Expression of Tpl-2 in colorectal carcinogenesis

AIM: To analyze the relationship between Tpl-2 (tumor progression locus 2)expression and clinicopathological parameters of colorectal carcinoma by investigating the expression of Tpl-2 in adjacent normal mucosa, colorectal adenomas and colorectal carcinoma. METHODS: Tpl-2 expression in normal mucosa, adenoma and carcinoma was examined and compared in a set of tissue microarrays by immunohistochemistry. The potential relationship between Tpl-2 expression and clinicopathological features was analyzed. RESULTS: The expression of Tpl-2 in carcinoma was significantly increased compared to the adenoma and normal mucosa (P<0.01). No significant difference was detected between the adenoma and normal mucosa (P>0.05). Meanwhile, the correlation between Tpl-2 expression and lymph node metastasis (N stage) and TNM stage (P<0.05) was observed. However, the correlation between the Tpl-2 expression and clinicopathological features of colorectal cancer including sex, age, body mass index (BMI), tumor size, histological differentiation, invasive depth (T stage),distant metastasis(M stage) and K-ras mutation (P>0.05) was not found. CONCLUSION: Tpl-2 has a relevance to the development of colorectal cancer as a promotive factor in the colorectal carcinogenesis.     

CHOP expression and its correlation with proliferative/apoptotic ratio in colorectal adenoma-carcinoma sequence under same genetic background

AIM：To investigate the expression of C/EBP homologous protein (CHOP) and its correlation with proliferative/apoptotic ratio (PAR) in colorectal adenoma-carcinoma sequence under the same genetic background. ME-THODS: Four kinds of tissue samples under the same genetic background from 23 patients, including normal colorectal tissue, adenoma with low-grade intraepithelial neoplasia, adenoma with high-grade intraepithelial neoplasia and colorectal adenocarcinoma samples, were collected. TUNEL method and Ki-67 immunohistochemistry were applied to determine the PAR. The expression of CHOP was detected by immunohistochemistry SABC method. RESULTS：(1) Under the same genetic background, the level of CHOP expression is significantly higher in colorectal adenocarcinoma than that in the adenoma with high-grade intraepithelial neoplasia, the adenoma with low-grade intraepithelial neoplasia and the normal mucosa. The level of CHOP expression was significantly higher in the adenoma with high-grade intraepithelial neoplasia than that in the adenoma with low-grade intraepithelial neoplasia and the normal mucosa. The level of CHOP expression was significantly higher in the adenoma with low-grade intraepithelial neoplasia than that in normal mucosa. (2) Under the same genetic background, PAR was significantly higher in the colorectal adenocarcinoma than that in the adenoma with high-grade intraepithelial neoplasia, the adenoma with low-grade intraepithelial neoplasia and the normal mucosa. PAR was significantly higher in the adenoma with high-grade intraepithelial neoplasia than that in the adenoma with low-grade intraepithelial neoplasia and the normal mucosa. PAR was significantly higher in the adenoma with low-grade intraepithelial neoplasia than that in the normal mucosa. (3) CHOP levels were positively correlated with PAR in the adenoma with low-grade intraepithelial neoplasia, adenoma with high-grade intraepithelial neoplasia and colorectal adenocarcinoma. CONCLUSION：CHOP expression and PAR continuously increased and positively correlated along the adenoma-carcinoma sequence, indicating that endoplasmic reticulum stress mediates the carcinogenesis of colorectal adenomas.     

Expression of ERK5 in colorectal cancer tissues and its correlation with distant metastasis in colorectal cancer patients

AIM: To investigate the expression of extracellular signal-regulated kinase 5 (ERK5) in primary colorectal cancer (CRC) and adjacent normal mucosa, and to analyze the relationship between ERK5 expression and clinicopathological parameters for exploring the functions of ERK5 in the occurrence and development of CRC. METHODS: The expression of ERK5 in carcinoma tissues and normal mucosa was examined by a set of tissue microarrays and the method of immunohistochemistry. The potential relationship between ERK5 expression and clinicopathological features was also analyzed. RESULTS: ERK5 expression was significantly higher in CRC tissues (134/338, 39.6%) than that in normal tissues (21/80, 26.2%; P<0.05). Overexpression of ERK5 in CRC tissues was significantly correlated with distant metastasis (P<0.05). However, no correlation between ERK5 expression and age at surgery, sex, tumor location, the depth of invasion, lymph node metastasis, TNM staging or differentiation grade was found (P>0.05). According to the Kaplan-Meier analysis, there is no significant difference in 5-year overall survival between the patients with ERK5 expression at high level and at low level. CONCLUSION: ERK5 protein is highly expressed in CRC with distant metastasis. This may be a promotive factor in the process of distant metastasis.     

Expression and significance of T-cell immunoglobulin and ITIM domain in colorectal cancer

AIM: To study the expression and clinical significance of T-cell immunoglobulin and ITIM domain (TIGIT) in colorectal cancer. METHODS: The patients with colorectal cancer (n=80) from January 2016 to June 2018 were selected. The expression of TIGIT and CD155 in the colorectal cancer tissues and adjacent normal tissues were detected by immunohistochemical staining method. The expression of TIGIT and CD155 was also determined by Western blot and ELISA. RESULTS: The positive expression rates of TIGIT and CD155 were 78.8% (63/80) and 83.8% (67/80) in the colorectal cancer tissues, significantly higher than that in the paracancerous tissues of 8.8% (7/80) and 18.8% (15/80), respectively (P<0.05). There was a positive correlation between TIGIT and CD155 expression (r=0.867, P<0.01). The expression levels of TIGIT and CD155 were increased as the stage evolved. The positive rates of TIGIT and CD155 in the colorectal cancer tissues were correlated with the degree of differentiation, pathological stage and lymph node metastasis (P<0.05). CONCLUSION: TIGIT and CD155 are correlated with the occurrence and development of colorectal cancer, and can be used as one of the prognostic indicators of colorectal cancer.     

Expression and function of PTEN in fibroblast-like synoviocytes of rheumatoid arthritis

AIM: investigate the expression of PTEN gene in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA).METHODS: FLS were isolated from synovial tissue obtained from the patients with RA, osteoarthritis (OA) or joint trauma. The mRNA expression of PTEN was detected by RT-qPCR. The protein levels of PTEN, p-Akt (Thr308) and total Akt were determined by Western blotting. The phosphorylation status of Akt was analyzed by the protein ratio of p-Akt (Thr308)/total Akt.RESULTS: The mRNA expression of PTEN was significantly lower in RA-FLS than that in OA-FLS and joint trauma-FLS (P < 0.01), while no statistically significant difference was observed between that in OA-FLS and joint trauma-FLS (P < 0.05). Similarly, the protein expression of PTEN in the RA-FLS was much lower than that in the OA-FLS and joint trauma-FLS (P < 0.05), and there was no difference between the latter 2 groups. Moreover, the phosphorylation level of Akt (Thr308) in the RA-FLS was significantly higher than that in the other 2 control groups (P < 0.01), and that in OA-FLS was much lower than that in the joint trauma-FLS (P < 0.01). Finally, Pearson correlation analysis between the phosphorylation level of Akt (Thr308) and PTEN protein expression in the RA-FLSs showed a significant negative correlation (r=-0.994 5, P < 0.01).CONCLUSION: The mRNA and protein expression of PTEN are both decreased in the RA-FLS, which may contribute to the increased phosphorylation level of Akt (Thr308).     

Expression and clinical significance of chemokine receptor CXCR6 in primary colorectal cancer

AIM: To investigate the expression of chemokine receptor CXCR6 in primary colorectal cancer and determine the association between CXCR6 expression and synchronous liver metastasis/prognosis. METHODS: The colorectal cancer tissues from 143 patients were collected from August 2004 to December 2008 in the First Affiliated Hospital, Sun Yat-sen University. Twenty-night cases of the adjacent normal colorectal tissues were enrolled as controls. The expression of CXCR6 was detected by immunohistochemistry and the mean intergrated absorbance ( mIA ) was calculated by Image-Pro Plus 6.0 software. The relationship between CXCR6 expression and synchronous liver metastasis/prognosis was analyzed. RESULTS: The CXCR6 staining was mainly positive in colorectal cancer tissues but not in adjacent normal colorectal tissues. The mIA of CXCR6 in colorectal cancer was 1.54±0.04 (range: 0.41~2.84), and was 1.63±0.05 and 1.41±0.08 (P<0.05) in the cases with (n=83) or without (n=60) synchronous liver metastasis, respectively. According to the mean mIA of CXCR6 (1.54), the cases was divided into high CXCR6 group (mIA≥1.54) and low CXCR6 group ( mIA <1.54). The overall survival rate in high CXCR6 group was significantly lower than that in low CXCR6 group (P<0.05). In multivariate Cox regression models, age (P<0.05), lymph node metastasis (P<0.05) and synchronous liver metastasis (P<0.01) but not CXCR6 were identified as independent risk factors for poor outcome. In subgroup analysis, high CXCR6 expression was associated with poorer survival in the patients with stage I~III colorectal cancer (P<0.01) but not those with synchronous liver metastasis (P>0.05).CONCLUSION: CXCR6 in primary colorectal cancer tissues is associated with liver metastasis. It may become a potential target for the treatment of colorectal cancer liver metastasis.     

Effects of N-acetylcysteine on the lipopolysaccharide-induced MAPK phosphorylation in mouse liver

AIM: To investigate the effects of antioxidant N-acetylcysteine (NAC) on the lipopolysaccharide (LPS)-induced MAPK phosphorylation in mouse liver. METHODS: 54 male mice were divided into three groups: control (n=6), 0.9% sodium chloride 0.2 mL ip; LPS group (n=24): LPS 5 mg ip; NAC+LPS group (n=24): NAC 150 mg·kg-1·d-1 ip, for 3 d; LPS 5 mg ip after 1 h of NAC administration at 3rd day. The liver was excised with carbrital anesthesia after LPS or 0.9 % sodium chloride injection at 0.5 h, 1 h, 2 h and 6 h for GSH and MDA assays. The protein extracted from liver was assayed for the phosphorylation of MEK1/2, ERK1/2, p38 MAPK by Western blotting. TNF-α in liver was assayed by radioimmunoassay. RESULTS: MDA in the liver was decreased remarkably and the GSH in the liver was increased significantly by NAC pretreatment. The phosphorylation of MEK1/2, ERK1/2 and p38 MAPK in liver were inhibited significantly by NAC pretreatment after LPS challenge. Meanwhile, TNF-α in liver was decreased markedly. CONCLUSION: Reactive oxygen species plays a critical role in MAPK signaling during the LPS induced acute liver injury. NAC partially inhibits LPS-induced MAPK signaling by antioxidant effect and decreases TNF-α production.     

Study of annexin A2 expression in gastric cancer by proteomics and tissue microarray

AIM: To investigate the differential expression of annexin A2 (ANXA2) in gastric carcinoma and to analyze the relationship between ANXA2 expression and clinicopathological parameters of gastric carcinoma. METHODS: Pure gastric adenocarcinoma cells (GAC) and normal gastric epithelial cells (NGEC) in 15 patients with gastric cancer were acquired by laser capture microdissection (LCM). All peptide specimens after trypsin digestion were labeled with ¹⁸O/¹⁶O. Quantitatively identification of differential expression of the proteins betweem GAC and NGEC was performed by Nano-RPLC-MS/MS. The expression of ANXA2 in the 2 kinds of tissues was detected by Western blotting. Tissue microarray containing 75 pairs of gastric carcinoma and para-carcinoma tissues was used and the expression of ANXA2 in these specimens was detected by the method of immunohistochemistry (IHC). The relationship between ANXA2 expression and clinicopathological parameters of the pateints with gastric carcinoma was analyzed. RESULTS: A total of 78 differential proteins were identified and ANXA2 was up-expressed in GAC (2.32∶ 1), which was confirmed by Western blotting (P<0.01). The results of IHC showed that the correlations between the expression level of ANXA2 protein and invasive depth (T stage), lymph node metastasis (N stage), histological differentiation, TNM stage and the size of tumor were observed (P<0.01), but the correlations between the ANXA2 expression and sex, age and distant metastasis (M stage) were not found (P>0.05). CONCLUSION: The up-expressed ANXA2 may play an important role in the biological behavior of gastric cancer.     

Expression of miR-625-3p in colorectal carcinoma tissues and cells

AIM: To investigate the expression of microRNA-625-3p (miR-625-3p) in colorectal carcinoma (CRC) and its underlying mechanism. METHODS: Quantitative real-time PCR was employed to detect the levels of miR-625-3p expression in different CRC cell lines, CRC tissues and pair-matched adjacent normal tissues. The relationships between the expression levels of miR-625-3p and the patients' clinicopathological parameters were estimated. The effects of miR-625-3p on the apoptosis and the cell mitotic cycle of CRC cells were analyzed with propidium iodide staining and flow cytometry. The effect of miR-625-3p on the apoptosis-related proteins was analyzed by Western blot. RESULTS: The expression level of miR-625-3p in the CRC tissues was higher than that in the pair-matched adjacent normal tissues (P<0.05). The expression of miR-625-3p in the CRC tumor tissues was significantly correlated with the tumor infiltrative depth, TNM stage and distant metastasis (P<0.05). The expression levels of miR-625-3p in CRC SW620 cells were higher than that in SW480 cells. The CRC cell mitotic cycle was significantly inhibited and cell apoptosis was significantly promoted when the expression of miR-625-3p was inhibited (P<0.05). The expression of Bax protein didn't change and the expression of Bcl-2 protein increased after miR-625-3p mimics were transfected into CRC SW620 cells(P<0.05). CONCLUSION: miR-625-3p may be a promising approach for the treatment of CRC by promoting cell proliferation and inhibiting apoptosis.     

Panaxadiol saponins induce activation of MAPK/ERK signaling pathway in bone marrow cells of aplastic anemia mice

AIM: To observe the effects of panaxadiol saponins (PDS) on up-regulation of MAPK/ERK signal pathway in bone marrow cells and increase in regulatory T (Treg) cells in spleen tissue of aplastic anemia (AA) mice, and to explore the mechanisms. METHODS: For preparation of immune-mediated AA model, BALB/c mice were exposed to sublethal dose (5.0 Gy) of[⁶⁰Co]-γ radiation, followed by transplantation of lymphocytes from DBA/2 donor mice. BALB/c mice (n=60) were randomly divided into 6 groups, including normal mouse group, AA model group, PDS treatment groups at low, medium and high doses, and cyclosporine group as positive control. PDS and cyclosporine were given by gavage for 14 d. The peripheral blood cell counts and bone marrow pathological examination were tested. The protein levels of MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 in the bone marrow cells were analyzed by Western blot and immunohistochemistry experiment. Flow cytometry was used to detect the proportion of Treg cells in spleen tissue of each group. RESULTS: The peripheral blood cell counts were significantly decreased in AA mouse group as compared with normal mouse group (P<0.05). The bone marrow sections showed markedly inhibition status of hematopoiesis and the decrease in cellularity. In response to PDS treatment, the peripheral blood cell counts and Treg cells in the spleen tissues of AA mouse treated with PDS were significantly increased in a dose-dependent manner (P<0.05). Treatment with PDS at medium and high doses up-regulated the protein levels of MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 in the bone marrow of AA mice (P<0.05). CONCLUSION: PDS is effective to enhance recovery of hematopoietic function in AA mice. This effect may be related to up-regulating multiple protein kinases of MAPK/ERK signal pathway in the bone marrow cells of AA mice. In addition, PDS has an impact on immune function of AA mice.     

Expression of EphA2 and ephrin-A1 in endometrial endometrioid adenocarcinoma and their relationship with angiogenesis

AIM: To investigate the expression of erythropoietin-producing hepatocellular receptor A2 (EphA2) and its ligand ephrin-A1 in endometrial endometrioid adenocarcinoma (EEA), and to analyze their relationship with angiogenesis of the tumor. METHODS: The CD34-stained microvessel density (MVD) and the expression of ephA2 and ephrin-A1 were detected by immunohistochemical assay in 56 cases of EEA, 20 cases of endometrial hyperplasia, 30 cases of normal proliferative endometrium and 30 cases of normal secretory endometrium. The correlations among the expression of EphA2 and ephrin-A1, MVD and clinicopathological features were analyzed. RESULTS: MVD and the expression of EphA2 and ephrin-A1 in EEA were significantly higher than those in the tissues from endometrial hyperplasia and normal endometrium (P<0.05). They were related to FIGO stage, histological differentiation, depth of myometrial invasion, lymphovascular invasion and progesterone receptor expression (P<0.05). A significant positive correlation between MVD and the expression of EphA2 and ephrin-A1 was observed by Spearman rank correlation test (r=0.476, P<0.05; r=0.501, P<0.05). CONCLUSION: Overexpression of EphA2 and its ligand ephrin-A1 in EEA may be involved in the angiogenesis and progesterone resistance.     

Downregulation of beta tropomyosin protein expression in colon adenocarcinoma

AIM: To investigate the cancer associated proteins and sensitive biomakers for early diagnosis in colon adenocarcinoma by using proteomic technique.METHODS: Two-dimensional gel electrophoresis was used to define patterns of protein expression in adenocarcinoma tissue from 8 patients with matched normal colonic mucosa. Proteins expressed differently of a 2-fold change were cut and analyzed by MALDI-TOF/TOF mass spectrometry. RESULTS: Two-dimensional protein maps of adenocarcinoma and normal colonic mucosa were gained successfully. Gel-analysis software identified an average of 3 289 spots in adenocarcinoma while 3 066 in normal mucosa and statistical filtering yielded 31 spots of a 2-fold change, 18 of which were identified by using mass spectrometry, including cytokeratin 8, cytokeratin 10, S100A6, beta tropomyosin (TMβ), protein disulfide isomerase, etc. Functional analysis revealed that these proteins were associated with adenocarcinoma cellular oncogenesia, proliferation, differentiation, metastasis and so on. The results of Western blotting validated that the expression level of TM β in colon adenocarcinoma was much lower than that in matched normal colonic mucosa.CONCLUSION: Proteomic analysis can identify the proteins with variance of colon adenocarcinoma versus normal colonic mucosa. Downregulation of TM β might serve as a new biomarker of colon adenocarcinoma.     

Effects of arginine vasopressin on subunit phosphorylation of GABAA receptors in rat preoptic area

AIM: To investigate the effects of arginine vasopressin (AVP) on the expression and phosphorylation of γ-aminobutyric acid (GABA) subtype A receptor (GABA_A receptor) subunits in the preoptic area (POA) of rats. METHODS: The rats were divided into AVP group (n=10), AVP+V1a receptor blocker group (n=10), V1a receptor blocker group (n=10) and control group (n=10). After intraperitoneal injection of AVP or V1a receptor antagonist for 0.5 h, the changes of the expression and phosphorylation of POA GABA_A receptor subunits (α, β and γ2) were analyzed by RT-qPCR and Western blot. RESULTS: Compared with control group, no significant change of GABA_A receptor subunit expression in the rats injected with AVP or V1a receptor antagonist was observed. AVP up-regulated the phosphorylation level of POA GABA_A receptor γ2 subunit (P<0.05), and significantly increased the expression and phosphorylation of protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ). CONCLUSION: Exogenous AVP has no effect on expression of POA GABA_A receptor subunits (α, β and γ2) and is involved in modulation of GABAergic synaptic transmission in the POA by activation of PKC and CaMKⅡ and phosphorylation of γ2 subunit via V1a receptor.     

Effect of KLF4 on viability,apoptosis of colorectal cancer cells and chemosensitivity to cisplatin

AIM:To investigate the effect of Krüppel-like factor 4 (KLF4) on the viability, apoptosis and cisplatin chemosensitivity of colorectal cancer cells. METHODS:KLF4 expression in colorectal cancer cell lines Caco2, SW480 and HCT116 was detected by Western blot. The SW480 cells were divided into pcDNA3.1 group (transfected with pcDNA3.1 empty plasmid), pcDNA3.1-KLF4 group (transfected with pcDNA3.1-KLF4 expression plasmid) and pcDNA3.1-KLF4+cisplatin group (treated with 1 mg/L cisplatin for 48 h after pcDNA3.1-KLF4 was transfected into SW480 cells). The protein levels of KLF4, p-IκBα, cyclin D1 and survivin were determined by Western blot. The cell viability was measured by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry. The content of reactive oxygen species(ROS) was measured by DCFH-DA probe. RESULTS:The expression of KLF4 in the colorectal cancer cells were significantly lower than that in the human colon mucosal epithelial NCM460 cells (P<0.05). Compared with pcDNA3.1 group, the protein expression of KLF4 in pcDNA3.1-KLF4 group was significantly increased (P<0.05). Compared with pcDNA3.1 group, the cell viability and the protein expression of cyclin D1 and survivin were significantly decreased, and the apoptotic rate, the content of ROS and the protein level of p-IκBα were significantly increased in pcDNA3.1 group (P<0.05). Compared with pcDNA3.1-KLF4 group, the cell viability and the expression of cyclin D1 and survivin proteins were significantly decreased, and the apoptotic rate, the content of ROS and the protein level of p-IκBα were significantly increased in pcDNA3.1-KLF4+cisplatin group (P<0.05). CONCLUSION:Upregulation of KLF4 gene expression in colorectal cancer cells reduces the cell viability, induces apoptosis and increases the chemosensitivity of the cells to cisplatin. The mechanism may be related to the enhancement of intracellular ROS content and down-regulaton of the phosphorylation level of IκBα, the key molecule of NF-κB signaling pathway.     

Expression and prognostic significance of SLP-2 in gastric cancer

AIM: To investigate the expression of the red cell membrane integration protein SLP-2 (stomatin-like protein 2) in gastric cancer tissues and to analyze its correlation with clinicopathological manifestations and prognosis. METHODS: One hundred and ninety gastric cancer tissue samples with detailed clinical information were collected from the Department of Pathology, Sun Yat-sen University Cancer Center. The protein expression of SLP-2 in ganstric cancer was detected by the method of immunohistochemistry. The relationships between SLP-2 expression and the clinicopathological manifestations were evaluated. RESULTS: The positive rate of SLP-2 in gastric cancer tissue was 63.2% (120/190). SLP-2 expression was relevant to infiltration depth, TNM stage and lymph node metastasis (P<0.05). However, no statistical difference was observed in the SLP-2 expression associated with sex, age, differentiation, tumor size and distant metastases. Kaplan-Meier survival curves revealed that increased expression of SLP-2 was associated with poor prognosis in gastric adenocarcinoma patients (P<0.01). Based on the univariate analysis, 7 factors were found to have statistical significance of associations with overall survival, including SLP-2 expression, lymph node metastasis, histological grade, tumor size, invasive depth, distant metastases and the 7th edition of the UICC TNM classification. Only the tumor size and the 7th edition of the UICC TNM classification were independent prognostic factors for overall survival in the multivariate analysis. CONCLUSION: SLP-2 is highly expressed in gastric adenocarcinoma tissues and may play an important role in tumor progression and metastasis. Although SLP-2 is not an independent prognostic factor, it may influence the prognosis of gastric cancer. Increased expression of SLP-2 can be used for predicting unfavorable prognosis in gastric adenocarcinoma patients.     

Expression of telomere regulating factor tankyrase in gastric adenocarcinoma

AIM:To detect the expression level of telomere regulating factor tankyrase and its clinical significance in the origin and development of gastric adenocarcinoma. METHODS:Quantitative Real-time PCR was used to measure the expression of tankyrase mRNA in cancerous and normal adjacent tissues from 16 patients with gastric adenocarcinoma. Immunohistochemistry was used to detect tankyrase protein expressions in 37 gastric adenocarcinoma samples and 13 normal controls. RESULTS:The expression of tankyrase mRNA in gastric adenocarcinoma ［median 1.44×10-2, range （3.88×10-5）-0.4847］ was significantly higher than that in normal adjacent mucosa ［median 1.0134×10-2, range 0-（4.933×10-2)］ (P<0.05). The protein expression level of tankyrase in gastric adenocarcinomas was significantly higher than that in normal controls (P<0.05). No significant difference was observed between different clinical stages and different pathological grades. CONCLUSION:Tankyrase was overexpressed in gastric adenocarcinomas. It may become one of the markers for detecting gastric adenocarcinomas.     

Expression of STC1 in colorectal cancer and its significance in tumor microenvironment

AIM:To explore the expression of stanniocalcin 1 (STC1) in colorectal cancer tissues and to analyze its relationship with prognosis, further to investigate the correlation between STC1 and tumor microenvironment. METHODS:The data of the STC1 mRNA expression were accessed by the UALCAN database (http://ualcan.path.uab.edu/index.html) and Oncomine database (https://www.oncomine.org). RT-qPCR and Western blot were used to detect the expression of STC1 in the clinical samples. OncoLnc (http://www.oncolnc.org/) analytical tool was adopted to evaluate the correlation of STC1 level and the prognosis of colorectal cancer. CoCl₂ was used to establish the hypoxia status and lipopolysaccharide (LPS) was utilized to establish inflammatory condition in the colorectal cancer HT-29 cells, respectively, and then STC1 expression was examined by RT-qPCR and Western blot. RESULTS:STC1 was over-expressed in the colorectal cancer tissues, and its high expression was positively correlated with poor prognosis of the colorectal cancer patients (P<0.01). In colorectal cancer HT-29 cells, treatment of CoCl₂ up-regulated the expression of STC1. Under the condition of inflammation by stimulating with LPS, the expression of STC1 was significantly increased. In the colorectal cancer, STC1 level was positively correlated with hypoxia-inducible factor-1α or inflammatory molecules expression, and the levels of tumor-infiltrating immune cells in colorectal cancer tissue. CONCLUSION:STC1 is over-expressed in the colorectal tumor tissue, and the high level of STC1 is an important prognosis indicator for colorectal cancer. Furthermore, STC1 is closely correlated with tumor microenvironment, especially with the tumor hypoxia and tumor immune regulation of colorectal cancer.     

MicroRNA-98 suppresses viability and invasion ability of colorectal can-cer cells by targeting EZH2

AIM: To study the target relationship between microRNA-98 (miR-98) and enhancer of Zeste homolog 2 (EZH2), and the effect of miR-98 on the viability and invasion ability of colorectal cancer cells.METHODS: The target relationship between EZH2 and miR-98 was predicted by TargetScan software and confirmed by dual-luciferase reporter assay. The miR-98 mimic and miR-98 inhibitor were transfected into human colorectal cancer SW480 cells and SW620 cells. The protein expression level of EZH2 was determined by Western blot. The cell viability was measured by MTT assay, and the invasion ability was detected by Transwell assay. EZH2 over-expression vector was transfected into the colorectal cancer cells, and the cell viability and invasion ability were measured.RESULTS: miR-98 targeted EZH2 and down-regulated EZH2 protein expression in the SW480 cells and SW620 cells. miR-98 over-expression significantly decreased, while miR-98 knockdown dramatically increased the viability and invasion ability of SW480 cells and SW620 cells. Additionally, EZH2 over-expression enhanced the viability and invasion ability of SW480 cells and SW620 cells.CONCLUSION: miR-98 inhibits the viability and invasion ability of SW480 cells and SW620 cells by targeting EZH2, which may provide new therapeutic target and method for colorectal cancer treatment.     

Metastasis-related function of osteopontin between two different colorectal cancer cell lines

AIM:To analyze the metastasis-related function of osteopontin(OPN) in colorectal cancer cell lines. METHODS:The sense- and antisense-osteopontin eukaryotic expression plasmids were transfected into Colo205 and SW480 cell lines. The metastatic function was detected by flow cytometry, immunohistochemistry, homogeneous and heterogeneous adhesion in different cell lines. RESULTS:High expression of OPN reduced E-cadherin expression and enhanced CD44v6 expression in colorectal cancer cells. Homogeneous adhesion was weakened, but heterogeneous adhesion was enhanced among these cells. CD44v6 expression was intensified to accelerate colorectal cancer cell adhering with ECM and invading into blood vessels and liver. CONCLUSION:OPN is one of potential and important factors in the process of colorectal cancer liver metastasis.     

HAPLN1 induces resistance to MTX in human colorectal cancer HT-29 cells though IKK/p65 pathway

AIM:To investigate the changes of hyaluronan and proteoglycan link protein 1 (HAPLN1) expression before and after resistance to methotrexate (MTX) in human colorectal cancer HT-29 cells and its effect on this drug resistance, and to explore the molecular mechanism in the process. METHODS:The drug-resistant HT-29/MTX cells were established by stepwise exposure of the cells to MTX, and then the HT-29/MTX cells were stably transfected with specific shRNA interference plasmid vectors targeting HAPLN1 and multidrug resistance-associated protein 2 (MRP2). The mRNA expression levels of HAPLN1 and MRP2 were measured by RT-PCR. CCK-8 assay was used to detect the viability of HT-29/MTX cells. The apoptosis rate was analyzed by flow cytometry. The protein levels of HAPLN1, MRP2, IκB kinase (IKK) α/β, p-IKKα/β (Ser176/Ser177), p65 and p-p65 (Ser536) were determined by Western blot. RESULTS:The HT-29/MTX cells had significantly higher mRNA and protein levels of HAPLN1 and MRP2 than HT-29 cells (P<0.05) with resistant factor of 463.756. HAPLN1 and MRP2 gene silencing significantly increased the cytotoxicity and apoptosis of HT-29/MTX cells induced by MTX (P<0.05). The IC₅₀ value was decreased from 15.304 μmol/L to 6.119 μmol/L and 7.801 μmol/L, respectively, and their reversal folds were 2.501 and 1.962, respectively. Silencing of HAPLN1 and IKK inhibitor IKK16 inhibited the phosphorylation of IKKα/β and p65 (P<0.05), and down-regulated the protein level of MRP2 in the HT-29/MTX cells (P<0.05). However, IKK16 did not affect the protein level of HAPLN1 in the HT-29/MTX cells.CONCLUSION:Knock-down of HAPLN1 gene expression reverses the resistance to MTX in human colorectal cancer HT-29/MTX cells possibly by blocking the IKK/p65 signaling pathway and thus down-regulating the expression of MRP2.