by qRT-PCR assay in 31 (15.34%，31/202) lymph nodes of 28 (70.00%， 28/40) patients. Thirty-nine lymph nodes from 10 patients with gastroduodenal ulcer were negative by qRT-PCR and HE staining. The degree of differentiation， depth of gastric mural invasion， and clinical stage had statistically significant correlation with the incidence of lymph node micrometastasis (P＜0.05). Conclusions qRT-PCR assay is a sensitive and specific method to detect lymph node micrometastasis in gastric cancer patients，and it has importantly clinical significance in evaluating clinical staging，prognosis and treatment prescription.
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.