Migration Of Colorectal Cells Research Articles

KCNJ14 knockdown significantly inhibited the proliferation and migration of colorectal cells

BackgroundThis study attempted to verify the potential of KCNJ14 as a biomarker in colorectal cancer (CRC).MethodsData on transcriptomics and DNA methylation and the clinical information of CRC patients were downloaded from The Cancer Genome Atlas and Gene Expression Omnibus databases. Biological information analysis methods were conducted to determine the role of KCNJ14 in the prognosis, diagnosis, immune cell infiltration, and regulation mechanism of CRC patients. The effect of KCNJ14 on the proliferation and migration of HCT116 and SW480 CRC cell lines was verified by in vitro experiments (MTT, colony-forming, wound healing, and transwell assays). Western blotting was performed to detect the effect of KCNJ14 on the levels of mTOR signalling pathway-related proteins.ResultsKCNJ14 expression was remarkably increased in CRC tissues and cell lines, which reduced the overall survival time of patients. KCNJ14 mRNA was negatively regulated by its methylation site cg17660703, which can also endanger the prognosis of patients with CRC. Functional enrichment analysis suggested that KCNJ14 is involved in the mTOR, NOD-like receptor, and VEGF signalling pathways. KCNJ14 expression was positively correlated with the number of CD4 + T cells and negatively correlated with that of CD8 + T cells in the immune microenvironment. KCNJ14 knockdown significantly reduced not only the proliferation and migration of CRC cell lines but also the levels of mTOR signalling pathway-related proteins.ConclusionsThis study not only increases the molecular understanding of KCNJ14 but also provides a potentially valuable biological target for the treatment of colorectal cancer.

BMAL1 promotes colorectal cancer cell migration and invasion through ERK- and JNK-dependent c-Myc expression.

Cancer metastasis is still a life threat to patients with colorectal cancer (CRC). Brain and muscle ARNT-like protein 1 (BMAL1) is an important biological proteins that can regulate the behavior of cancer cells and their response to chemotherapy. However, the role of BMAL1 in the tumorigenic phenotype of CRC remains unclear. Here, we aim to investigate the functional role and mechanisms of BMAL1 in CRC. The mRNA expression of BMAL1 was studied using the Cancer Genome Atlas (TCGA) databases. The protein level in clinical tissues was confirmed by immunohistochemistry (IHC). The effects of BMAL1 on the epithelial-to-mesenchymal transition (EMT) and proliferation of CRC cell lines (including BMAL1 overexpressed or silencing cells) were studied by Transwell, wound healing, CCK-8 and colony formation experiments. A series of experiments were conducted to demonstrate the mechanisms of BMAL1 regulating EMT and cancer proliferation in vitro and in vivo. We found that BMAL1 expression was closely related to the poor prognosis of CRC. BMAL1 overexpression promoted cell proliferation and migration. Mechanistically, we found that BMAL1 may activate the epithelial-to-mesenchymal transition (EMT) pathway and induce the β-catenin release further promotes the expression of oncogene c-Myc and the migration of colorectal cells by activating MAPK pathway. However, BMAL1 silencing achieved the opposite effect. In addition, blocking MAPK-signaling pathway with specific inhibitors of ERK1/2 and JNK can also downregulate the expressions of c-Myc in vitro. Taken together, these results suggested that the BMAL1/ c-Myc-signaling pathway may regulate the metastasis of CRC through the JNK/ERK1/2 MAPK-dependent pathway. Our study showed that BMAL1 promotes CRC metastasis through MAPK-c-Myc pathway. These results deepen our understanding of the relationship between BMAL1 and tumorigenic phenotypes, which may become a promising therapeutic target for BMAL1 overexpressing CRC.

MiR-451 regulates proliferation and migration of colorectal cells by targeting MIF

Objective: To investigate the effect and mechanism of miR-451 on the proliferation and migration of human colorectal cancer cell SW480 by targeting macrophage migration inhibitory factor (MIF). Methods: Microarray analysis was used to screen differentially expressed microRNAs and messenger RNA in SW480 cells. Real-time quantitative PCR (RT-qPCR) was used to detect the expressions of miR-451 and MIF in SW480 cells before and after transfection. Cell clone formation assay and Transwell assay were used to detect the proliferation and invasion of SW480 cells, respectively. Cell scratch assay was used to detect the migration ability of SW480 cells. The TargetScan database was used to analyze the correlation between miR-451 and MIF. Dual luciferase reporter gene was used to detect the interaction of miR-451 and MIF. MTT assay was used to detect the viability of SW480 cells. Results: Compared with human normal colorectal mucosal cell FHC (1.00), the expression of miR-451 was down-regulated in SW480 cells ( 0.36±0.18, P<0.001). Knockdown of miR-451 promoted proliferation, and migration of SW480 cells. Compared with that in FHC cells, MIF expression was up-regulated in SW480 cells (2.28±0.45, P<0.001). MIF down-regulation inhibited SW480 cell proliferation, invasion and migration. MiR-451 specifically bind to the MIF 3'UTR and regulated the expression of MIF. Overexpression of miR-451 reduced while overexpression of MIF increased the viability of SW480 cells. Overexpression of MIF promoted the proliferation and migration of SW480 cells (P<0.01), reversed the effect of miR-451 suppressed proliferation and migration of SW480 cells. Conclusion: MiR-451 may regulate proliferation and migration of human colorectal cancer cells by targeting MIF.

Tumor necrosis factor receptor associated factor 4 promotes invasion and metastasis of colorectal cancer cell line LoVo

Objective To study the effect of low expression of tumor necrosis factor receptor associated factor 4 (TRAF4) on invasion and migration of colorectal cancer cells. Methods Immunohistochemistry was used to detect the expression of TRAF4 in clinical specimens of colorectal cancer and the adjacent tissues. The expression levels of epithelial-mesenchymal transition (EMT)-related proteins [E-cadherin (E-cad), Snail and Slug] were detected by Western blotting. The Transwell assay was used to examine the invasive ability and migration ability of tumor cells LoVo after silencing TRAF4. Results Immunohistochemistry showed that TRAF4 expression was significantly higher in cancer tissues than in adjacent tissues (t=8.322, P<0.01). After transfection with TRAF4 small interfering RNA (siRNA), the expression of TRAF4 was significantly decreased (t=7.687, P<0.01), the expression of E-cad was increased (t=4.322, P<0.05), the expression of Snail and Slug was decreased (t=9.831, P<0.01, t=8.677, P<0.01), and the invasion and migration ability was obviously decreased (t=9.553, P<0.01, t=11.290, P<0.01). Conclusion Silencing TRAF4 can inhibit the invasion and migration of colorectal cancer cell LoVo. Key words: Colorectal cancer; Tumor necrosis factor receptor associated factor 4; Invasion; Migration; Epithelial-mesenchymal transition

Nicotinamide phosphoribosyl transferase promoting colorectal cancer invasion, metastasis and proliferation

Objective To investigate whether nicotinamide phosphoribosyl transferase (Nampt) can promote migration of colorectal cancer cells and its effect on proliferation. Methods The Transwell assay was used to examine the effect of Nampt on migration of SW480 and HT-29 cells. Western blotting was used to detect the expression of matrix metalloproteinase (MMP)-9 in SW480 and HT-29 cells. The effects of Nampt on cell cycle and DNA synthesis were tested after Nampt overexpression in SW480 and HT29 cells. Results The exogenous Nampt could promote the migration of SW480 and HT-29 cells in a dose-dependent manner, and simultaneously increase the protein expression of MMP-9 level in SW480 cells. The overexpression of Nampt in SW480 and HT29 cells could promote the entry of cells into the S phase and proliferation in SW480 cells (Nampt group: 51.21±0.78; blank control group: 31.47±0.90; and negative control group: 32.35±1.15, t=4.566, P<0.05) and HT-29 cells (Nampt group: 53.40±1.39; blank control group: 35.71±1.28; and negative control group: 33.15±2.04, t=5.432, P<0.05). Conclusion Nampt can promote the migration and proliferation of colon cancer cells. Key words: Nicotinamide phosphoribosyl transferase; Colorectal cancer; Migration; Proliferation

Effect of Galectin-3 on biological behavior of colorectal cancer cells

Objective To investigate the effects of Galectin-3 on the proliferation, migration and tumorigenesis of colorectal cancer cells. Methods The stable cell line of Galectin-3 knockout (KO) was established by CRISPR-Cas9 technique in SW480 cells and the cells without knockout of Galectin-3 were used as control group. The expression level of Galectin-3 in control group and Galectin-3 KO group were analyzed by Western blotting. The proliferation ability in control group and Galectin-3 KO group were analyzed by Edu method. The migration ability in control group and Galectin-3 KO group were analyzed by scratch test and Transwell experiment. and was used to analyze The growth of tumors cell in control group and Galectin-3 KO group were analyzed by in vivo tumorigenesis experiment. Results Two single guide RNA (sgRNA) targeting knockout Galectin-3 proteins were selected and Galectin-3 KO1 and Galectin-3 KO2 were obtained respectively. Compared with the control group [(52.19±12.09)%], the positive rate of EDU in the Galectin-3 KO1 group and Galectin-3 KO2 groups [(11.35±9.27)% and (14.39±7.88)%] decreased significantly (t=3.912, P<0.05; t=3.209, P<0.05). Compared with the control group [(81.32±9.81)%], the percentage of healing distance of scratches in Galectin-3 KO1 group and Galectin-3 KO2 group [(33.28±8.77)% and (30.19±9.10)%] decreased significantly (t=5.197, P<0.05; t=5.273, P<0.05). Compared with the control group [(201.33±19.81) cells], the average number of cell migration per visual field in Galectin-3 KO1 group and Galectin-3 KO2 group [(109.4±12.87) cells and (125.10±15.91) cells] decreased significantly (t=9.255, P<0.05; t=10.364, P<0.05). The growth rate of cells in Galectin-3 KO1 group and Galectin-3 KO2 group in nude mice was significantly lower than that in control group (P<0.05). Conclusion Detection of Galectin-3 protein level is helpful to judge the proliferation and migration of colorectal cells. Key words: Galectin-3; Colorectal cancer; Proliferation; Migration; Tumor formation

Role and mechanism of circular RNA protein arginine methyltransferase 5 in proliferation, migration of colorectal cancer cells

Objective To evaluate the role of circular RNA protein arginine methyltransferase 5 (circPRMT5) in the genesis and progression of colorectal cancer. Methods From January 2013 to December 2017, 96 patients with colorectal cancer who underwent radical resection in Department of General Surgery, Jiading District Central Hospital Affiliated to Shanghai Medical College of Health were collected. The expression of circPRMT5 in colorectal cancer tissues was examined by real-time polymerase chain reaction (RT-PCR). The correlation between circPRMT5 expression level and age, gender, tumor size, tumor location, pathological differentiation, TNM stage, lymph node metastasis of patients with colorectal cancer was analyzed. The SW620 and LOVO cells were divided into control group, circPRMT5-lenti group and circPRMT5-shRNA-lenti group according to different interventions. The effects of circPRMT5 expression level on viability, apoptosis, mitochondrial membrane potential and migration of SW620 and LOVO cells were detected. The influence of circPRMT5 expression level on E-cadherin, Slug, N-cadherin and vimentin was determined by Western blotting method. The potential target miRNA of circPRMT5 was predicted by Starbase V2.0. Student′s t test, analysis of variance and chi-square test were performed for statistical analysis. Results The results of RT-PCR showed that the expression of circPRMT5 in colorectal cancer tissues was higher than that of adjacent cancer tissues (2.167±0.345 vs. 1.103±0.144), and the difference was statistically significant (t=26.847, P<0.01). The circPRMT5 expression level was positively correlated with tumor size, TNM stage, lymph node metastasis and distant metastasis (χ2=6.010, 10.971, 5.321 and 6.272, all P<0.05). The upregulation of circPRMT5 expression could promote proliferation and migration of SW620 and LOVO cells. The circPRMT5 downregulation could inhibit cell proliferation, induce apoptosis and decrease mitochondrial membrane potential. The results of Western blotting indicated that, compared with those of control group, the expression of Slug, N-cadherin and vimentin increased in circPRMT5-lenti group (1.023±0.038 vs. 2.105±0.042, 1.051±0.309 vs. 2.277±0.111, 1.055±0.040 vs. 2.002±0.537, respectively), however the expression of E-cadherin decreased (2.074±0.214 vs. 0.627±0.023), and the differences were statistically significant (t=31.817, 22.065, 14.536 and 9.148, all P<0.01). Compared with the control group, the expression of Slug, N-cadherin and vimentin decreased in circPRMT5-shRNA-lenti group (1.023±0.038 vs. 0.585±0.023, 1.051±0.309 vs. 0.616±0.043, 1.055±0.040 vs. 0.537±0.022), while the expression of E-cadherin increased (2.074±0.214 vs. 2.756±0.148), and the differences were statistically significant (t=-13.795, -14.252, -11.794 and -13.116, all P<0.05). A total of 21 miRNAs might have potential binding sites with circPRMT5 predicted by Starbase V2.0 software. The expression of miRNA4735-3p, miRNA202-3p, miRNA326, let-7i-5p and miRNA4500 was negatively correlated with circPRMT5 expression in both SW620 and LOVO cells confirmed by RT-PCR. Conclusion CircPRMT5 is an important oncogenic gene in the genesis and progress of colorectal cancer, and may have certain potential application prospect in the research and development for colorectal cancer. Key words: Colorectal neoplasms; circPRMT5; Epithelial-mesenchymal transition

Effect of miR-26b on the invasion and metastasis of colorectal cancer

To investigate the role of miR-26b in the invasion and metastasis of colorectal cancer. Data of public chip databases were extracted to analyze the relationship between miR-26b expression and lymph node metastasis. Two types of colorectal cancer cell lines, Caco2 and DLD1, were selected, and the miR-26b-high colorectal cancer cell line was constructed using the method of lentivirus infection. The effects of up-regulating miR-26b expression on the invasion and metastasis of colorectal cancer cells were analyzed by Transwell migration and invasion experiment and wound healing assay. The effect of up-regulating miR-26b expression on stem cell phenotype of colorectal cancer cells was analyzed by sphere-formation assay. The microarray detection results showed that the expression of miR-26b in tumor tissues of patients with lymph node metastasis was significantly higher than those without lymph node metastasis[(12.04±0.20) vs. (11.31±0.19), t=2.646, P = 0.010]. In the in vitro experiment section, the Transwell experiment results showed that the number of invasive cells [(16.40±1.36) vs. (3.80±0.86), t=7.814, P=0.000] and migrating cells [(33.40±2.93) vs. (8.80±2.40), t=6.505, P=0.000] in miR-26b-high colorectal cancer cells was significantly higher as compared to miR-26b-low cells(all P<0.05). Would healing assay also confirmed that the migration speed of miR-26b-high colorectal cancer cells was significantly accelerated. Both the rate and the density of sphere formation were higher in miR-26b-high colorectal cancer cells than those in miR-26b-low colorectal cancer cells [Caco2:(168.3±11.7) vs. (54.2±10.8), t=7.185,P=0.002; DLD1:(4 076.0±409.8) vs.(1 613.0±210.1), t=5.349, P=0.006]. miR-26b may promote the invasion and metastasis of colorectal cancer by accelerating the migration and invasion of colorectal cancer cells and enhancing the stem cell phenotype of tumor cells.

Effects of secreted frizzled-related proteins 2 and β-catenin on proliferation, migration and invasion of colorectal cancer cells

Objective To observe the effects of secreted frizzled-related proteins 2 (sFRP2) and β-catenin on proliferation, migration and invasion of colorectal cancer cells. Methods The pcDNA3.1 plasmid containing sFRP2 gene sequence was constructed to overexpress sFRP2 in HCT116 by transfection. The expression level of sFRP2 in HCT116 cells after transfection was detected by Western blotting. Cell counting kit-8 (CCK-8) assay was used to detect the effect of sFRP2 upregulation on the proliferation of colorectal cancer cells; Wound-healing assay was used to detect the effect of sFRP2 up-regulation on the migration of colorectal cancer cells; Transwell assay was used to detect the effect of sFRP2 up-regulation on the invasion ability of colorectal cancer cells. Results After plasmid transfection, the expression of sFRP2 and β-catenin significantly increased. The ratio of sFRP2 protein to β-actin was 0.62±0.03 vs. 0.21±0.02 (t=25.430, P=0.001), and the ratio of β-catenin to β-actin was 0.32±0.02 vs. 0.17±0.01 (t=15.000, P=0.001), and the difference was statistically significant. The absorbance values at 12, 24, 36, 48 and 60 h in sFRP2 transfection group, control group and empty plasmid group were measured to be 0.213±0.015 vs. 0.215±0.018 vs. 0.218±0.012, 0.345±0.053 vs. 0.351±0.041 vs. 0.362±0.038, 0.382±0.058 vs. 0.413±0.051 vs. 0.433±0.048, 0.441±0.079 vs. 0.503±0.033 vs. 0.534±0.053, and 0.482±0.034 vs. 0.594±0.045 vs. 0.628±0.062. As compared with the control group and the empty plasmid group, the cell proliferation rate in sFRP2 transfection group was significantly decreased (12 h: t=0.016, P=0.988 and t=0.041, P=0.970; 24 h: t=0.155, P=0.884 and t=0.452, P=0.675; 36 h: t=0.695, P=0.525 and t=1.173, P=0.306; 48 h: t=1.254, P=0.278 and t=1.693, P=0.166; 60 h: t=3.440, P=0.026 and t=3.576, P=0.023). The difference in the distances between the both sides of scratches at 0 h and 48 h in the blank group was used as control. The distance in the blank group, empty plasmid group, and sFRP2 transfection group was 1.00±0.00, 0.91±0.06, and 0.61±0.04, respectively. The distance in sFRP2 transfection group was farther than blank group and empty plasmid group, and the migration speed was significantly slower (t=16.890, P=0.001 and t=7.206, P=0.002); The number of transmembrane cells in the sFRP2 transfection group was (75.69±7.19) cells, significantly less than that in the control group [(195.39±8.68) cells, t=25.459, P=0.001], indicating the invasive ability of the cells was significantly decreased in the sFRP2 transfection group. Conclusion The up regulation of sFRP2 significantly inhibited the proliferation, migration and invasion of colorectal cancer cell line HCT116. Key words: Colorectal cancer; Secreted Frizzled-related proteins 2; β-catenin; Proliferation; Migration; Invasion

Effects of PinX1 gene on migration and invasion of colon cancer cells and the molecular mechanisms

Objective To observe the function of telomerase inhibitor 1 (PinX1) gene in cell migration and invasion and its possible molecular mechanisms in the colon cancer cell lines. Methods The PinX1 interfering lentivirus (sh-PinX1) and its negative control (sh-con) were transfected into HCT116 and SW480 colon cancer cells. The protein expression levels of PinX1, matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blotting. The alterations in the ability of the migration and invasion of colorectal cancer cells (HCT116 and SW480) were investigated by Transwell migration and invasion assays. The MMPs activities in the two colon cancer cell lines were examined by gelatin zymography. Results Western blotting showed that PinX1 protein level was decreased by interference of PinX1 gene in colon cancer cell lines: for HCT116 [(40 459±154) vs. (3 135±24), P=0.001]; for SW480 (54 709±202) vs. (6 537±55), (P=0.000). Migration assay showed that the decreased PinX1 increased the ability of migration in colon cancer cells [116-sh-con group (100.0±5.8) vs. 116-sh-PinX1 group (217.3±11.6), P=0.000; 480-sh-con group (193.0±6.6) vs. 480-sh-PinX1 group (407.0±6.6), P=0.004]. Invasion assay showed that decreased PinX1 increased the invasive ability in colon cells [116-sh-con group (71.7±3.3) vs. 116-sh-PinX1 group (170.0±5.8), P=0.000; 480-sh-con group (58.3±4.4) vs. 480-sh-PinX1 (130.0±5.8), P=0.001]. Western blotting showed that MMP-2 protein level was increased by interference of PinX1 gene in colon cancer cells [116-sh-PinX1 group (58 549±220) vs. 116-sh-con group (3 819±31), P=0.000); 480-sh-PinX1 group (56 540±207) vs. 480-sh-con group (2 909±33), P=0.001]. Western blotting showed that PinX1 did not affect the MMP-9 expression [116-sh-PinX1 group (8 499±769) vs. 116-sh-con group (8 499±769, P=0.390); 480-sh-PinX1 group (4 008±290) vs. 480-sh-con group (3 907±256), P=0.808]. Gelatin zymography assay showed that low expression PinX1 suppressed the activity of MMP-9 [116-sh-PinX1 group (12 699±284) vs. 116-sh-con group (5 571±91, P=0.000); 480-sh-PinX1 group (14 418±296) vs. 480-sh-con group (8 704±55), P=0.000]. Conclusion PinX1 could increase the ability of migration and invasion by activating MMP-2 signaling pathway in colon cancer cells. Key words: PinX1; Colon cancer; Migration; Invasion; Matrix metalloproteinase-2

SU6668 inhibits the proliferation and motility of colorectal cancer cells by inducing cycle arrest, and promotes their apoptosis

Background: The multi-targeted angiogenesis inhibitor SU6668 has demonstrated promising therapeutic effects on the progression of hematologic malignancies and certain solid tumors. Previous studies reported that SU6668 inhibited the liver metastasis of both human and mouse colon cancer xenografts via its antiangiogenic effect. This study was conducted to determine the effect of SU6668 on the proliferation, migration, and invasion of colorectal cancer cells and their rates of apoptosis. Methods: SW480 and SW620 cells were treated with various concentrations of SU6668 (15, 30, and 45 μM, respectively), and their proliferation was examined by CCK-8 and EdU assays. Changes in the clone formation ability of colorectal cancer cells treated with SU6668 were assessed with colony formation assays. The effects of SU6668 on cell cycle progression and apoptosis were detected by flow cytometry. Hoechst staining was used to determine the rates of apoptotic cell death. Western blot was used to analyze the relative levels of Bax, caspase-3 and Bcl-2 protein expression. Matrigel invasion assays were used to assess the effects of SU6668 on colorectal cancer cell migration and invasion capabilities. Results: SU6668 inhibited the proliferation and clone formation ability of colorectal cancer cells in a dose- and time-dependent manner. SU6668 also induced G0/G1 cell cycle arrest and cell apoptosis. The expression levels of apoptosis-related proteins (Bax and caspase-3) were enhanced, while the level of Bcl-2 expression was significantly decreased in SU6668-treated colorectal cancer cells. SU6668 significantly inhibited the migration and invasion of colorectal cancer cells. Conclusions: SU6668 can inhibit the proliferation, migration, and invasion of colorectal cancer cells, as well as their ability for colony formation. SU6668 inhibited cell proliferation by inducing G1 cell cycle arrest and induced apoptosis by activating Bax and caspase-3.

Study on the inhibitory effect of miR-199b on colorectal cancer metastasis

Objective To investigate the expression of miR-199b in colorectal cancer and the effect of miR-199b on metastasis of colorectal cancer. Methods The expression of miR-199b in colorectal cancer was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The miR-199b-overexpressed human colon tumor-116 (HCT-116) stable cell lines was constructed by lentivirus assay. The effects of miR-199b on the migration and invasion of colorectal cancer were confirmed by Transwell assay. Bioinformatics, luciferase reporter assay, and enzyme linked immunosorbent assay (ELISA) were performed to verify the potential target genes of miR-199b. Results miR-199b was at lower level in cancerous tissues comparing to the corresponding noncancerous tissues. Up-regulation of miR-199b significantly blocked the migration and invasion of colorectal cancer cells. Co-transfection of miR-199b and VEGFA wild type (WT) 3'untranslated region (3'UTR) could inhibit the luciferase activity, while there was no significant difference when co-transfection with mutant (MT) 3'UTR. The protein level of vascular endothelial growth factor A (VEGFA) after overexpression of miR-199b was repressed, which was validated by ELISA. Conclusions miR-199b was a new colorectal cancer suppressor miRNA, which inhibited tumor migration and invasion by negatively regulating VEGF. Key words: MicroRNAs/BI/GE; Colorectal neoplasms/PA/ME/GE; Neoplasm invasiveness; Neoplasm metastasis

MicroRNA-140 suppresses the migration and invasion of colorectal cancer cells through targeting Smad3

To investigate the effect of microRNA-140 (miR-140) on the migration and invasion of colorectal cancer (CRC) cells and the possible mechanism. miR-140 mimics, miR-140 specific inhibitor or small interfering RNA (siRNA) against Smad3 were transfected into human CRC cell line RKO cells respectively, using Oligofectamine or Lipofectamine2000. Quantitative real-time PCR (real-time PCR) was used to measure the expression levels of miR-140 and Smad3 mRNA. Smad3 protein was analyzed by Western blot. The in vitro cell migrating and invasive abilities were determined by wound-healing and Transwell chamber assay after up-regulating or down-regulating miR-140 or knocking down Smad3. The Western blot assays showed that the Smad3 protein level was significantly reduced after up-regulating miR-140 (0.04 ± 0.01), compared with that of (0.47 ± 0.02, P < 0.05) in the control group and that of (0.52 ± 0.06) in the negative control group (P < 0.05 for both). The results of real-time PCR indicated that no significant difference was found in the levels of Smad3 mRNA between miR-140 transfection and NC groups (1.11 ± 0.13 vs. 1.00 ± 0.06, P > 0.05). The wound-healing assay showed that the migrating ability was dramatically attenuated by miR-140 compared with that in the control and NC groups, whereas no significance was found when compared with that of the Smad3 siRNA transfected cells. The number of cells migrating through Transwell chamber without matrigel in the miR-140 group was (76.2 ± 4.4), remarkably lowered than that in the control (267.1 ± 4.9) and NC (336.1 ± 5.7) groups (P < 0.05 for both), but no significant difference between the miR-140 (76.2 ± 4.4) and Smad3 siRNA (83.5 ± 7.3) groups. Transwell chamber with matrigel assay showed that number of cells penetrating through the membrane was (109.5 ± 7.4) in the miR-140 group, significantly lower than that in the control (403.1 ± 5.1) and NC (392.6 ± 8.4) groups (P < 0.05 for both), while Smad3 siRNA transfection had a similar effect (138.8 ± 3.6)(P > 0.05). Down-regulation of miR-140 increased the level of smad3 protein expression, and partially reversed the inhibition of the cell migration and invasion mediated by miR-140. Co-transfection of miR-140 inhibitor and Smad3 siRNA had no significant effect on the Smad3 protein expression and the abilities of cell migration and invasion. miR-140 regulates the Smad3 expression at the post-transcriptional level. miR-140 suppresses the migrating and invasive abilities of CRC cells, possibly through down-regulation of Smad3. The findings of this study suggest that miR-140 may have a unique potential as a possible biomarker candidate for diagnosis and therapy of tumor metastasis.

APRIL mediates migration and invasion of colorectal cancer cells via regulation of matrix metalloproteinases

Objective To investigate the effects of a proliferation-inducing ligand(APRIL) on migration and invasion of colorectal cancer (CRC) and matrix metalloproteinases (MMPs) expression in order to observe the role of APRIL in CRC metastasis.Methods The siRNA plasmid vector targeting APRIL gene (siRNA-APRIL) was transfected into SW480 cells and recombinant human APRIL(rhAPRIL) was used to stimulate HCT-116 cells.Tumor cell migration and invasion were measured by Transwell chambers.RT-PCR and ELISA were applied to examine the expression level of MMPs.Results Metastatic and invasive capacities of siRNA-APRIL transfected SW480 were significantly inhibited,and these capacities of APRIL stimulated HCT-116 cells were significantly enhanced compared with their respective controls( all P＜0.05 ),accompanied with the alterations of MMPs mRNA and secreted protein expression( P＜0.05).The number of invading cells of SW480 control and rhAPRIL stimulated HCT-116 was significantly decreased by a MMP inhibitor GM6001 ( P＜0.05 ).Conclusion APRIL facilitates migration and invasion of CRC via regulation of MMPs,which suggests that APRIL might be used as a new target for the intervention and treatment of CRC metastasis. Key words: A proliferation-inducing ligand (APRIL) ; Colorectal cancer (CRC) ; Matrix Metalloproteinase (MMP); Invasion; Metastasis

Effect of von Willebrand factor on the biological characteristics of colorectal cancer cells

To study effect of von Willebrand factor (vWF) on the proliferation, adhesion and migration of human colorectal cancer cells. Human colorectal cancer cell line SW480 was cultured in vitro, and the expression of vWF in SW480 cells was detected by immunocytochemistry. SW480 cells were treated with vWF antibody (vWFAb), and the morphological change was examined by inverted microscope. Cell proliferation and ability to adhere extracellular matrix IIII( collagen were detected with MTT. Migration ability of SW480 cells was assayed by Transwell. The human colorectal cancer cell line SW480 expressed vWF which was mainly in nucleus and slightly in cytoplasm. vWFAb significantly inhibited the proliferation ability of SW480 cells in dose- and time-dependent manner (P<0.05). After vWFAb treatment, SW480 cells adhesion decreased significantly (P<0.05), and transmembrane migration of cells significantly decreased (54.60+/-11.01 vs 97.27+/-10.01, P<0.01). Human colorectal cancer cells can express vWF. vWF in human colorectal cancer cells plays an important role in promoting proliferation, adhesion, and migration.

The expression of chemokine receptor CXCR4 in the colorectal carcinoma cell lines and its chemotaxis assay

Objective To investigate the expression of chemokine receptor CXCR4 in colorectal carcinoma cell lines and its chemotaxis assay for an exploration of metastasis mechanism in colorectal carcinoma. Methods Colorectal carcinomas cell line HT29 and SW480 cells were cultured. The expression of CXCR4 receptor in the cell lines was detected by using immunohistochemistry, RT-PCR and Western blot. In vitro,chemotaxis chamber was used to test the chemotactic effect of SDF-lα on HT29 and SW480 cells. Results 48.7% HT-29 and 52.6% SW480 expressed CXCR4 receptor in every 100 cells by immunohistochemistry. Chemotaxis assay demonstrated that the migrating carcinomas cells induced by SDF-1 (HT-29:27. 6 ±3. 9; SW480:25.3 ±4.4) were more than those in blank control group (HT-29:9.6 ±2.5;SW480:9.8 ±2.1) and antibody group (HT-29:9.1 ± 3.1; SW480:9.3 ± 2.0). Conclusion HT29 and SW480 cell line could express chemokine receptor CXCR4. SDF-1 could chemoattract the migration of the colorectal carcinoma cells. Key words: Colorectal carcinomas; Chemokine receptor; Metastasis