OBJECTIVE: To explore the role of miR-593 in regulating the proliferation of colon cancer cells and the molecular mechanism. METHODS: Bioinformatics analysis identified PLK1 as the possible target gene of miR-593. Luciferase assay was employed to verify the binding between miR-593 and PLK1, and qRT-PCR and Western blotting were used to verify that PLK1 was the direct target gene of miR-593. CCK-8 assay was performed to test the hypothesis that miR-593 inhibited the proliferation of colon cancer cells by targeting PLK1. RESULTS: Luciferase assay identified the specific site of miR-593 binding with PLK1. Western blotting showed a significantly decreased expression of PLK1 in the colon cancer cells transfected with miR-593 mimics and an increased PLK1 expression in the cells transfected with the miR-593 inhibitor as compared with the control cells ( < 0.05). The results of qRT-PCR showed no significant differences in the expression levels of PLK1 among the cells with different treatments ( > 0.05). The cell proliferation assay showed opposite effects of miR-593 and PLK1 on the proliferation of colon cancer cells, and the effect of co-transfection with miR-593 mimic and a PLK1-overexpressing plasmid on the cell proliferation was between those in PLK1 over-expressing group and miR-593 mimic group. CONCLUSIONS miR-593 inhibits the proliferation of colon cancer cells by down-regulating PLK1 and plays the role as a tumor suppressor in colon cancer.
Binding Sites ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; metabolism ; pathology ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; In Vitro Techniques ; MicroRNAs ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sincalide ; metabolism ; Transfection

OBJECTIVE: To investigate the expression of cytokine signal suppressor 3 (SOCS3) in colon cancer tissue and the mechanism by which SOCS3 regulates the proliferation and invasion of colon cancer. METHODS: We collected the specimens of tumor tissues and paired adjacent tissues from 80 patients with colon cancer undergoing radical resection in our hospital between July, 2014 and May, 2017, and the expression of SOCS3 in the tissue samples was analyzed using Western blotting. We also transfected colon cancer cell line SW480 with a SOCS3-overexpressing plasmid or a small interference RNA (siRNA) for SOCS3 knockdown, and the changes in the cell proliferation and invasion capacity were evaluated using CCK-8 assay and Transwell assay, respectively. The effect of demethylation and IL-6 treatment on SOCS3 expression and the proliferation and invasion of SW480 cells were observed. RESULTS: Colon cancer tissues showed a lowered expression of SOCS3 compared with the adjacent tissues. Over-expression of SOCS3 significantly inhibited while SOCS3 knockdown obviously promoted the proliferation and invasion of SW480 cells . Demethylation treatment up-regulated SOCS3 expression and inhibited the proliferation and invasion capacity of SW480 cells; IL-6 treatment of the cells caused the reverse changes. CONCLUSIONS SOCS3 participates in the development and progression of colon cancer and serves as a potential target for colon cancer treatment. In patients with colon cancer, the low expression of SOCS3 possibly as a result of methylation may promote the proliferation and invasion of the cancer cells.
Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; etiology ; pathology ; Cytokines ; Demethylation ; Disease Progression ; Humans ; Interleukin-6 ; pharmacology ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; RNA, Small Interfering ; Signal Transduction ; Suppressor of Cytokine Signaling 3 Protein ; genetics ; metabolism ; Transfection

OBJECTIVE: To study the expression of miR-454-3p in colon cancer and its effect on colon cancer proliferation, invasion and hepatic metastasis. METHODS: Specimens of tumor tissues and paired adjacent tissues were collected from 20 patients with colorectal cancer for detecting the expression levels of miR-454-3p using in situ hybridization. Colon cancer cell line SW480 was transfected with a lentiviral vector to induce miR-454-3p over-expression, and the changes in cell proliferation and invasion were observed using cell counting kit-8 (CCK-8), clone formation assay and Transwell experiment. The effect of miR- 454-3p over-expression on hepatic metastasis of colon cancer was assessed in BALB/c mouse models. RESULTS: The results of in situ hybridization showed a significantly increased expression level of miR-454-3p in colon cancer tissues as compared with normal colon tissues ( < 0.05). In SW480 cells, over-expression of miR-454-3p significantly promoted the cell proliferation and invasion ( < 0.05). In BALB/c mice, SW480 cells over-expressing miR-454-3p showed a significantly higher potential for liver metastases than the control cells ( < 0.05). CONCLUSIONS miR-454-3p is overexpressed in the tumor tissues in patients with colorectal cancer and participates in the progression of colorectal cancer by promoting cancer cell proliferation, invasion, and liver metastasis. miR-454-3p may serve as a novel biomarker for colorectal cancer diagnosis and prognostic evaluation.
Animals ; Biomarkers, Tumor ; metabolism ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colon ; metabolism ; Colonic Neoplasms ; metabolism ; pathology ; Disease Progression ; Humans ; Liver Neoplasms ; secondary ; Mice ; Mice, Inbred BALB C ; MicroRNAs ; metabolism ; Neoplasm Invasiveness ; Sincalide ; metabolism

OBJECTIVE: To investigate the expressions of secreted frizzled-related protein 4 (SFRP4) in stage Ⅱ DNA mismatch repair-deficient (dMMR) and mismatch repair- proficient (pMMR) colorectal cancers and explore their clinical significance. METHODS: We collected fresh stage Ⅱ colon cancer tissues with different MMR status detected by immunohistochemistry (IHC). The differentially expressed mRNAs between dMMR and pMMR tumors were identified by Affymetrix Human oeLncRNA gene chip, and the expression of SFRP4 in these cancer tissues and in colorectal cancer cell lines were detected using Western blotting and real- time quantitative PCR. The apoptosis rates of HCT116 cells with and without siRNA- mediated transient SFRP4 knockdown were determined using flow cytometry. We further investigated the expression pattern of Ki-67 and its correlation with SFRP4 expression. RESULTS: Compared with pMMR colon cancer tissues or cells, both dMMR colon cancer tissues (=0.014) and cells (=0.0079) showed significantly increased expression of SFRP4, which was in negative correlation with Ki-67 (=0.041). In HCT116 cells, transient SFRP4 knockdown resulted in decreased cell apoptosis, including both early apoptosis (=0.003) and late apoptosis (=0.024). CONCLUSIONS Up-regulation of SFRP4 in dMMR stage Ⅱ colon cancer promotes apoptosis and inhibits proliferation of the cancer cells, and may improve the prognosis of dMMR colon cancer.
Apoptosis ; Cell Proliferation ; Colon ; metabolism ; pathology ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; DNA Mismatch Repair ; Gene Knockdown Techniques ; HCT116 Cells ; Humans ; Ki-67 Antigen ; metabolism ; Prognosis ; Proto-Oncogene Proteins ; genetics ; metabolism ; Up-Regulation

OBJECTIVETo investigate the relationship between the expression of DDX39 protein and prognosis in colorectal cancer.

METHODSClinical data and paraffin specimens of postoperative tumor tissue from 824 patients with primary colorectal cancer who received first surgical treatment at the Department of Colorectal Surgery of Changhai Hospital of Navy Military Medical University from January 2010 to December 2011 were collected. Paraffin samples of paracancerous tissues of 38 patients were served as controls. At the same time, samples of normal rectal mucous membrane from 37 cases after procedure of prolapse and hemorrhoids, and samples of colorectal adenoma from 33 cases after endoscopic treatment were enrolled in this study. All the specimens were made as the tissue microarray, and the expression of DDX39 protein was detected by immunohistochemistry. The expression of DDX39 in the epithelium and stroma was evaluated with the average staining intensity (H-Score) and the number of positive cells. It was defined as high expression in the epithelium that the H-Score was greater than or equal to 200. It was defined as high expression in the stroma that the number of positive cells was greater than or equal to 50 in 200 times the field of vision. Relationship of different DDX39 expression levels with clinicopathological parameters and prognosis of colorectal cancer was analyzed.

RESULTSThe expression of DDX39 in colorectal cancer tissues was lower than that in normal tissues, paracancerous tissues and adenomatous tissues, whether it is in the epithelium or in the stroma [DDX39 expression in the epithelium: normal tissues 253.2±64.1, paracancerous tissues 238.8±79.2, adenomatous tissues 259.4±51.6, colorectal cancer tissues 194.2±76.5 (P=0.000, P=0.005, P=0.000, respectively); DDX39 expression in the stroma: normal tissues 110.1±64.8, paracancerous tissues 106.0±49.2, adenomatous tissues 108.5±79.1, colorectal cancer tissues 54.1±34.7(all P=0.000)]. Among the cases of colorectal cancer, there were 541 cases of high DDX39 expression and 283 cases of low DDX39 expression in the epithelium; there were 424 cases of high DDX39 expression of and 400 cases of low DDX39 expression in the stroma. The high DDX39 expression and low DDX39 expression in epithelial and stromal of colorectal cancer were related respectively with tumor location (P=0.006, P=0.016), degree of tumor differentiation (P=0.002, P=0.064), TNM stage (P=0.021, P=0.000), serum CEA level (P=0.003, P=0.005), serum CA199 level (P=0.040, P=0.005) and tumor recurrence and metastasis (P=0.000, P=0.000). All the colorectal cancer cases were followed up for (41.6±15.7) months after operation. The 5-year overall survival (OS) and disease-free survival (DFS) rates of the cases with epithelial low DDX39 expression were 84.1% and 61.5%, and both were significantly lower as compared to those with epithelial high DDX39 expression (95.4% and 88.2%, P=0.000, P=0.000). The 5-year OS and DFS rates of the stroma low DDX39 expression were 86.8% and 66.8%, and both were significantly lower as compared to those with stroma high DDX39 expression (96.1% and 90.6%, P=0.000, P=0.000). Cox multivariate analysis showed that tumor differentiation (OS:HR=0.252, 95%CI: 0.128 to 0.497, P=0.000; DFS:HR=0.266, 95%CI: 0.134 to 0.530, P=0.000), DDX39 expression level in epithelium (OS: HR =0.229, 95%CI: 0.138 to 0.382, P=0.000; DFS: HR =0.266, 95%CI: 0.158 to 0.446, P=0.000), and DDX39 expression level in stroma (OS: HR =0.331, 95%CI: 0.188 to 0.582, P=0.000; DFS:HR=0.326, 95%CI: 0.184 to 0.578, P=0.000) were independent influencing factors of overall or disease-free survival in patients with colorectal cancer.

CONCLUSIONThe low expression of DDX39 protein suggests poor prognosis and DDX39 is expected to be a new prognostic marker of colorectal cancer.

Biomarkers, Tumor ; metabolism ; Colonic Neoplasms ; Colorectal Neoplasms ; metabolism ; pathology ; DEAD-box RNA Helicases ; metabolism ; Disease-Free Survival ; Humans ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Prognosis

OBJECTIVETo investigate the effect and its possible mechanism of interleukin-17 (IL-17) on invasion and metastasis of human colon cancer cells.

METHODSIL-17 was added into the culture media of human colon cancer cells SW480 and LOVO. Cells were divided into 4 groups: SW480 control group (SW480 cells), LOVO control group (LOVO cells), SW480 experiment group (50 μg/L IL-17+SW480 cells), and LOVO experiment group (50 μg/L IL-17+LOVO cells). Cell growth was measured by CCK-8 assay. The proliferation rate(%)=[(Aexperiment group-Ablank)/(Acontrol group-Ablank)]×100%). The ability of cell invasion and migration was measured by transwell assay. Real time-PCR was used to detect mRNA expression of VEGF and MMP-9. Western blot was performed to detect protein expression of STAT3, p-STAT3, VEGF and MMP-9. Enzyme-linked immunosorbent assay (ELISA) was applied to measure the protein content of VEGF and MMP-9 in the supernatant.

RESULTSAfter cultivation for 24, 48 and 72 hours, CCK-8 assay revealed that the proliferation rate of SW480 was 1.18%±0.07%, 1.42%±0.09%, and 1.62%±0.08%; the proliferation rate of LOVO was 1.13%±0.02%, 1.32%±0.05% and 1.73%±0.02% in experiment group. Transwell experiments showed that after cultivation with IL-17 for 24 hours, number of invasion cell in experimental groups (SW480: 34.00±0.45, LOVO: 41.60±0.51) was higher as compared to corresponding control groups (SW480: 4.53±0.14; LOVO: 3.67±0.33) with significant differences (SW480: t=-76.026, P=0.001; LOVO: t=-81.580, P=0.005). The number of migration cell in experimental groups (SW480: 36.40±0.51, LOVO: 46.40±0.68) was higher as compared to corresponding control groups (SW480: 7.83±0.69; LOVO: 6.67±0.48) with significant differences (SW480: t=-51.542, P=0.003; LOVO: t=-49.265, P=0.005). Real-time PCR results revealed that after cultivation with IL-17 for 24 hours, VEGF and MMP-9 mRNA relative expression levels in experimental groups (SW480: VEGF:1.53±0.12, MMP-9: 2.44±0.23; LOVO: VEGF: 2.96±0.35, MMP-9: 3.38±0.55) were higher than those in control groups (both 1) with significant differences (VEGF: t=3.799, P=0.043; MMP-9: t=5.254, P=0.039). Western blot illustrated that after cultivation with IL-17 for 24 hours, STAT3, p-STAT3, VEGF and MMP-9 proteins relative expression levels in experimental groups were significantly higher that those in control groups (SW480:STAT3: t=3.233, P=0.023; p-STAT3: t=3.954, P=0.032; VEGF: t=3.201, P=0.025; MMP-9: t=3.154, P=0.029; LOVO: STAT3: t=3.788, P=0.012; p-STAT3: t=2.662, P=0.040; VEGF: t=4.118, P=0.035; MMP-9: t=4.268, P=0.030). ELISA indicated that content of VEGF and MMP-9 in the supernatant of experimental groups (SW480: VEGF 5 491.41±63.22, MMP-9: 21.43±1.35. LOVO: VEGF: 8 631.46±129.59, MMP-9: 178.32±3.20) were higher than those in control groups (SW480: VEGF:4 456.32±87.56, MMP-9:18.57±2.44. LOVO: VEGF: 8 122.38±108.66, MMP-9: 163.22±6.89) with significant differences (SW480: VEGF: t=6.993, P=0.037; MMP-9: t=5.587, P=0.040. LOVO: VEGF: t=7.013, P=0.044; MMP-9: t=6.762, P=0.043).

CONCLUSIONIL-17 may be able to activate STAT3 signal transduction pathway in vitro through up-regulation of VEGF and MMP-9 expression, thereby enhancing the invasion and migration of colon cancer SW480 and LOVO cells.

Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colonic Neoplasms ; pathology ; Humans ; Interleukin-17 ; pharmacology ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Real-Time Polymerase Chain Reaction ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Up-Regulation ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo explore the effect of colon cancer cell-derived interleukin-1α on the migration and proliferation of human umbilical vein endothelial cells as well as the role of IL-1α and IL-1ra in the angiogenesis process.

METHODSWestern blot was used to detect the expression of IL-1α and IL-1R1 protein in the colon cancer cell lines with different liver metastatic potential. We also examined how IL-1α and IL-1ra influence the proliferation and migration of umbilical vascular endothelial cells assessed by PreMix WST-1 assay and migration assay, respectively. Double layer culture technique was used to detect the effect of IL-1α on the proliferation and migration of vascular endothelial cells and the effect of IL-1ra on the vascular endothelial cells.

RESULTSWestern blot analysis showed that IL-1α protein was only detected in highly metastatic colon cancer HT-29 and WiDr cells, but not in the lowly metastatic CaCo-2 and CoLo320 cells.Migration assay showed that there were significant differences in the number of penetrated cells between the control (17.9±3.6) and 1 ng/ml rIL-1α group (23.2±4.2), 10 ng/ml rIL-1α group (31.7±4.5), and 100 ng/ml rIL-1α group (38.6±4.9), showing that it was positively correlated with the increasing concentration of rIL-1α (P<0.01 for all). The proliferation assay showed that the absorbance values were 1.37±0.18 in the control group, and 1.79±0.14 in the 1 ng/ml rIL-1α group, 2.14±0.17 in the 10 ng/ml rIL-1α group, and 2.21±0.23 in the 100 ng/ml rIL-1α group, showing a positive correlation with the increasing concentration of rIL-1α(P<0.01 for all). IL-1ra significantly inhibited the proliferation and migration of vascular endothelial cells (P<0.01). The levels of VEGF protein were (1.697±0.072) ng/ml, (3.507±0.064)ng/ml and (4.139±0.039)ng/ml in the control, HUVECs+ IL-1α and HUVECs+ HT-29 co-culture system groups, respectively, showing a significant difference between the control and HUVECs+ 10 pg/ml rIL-1α groups and between the control and HUVECs+ HT-29 groups (P<0.01 for both).

CONCLUSIONSOur findings indicate that colon cancer cell-derived IL-1α plays an important role in the liver metastasis of colon cancer through increased VEGF level of the colon cancer cells and enhanced vascular endothelial cells proliferation, migration and angiogenesis, while IL-1ra can suppress the effect of IL-1α and inhibit the angiogenesis in colon cancer.

Blotting, Western ; Caco-2 Cells ; Cell Line, Tumor ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Coculture Techniques ; Colonic Neoplasms ; blood supply ; metabolism ; pathology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Interleukin 1 Receptor Antagonist Protein ; metabolism ; physiology ; Interleukin-1alpha ; metabolism ; physiology ; Liver Neoplasms ; secondary ; Neovascularization, Pathologic ; etiology

OBJECTIVETo explore the relationship between DNA mismatch repair (MMR) and clinicopathologic features and prognosis in patients with stages II and III colon cancers.

METHODSThe clinical and pathological data of 440 patients with stage II/III colon cancer after radical resection were retrospectively reviewed and analyzed. Immunohistochemical staining was used to assess the expression of MMR proteins (MLH1, MSH2, MSH6 and PMS2), and the correlation between DNA MMR and clinicopathological features and prognosis of colon cancers was analyzed.

RESULTSOf the 440 tumor samples tested for DNA mismatch repair status, 90 (20.5%) demonstrated defective DNA mismatch repair and 350 (79.5%) had proficient DNA mismatch repair. Defective DNA mismatch repair (dMMR) was associated with young patients (≤ 60), proximal colon cancer, stage II, poorly differentiated adenocarcinoma and mucinous adenocarcinoma (P<0.05 for all). Among the 440 patients, 126 (28.6%) cases had recurrence or metastasis and 93 (21.1%) died during the median follow-up of 61.0 months. The five-year disease-free survival (DFS) rate was 82.2% among the patients with tumor exhibiting dMMR, significantly higher than that in patients with tumors exhibiting pMMR (68.9%, P=0.02). The univariate and mutlivariate analyses showed that the MMR status is an independent factor affecting 5-year disease-free survival and overall survival (OS) in colon cancer patients (P<0.05 for both).

CONCLUSIONSDefective DNA mismatch repair (dMMR) is associated with patients with proximal colon cancer, stage II and poorly defferentiated adenocarcinoma and mucinous adenocarcinoma. The prognosis for patients with dMMR is better than those with pMMR. dMMR may be a useful biomarker for the prognosis of colon cancer.

Adaptor Proteins, Signal Transducing ; metabolism ; Adenocarcinoma ; genetics ; metabolism ; mortality ; pathology ; Adenocarcinoma, Mucinous ; genetics ; metabolism ; mortality ; pathology ; Adenosine Triphosphatases ; metabolism ; Age Factors ; Analysis of Variance ; Colonic Neoplasms ; genetics ; metabolism ; mortality ; pathology ; DNA Mismatch Repair ; DNA Repair Enzymes ; metabolism ; DNA-Binding Proteins ; metabolism ; Disease-Free Survival ; Humans ; Mismatch Repair Endonuclease PMS2 ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; metabolism ; Neoplasm Recurrence, Local ; Nuclear Proteins ; metabolism ; Prognosis ; Retrospective Studies ; Survival Rate

The aim of this study is to evaluate anti-tumor activities and mechanism of a novel kinase inhibitor ZLJ213 which targeted Aurora A and vascular endothelial growth factor receptor (VEGFR) in vitro and in vivo against human colon cancer. Results showed that ZLJ213 inhibited cell proliferation and induced cell cycle arrest and apoptosis of HCT1 16 and SW48 cell lines. In HCT116-derived xenograft, ZLJ213 dosed at 100 mg · kg(-1) inhibited tumor growth by 73.24%. The IC50 of ZLJ213 on the expression of p-Aurora A was 0.258 µmol · L(-1) analyzed by ELISA. Under the concentration of 0.08 µmol · L(-1), ZLJ213 could inhibit the activities of Aurora A, Histone H3 and VEGFR of HCT116 and SW48 cell lines. Simultaneously, ZLJ213 induced activation of Caspase 3 and PARP cleavage. Above data suggested that ZLJ213 had the ability to inhibit cell proliferation and induce cell apoptosis both in vitro and in vivo in colon cancer, and down-regulate the expression of p-Aurora A and p-VEGFR. ZLJ213 might be a potential therapeutic agent against colon cancer.
Animals ; Apoptosis ; Aurora Kinase A ; antagonists & inhibitors ; Cell Cycle Checkpoints ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Colonic Neoplasms ; pathology ; Humans ; Protein Kinase Inhibitors ; pharmacology ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; Xenograft Model Antitumor Assays

Animals ; Cell Adhesion Molecules ; metabolism ; Colonic Neoplasms ; pathology ; Female ; Humans ; Liver Neoplasms ; secondary ; Protein-Serine-Threonine Kinases ; Proto-Oncogene Proteins ; metabolism