Cases Of Normal Colorectal Mucosa Research Articles

Differential expression of PLAC1 and Netrin-1 in liver metastasis of colorectal cancer and its predictive value

ObjectiveTo explore the differential expression of placental specific gene 1 (PLAC1) and neurite guidance factor 1 (netrin-1) in colorectal cancer (CRC) liver metastasis and its predictive value.MethodsParaffin specimens of primary CRC were selected, including 60 simple colorectal cancer specimens and 47 liver metastasis specimens. At the same time, 40 cases of normal colorectal mucosa were taken as the control group. The expression of PLAC1 and Netrin-1 in tissue was detected by immunohistochemistry (IHC). The correlation between PLAC1 and Netrin-1 expression and clinicopathological characteristics of patients with CRC liver metastases was analyzed. Logistic analysis was adopted to analyze the influencing factors of liver metastasis in CRC. A prediction model was established and ROC curve was used to detect the discrimination of the prediction model. The clinical value of PLAC1 and netrin-1 in predicting liver metastasis of CRC was analyzed using ROC curve. The relationship between the expression of PLAC1 and netrin-1 and the prognosis of CRC patients with liver metastasis was analyzed using Kaplan Meier survival curve.ResultsThe positive staining of PLAC1 and netrin-1 was mainly located in the cytoplasm by IHC detection. Positive expression of PLAC1 and netrin-1 in CRC tissues was markedly higher than that in normal colorectal mucosal epithelium (P < 0.05). Positive expression of PLAC1 in metastatic group was higher than that in non-metastatic group without significant difference (P > 0.05). The metastasis group had much higher positive expression of netrin-1 than the non-metastasis group (P < 0.05). The content of PLAC1 in the tissues of CRC with liver metastasis had a close relationship with differentiation degree and lymph node metastasis (P < 0.05). The expression of Netrin-1 in the tissues of CRC with liver metastasis was associated with Dukes stage, differentiation degree and lymph node metastasis (P < 0.05). Logistic regression analysis showed that Dukes stage, differentiation, lymph node metastasis, CEA, Alb and D-dimer were the independent risk factors for liver metastasis of CRC (P < 0.05). The model was constructed according to the regression coefficients and constant terms, and the discrimination of the prediction model was evaluated using ROC curve, with the AUC of 0.903 (95% CI 0.831 ~ 0.975), the sensitivity of 93.80%, the specificity of 80.00%, and the Jordan index of 0.738. The AUC of PLAC1 and netrin-1 alone and combined detection to predict liver metastasis of CRC were 0.805, 0.793 and 0.921, respectively. The survival time of patients with positive PLAC1 and netrin-1 expression were sharply shorter than that of the patients with negative expression (P < 0.05).ConclusionsThe expression of PLAC1 and netrin-1 was strongly increased in CRC with liver metastasis, which had a certain clinical value in predicting liver metastasis of CRC. Dukes stage, differentiation degree, lymph node metastasis, CEA, Alb and D-dimer were independent risk factors for liver metastasis of CRC, and the model based on these indicators had good discrimination for effectively evaluating the risk of liver metastasis in CRC.

Association of Helicobacter bilis Infection with the Development of Colorectal Cancer

The role of Helicobacter_bilis (H.bilis) in the development of inflammatory bowel disease (IBD) and colitis-associated carcinogenesis (CAC) has seldom been investigated. We examined the abundance of H.bilis in 58 colorectal cancers (CRCs), 20 IBDs, 40 cases of normal colorectal mucosa (NCs), and 20 adenomas (ADs) by 16S rRNA fluorescence in situ hybridization (FISH). Number of CD4+CD45RB+T cell and expression of IFN-γ and TNF-α in these tissues was determined by immunofluorescence. The abundance of H.bilis was significantly higher in CRCs than that in IBDs (P = 0.006), ADs (P < 0.001) and NCs (P < 0.0001). The abundance of H.bilis in IBDs was significantly higher than that in ADs (P = 0.013). Moreover, the average number of CD4+CD45RB+T cell was significantly higher in CRCs than that in IBDs (P = 0.017) and NCs (P = 0.009). In addition, there was a positive correlation between the H.bilis abundance and density of CD4+CD45RB+T cells in 30 colorectal tissues (P < 0.0001). The frequency of co-staining for CD4+CD45RB+T cells and IFN-γ was significantly higher in H.bilis positive group than that in H.bilis negative group (P = 0.002). H.bilis may play a role in the initiation of IBD and CAC, possibly through promoting the transformation of T cells into CD4+CD45RB+T cells and increasing the expression of proinflammatory cytokines IFN-γ.

Expression of Raptor and Rictor and their relationships with angiogenesis in colorectal cancer.

Mammalian target of rapamycin (mTOR) has two subtypes, i.e., mTORC1 and mTORC2, which contain the Raptor and Rictor core molecules, respectively. The effect of Raptor and Rictor on hypoxia inducible factor (HIF)-1α, HIF-2α, and vascular endothelial growth factor (VEGF) in colorectal cancer (CRC) is unclear. In this work, we investigated the correlations among Raptor, Rictor, HIF-1α, HIF-2α, and VEGF expression in CRC. We subsequently analyzed the clinicopathological features of patients. Immunohistochemistry, western blot, and RT-PCR analyses were performed to detect the expression of Raptor, Rictor, HIF-1α, HIF-2α, and VEGF in 120 cases of CRC and 60 cases of normal colorectal mucosa. CD34 was used to label microvascular density (MVD), which was found to be higher in patients with positive Raptor or Rictor than in those with negative Raptor or Rictor. The positive rates of Raptor, Rictor, HIF-1α, HIF-2α, and VEGF in CRC were significantly higher than in normal colorectal mucosa. Raptor expression was positively correlated with HIF-1α and VEGF but not with HIF-2α expression. By contrast, Rictor expression was positively correlated with HIF-2α and VEGF but not with HIF-1α expression. Survival analysis further indicated that Raptor, Rictor, HIF-1α, HIF-2α, VEGF and lymph node metastasis were independent prognostic factors in CRC. To conclude, Raptor and Rictor expression was related to the initiation and development of CRC and angiogenesis in different ways. The combined detection of Raptor and Rictor is important for patients with colorectal carcinoma in prognosis and optimal therapy.

Expressions and clinical significances of Eag1 and VEGF in colorectal cancer tissues

Objective To study the expressions of Eag1 and vascular endothelial growth factor (VEGF) in colorectal cancer, and to explore the relationships between Eag1 and VEGF expressions and clinicopathologic features of colorectal cancer. The correlation between Eag1 and VEGF and their clinical significance in colorectal cancer were explored. Methods A total of 73 cases of colorectal cancer tissues, 25 cases of colorectal adenoma and 10 cases of normal colorectal mucosa from January 2015 to December 2016 in Hunan Provincial People′s Hospital were collected. The expressions of Eag1, VEGF protein and mRNA in 73 cases of colorectal cancer tissues, 25 cases of colorectal adenoma and 10 cases of normal colorectal mucosa were detected by immunohistochemistry and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The relationships between the two factors and the clinical pathological features of patients with colorectal cancer were analyzed. Results Immunohistochemistry showed that the positive rate of Eag1 in colorectal cancer group was 86.30% (63/73), which was significantly higher than that in colorectal adenoma group (20.00%, 5/25) and normal colorectal mucosa group (10.00%, 1/10), with significant differences (χ2=38.539, P<0.001; χ2=24.874, P<0.001). The positive rate of VEGF in colorectal cancer group was 89.04% (65/73), which was significantly higher than that in colorectal adenoma group (56.00%, 14/25) and normal colorectal mucosa group (30.00%, 3/10), with significant differences (χ2=10.980, P<0.001; χ2=16.911, P<0.001). There was a significant positive correlation between Eag1 and VEGF in colorectal cancer and colorectal adenoma tissues (c=0.580, P=0.010; c=0.669, P<0.001). The results of RT-qPCR showed that the expressions of Eag1 and VEGF mRNA in colorectal cancer group, colorectal adenoma group and normal colorectal mucosa group were 4.50±0.34, 3.14±0.44; 1.42±0.22, 1.61±0.15 and 1.00±0.16, 1.00±0.53, respectively, and the differences were statistically significant (F=15.546, P=0.014; F=5.363, P=0.025). The relative expression levels of Eag1 and VEGF mRNA in colorectal cancer group was about 4.50 times (t=31.851, P=0.003) and 3.14 times (t=6.870, P=0.014) than those in normal colorectal mucosa group. The mRNA expression levels of Eag1 and VEGF in colorectal cancer were correlated with degree of differentiation (F=840.725, P<0.001; F=102.950, P<0.001), depth of invasion (t=4.754, P<0.001; t=8.557, P<0.001), Duke staging (F=179.902, P<0.001; F=55.911, P<0.001) and lymph node metastasis (t=20.337, P<0.001; t=25.218, P<0.001). But they were not related to age (t=0.196, P=0.845; t=0.534, P=0.595) and sex (t=0.409, P=0.684; t=1.078, P=0.285). Conclusion Eag1 and VEGF are highly expressed in colorectal cancer tissues with positive correlation, and are closely related to the degree of differentiation, depth of invasion, Duke staging and lymph node metastasis of colorectal cancer. Both of them play important roles in the occurrence and development of colorectal cancer, which is expected to provide favorable research directions to the early diagnosis, treatment and prognosis of colorectal cancer. Key words: Colorectal neoplasms; Vascular endothelial growth factors; Eag1

Diagnostic value of SATB2, CK7 and CK20 in colorectal cancer

To study the diagnostic value of SATB2, together with CK7 and CK20, in colorectal cancer. Immunohistochemical study for SATB2, CK7 and CK20 was carried out in 210 cases of colorectal cancer tissue, 100 cases of non-colorectal cancer tissue, 90 cases of lymph node metastases and 50 cases of normal colorectal mucosa. The sensitivity and specificity of CK20+/CK7- immunophenotype for diagnosis of colorectal adenocarcinoma were 78.1% and 92.0%, respectively. When triple markers were used, the immunophenotype CK20+/CK7-/SATB2+ had a sensitivity of 57.1% and a specificity of 98.0%. When combining the immunophenotype of SATB2+/CK7- or CK20+/CK7-, the sensitivity was 85.7% and specificity was 90.0%. A panel of immunohistochemical markers SATB2, CK7 and CK20 could increase the specificity for diagnosis of colorectal adenocarcinoma significantly. SATB2 is considered as a useful adjunct in this respect.

Abstract 1064: RASSF10 suppresses colorectal cancer growth by activating p53 signaling and sensitizes colorectal cancer cell to docetaxel

Abstract RASSF10 has previously been reported to be frequently methylated in a number of malignancies. To understand the importance of RASSF10 inactivation in colorectal carcinogenesis, eight colorectal cancer cell lines, 89 cases of primary colorectal cancer and 5 cases of normal colorectal mucosa were examined. Methylation specific PCR, western blot, siRNA, gene expression array and xenograft mice were employed. The expression of RASSF10 was was regulated by promoter regional methylation in colorectal cancer cells. RASSF10 was methylated in 60.7% (54/89) of primary colorectal cancers and was positively associated with tumor stage (p&amp;lt;0.05) and metastasis (p&amp;lt;0.05). Restoration of RASSF10 led to inhibition of colorectal cancer cell proliferation in vitro and in vivo and increased apoptosis. Gene expression arrays discovered RASSF10 inhibition of MDM2 expression as a mediator of these effects, which was confirmed with RT- PCR and western blot. RASSF10 was shown to activate P53 signaling in RKO and HCT116 cells after UV exposure, and sensitized these cells to docetaxel. In conclusion, our study demonstrates RASSF10 is frequently methylated in human colorectal cancer leading to loss of expression. RASSF10 normally suppresses human colorectal cancer growth by activating P53 signaling in colorectal cancer, and restored expression sensitizes colorectal cancer to docetaxel. Citation Format: Jing Guo, Yage Yang, Yunsheng Yang, Enqiang Linghu, Qimin Zhan, Malcolm V. Brock, James G. Herman, Bingyong Zhang, Mingzhou Guo. RASSF10 suppresses colorectal cancer growth by activating p53 signaling and sensitizes colorectal cancer cell to docetaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1064. doi:10.1158/1538-7445.AM2015-1064

RASSF10 suppresses colorectal cancer growth by activating P53 signaling and sensitizes colorectal cancer cell to docetaxel.

RASSF10 has previously been reported to be frequently methylated in a number of malignancies. To understand the importance of RASSF10 inactivation in colorectal carcinogenesis, eight colorectal cancer cell lines, 89 cases of primary colorectal cancer and 5 cases of normal colorectal mucosa were examined. Methylation specific PCR, western blot, siRNA, gene expression array and xenograft mice were employed. The expression of RASSF10 was regulated by promoter regional methylation in colorectal cancer cells. RASSF10 was methylated in 60.7% (54/89) of primary colorectal cancers and was positively associated with tumor stage (p < 0.05) and metastasis (p < 0.05). Restoration of RASSF10 led to inhibition of colorectal cancer cell proliferation in vitro and in vivo and increased apoptosis. Gene expression arrays discovered RASSF10 inhibition of MDM2 expression as a mediator of these effects, which was confirmed with RT-PCR and western blot. RASSF10 was shown to activate P53 signaling in RKO and HCT116 cells after UV exposure, and sensitized these cells to docetaxel. In conclusion, our study demonstrates RASSF10 is frequently methylated in human colorectal cancer leading to loss of expression. RASSF10 normally suppresses human colorectal cancer growth by activating P53 signaling in colorectal cancer, and restored expression sensitizes colorectal cancer to docetaxel.

Upregulation of nemo-like kinase is an independent prognostic factor in colorectal cancer.

To investigate the expression and oncogenic role of nemo-like kinase (NLK) in colorectal cancer. Expression of NLK protein was assessed by immunohistochemistry in tissue specimens from 56 cases of normal colorectal mucosa, 51 cases of colorectal adenoma, and 712 cases of colorectal cancer. In addition, NLK expression was knocked down using a lentivirus carrying NLK small hairpin RNA in colorectal cancer cells. Cell viability methylthiazoletetrazolium assays, colony formation assays, flow cytometry cell cycle assays, Transwell migration assays, and gene expression assays were performed to explore its role on proliferation and migration of colorectal cancer. Expression of NLK protein progressively increased in tissues from the normal mucosa through adenoma to various stages of colorectal cancer. Overexpression of NLK protein was associated with advanced tumor-lymph node-metastasis stages, poor differentiation, lymph node and distant metastases, and a higher recurrence rate of colorectal cancer (P < 0.05). Multivariate analyses showed that NLK expression was an independent prognostic factor to predict overall survival (hazard ratio 2.57, 95% confidence interval: 1.66-3.98; P < 0.001) and disease-free survival (hazard ratio 1.96, 95% confidence interval: 1.40-2.74: P < 0.001) of colorectal cancer patients. Furthermore, knockdown of NLK expression in colorectal cancer cell lines reduced cell viability, colony formation, and migration, and arrested tumor cells at the G0/G1 phase of the cell cycle. At the gene level, knockdown of NLK expression inhibited matrix metalloproteinase-2 expression in colorectal cancer cells. NLK overexpression is an independent prognostic factor in colorectal cancer and knockdown of NLK expression inhibits colorectal cancer progression and metastasis.

Clinicopathological significance of X-ray repair cross complementing gene 1 expression in colorectal carcinoma

Objective To explore the clinicopathological value of X-ray repair cross complementing gene 1(XRCC1) expression in colorectal carcinoma. Methods The XRCC1 gene expression in 107 cases of colorectal carcinomas, 25 cases of adjacent mucosa and 36 cases of normal colorectal mucosa was detected immunohistochemically, and the correlation between the expression and clinicopathological factors was analyzed. Results The positive rates of XRCC1 expression in colorectal carcinoma and adjacent mucosa,87.8 % (94/107) and 84.0 % (21/25) respectively, were significantly higher than that in the normal colorectal mucosa [27.8 %(10/36) (P=0.000, P =0.000)]. In colorectal carcinoma, the positive rate of XRCC1 expression in the group of poor differentiation [44.4 % (4/9)] was significantly lower than that in the groups of moderate and well differentiation [94.8 % (50/58)(P=0.000) and 87.5 %(35/40) (P=0.015)], while the positive rate of XRCC1 expression was not related to sex, age, location, infiltration depth and lymph node metastasis (P ＞0.05). Conclusion The results indicated that XRCC 1 protein can be used as a marker to diagnose colorectal carcinoma. Key words: Intestinal neoplasms; X-ray repair cross complementing gene 1; Immunohistochemistry

Transgelin as a suppressor is associated with poor prognosis in colorectal carcinoma patients

We performed comparative proteomic analysis of colorectal cancer to investigate potential target proteins correlated with carcinogenesis and prognosis. Among them, transgelin, a 22 kDa protein also called SM22, was identified as a novel tumor suppressor protein, but little is known about this protein in tumors so far. A remarkable reduced expression of transgelin was found in colorectal cancer samples compared with normal colorectal mucosa. The effect of 5-aza-2′-deoxycytidine as a demethylation agent would obviously restore the original expression level of transgelin, implicating DNA hypermethylation of transgelin is important in the regulation of transgelin transcription in colorectal cancer. As a control, the investigation at cell line level confirms that transgelin protein comes from epithelium but not mesenchymal cells. Further, immunohistochemical staining for transgelin was performed on paraffin sections of 62 and 126 cases of normal colorectal mucosa and colorectal cancer specimens, respectively. As compared to normal colorectal tissue, we observed a significantly lower transgelin expression in colorectal cancer samples (P<0.001). Survival analysis demonstrated that patients without transgelin expression had shorter overall survival, whereas patients with transgelin expression had better survival (P=0.006). Multivariate analysis showed that negative transgelin expression was an independent prognostic indicator for patient's survival. Our results suggest that transgelin as a suppressor may serve as important biomarker of malignancy. Loss of transgelin involves gene promoter hypermethylation and is closely associated with poor overall survival in colorectal cancer patients.

Localization of CEA messenger RNA by in situ hybridization in normal colonic mucosa and colorectal adenocarcinomas.

Carcinoembryonic antigen (CEA) mRNA expression was studied in 14 cases of normal colorectal mucosa and colonic adenocarcinomas using in situ hybridization with a 32P-labelled cDNA probe to the unique 3'-untranslated region of CEA. This approach has the advantage that the target mRNA remains in the cell of origin, whereas there is considerable ambiguity in immunocytochemistry data for CEA because the protein is secreted. Furthermore, the specific cDNA probe overcomes potential problems of immunological cross-reactivity with other members of the CEA family. The results demonstrated that abundant, heterogeneously distributed CEA mRNA was present in colorectal adenocarcinomas, with the highest levels in cells lining glandular structures. Parallel immunohistochemistry with anti-CEA monoclonal antibody A5B7 showed that the regions of tumours with the highest levels of CEA mRNA also had the highest CEA protein levels, suggesting that the heterogeneous distribution reflects CEA expression rather than differential secretion of the protein. In the normal colonic mucosa, CEA mRNA expression was observed in surface epithelial cells and goblet cells of the upper crypts, with very low hybridization in the mid crypt and at the base. This crypt-surface distribution was identical to that observed for CEA protein. In situ hybridization therefore confirms that high levels of CEA mRNA are expressed in differentiated surface epithelial cells of the normal colon.