Inhibitory effects of nm23-H1 gene on proliferation and invasion of A549 cell line

AIM:To study the inhibitory effects of nm23-H1 gene on proliferation and invasion of human lung adenocarcinoma A549 cell line. METHODS:Recombinant eukaryotic expression vector pcDNA3.1-nm23-H1 containing full length of human nm23-H1 cDNA was constructed and transfected into a human lung adenocarcinoma A549 cell line by lipofectamine. Cell strain that expressed nm23-H1 stably was screened out by G418 and named pcDNA-nm23-A549. Expression of nm23-H1 was identified by RT-PCR and immunohistochemistry. Growth curves were drawn to detect the inhibitory effects on cell proliferation. Cell cycle of pcDNA-nm23-A549 was examined by flow cytometry. Atomic force microscopy was used to observe the filopodia on the surface of the cells. RESULTS:Introduction of nm23-H1 obviously inhibited the proliferation of A549. Expression of nm23-H1 did not induce apotosis in A549 cells but increased the percentage of phase G₁ cells and decreased phase S cells. Meanwhile, phase G₁ to phase S transition was restrained. Filopodia in the cell surface was much fewer and its structure changed in cells transformed. CONCLUSION:nm23-H1 is capable of inhibiting A549 proliferation and decreasing its metastatic ability, probably by interfering with cell cycle and cell surface structure.     

αvβ6-ERK pathway participates in norcantharidin-induced apoptosis of HT-29 colon cancer cells

AIM: To investigate the pharmacological mechanism of norcantharidin (NCTD)-induced apoptosis of HT-29 colon cancer cells. METHODS: Hoechst 33258 staining was used to analyze the apoptosis of HT-29 cells treated with NCTD. The effects of NCTD on the expression of integrin in HT-29 cells were determined by flow cytometry. The effects of several functional blocking antibodies on HT-29 cells were detected by MTT method. The expression and the phosphorylation of mitogen activated protein kinases (MAPKs) in HT-29 cells were measured by Western blotting. Co-immunoprecipitation assay was used to detect the activity of αvβ6-extracellular signal-regulated kinase (ERK) direct linkage in HT-29 cells.RESULTS: NCTD induced the apoptosis of HT-29 colon cancer cells. The expression of integrin αvβ6 in HT-29 cells treated with NCTD was reduced, but the expression of αvβ3 and αvβ5 was not changed. A function-blocking antibody to αvβ6,10D5,strengthened the growth inhibitory effect of NCTD on HT-29 cells ,but LM609 (a function-blocking antibody to αvβ3) and P1F6 (a function-blocking antibody to αvβ5) did not. The level of phosphorylated ERK (p-ERK) was decreased substantially after treated with NCTD in a dose-and time-dependent manner. NCTD also affected the association of αvβ6 and ERK. CONCLUSION: NCTD decreases the expression of integrin αvβ6 and interferes with the phosphorylation of ERK. As a result, the formation of αvβ6-ERK direct linkage is affected and the signal transduction mediated by αvβ6 is disturbed. The mechanism of NCTD-induced HT-29 cell apoptosis is involved in the αvβ6-ERK signaling pathway.     

Effects of antioxidant and NF-κB on the induction of IL-8 in human colon cancer cell line HT-29 cells in vitro

AIM: To investigate the role of nuclear factor κB (NF-κB) in the induction of IL-8 gene by TNF-α in colon cancer cells and the effect of antioxidant on the induction of IL-8. METHODS: ELISA was used to detect the concentrations of IL-8. IL-8 mRNA was analyzed by using RT-PCR. NF-κB in the cell nuclei was detected with electrophoretic mobility shift assay. RESULTS: (1) IL-8 production and IL-8 mRNA expression induced by TNF-α was blocked by pyrrolidine dithiocarbamate (PDTC). (2) TNF-α triggered the activation and translocation of NF-κB and PDTC inhibited the activation of NF-κB induced by TNF-α. CONCLUSION: The induction of IL-8 gene and protein by TNF-α is dependent on the activation of NF-κB. Antioxidants may inhibit the induction of IL-8 gene and protein through inhibiting NF-κB activation.     

Effect of EGCG on invasion of breast cancer cells and its possible mechanism

AIM: To study the effects of (-)-epigallocatechin-3-gallate (EGCG), a tea extract, on the invasion and metastasis of breast cancer cell line MDA-MB-231 and the possible mechanisms in vitro. METHODS: The expression of MUC1 in breast cancer cells treated with or without EGCG was detected by immunohistochemistry. The effect of EGCG on invasion of MDA-MB-231 cells was evaluated using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane (Matrigel). Gelatin zymography was performed to detect the secretion of collagenase-Ⅳ. RESULTS: EGCG reduced the expression of MUC1, significantly suppressed the invasion of tumor cells to basement membrane and reduced the secretion of collagenase-Ⅳ. CONCLUSION: In vitro, EGCG may suppress invasion, metastasis, and collagenase-Ⅳ secretion in MDA-MB-231 cells by inhibiting the production of MUC1.     

Effect of curcumin on expression of p21 and CD44V6 in human breast cancer xenografts

AIM:To study the effect of curcumin on the expression of p21 and CD44V₆ in breast carcinoma in nude mice.METHODS:Nude mice were xenografted with human breast cancer cell line MCF-7 and randomly divided into 2 groups (n=4 in each group): control group and curcumin group. In latent period,the percentage of tumor development was observed. Tumors were measured and the surface areas were calculated. RT-PCR was performed to detect the expression level of cyclin D1,p21 and CD44V₆ mRNA. RESULTS:The tumor surface areas in the curcumin group were significantly lower than those in control group. In curcumin treatment group,the expression of p21 was up-regulated while cyclin D1 was nearly not changed. The expression of CD44V₆ was significantly down-regulated in curcumin group.CONCLUSION:Curcumin inhibits the expression of CD44V₆ and up-regulates the expression of p21 in nude mice bearing human breast cancer cell line MCF-7.     

Study on genetic instability of nm23-H1 gene and expression of hMLH1,hMSH2 in sporadic gallbladder carcinoma

AIM: To examine the MSI and LOH of locus D17S396 and their influence on the expression of nm23-H1 in gallbladder tumors,and to examine the protein expression of hMLH1/hMSH2,which may provide experimental evidence for the tumor occurrence and metastasis.METHODS: Techniques such as DNA extraction,CR-SSCP,ordinary silver stain were used to study MSI and LOH of locus D17S396.Envision IHC was used to assess the expression of nm23-H1 and hMLH1/hMSH2.RESULTS: ① The frequency of heredity instability of gallbladder carcinoma was 42.55%.The frequency of LOH in liver and lymph node metastasis cases and in stage Nevin IV and V was significantly higher than that without metastasis and stage I,II and III.However,the frequency of MSI showed contrary correlation with some clinicopathologic characteristics.② The expression of nm23-H1 was 46.81%.The case with lymph node metastasis and Nevin stage IV and V showed significantly lower expression than that without lymph node metastasis and stage I,II and III.③ The expressions of hMLH1 and hMSH2 were 51.06% and 42.55% respectively.hMLH1 in lymph node and liver metastasis cases and in stage Nevin IV and V were significantly lower than that without metastasis and in stage I,II and III.④ Positive frequency of hMLH1 in MSI positive group was higher than that in MSI negative group.The positive frequency of nm23-H1 and hMSH2 protein in LOH positive group was lower than that in negative group.CONCLUSION: The heredity instability of nm23-H1 gene may be implicated pathogenesis and progression of gallbladder carcinoma.Both MSI and LOH of nm23-H1 control the development of gallbladder carcinoma independently in different paths.Abnormal expression of hMLH1/hMSH2 may be a molecule marker in early stage of gallbladder carcinoma.     

High mobility group box 1 protein promotes proliferation,migration and invasion of colon cancer cells

AIM: To investigate the expression of high mobility group box 1 protein (HMGB1) in colon can-cer cells, and to determine its regulatory roles in colon cancer cell proliferation, migration and invasion. METHODS: qPCR and Western blot were used to quantify the mRNA and protein expression levels of HMGB1 in human colon cancer SW620 cells and normal colonic epithelial FHC cells. HMGB1 shRNA was transfected into the SW620 cells to establish the stable HMGB1-downregulating colon cancer cells (shHMGB1 group), and negative control (shNC) group and blank control (blank) group were also set up. The proliferation, migration and invasion of the cells were determined by CCK-8 assay, colony formation experiment and Transwell chamber assays. Western blot was used to determine the protein levels of p-ERK, ERK, c-Myc, matrix metalloproteinase (MMP)-2/9, E-cadherin, N-cadherin, Bcl-2 and Bax. RESULTS: Both of the mRNA and protein levels of HMGB1 in colon cancer cells were higher than those in the normal colonic epithelial cells (P<0.05). HMGB1 gene was successfully knocked down in SW620 cells. Compared with blank group and shNC group, the proliferation, migration and invasion abilities of the cells in shHMGB1 group were significantly inhibited (P<0.05). The protein levels of MMP-2, MMP-9, N-cadherin, c-Myc, Bcl-2 and p-ERK were reduced notably, while the expression of Bax protein was increased (P<0.05) in shHMGB1 group compared with shNC group and blank group.CONCLUSION: HMGB1 effectively promotes the proliferation, migration and invasion of colon cancer cells through ERK/c-Myc signaling pathway.     

Effect of HMGB1 expression knockdown on invasion ability of breast cancer cells induced by TNF-α

AIM:To investigate the effect of high-mobility group box-1 (HMGB1) expression knockdown on the invasion ability of breast cancer cells induced by tumor necrosis factor-α (TNF-α). METHODS:HMGB1 siRNA was used to transfect into the breast cancer MDA-MB-231 cells. The expression of HMGB1 at mRNA and protein levels was determined by RT-qPCR and Western blot. After the MDA-MB-231 cells with HMGB1 expression knockdown were treated with TNF-α, the apoptosis rate was analyzed by flow cytometry, the cell invasion ability was measured by Transwell assay, and the cell migration ability was detected by cell scratch test. The protein expression of E-cadherin, MMP-2, N-cadherin, MMP-9 and Bax was determined by Western blot. RESULTS:The expression of HMGB1 at mRNA and protein levels in the MDA-MB-231 cells transfected with HMGB1 siRNA was significantly lower than that in the non-transfected cells (P<0.05). The apoptosis rate in the cells was increased after TNF-α treatment, and the cell invasion and migration abilities were also increased. The protein level of E-cadherin in the cells was decreased, the protein level of N-cadherin was increased, and the protein levels of MMP-2, MMP-9 and Bax were also increased (P<0.05). After the MDA-MB-231 cells with HMGB1 expression knockdown were induced by TNF-α, the apoptotic rate was increased, the invasion and migration abilities were decreased, the protein levels of E-cadherin and Bax were increased, and the protein levels of N-cadherin, MMP-2 and MMP-9 were decreased, as compared with the cells only induced by TNF-α without knockdown of HMGB1 expression (P<0.05). CONCLUSION:Knockdown of HMGB1 expression enhances the apoptosis of breast cancer cells induced by TNF-α, and inhibited the cell invasion, migration and epithelial-mesenchymal transition induced by TNF-α. The mechanism may be related with the changes of protein expression of MMP-2, MMP-9 and Bax.     

Effect of Wip1 gene silencing on chemotherapy sensitivity of human colon cancer cells

AIM: To observe the inhibitory effect of siRNA targeting to Wip1 gene on the Wip1 gene expression in the colon cancer cells and to investigate the influence of Wip1 gene silencing on the chemotherapy sensitivity of colon cancer cells. METHODS: Wip1-811 siRNA targeting to Wip1 gene was transfected into RKO colon cancer cells with high expression of Wip1 gene. The mRNA expression of Wip1 was measured by real-time PCR. The protein level of Wip1 was detected by Western blotting. The viability of RKO colon cancer cells was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Wip1-811 siRNA efficiently inhibited the expression of Wip1 at mRNA and protein levels. The enhanced chemotherapy sensitivity of RKO colon cancer cells was observed after inhibition of Wip1 gene expression. The viability of RKO colon cancer cells was decreased from (89.4±6.6)% to (74.7±3.9)% after treated with 5-fluorouracil (P<0.05) and decreased from (77.9±2.4)% to (66.7±2.9)% after treated with oxaliplatin (P<0.05). The cell apoptotic rate was increased from (7.7±0.5)% to (12.3±3.2)% and from (14.7±2.1)% to (34.0±2.1)% when RKO colon cancer cells were treated with 5-fluorouracil and oxaliplatin, respectively (P<0.05). CONCLUSION: Wip1 gene silencing enhances chemotherapy sensitivity of colon cancer cells.     

Effects of NOB1 gene silencing on drug sensitivity and invasion and migration abilities of human colon cancer SW480 cells

AIM:To investigate the effect of NOB1 gene expression knock-down by transfection of small interfering RNA (siRNA) on the viability, drug sensitivity, apoptosis, cell cycle distribution, and invasion and migration abilities of human colon cancer SW480 cells. METHODS:NOB1 siRNA was transfected into SW480 cells using Lipofectamine 3000. The mRNA and protein levels of NOB1 in the SW480 cells were determined by real-time PCR and Western blot. The cell viability and sensitivity to different chemotherapeutic drugs (cisplatin, 5-fluorouracil, oxaliplatin and capecitabine) were detected by MTT assay after knock-down of NOB1 gene expression in the SW480 cells. The apoptosis and cell cycle distribution of SW480 cells were analyzed by flow cytometry. The invasion and migration abilities of SW480 cells were detected by Transwell assay. RESULTS:After transfection with NOB1 siRNA, the mRNA and protein levels of NOB1 in the SW480 cells were significantly decreased (P<0.05). Compared with control group and control siRNA group, the viability of SW480 cells in NOB1 siRNA group was significantly decreased at 24~72 h. The half maximal inhibitory concentrations of the chemotherapy drugs cisplatin, 5-fluorouracil, oxaliplatin and capecitabine were significantly decreased. The apoptotic rate was significantly increased and the cell cycle were blocked. The cell invasion and migration abilities were significantly reduced (P<0.05). CONCLUSION:Knock-down of NOB1 gene expression inhibits the viability and invasion and migration abilities of colon cancer SW480 cells, and promotes drug sensitivity and apoptosis. NOB1 may be a new target for diagnosis and treatment of colon cancer.     

Effects of G6PD silencing by siRNA on expression of HIF-1α in human colon cancer cells

AIM: To study the effects of glucose-6-phosphate dehydrogenase (G6PD) silencing by small interference RNA(siRNA) on the levels of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and hypoxia-inducible factor 1α(HIF-1α) under hypoxia in human colon cancer cell line LoVo.METHODS: Specific siRNA expression vector targeting G6PD gene was constructed. The recombinant plasmid was identified by restriction endonuclease and DNA sequencing, and then transfected into LoVo cells. The effects of G6PD silencing were evaluated by detecting the activity and mRNA expression of G6PD. LoVo cells were cultured in vitro under hypoxic condition. NADPH levels were determined.The mRNA and protein levels of HIF-1α were analyzed by RT-PCR and Western blotting,respectively. RESULTS: The recombinant plasmid for G6PD silencing by siRNA was successfully constructed and transfected into LoVo cells. Compared with untransfected cells,the mRNA expression of G6PD in transfected cells was decreased by 43% and G6PD activity was decreased by 63.5%. Under hypoxic condition, the level of NADPH in transfected cells was significantly decreased (41% vs 100%, P<0.05).HIF-1α protein was also decreased significantly but its mRNA expression had no change as compared with the control cells. CONCLUSION: G6PD silencing by siRNA decreases NADPH level, resulting in the decline of HIF-1α stability in cancer cells under hypoxic condition. By this mechanism, G6PD silencing can influence the hypoxic responses in cancer.     

Effect of toosendanin on invasion and migration of human ovarian cancer cells

AIM: To investigate the effect of toosendanin (TSN) on invasion and migration abilities of human ovarian cancer cells and the related mechanism. METHODS: The human ovarian cancer cell lines CAVO-3 and SKVO-3 were treated with TSN at different concentrations. The cell viabilty at 12, 24, 48, 72 and 96 h after TSN treatment was measured by CCK-8 assay. Scratch wound healing assay and Transwell assay were employed to measure the invasion and migration abilities of CAVO-3 cells. The protein expression of nuclear factor-κB (NF-κB) p65, E-cadherin, N-cadherin, vimentin and Snail was determined by Western blot. RESULTS: TSN significantly inhibited the viability of CAVO-3 and SKVO-3 cells (P<0.05). Compared with control group, the migration and invasion abilities of CAVO-3 cells in TSN group decreased significantly (P<0.05). In addition, the expression of NF-κB p65 and E-cadherin protein increased notably, followed with N-cadherin, vimentin and Snail protein decreased significantly (P<0.05). However, the inhibitor of NF-κB BAY11-7082 reversed the impact above. Compared with TSN group, the migration and invasion abilities in TSN+BAY11-7082 group increased significantly (P<0.05). The protein expression of E-cadherin also decreased notably, followed with the protein expression of N-cadherin, vimentin and Snail increased significantly (P<0.05). CONCLUSION: TSN inhibits the invasion and migration abilities of human ovarian cancer cells, which is related to the inhibition of epithelial-mesenchymal transition process mediated by NF-κB/Snail signaling pathway.     

Effect of inhibiting lncRNA LINC01503 on viability,migration and invasion of lung cancer cells by targeted regulation of miR-335-5p

AIM To investigate the effects of long non-coding RNA (lncRNA) LINC01503 on the viability, migration and invasion of lung cancer cells and its mechanism. METHODS Human lung carcinoma H1299 cells were divided into si-NC group (transfected with si-NC), si-LINC01503 group (transfected with si-LINC01503), pcDNA group (transfected with pcDNA), pcDNA-LINC01503 group (transfected with pcDNA-LINC01503), miR-NC group (transfected with miR-NC), miR-335-5p group (transfected with miR-335-5p mimics), si-LINC01503+anti-miR-NC group (co-transfected with si-LINC01503 and anti-miR-NC), si-LINC01503+anti-miR-335-5p group (co-transfected with si-LINC01503 and anti-miR-335-5p), miR-NC+WT-LINC01503 group (co-transfected with miR-NC and WT-LINC01503), miR-NC+MUT-LINC01503 group (co-transfected with miR-NC and MUT-LINC01503), miR-335-5p+WT-LINC01503 group (co-transfected with miR-335-5p and WT-LINC01503) and miR-335-5p+MUT-LINC01503 group (co-transfected with miR-335-5p and MUT-LINC01503). The expression of miR-335-5p and LINC01503 was detected by RT-qPCR. Western blot was used to detect protein expression. MTT assay was used to detect cell viability. Transwell assay was used to detect the migration and invasion abilities. Dual-luciferase reporter assay was used to confirm the targeted relationship between LINC01503 and miR-335-5p. RESULTS Compared with normal tissues, the expression of LINC01503 was significantly increased in the lung cancer tissues, and the expression of miR-335-5p was significantly decreased (P<0.05). Compared with stage I/II , the expression level of LINC01503 in the lung cancer tissues of stage III/IV was significantly increased, and the expression of miR-335-5p was significantly decreased (P<0.05). The patients with high expression of LINC01503 had lower short-term survival rates than those with low expression of LINC01503 (P<0.05). Compared with normal human bronchial epithelial cell line BEAS-2B, the expression of miR-335-5p in lung cancer cell lines H1299, A549 and SPC-A-1 were significantly decreased, and the expression of LINC01503 was significantly increased (P<0.05). Over-expression of miR-335-5p and inhibition of LINC01503 expression inhibited the viability, migration and invasion of H1299 cells, and inhibited the protein expression of cyclin D1, matrix metalloproteinase-2 （MMP-2） and MMP-9 (P<0.05). LINC01503 targeted and regulated miR-335-5p expression, and interfering with miR-335-5p expression reversed the inhibitory effect of inhibiting LINC01503 expression on the viability, migration and invasion of H1299 cells. CONCLUSION Inhibition of lncRNA LINC01503 inhibits the viability, migration and invasion of lung cancer cells. The mechanism may be related to the targeted regulation of miR-335-5p.     

miR-105 inhibits cell proliferation,migration and invasion abilities in non-small-cell lung cancer cells by targeting and negative regulation of KIFC1

AIM:To study the effects of microRNA-105(miR-105) on the cell proliferation, migration and invasion abilities of non-small-cell lung cancer (NSCLC) H460 cells, and further to explore its mechanism. METHODS:The expression of miR-105 and kinesin family member C1 (KIFC1) mRNA in the NSCLC tissues and adjacent tissues and cells was detected by RT-qPCR. The protein expression of KIFC1 in the NSCLC tissues, adjacent normal tissues and cells was determined by Western blot. The H460 cells were divided into miR-105 group (transfection with miR-105 mimics), miR-negative control (NC) group (transfection with miR-NC), inhibitor-NC group (transfection with NC of inhibitor), inhibitor-miR-105 group (transfection with miR-105 inhibitor), si-NC group (transfection with NC siRNA), si-KIFC1 group (transfection with KIFC1 siRNA), miR-105+vector group (miR-105 mimics and pcDNA 3.1 co-transfection) and miR-105+KIFC1 group (miR-105 mimics and pcDNA 3.1-KIFC1 co-transfection). The cell proliferation was measured by MTT assay and colony formation assay. The migration and invasion abilities were detected by Transwell methods. The relative luciferase acitivity was evaluated by double luciferase reporter assay. RESULTS:Compared with the adjacent tissues, the expression of miR-105 was significantly decreased and the expression of KIFC1 was significantly increased in NSCLC tissues (P<0.05). Compared with human normal embryonic lung fibroblasts MRC-5, the expression of miR-105 in the H460 cells was significantly decreased, and the expression of KIFC1 was significantly increased (P<0.05). miR-105 inhibited the relative luciferase activity of H460 cells with wild-type KIFC1 and negatively regulated the protein expression of KIFC1. Over-expression of miR-105 and knockdown of KIFC1 expression significantly inhibited the proliferation, migration and invasion abilities of H460 cells. Over-expression of KIFC1 reversed the inhibitory effect of miR-105 on the cell proliferation, migration and invasion abilities of H460 cells. CONCLUSION:miR-105 inhibits the proliferation, migration and invasion abilities of NSCLC cells. The mechanism may be related to targeting and negatively regulating expression of KIFC1.     

Effect of DEC1 gene over-expression on proliferation and invasion abilities of human esophageal cancer ECA109 cells

AIM: To investigate the effect of DEC1 gene over-expression on the proliferation and invasion abilities of human esophageal cancer ECA109 cells.METHODS: ECA109 cells were transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group). The mRNA and protein levels of DEC1, cyclin D1 and MMP-9 were evaluated by real-time PCR and Western blot, respectively. The effects of DEC1 over-expression on the proliferation and invasion abilities of the ECA109 cells were evaluated by CCK-8 assay, colony formation assay and Transwell test respectively.RESULTS: The DEC1 expression level in ECA109 cells in DEC1 group was significantly higher than that in vector group (P<0.01), but the levels of MMP9 and cyclin D1 expression were opposite (P<0.01). However, both the proliferation and invasion abilities of ECA109 cells in DEC1 groups decreased significantly as compared with those in vector group (P<0.05).CONCLUSION: The over-expression of DEC1 significantly inhibits the proliferation and invasion of ECA109 cells, which may be involved in the expression of cyclin D1 and MMP9.     

Effect of oridonin on invasion and migration of human lung cancer NCI-H460 cells

AIM：To investigate the effect of oridonin on the invasion and migration of human lung cancer NCI-H460 cells. METHODS：NCI-H460 cells were divided into high-dose (HD), middle-dose (MD) and low-dose (LD) oridonin groups (cultured with 40, 20 and 10 μmol/L of oridonin, respectively, as experimental groups), and normal (N) group (treated without oridonin as control). The cell growth was observed. The cell proliferation was detected by MTT assay. Boyden chamber was used to determine the cell invasive capacity. The cell migration was also measured. The levels of MMP-2 and MMP-9 were assayed by Western blotting. RESULTS：The cell counts in the experimental groups were lower than that in N group. The cell proliferation was inhibited as the inhibitory rates were 48.94%, 36.17% and 19.15% for HD group, MD group and LD group, respectively. The numbers of the invasive cells were 26.67±5.16 for HD group, 36.17±5.08 for MD group, and 44.33±5.50 for LD group. The migration rates in the experimental groups were lower than that in N group. The expression of MMP-2 and MMP-9 decreased dependent on the oridonin dose as follows: HD group < MD group < LD group < N group. CONCLUSION：Oridonin inhibits the invasion and migration of NCI-H460 lung cancer cells, and reduces the expression of MMP-2 and MMP-9.     

Effect of TRIM44 silencing on proliferation of hepatocellular carcinoma cells and its molecular mechanism

ATM: To investigate the effect of tripartite motif-containing protein 44 (TRIM44) on the proliferation of hepatocellular carcinoma (HCC) cells and to study the molecular mechanism. METHODS: The expression of TRIM44 at mRNA and protein levels in normal liver tissues, HCC tissues, adjacent nontumor liver tissues, immortalized hepatocytes and hepatoma cell lines was determined by RT-qPCR and Western blot, respectively. The silencing of TRIM44 was conducted by transfection of vector expressing shRNA targeting TRIM44 (shTRIM44) in the HCC cells, and the protein level of TRIM44 was measured by Western blot. The viability of the HCC cells was analyzed by MTS assay. The DNA synthesis of HCC cells was detected by Click-iT EdU Imaging Kit. The ability of anchorage-independent growth was determined by the method of colony formation on the soft agar. The effects of TRIM44 on the total protein and phosphorylation of mammalian target of rapamycin (mTOR) levels were measured by Western blot. The HCC cells were transfected with shTRIM44 and treated with mTOR agonist MHY1485, and the cell viability was analyzed by MTS assay. RESULTS: The mRNA and protein levels of TRIM44 in the HCC tissues were significantly higher than those in the adjacent nontumor liver tissues and normal liver tissues. In addition, the mRNA and protein levels of TRIM44 in the hepatoma cell lines were significantly higher than those in the immortalized hepatocytes. TRIM44 silencing significantly inhibited the viability of HCC cells and reduced the abilities of DNA synthesis and anchorage-independent growth of the HCC cells. TRIM44 silencing decreased the phosphorylation level of mTOR protein. MHY1485 significantly antagonized the inhibitory effect of TRIM44 silence to the viability of HCC cells. CONCLUSION: TRIM44 silencing inhibits the proliferation of HCC cells possibly through down-regulating the activity of mTOR.     

Synergistic anti-proliferation effect of aspirin and 5-fluorouracil on colon cancer cells and its mechanism

AIM：To investigate the synergistic anti-proliferation effect of aspirin and 5-fluorouracil on the colon cancer cells and its mechanism. METHODS：Colon cancer cells were divided into 4 groups: control group, aspirin group, 5-fluorouracil group and aspirin+5-fluorouracil group. Synergistic anti-proliferation effect of aspirin and 5-fluorouracil on the colon cancer cells was observed by MTT assay. Apoptosis-inducing effect and mechanism were detected by Hoechst 33258 staining, caspase activity assay and flow cytometry analysis. The mRNA and protein levels of apoptosis-related proteins were evaluated by real-time PCR and Western blotting. RESULTS：5-Fluorouracil inhibited proliferation of HCT116 and SW620 colon cancer cells effectively, and low concentration of aspirin exerted synergistic inhibitory effect. 5-Fluorouracil induced apoptotic morphology and increased caspase activity and sub-G1 phase in HCT116 cells. The synergistic effect of aspirin obviously enhanced apoptotic ratio and caspase activity. Moreover, 5-fluorouracil inhibited the mRNA and protein expression of Bcl-2, which was amplified by low concentration of aspirin. CONCLUSION：Aspirin and 5-fluorouracil had a synergistic anti-proliferation effect on the colon cancer cells through apoptosis pathway.