Correlation between hypermethylation and expression of Syk gene in colorectal cancer cell lines

AIM:To explore the relationship of methylation status and expression of spleen tyrosine kinase (Syk) gene in colorectal cancer cell lines. METHODS:Bisulfite sodium modification sequencing, methylation specific PCR and Western blotting were used to detect the methylation status and expression of Syk gene in 23 colorectal cancer (CRC) cell lines. Luciferase assay was applied to measure the activity of promoter with or without methylation in CpG islands. Meanwhile, methylation status and expression level were compared in Syk(-) HCT-15 cell line before or after 5-Aza-CdR administration. RESULTS:(1) Among 23 cell lines, loss expression of Syk gene in 9 cell lines due to hypermethylation in promoter, and 14 expression with unmethylation status were observed. The total methylation rate was 39.2%. (2) Microsatellite instability was found in 7 of 9 cell lines with promoter hypermethylation, 4 of 14 were wihtout hypermethylation. The difference between methylation and unmethylation group was significant (P<0.05). (3) 5-Aza-CdR restored methylated-Syk gene promoter activity. Compared to methylated-promoter, luciferase activities increased to 4.5 and 4.7 folds with Syk full size promoter and unmethylated-promoter, respectively. (4) 5-Aza-CdR restored methylated-Syk gene expression and the effect had time-course dependence. CONCLUSION:Hypermethylation of CpG islands in Syk gene promoter silences Syk expression in CRC cell lines, and 5-Aza-CdR restores Syk expression by demethylation.     

Methylation of TIMP-3 gene and their relationships with their clinical-pathological characteristics of colorectal cancers

AIM: To investigate whether methylation of the TIMP-3 gene is associated with clinical-pathological characteristics, recurrence and metastasis of the colorectal cancer. METHODS: Nest methylation specific PCR (nMSP) and RT-PCR techniques were used to detect methylation of TIMP-3 gene and its mRNA expression in the colorectal cancer specimen and adjacent non-cancerous tissues. RESULTS: The expression of TIMP-3 mRNA in tumor tissues was distinctly reduced (P<0.01). The expression of TIMP-3 mRNA in those without lymph node metastasis was higher than those with lymph node metastasis (P<0.01). The patients with Duke's C, D and lymph node metastasis were more to contain methylated TIMP-3 compared to those with Duke's A, B and no lymph node metastasis (P<0.05). Statistical differences in pathological characteristics such as tumor site, Duke's stage, histological differentiation and type between TIMP-3 methylation positive group and negative group were observed (P<0.05). CONCLUSION: Methylation of the TIMP-3 gene promoter usually occurs in the proximal site, infiltrating type, poor cellular differentiation, lymph node metastasis and advanced stage of colorectal cancers patients.     

Application of multiple gene methylations in plasma for diagnosis of lung cancer

AIM: To determine the aberrant methylation status in the gene promoter regions of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3by detecting the plasma specimens and the value of their combined detection for diagnosis of lung cancers. METHODS: Nest methylation specific PCR (nMSP) was used to detect the promoter methylation status of the 5 genes in the plasma from 106 normal controls， lung cancer tissues, lung benign tissues and the plasma from 106 patients with lung cancers. Multiple displacement amplification (MDA) was used to amplify modified genomic DNA to solve the problem of insufficient of plasma DNA template. RESULTS: The positive rates of promoter methylation of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3in the lung cancer tissues were 51.9%, 44.3%, 54.7%, 36.8%, 24.5%, respectively, and those in the plasma were 46.2%, 41.5%, 50.9%, 31.1%, 19.8%, respectively. The results of the Kappa consistency check showed that the lung cancer tissues and the plasma had obviously coherence in the methylation status of the 5 gene promoter regions. Combination of DLEC1, CDH13, RASSF1A, and SEPT9 had a higher diagnostic efficiency than the others, as their ACC value was 0.8208 and youden index was 0.6415 (with the sensitivity of 81.13% and the specificity of 83.02%). CONCLUSION: Combination detection of promoter methylation of lung cancer-related genes in the plasma is expected to apply to the early diagnosis of lung cancer.     

Expression and clinical significance of Bmi-1 in colorectal cancer

AIM:This study was to investigate the expression and significance of Bmi-1 in colorectal carcinoma (CRC), and to explore the effect of Bmi-1 on Ki67 expression in human CRC.METHODS:The samples from sixty CRC, thirty adenomas and twenty normal colorectal mucosal tissues were used in this study.The expression of Bmi-1 protein was detected by immunohistochemistry.The clinicopathological features and survival rate of patients were also analyzed.RESULTS:The overexpression of Bmi-1 was respectively 25.0％, 6.7％and 0% in CRC and adenomas as well as normal colorectal mucosal tissues.The results showed that the expression of Bim-1 was significantly higher in CRC, compared with that in adenomas and normal colorectal mucosal tissues (P<0.05).The overexpression of Bmi-1 protein in CRC was obviously associated with distant metastasis and TNM stage (P<0.05), but not with gender, age, tumor size, tumor site, histological type, differentiation degree and lymph node metastasis (P>0.05).Kaplan-Meier survival analysis showed that the overexpression of Bmi-1 reduced significantly survival of CRC patients (P<0.05).No statistical relation between expression of Bmi-1 and Ki67 in CRC was observed.CONCLUSION:The overexpression of Bmi-1 protein is significantly correlated with tumorigenesis, metastasis and prognosis of CRC.Bmi-1 might be regarded as a parameter in evaluating prognosis of CRC.     

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.     

Relationship between expression level of TIMP-3 and invasion/metastasis of hepatocellular carcinoma

AIM: To detect the levels of tissue inhibitor of metalloproteinase-3 (TIMP-3) in both plasma and the tissue of hepatocellular carcinoma (HCC), and to elucidate their association with clinical features.METHODS: Plasma protein levels of TIMP-3 in 56 HCC patients and 30 cases of controls were detected by ELISA.The mRNA and protein levels of TIMP-3 in 30 HCC tissue samples with their portal vein tumor embolus and lymphatic metastasis tissues, and in normal liver tissues from 30 controls were detected by RT-PCR and Western blotting.The relationship between mRNA and protein levels and their clinic-pathological data were analyzed.RESULTS: The plasma TIMP-3 protein levels in the extrahepatic metastasis patients were obviously lower than those in the non-extrahepatic metastasis patients (P<0.05).The mRNA levels of TIMP-3 in normal liver, carcinoma in situ, portal vein tumor embolus and lymphatic metastasis tissues were 0.78±0.09, 0.52±0.09, 0.42±0.07 and 0.40±0.08, respectively, with significant differences among them (P<0.05).The protein levels of TIMP-3 in these 4 kinds of tissues were 115.08±8.60, 77.04±8.83, 64.43±3.80 and 62.80±3.73, respectively, also with significant differences among them (P<0.05).CONCLUSION: The expression of TIMP-3 significantly decreases in the carcinoma in situ tissues of HCC patients, and decreases more obviously in the portal vein tumor embolus and lymphatic metastasis tissues, indicating that low expression of TIMP-3 may play an important role in HCC invasiveness and metastasis.     

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.     

Association between survivin promoter -31C/G polymorphism and genetic susceptibility to sporadic colorectal cancer

AIM: To investigate the association between -31C/G polymorphism in the promoter of survivin gene and the susceptibility to sporadic colorectal cancer (CRC) in southern Chinese population. METHODS: survivin -31C/G genotypes were determined by PCR-RFLP in 711 healthy controls and 702 CRC cases. RESULTS: The number of CRC patients carrying with CC genotype was much higher than that of controls (36.5 % vs 26.2%,2 =17.89,P<0.01). Compared to CC genotypes, CG, GG genotypes and G allele carriers had a significantly decreased risk of CRC, with the decrease being 0.61-fold (95% confidence interval=0.46-0.80, P<0.01), 0.52-fold (95% confidence interval=0.38-0.71,P<0.01) and 0.58-fold (95% confidence interval=0.45-0.74, P<0.01), respectively. CONCLUSION: survivin gene -31C/G polymorphism is associated with sporadic CRC risk, the G variant genotype is the independent protective factors against sporadic CRC in southern Chinese population.     

Effect of long non-coding RNA MEG3 on invasion and migration of colorectal cancer cells

AIM: To investigate the expression of long non-coding RNA maternally expressed gene 3(MEG3) in colorectal cancer(CRC) cells, and to observe the effect of MEG3 on the invasion and migration of CRC cells. METHODS: The levels of MEG3 in human normal colon cell NCM460 and CRC cells SW48 and LoVo were detected by real- time PCR. MEG3 was over-expressed by plasmid transfection, and the effects of MEG3 on the invasion and migration of SW48 and LoVo cells were analyzed by Transwell assay and wound healing assay. The expression of matrix metalloproteinase(MMP) family proteins was determined by Western blotting. RESULTS: The level of MEG3 was down-regulated in CRC cells compared with normal colon cell NCM460. The invasion and migration of CRC cells were reduced after MEG3 over-expression. Transwell invasion and migration assays showed that the numbers of transmembrane SW48 and LoVo cells were smaller in MEG3 over-expression group than control group(CONCLUSION: The expression of MEG3 is down-regulated in CRC cells. Over-expression of MEG3 inhibits the invasion and migration of CRC cells. TIMP-2, MMP-2 and MMP-9 might play an important role in this regulation.     

Effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells

AIM: To observe the effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells. METHODS: Both hepatocellular carcinoma cells (H2M) and epidermoid carcinoma cells (A431) with methylation of TIMP-3 promoter gene were treated with 5-Aza-2'-deoxycytidine (5Aza-dc). Invasion ability and motility of the cells were detected by Transwell experiments. Expressions of TIMP-3 protein and mRNA were detected by Western blotting and RT-PCR, respectively. TIMP-3 gene promoter methylation was detected by methylation-specific PCR (MSP). RESULTS: ① Invasion ability and motility of H2M and A431 cells were declined after treatment with 5Aza-dc; ② After treatment with 5Aza-dc, the expression of TIMP-3 protein and mRNA were increased in H2M and A431 cells; ③ After treatment with 5Aza-dc, methylation of TIMP-3 promoter gene was not detectable in the cell lines. CONCLUSIONS: 5Aza-dc induces demethylation in TIMP-3 promoter gene, restores TIMP-3 gene and protein expression, and inhibits invasion ability and motility of the carcinoma cells.     

Analyzing clinical characteristic of miR-196b in human colorectal cancer using TCGA and effect of miR-196b on 5-FU resistance of CRC cell line

AIM: To investigate the clinical characteristics of microRNA (miR)-196b in colorectal cancer (CRC) and to study its biological function in 5-fluorouracil (5-FU) resistance. METHODS: miRNA sequence dataset and the corresponding clinical data of CRC patients were downloaded from The Cancer Genome Atlas (TCGA). Expression level and clinical characteristics of miR-196b in CRC patients were analyzed using SPSS 17.0. CRC cell line overexpres-sing miR-196b was established using transient transfection method. MTS test was used to evaluate the effect of miR-196b overexpression on 5-FU resistance. RESULTS: miR-196b expression was associated with lymph node metastasis and TNM stage (P<0.05), but not related with age and sex. Lymph node metastasis and distant metastasis were independent prognostic factors for rectal patients (P<0.05). The expression level of miR-196b was not associated with survival condition of rectal patients. The viability of the cells overexpressing miR-196b treated with different concentrations of 5-FU was significantly higher than that in control group (P<0.05). CONCLUSION: miR-196b may be a potential biomarker of TNM stage and lymph node metastasis in CRC. miR-196b increases the 5-FU resistance of CRC cells.     

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.     

Hypermethylation of SFRP genes in esophageal squamous cell cancer

AIM: To investigate the promoter methylation of secreted frizzled-related protein(SFRP) genes in esophageal squamous cell cancer(ESCC). METHODS: The methods of methylation-specific PCR(MS-PCR) and RT-PCR were applied to examine the CpG methylation of the SFRP promoter and the mRNA expression of SFRP genes,respectively, in 78 samples of ESCC and corresponding adjacent non-cancer tissues. The protein expression of β-catenin was determined by immunohistochemistry.RESULTS: In the ESCC tissues, the frequencies of promoter methylation in SFRP1 , SFRP2 , SFRP4 and SFRP5 genes were 65.4%(51/78), 69.2%(54/78), 62.8%(49/78) and 52.6%(41/78),respectively, significantly higher than those in the adjacent tissues(P<0.01). The hypermethylation of these genes had no correlation with clinical stage and pathological classification in ESCC tissues(P>0.05). The frequency of simultaneous methylation of the 4 genes was correlated with the clinical stage(P<0.05). The positive rates of mRNA expression of the 4 genes in ESCC tissues were 42.3%(33/78), 46.2%(36/78), 50.0%(39/78) and 39.7%(31/78), respectively lower than those in the adjacent tissues(P<0.01). The mRNA expression of SFRP genes and the ectopic expression of β-catenin were correlated with the methylation frequency of SFRP genes(P<0.01).CONCLUSION: Promoter methylation of SFRP1 , SFRP2 , SFRP4 and SFRP5 genes was a frequent event in ESCC, indicating a contribution to the pathogenesis of ESCC through aberrant canonical Wnt/β-catenin signaling pathway. Combination analysis of methylation status in SFRP genes may has definite value on estimating prognosis of ESCC.     

Relationship between the expression of MMP-2, TIMP-2 and VEGF-C in hepatocellular carcinoma with or without lymph node metastasis

AIM: To investigate the relationship between the expression of MMP-2, TIMP-2 and VEGF-C in hepatocellular carcinoma (HCC) with or without lymph node metastasis. METHODS: The expression of MMP-2, TIMP-2 and VEGF-C in 44 cases of HCC were examined using immunohistochemistry methods (SP).RESULTS: The positive expression of MMP-2, TIMP-2 and VEGF-C was associated with lymph node metastasis of HCC (P<0.05). CONCLUSIONS: There was a significant correlation between the high-expression of MMP-2 and VEGF-C, and the low-expression of TIMP-2 in lymph node metastasis of HCC. VEGF-C is an important factor promoting lymph node metastasis of HCC. The expression of MMP-2, TIMP-2 and VEGF-C may have important value in determining lymph node metastasis and prognosis of HCC.     

Methylation and mRNA expression of TIG1 gene in esophageal squamous-cell carcinoma tissues

AIM: To investigate the expression and promoter methylation of tazarotene-induced gene-1 (TIG1) in esophageal squamous-cell carcinoma (ESCC) tissues. METHODS: The methods of methylation-specific PCR and real-time fluorescence quantitative PCR were applied to examine the methylation and mRNA expression of TIG1, respectively, in 43 cases of ESCC tissues, 20 cases of paracancerous tissues and 15 cases of normal tissues. RESULTS: The frequency of promoter methylation of TIG1 gene in ESCC tissues was 25.6% (11/43), which was significantly higher than that in the paracancerous tissues (5.0%, 1/20) and normal tissues (0/20). The hypermethylation of TIG1 gene in these tissues had no correlation with sex, age and clinical stage of the patients. However, it was correlated with the pathological stage (P<0.01) and lymph node metastasis (P<0.05). The mRNA expression of TIG1 in ESCC tissues was significantly lower than that in paracancerous tissues (P<0.05) and normal tissues (P<0.01). However, the expression level of TIG1 mRNA in methylated tissues was significantly lower than that in unmethylated tissues (P<0.01). CONCLUSION: Promoter methylation may be an important mechanism of TIG1 gene inactivation in ESCC, which was related to lymph node metastasis and TNM stage of esophageal carcinoma.     

Microarray analysis of tumor metastasis-related gene expression in PSMA-silencing prostate cancer cells

AIM: To investigate the functions of prostate-specific membrane antigen (PSMA) in prostate cancer metastasis. METHODS: Specific siRNA to knock down PSMA expression was designed and transfected into LNCaP cells. The tumor metastasis gene chip was also used to analyze the differential expression of 84 genes related to cancer metastasis. RESULTS: Specific siRNA was successfully designed and constructed and the gene expression of PSMA in LNCaP cells was knocked down. The RNAi efficiency was more than 75% at mRNA level and more than 68% at protein level. The results of the tumor metastasis gene chip indicated that 10 genes were up-regulated (such as CDH6 and CXCL12) and 4 genes were down-regulated (such as CCL7 and MDM2) in the LNCaP cells treated with PSMA siRNA. CONCLUSION: The PSMA is involved in the regulatory pathways in prostate cancer metastasis.     

Expression of IL-1α and TNF-α modified by Toll-like receptor 4 genetic polymorphism is associated with colorectal carcinoma

AIM: To investigate the association of D299G, T399I and A896G polymorphisms of Toll-like receptor 4 (TLR4) and colorectal carcinoma (CRC). METHODS:
The genotypes of these 3 loci among 268 patients with CRC and 268 healthy controls were determined by polymerase chain reaction-restriction fragment lengthy polymorphism (PCR-RFLP). The protein levels of IL-1α, IL-8, TGF-β and TNF-α in the homogenate of CRC biopsies were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: No significant difference of the genotype frequencies of TLR4 A896G and D299G between the cases and the controls was observed. CT combined TT genotype of T399I was significantly associated with increased CRC risk. The individuals with the T allele of T399I showed a 1.843-fold increase in CRC risk as compared with the C allele. The concentrations of IL-1α and TNF-α in CRC biopsies were significantly elevated in the individuals with the genotype of T399I CT combined with TT as compared with the genotype of CC. CONCLUSION: TLR4 T399I promotes the development of CRC by modifying the expression of IL-1α and TNF-α in CRC tissues.     

Rab11-FIP4 regulates migration and invasion of colorectal cancer cells through IGF1R

AIM To investigate the role, clinical implications and the underlying mechanisms of Rab11-family interacting protein 4 (Rab11-FIP4) in colorectal cancer (CRC). METHODS The expression levels of Rab11-FIP4 in CRC tissues and corresponding paracancerous tissues were compared by immunohistochemistry, Western blot and RT-qPCR. The above methods were also used to detect the expression levels of Rab11-FIP4 in CRC cells under normal environment and hypoxia. The patiens were divided into Rab11-FIP4 high expression group (n=61) and Rab11-FIP4 low expression group (n=39) according to the immunohistochemical staining score.The overall survival and recurrence time of the 2 groups were compared by Kaplan-Meier survival analysis. HCT116 and LoVo cells with stable over-expression of Rab11-FIP4 were constructed using a lentiviral system. The cytological characteristics effects of Rab11-FIP4 over-expression in CRC cells were examined by CCK-8 assay, clonogenic assay and the Transwell assay. The co-immunoprecipitation was used to detect the correlation between Rab11-FIP4 and insulin-like growth factor 1 receptor (IGF1R). Human phosphokinase array was performed to investigate the signaling pathhway affected by IGF1R in CRC cells with increased expression of Rab11-FIP4. The relationship between Rab11-FIP4 and hypoxia-inducible factor 1α (HIF-1α) was analyzed by tissue microarrays and dual-luciferase reporter assay. RESULTS Rab11-FIP4 expression was up-regulated in CRC tissues and high expression of Rab11-FIP4 was associated with poor prognosis of the patients with CRC (P<0.05). Over-expression of Rab11-FIP4 promoted the viability, migration and invasion of CRC cells (P<0.05). High expression of Rab11-FIP4 regulated ERK1/2 and AKT signaling pathway via IGF1R (P<0.05). Hypoxia promoted the activation of HIF-1α on the Rab11-FIP4 promoter, thereby up-regulating the expression of Rab11-FIP4 (P<0.05). CONCLUSION Rab11-FIP4 may act as an oncogene to regulate migration and invasion of colorectal cancer cells through IGF1R.