Effects of agglutination on tissue factor,D-dimer and endothelial cells in rats

ATM: to explore the effects of agglutination on tissue factor (TF), D-dimer and endothelial cells in vivo. METHODS: Forty-two SPF SD rats (male and female, 180~200 g) were randomly divided into normal saline control group, and different concentrations (5, 10 and 20 g/L) of phytohemagglutinin (PHA) groups and inactivated PHA groups, with 6 rats in each group. The rats were injected with normal saline, PHA and inactivated PHA through the tail vein respectively. The plasma contents of TF and D-dimer were detected by ELISA. The pulmonary capillary endothelial cells of the rats were observed under electron microscope. RESULTS: The plasma levels of TF and D-dimer were significantly higher in all PHA groups than those in normal saline group and 3 corresponding inactivated PHA groups. Compared with normal saline group, the pulmonary capillary endothelial cells of the rats in each PHA group were damaged, characterized by endothelial cell swelling and dissolution, cytoplasm osteoporosis, unclear boundary, mitochondrial swelling and thick basement membrane. In all inactivated PHA-groups, the structures of the endothelial cells were normal. CONCLUSION: PHA-induced agglutination damages capillary endothelial cells and causes the disorders of the coagulation-fibrinolysis system.     

Effect of tissue factor on intravascular migration of tumor cells

AIM: To evaluate the effect of tissue factor on intravascular migration of tumor cells.METHODS: Expression of tissue factor in tumor cells (HT1080) was analyzed by flow cytometry and immunofluorescence staining. An in vitro model was used to observe intravascular migration of tumor cells. RESULTS: High expression of tissue factor was observed in tumor cells (HT1080). The antibody for tissue factor inhibited intravascular migration of tumor cells. CONCLUSIONS: Tissue factor stimulated tumor metastasis through promoting intravascular migration of tumor cells.     

Screening of tissue factor targeting peptides alternately from phage display peptide library

AIM: To screen tissue factor (TF) targeting peptides by establishment of a new and effective phage display method to acquire the peptides specifically bound to the transmembrane receptor. METHODS: Five rounds of panning were alternately conducted by targeting TF and HT-29 cell line which showed the detectable TF expression (screen for targeting receptor and cell alternately with phage display, STRCA). The 30 phage clones were assessed by enzyme-linked immunosorbent assay (ELISA). DNA sequencing was performed for the phage clones. The affinity of synthetic peptides was verified with competitive inhibition ELISA. The repeated experiment was conducted to verify the reliability of the results. RESULTS: The phages were effectively enriched after 5 rounds of panning with the improvement of the recovery rate from (2.25×10^-4)% to (1.32×10^-2)%. In 30 individual phages, ELISA positive rate was 76.7%, and the repetition of A, B, C and D peptides showed 23.3% (7/30), 23.3% (7/30), 26.7% (8/30) and 10.0% (3/30),respectively. E peptide constructively consisted of A and B. The five synthetic peptides were verified by ELISA, and the IC₅₀ of each peptide showed 3.25 nmol/L, 6.72 mol/L, 3.24×10³ mol/L, 2.08×10² mol/L and 45.77 mol/L,respectively. The positive phages were selected again in the second experiment to compare the results of the first experiment and the repeatability was 33.3%. CONCLUSION: STRCA can select TF targeting peptides with high affinity, which has the potential to become a therapeutic screening of transmembrane receptor-binding peptides.     

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

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

Regulatory effect of siRNA interference targeting BMI-1 gene on proliferation of bladder cancer EJ cells

AIM: To investigate whether siRNA-mediated BMI-1 gene silencing inhibits the proliferation of EJ cells by detecting the expression of BMI-1, p16^INK4a and p14^ARF genes at mRNA and protein levels in bladder cancer EJ cells and normal bladder transitional epithelium cells. METHODS: The protein expression and localization of BMI-1, p16^INK4a and p14^ARF in EJ cells were determined by cellular immunofluorescence. An siRNA targeting BMI-1 gene was synthesized and transfected into bladder carcinoma EJ cells by liposomes. The mRNA expression of BMI-1, p16^INK4a and p14^ARF was detected by real-time PCR and the protein levels were measured by Western blotting in bladder cancer EJ cells and normal bladder transitional epithelium cells. Cell survival was analyzed by CCK-8 assay. Cell apoptosis were examined by flow cytometry. RESULTS: The mRNA and protein expression of BMI-1 in EJ cells was higher than that in bladder transitional epithelium cells. However, the expression of p16^INK4a and p14^ARF were opposite.While BMI-1 gene in EJ cells was silenced by siRNA, the mRNA and protein expression of BMI-1 were declined whereas the expression of p16^INK4a and p14^ARF was increased. The viability of the EJ cells was decreased and the apoptotic cells were increased when BMI-1 gene was silenced. CONCLUSION: The expression of BMI-1 is inversely correlated with the expression of p16^INK4a and p14^ARF in bladder transitional cell carcinoma EJ cells. The siRNA-mediated BMI-1 gene silencing in bladder cancer EJ cells inhibits the cell growth and up-regulates the expression of p16^INK4a and p14^ARF in vitro.     

Let-7a-3p inhibits activity of cancer stem cells in human lung cancer A549 cells by targeting IGF1R

AIM:To study the effect of let-7a-3p on the activity of cancer stem cells in human lung cancer A549 cells and its molecular biological mechanism. METHODS:The exepression levels of let-7a-3p in lung cancer cell lines A549, NCI-H1299, SPC-A1, H1650 and HCC-827, and human normal bronchial epithilial cell line BEAS-2B were compared by RT-qPCR. The lung cancer A549 cells were transfected with let-7a-3p mimic and negative control mimic, as let-7a-3p group and negative control group, respectively, and non-transfected control group was also set up. The content of let-7a-3p in each group was detected by RT-qPCR. Tumor sphere formation assay was used to detect the tumor sphere formation ability in 3 groups of the cancer stem cells. The proportion of cancer stem cells was detected by flow cytometry. The protein levels of NANOG, OCT4 and insulin-like growth factor 1 receptor (IGF1R) were determined by Western blot. The target gene of let-7a-3p was predicted by the bioinformatic method. The relationship between let-7a-3p and IGF1R was analyzed by double luciferase assay. Western blot was used to detect whether IGF1R over-expression antagonized the inhibitory effect of let-7a-3p on the activity of cancer stem cells. A subcutaneous transplantation tumor model was also established and the effect of let-7a-3p in vivo was observed. RESULTS:The expression level of let-7a-3p in the lung cancer cell lines was significantly lower than that in the normal bronchial epithelial cell line (P<0.01). The expression level of let-7a-3p in the A549 cells of let-7a-3p group was significantly up-regulated compared with non-transfected group (P<0.01). The number of tumor spheres in let-7a-3p group was significantly lower than that in non-transfected group. The percentage of CD133⁺ cells in let-7a-3p group was significantly lower than that in non-transfected group (P<0.01). The protein expression of NANOG and OCT4 in let-7a-3p group was significantly lower than that in non-transfected group (P<0.01). Bioinformatic prediction showed that let-7a-3p complementarily matched the 3'-UTR of IGF1R, and IGF1R might be the target gene of let-7a-3p. Luciferase assay confirmed that IGF1R was the direct downstream target gene of let-7a-3p. The protein expression of IGF1R in let-7a-3p group was significantly decreased (P<0.01). Subcutaneously transplantated tumor in let-7a-3p group was significantly smaller than that in non-transfected group. CONCLUSION:Let-7a-3p may affect the expression of lung cancer stem cell-related proteins and inhibit the potential of lung cancer stem cells by down-regulating its downstream target gene IGF1R.     

Differentiation induction of human hepatocellular carcinoma BEL-7402 cells with tissue extracts of injured liver

AIM: To observe the effects of tissue extracts of injured liver on BEL-7402 cells,and explore a novel strategy of tumor therapy by differentiation induction. METHODS: Differentiation induction of human hepatocellular carcinoma cell line BEL-7402 was carried out with liver tissue extracts from an animal model of liver injury. The changes of cell biological characteristics, such as morphological features of the cells, MTT growth curves and cell cycle distribution, were dynamically observed. RESULTS: After exposed to the tissue extracts of injured liver, the number of mitotic cells was decreased, and the speed of growth and the proliferation of carcinoma cells were slowed down dramatically. The percentage of the cells in G₀/G₁ phase was increased, while the cells in S phase was decreased. The level of proliferation index (PI) also declined. CONCLUSION: The tissue extracts of injured liver affect the differentiation status of human hepatocellular carcinoma cell line BEL-7402, promote the cell differentiation and reduce the tumor characteristics. The tissue extracts of injured liver possess an important potential as a tumor differentiation inducer.     

Synergic antitumor effect of verapamil and tumor necrosis factor alpha

AIM: To study the synergic antitumor effect of verapamil (VPL) and tumor necrosis factor alpha (TNFα). METHODS: In the presence of verapamil or verapami plus tumor necrosis factor, the proliferation, the expressions of c-myc and PCNA, the mean vessel density(MVD)of human hepatoma cell line(HepG₂) were studied in vivo and in vitro. RESULTS: It was demonstrated that the growth of HepG₂ was inhibited remarkably, under the presence of verapamil and TNFα simutaneously, the expressions of c-myc, PCNA and the MVD were also decreased.CONCLUSION:The calcium channel blocker can inhibit the proliferation of tumor and has a remarkable synergy with TNF.     

Anti-angiogenic effect of thymoquinone on breast cancer

AIM: To investigate the anti-angiogenic effect of thymoquinone on human breast cancer and the possible mechanism. METHODS: The activities of human umbilical vein endothelial cells (HUVECs) and human breast cancer MCF-7 cells were assessed by CCK-8 assay. The apoptotic rate was measured by flow cytometry. Furthermore, the assay of capillary tube formation was used to observe the effect of thymoquinone on the tube formation of HUVECs. The protein levels of cleaved caspase-3, p-ERK and p-AKT were detected by Western blot. MCF-7 cells were subcutaneously injected into nude mice to establish breast xenograft tumors. After 3 weeks of implantation, the mice were randomized into control group and thymoquinone group. After the mice were sacrificed, immunohistochemistry was used to detect the expression of CD31 in the tumors, and the TUNEL test kit was used to explore cell apoptosis in the tumor tissues. RESULTS: Thymoquinone at concentrations of 20~80 nmol/L exerted no growth inhibitiory effect on MCF-7 cells. However, the cell viability of HUVECs were (66.1±8.3)％, (53.7±3.4)％ and (41.6±4.9)％ when the concentrations of thymoquinone were 20, 40 and 80 nmol/L, respectively. The apoptotic ratio of MCF-7 cells were (2.6±0.3)％, (2.4±0.3)％ and (4.6±0.4)％ and the apoptotic ratio of HUVECs were (21.5±3.7)％, (23.8±2.9)％ and (27.8±3.1)％ when the concentrations of thymoquinone were 20, 40 and 80 nmol/L, respectively. HUVECs were more sensitive to thymoquinone-induced apoptosis and inhibition in the cell activity than MCF-7 cells. Incubation of HUVECs with diffe-rent concentrations of thymoquinone (20, 40 and 80 nmol/L) for 1 h decreased their tube formation capacity (P<0.05). The protein level of cleaved caspase-3 was up-regulated, but the phosphorylation of AKT and ERK were inhibited in a concentration-dependent manner. The immunohistochemical analysis of CD31 showed significant difference of the integral absorbance between control group and thymoquinone group, and the TUNEL-positive cells in thymoquinone group was significantly more than that in control group. CONCLUSION: Thymoquinone has the anti-angiogenic effect on breast cancer both in vitro and in vivo, which may be related to the decreases in p-ERK and p-AKT.     

Effect of transfection of tumor necrosis factor α gene and its mutants on tumorigenicity of H22 tumor cells in vivo

AIM: To compare the tumorigenecity of H22 cells transfected with TNF-α gene and its mutants(secreted TNF-α mutant, S-TNF^m, transmembrane TNF-α mutant, TM-TNF^m and wild type of TNF-α, Wt-TNF) in vivo. METHODS: Three kinds of mouse liver cancer cell line H22 expressing TNF-α and its two mutants were mixed with untransfected H22 at different effector/target ratio separately. The growth of tumor was examined after injection of 2.5×10⁵(100 μL) mixed H22 tumor cells into mice. The lymphocyte infiltration in the site of tumor and the expression of Fas on tumor cells were detected by immunohistochemistry. RESULTS: The tumorigenecity of H22 cells transfected with TNF-α gene and its mutants was significantly weakened( P <0.01). Besides the cytotoxicity of the both forms of TNF, TM-TNF was found to induce the expression of death receptor Fas by tumor cells and S-TNF was shown to promote frank lymphocyte infiltration in the site of tumor. Furthermore, a transient decrease in body weight was found in mice inoculated with H22/S-TNF^m. CONCLUSION: The tumorigenecity of tumor cells was reduced by transfection with TM-TNF or S-TNF gene. It results from the cytotoxicity of TNF and their activation of tumorcidal mechanisms in vivo. TM-TNF may induce tumor cell apoptosis via the Fas pathway while S-TNF may exert its antitumor effect by recruiting and activating lymphocytes in the site of tumor.     

Construction of a lentiviral RNA interference vector targeting rat myocardin and its effect on vascular smooth muscle cell differentiation

AIM: To construct a lentiviral RNA interference(RNAi)vector targeting rat myocardin mRNA and to investigate its effect on the differentiation of vascular smooth muscle cells(VSMCs).METHODS: Three pairs of dsDNA targeting rat myocardin mRNA were designed, synthesized and cloned into lentiviral vector pGCSIL-GFP to generate pGCSIL-GFP-shMyocd lentvirus. A Flag-tagged myocardin-overexpression vector pEGFP-N1-Myocd was constructed with pEGFP-N1/X124G. After these two vectors were cotransfected into 293T cells, the flag protein was assessed by Western blotting to analyze the knockdown effect of pGCSIL-GFP-shMyocd. The expression of myocardin and SM22α was also detected by RT-PCR and Western blotting after the pGCSIL-GFP-shMyocd viruses were transfected into primary cultured rat aortal VSMCs.RESULTS: The rat myocardin lentviral RNAi vector pGCSIL-GFP-shMyocd and myocardin-overexpression vector pEGFP-N1-Myocd were successfully constructed. After these two kinds of vectors were cotransfected into 293T cells,the No.1 interfering vector displayed the highest inhibitory effect on flag expression.After the No.1 lentvirus at the titer of 1×10¹² TU/L was transfected into VSMCs, the myocardin and SM22α expression was significantly attenuated. CONCLUSION: The lentiviral pGCSIL-GFP-shMyocd RNAi vector is successfully constructed, which is useful for further study regarding the molecular mechanism of the phenotypic switching in VSMCs under special pathological conditions such as atherosclerosis. Inhibition of myocardin expression in VSMCs leads to the decrease in the expression of differentiation marker, and implies a crucial role of myocardin in VSMCs differentiation.     

Construction of siArid2 recombinant adenovirus and its effect on proliferation of hepatocarcinoma cells

AIM: To observe the effects of Arid2gene on cell proliferation and cell cycle by interference of endogenous Arid2 expression in hepatoma cells. METHODS: Three pairs of shRNA targeting Arid2gene were cloned into a shuttle vector to construct recombinant adenovirus plasmids. HEK293 cells were transfected with the recombinant adenovirus plasmids. After several rounds of the package and amplification, the high-titer adenoviruses AdsiArid2-1~3 were obtained. To verify the inhibitory effects of AdsiArid2 adenoviruses, Western blotting was used to detect the endogenous Arid2 protien expression in SMMC-7721 cells. Cell growth and cell cycle analysis were carried out by MTS assay and flow cytometry. RESULTS: High- titer recombinant adenovirus of siArid2 were successfully obtained, and named AdsiArid2-1~3, among which the AdsiArid2-3 had the best inhibitory effects. MTS assay showed that the absorbance values at 490 nm were increased at 72 h and 96 h after transduction compared with the mock and Adsicontrol groups. These data indicated that knockdown of Arid2 promoted the proliferation rate of SMMC-7721 cells(P<0.05). Moreover, the flow cytometry analysis revealed that the G1-phase distribution at 72 h in AdsiArid2 group was lower than that in mock group and Adsicontrol group. In contrast, the S-phase distribution in AdsiArid2 group was much higher than that in mock group and Adsicontrol group. CONCLUSION: The recombinant plasmids and recombinant adenovirus were successfully constructed. shRNA-mediated knockdown of Arid2 promotes the proliferation and the transition from G1 phase to S phase of hepatoma cells.     

Effects of lipopolysaccharide,IL-6 and TNFα on the tissue factor expression of astrocytes

AIM:To study the effects of lipopolysaccharide(LPS), interleukin-6(IL-6)and tumor necrosis factor α (TNFα) on tissue factor(TF) expression of astrocytes. METHODS:Astrocytes were identified with anti-glial fibrillary acidic protein antibody. The TF activity of cell lysate was measured with one stage clotting assay. RESULTS:TF activity of astrocytes of LPS,IL-6,TNFα groups were obviously higher than that of the control group(P <0.05); While LPS,IL-6 and TNFα were combined with trifluoperazine or H₇, their inductive effects were inhibited. CONCLUSION:LPS,IL-6 and TNFα promoted the TF expression of astrocytes and its mechanisms may connected with Calcium/Camodulin and protein kinase C pathway.     

Hsa-miR-218 inhibits growth of cervical cancer HeLa cells by targeting to LASP1

AIM: To study the effect of hsa-miR-218 on cervical cancer HeLa cell growth and the underlying molecular mechanism.METHODS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed. pmiR-218 was transfected into HeLa cells. The number of viable HeLa cells was counted by the method of Trypan blue exclusion. The inhibitory rate of cell activity was detected by WST-8 assay. The expression of LIM and SH3 protein 1(LASP1) at mRNA and protein levels was determined by real-time PCR and Western blot. The interaction between miR-218 and LASP1 was examined using a luciferase reporter assay.RESULTS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed successfully and confirmed by DNA sequencing. Over-expression of miR-218 inhibited the activity of HeLa cells with the inhibitory rates of 15%, 26% and 65% at 24 h, 48 h and 72 h, respectively. The difference between transfection group and blank control/negative control group was statistically significant. The luciferase activity was reduced when co-transfection with miR-218 mimics and LASP1-3,UTR plasmid. The relative expression of miR-218 was increased after transfection with pmiR-218. Over-expression of miR-218 down-regulated the LASP1 expression at mRNA and protein levels by 25% and 75% respectively. Compared with blank control group and negative control group, the difference was statistically significant(P<0.05).CONCLUSION: pmiR-218 effectively inhibits the growth of HeLa cells in a time-dependent manner. miR-218 targets to the 3,UTR of LASP1, thus down-regulating the expression of LASP1 in HeLa cells.