Thiazol Research Articles

Antimicrobial, Antioxidant and Antiproliferative Activities of Novel Quinolones

AbstractThe compound (2) formed by esterification of dimethyl morpholine (1) was converted to acetohydrazide (3). Subsequently Schiff bases (4 a–d) and carboxy(thio)amide derivatives (5 a–e) were synthesized. Then 1,2,4‐triazole (6 a–e), thia(oxa)zolidine (7 c,e) and thia(oxa)zol (8 c,e) derivatives were obtained by ring closure from carboxy(thio)amides. Mannich bases, which are containing quinolone were synthesized from 1,2,4‐triazoles. The structures of newly synthesized compounds were illuminated by spectroscopic methods. Their antimicrobial (MIC method), antioxidant (DPPH, FRAP, and CUPRAC methods), and anticancer activities (MTT method) were examined. Results showed that most of the compounds exhibited good antimicrobial (<0.03–31.25 μg/mL with MIC values) and antioxidant activities (IC50=0.001–0.004 with DPPH values). Also, some of the compounds have been found to have antiproliferative effects on the prostate (PC‐3), liver (Hep3B), and breast (MCF‐7) human cancer cells, and also these compounds did not have a cytotoxic effect on a normal cell.

Study on the mechanism of long non-coding RNA migration inhibitory factor antisense RNA1 in regulating proliferation, invasion and metastasis of non-small cell lung cancer

Objective To explore the molecular mechanism by which long non-coding RNA (lncRNA) migration inhibitory factor antisense RNA1 (MIF-AS1)/microRNA (miRNAM miR)-370-3p/mitogen activated protein kinase 9 (MAP3K9) molecular axis regulates proliferation, invasion and migration of non-small cell lung cancer (NSCLC). Methods Twenty cancer samples of NSCLC patients were collected from Henan Provincial People’s Hospital from May 2017 to January 2019. There were 12 males and 8 females, aged (52.37±10.34) years. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the expression levels of MIF-AS1, miR-370-3p and MAP3K9 in cancer tissues and 9 cell lines. NSCLC A549 cells were transfected with si-MIF-AS1, si-NC, si-MIF-AS1+ anti-miR-370-3p or si-MIF-AS1+ pcDNA-MAP3K9, respectively. Methyl thiazol tetrazolium (MTT) assay and Transwell assay were used to detect the proliferation, migration and invasion of transfected cells. Western blotting was used to detect the expression of Cyclin D1, p21, p27, matrix metalloproteinase (MMP)-2, MMP-9 and MMP-14 proteins. The dual luciferase reporter gene verified the targeting regulation of MIF-AS1 on miR-370-3p and miR-370-3p on MAP3K9. Results The expression of MIF-AS1 and MAP3K9 in NSCLC tissues (2.49±0.25 and 2.24±0.22) and A549 cells (2.54±0.24 and 2.31±0.23), H1299 cells (2.11±0.21 and 2.44±0.24) and PC-9 cells (2.26±0.23 and 2.16±0.22) was significantly higher than that in adjacent tissues (1.00±0.09 and 1.02±0.09) and normal lung epithelial cells BEAS-2B (1.01±0.09 and 1.00±0.09) (t=25.078, P<0.05; F=94.367, P<0.05). The expression of miR-370-3p in NSCLC tissues (0.42±0.04), A549 cells (0.34±0.03), H1299 cells (0.53±0.05), and PC-9 cells (0.44±0.04) was significantly lower than that in adjacent tissues (1.01±0.08) and BEAS-2B cells (1.00±0.08) (t=29.500, P<0.05; F=269.552, P<0.05). Transfection with si-MIF-AS1 significantly inhibited NSCLC cell proliferation (24 h: 0.27±0.03, 48 h: 0.36±0.03, 72 h: 0.48±0.04), invasion (24.33±3.14), migration (32.46±3.34) ability (P<0.01), up-regulated p21 (0.64±0.06), p27 (0.77±0.07) protein expression (t=17.441, P<0.05; t=17.726, P<0.05) and down-regulated Cyclin D1 (0.29±0.03), MMP-2 (0.25±0.03), MMP-9 (0.34±0.03) and MMP-14 (0.29±0.03) protein expression (t=17.732, P<0.05). The dual luciferase reporter gene confirmed that MIF-AS1 was bond to miR-370-3p and miR-370-3p was bond to MAP3K9. Transfection with si-MIF-AS1+ anti-miR-370-3p or si-MIF-AS1+ pcDNA-MAP3K9 inhibited the effects of si-MIF-AS1 on proliferation, invasion and migration of NSCLC cells. Conclusion LncRNA MIF-AS1 promotes proliferation, migration and invasion of NSCLCs by regulating miR-370-3p/MAP3K9 molecular axis. Key words: Non-small cell lung cancer; Long non-coding RNA migration inhibitory factor antisense RNA1; MicroRNA-370-3p; Mitogen activated protein kinase 9; Proliferation; Migration; Invasion

Rosuvastatin regulates osteogenic differentiation of mesenchymal stem cells through phosphatidylinositol-3-kinase/protein kinase B

Objective To investigate the effects of rosuvastatin on proliferation, osteogenic differentiation and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) expression of mesenchymal stem cells (MSCs). Methods MSCs were isolated. Rosuvastatin at concentrations of 1×10-11, 1×10-9 and 1×10-7 mol/L were added to the experimental groups, and dimethylsurfoxide (DMSO) served as control. Cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay. After 3, 7, 14 and 21 days of osteogenic induction, alkaline phosphatase (ALP) quantitative analysis, alizarin red staining and quantitative analysis, real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were performed. T-test was used for comparison between two groups, and ANOVA for more than two groups. Results At 24 h and 48 h, the proliferation of MSCs in high concentration group was significantly higher than in the control group (24 h: 3.57±0.24 vs. 3.14±0.14, t=-3.851, P<0.05; 48 h: 4.19±0.18 vs. 3.44±0.11, t=-6.780, P<0.05). Until 72 h, the proliferation with 1×10-9 mol/L rosuvastatin was also significantly increased (5.42±0.13 vs. 4.47±0.16, t=-8.700, P<0.05). At 3rd day after induction culture, 1×10-7 mol/L rosuvastatin significantly enhanced the genes expression of BMP2, Runx 2, OPN 2, OPN and ColⅠ (BMP2: 2.1±0.2 vs. 1.0±0.2, t=-6.736, P<0.05; Runx2: 5.2±0.6 vs. 1.0±0.1, t=-11.959, P<0.05; OPN: 1.8±0.4 vs. 1.0±0.2, t=-3.098, P<0.05; ColⅠ: 1.9±0.3 vs. 1.0±0.3, t=-3.674, P<0.05); 1×10-7 mol/L rosuvastatin could increase the expression of p-Akt/PI3K. The ALP activity after adding 1×10-9 or 1×10-7 mol/L rosuvastatin was significantly increased at 14th and 21st day (14 d: 12.07±1.67, 18.32±2.26 vs. 3.43±0.36, F=7.200, P<0.05; 21 d: 10.74±1.27, 14.71±3.18 vs. 5.76±0.63, F=6.489, P<0.05). The quantitative results of alizarin red were similar to those of the staining. Meanwhile, the quantitative values of alizarin red in the 1×10-9 and 1×10-7 mol/L groups were also significantly increased (14 d: 5.6±0.8, 6.2±1.2 vs. 1.0±0.2, F=5.600, P<0.05; 21 d: 5.8±1.1, 7.1±1.8 vs. 2.4±0.3, F=5.956, P<0.05). Conclusion Rosuvastatin promotes proliferation and osteogenic differentiation of MSCs, which may be related to PI3K/Akt signaling. Key words: Rosuvastatin; Phosphatidylinositol-3-kinase/protein kinase B signaling pathway; Mesenchymal stem cells; Osteogenic differentiation

Effects of long-chain non-coding HIF2PUT on migration and invasion of osteosarcoma cells

Objective To observe the effects of long-chain non-coding RNA (lncRNA) HIF2PUT on the proliferation, migration and invasion of osteosarcoma cells. Methods Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of lncRNA HIF2PUT in 47 cases of osteosarcoma tissues (28 males and 19 females, the oldest was 45 years old, the youngest was 8 years old with an average of 20.24 years old) and 2 osteosarcoma cell lines (MG63 and HOS). After screening for stable transfectants by lentivirus, the methyl thiazol tetrazolium (MTT) assay and Transwell assay were applied to detect the effects of lncRNA HIF2PUT on proliferation, migration and invasion of osteosarcoma cells. According to the cell transfection, the experimental group (overexpressed lncRNA HIF2PUT group), the blank transfection group and the blank control group were respectively set. Data were expressed as mean±standard deviations (SD), and SPSS 21.0 software was used for statistical analysis. Results The expression of lncRNA HIF2PUT in 47 cases of osteosarcoma (39.56±0.48) was significantly lower than that in normal tissues (79.56±1.16, t=3.516, P<0.05). The expression level of lncRNA HIF2PUT in MG63 cells was significantly higher than that in HOS cells. Cell function experiments showed that lncRNA HIF2PUT had a significant effect on the proliferation, migration and invasion of osteosarcoma cells. Proliferation of MG63 and HOS cells was significantly inhibited by lncRNA HIF2PUT, and the proliferation index was significantly lower in lncRNA HIF2PUT group than in blank transfection group and blank control group (t=4.785, P<0.05). For the migration experiment, the migrating number of MG63 cells was (154.36±13.85), (196.32±13.52) and (258.63±12.35), respectively (t=3.968, P<0.01), and that of HOS cells was (131.24±10.58), (247.36±17.52) and (224.52±14.52) respectively (t=3.052, P<0.01). For the invasion experiment, the penetrating number of MG63 cells was (73.50±13.89), (98.63±12.57) and (112.30±15.29), respectively (t=23.178, P<0.01), and that of HOS cells was (70.74±13.49) (98.40±16.46) and 89.74±12.47, respectively (t=14.275, P<0.01). The migration and invasion ability of MG63 and HOS cells in lncRNA HIF2PUT group was lower than that in blank transfection group and blank control group. Conclusion The expression of lncRNA HIF2PUT was significantly low in osteosarcoma tissues and MG63 and HOS cell lines. The expression of lncRNA HIF2PUT significantly inhibited the proliferation, migration and invasion of osteosarcoma cells. Key words: Osteosarcoma; Long-chain non-coding RNA; Migration; Invasion

Expression of prostate cancer-associated transcript 6 in serum of patients with liver cancer and the effect of its interference on biological function of liver cancer cells

Objective To investigate the expression of prostate cancer-associated transcript 6 (PCAT6) in the serum of hepatocarcinoma patients and its effect on the proliferation, migration, invasion and apoptosis of Hep3B cells. Methods The qPCR method was used to detect the level of long non-coding RNA (lncRNA) PCAT6 in serum and THLE-3, Hep3B and HepG2 cells. The Hep3B cells were randomly divided into blank control group (NC group), negative empty vector transfection group (si-con group) and lncRNA PCAT6 silencing group (si-PCAT6 group). Western blotting was used to detect the expression of matrix metalloproteinase (MMP)-2, MMP-9, cleaved cysteinyl aspartate-specific protease (Caspase)-3, cleaved Caspase-9, phosphatidylinositol 3 kinase (PI3K), phosphorylated-PI3K (p-PI3K), protein kinase B (Akt), and phosphorylated Akt (p-Akt) proteins. The cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay. The apoptosis was examined by flow cytometry, and the migration and invasion was tested detected by Transwell chamber method. The SPSS 21.0 software was used for statistical analysis, and the measurement data were expressed as mean±standard deviation (SD). Results The expression level of lncRNA PCAT6 in patients with moderate or high differentiation (60.94%) was significantly higher than that in patients with low differentiation (6.25%) (χ2=22.968, P<0.01). The percentage of patients with high expression of lncRNA PCAT6 in T-stage Ⅱ-Ⅳ (57.81%) was significantly higher than that in stage Ⅰ (9.38%) (χ2=8.529, P<0.01). The expression of lncRNA PCAT6 in Hep3B cells (3.72±0.67) and HepG2 cells (3.38±0.53) was significantly higher than that in THLE-3 cells (1.03±0.14) (t=9.322 and 8.144, P<0.05). The expression of lncRNA PCAT6 (0.21±0.11) in si-PCAT6 group was significantly lower than that in si-CON group (0.96±0.15) (t=9.915, P<0.05), and the cell viability was significantly reduced at 24 h, 48 h and 72 h (t=3.280, 6.144 and 6.373, P<0.05). Western blotting showed that the expression of cleaved Caspase-3, cleaved Caspase-9, p-pi3k and p-Akt increased significantly (t=11.408, 14.628, 8.683 and 9.585, P<0.01), and the expression of MMP-2 and MMP-9 decreased significantly in si-PCAT6 group (t=10.568 and 10.814, P<0.01). The apoptosis rate of Hep3B cells increased significantly (t=21.075, P<0.01), and the migration and invasion of Transwell cells decreased significantly (t=12.816 and 12.707, P<0.01). Conclusion The serum lncRNA PCAT6 is highly expressed in HCC patients, which is related to the degree of HCC differentiation and T stage. Silencing lncRNA PCAT6 can inhibit the biological behaviors of HCC probably by inhibiting the Akt signaling pathway activation. Key words: Long non-coding RNA; Prostate cancer-associated transcript 6; Liver cancer; Biological function

Effect of trim28 (ser473) phosphorylation on viability of hepatocellular carcinoma cells during DNA damage

Objective To observe effect of Trim28 (ser473) phosphorylation on viability of hepatocellular carcinoma (HCC) cells during DNA damage. Methods To divide Hepg2 cells into 2 groups as SB203580+ UV group and UV group. Hepg2 cells were modeled for DNA damage by UV (Ultraviolet) irradiation. SB203580+ UV group was treated with Trim28 phosphorylation inhibitor SB203580 after UV irradiation. Expression of Trim28 and phosphorylation-Trim28 ser473 (P-Trim28 ser473)were detected by westernblot experiment. The viability of Hepg2 cells were observed by methyl thiazol tetrazolium (MTT) experiment. Results Compared with UV group (0.94±0.13), the expression value of Trim28 in SB203580+ UV group had no significant difference (t=-0.190, P>0.05). Value of phosphorylation-Trim28 in SB203580+ UV group (0.32±0.04) was obviously lower than UV group (0.72±0.05), the difference had statistical meaning (t=8.500, P<0.05); The 24, 48 and 72 hours after UV irradiation, the cell survive rate of UV group [(95±7)%, (62±10)%, (35±6)%] is higher than SB203580+ UV group [(85±7)%, (37±4)%, (20±3)%]. The difference has statistical meaning (t=-2.326, -5.212, -4.577, P<0.05). Conclusion The viability of HCC cells during DNA damaged can be reduced by inhibition for Trim28 phosphorylation. Key words: Trim28; Phosphorylation; Cell survival

Expression of long chain non coding RNA gastric cancer-associated transcript 3 in hepatocellular carcinoma and its effect on biological characteristics of HepG2 cells

Objective To investigate the expression of long chain non coding RNA (lncRNA) gastric cancer-associated transcript 3 (GACAT3) in hepatocellular carcinoma and its effect on proliferation, invasion and migration of HepG2 cells and its potential mechanism. Methods real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect the differential expression of lncRNA GACAT3 in liver cancer tissue, adjacent tissues, PLC/PRF/5, Hep3B, HepG2 cell lines and normal liver cells THLE-3. Negative control small interfering RNA (siRNA) and lncRNA GACAT3 siRNA were transfected into HepG2 cells, after 48 h, Real-time PCR was used to detect the expression of lncRNA GACAT3 in transfected cells. Methyl thiazol tetrazolium (MTT) assay was used to detect cell proliferation in each group at 24, 48 and 72 h. The cell migration and invasion were detected by cell scratches experiments and Transwell assay, and the expression of Wnt/β-catenin signaling pathway protein was detected by Western blotting. Results The expression of lncRNA GACAT3 in hepatocellular carcinoma tissues (3.10±1.93) was significantly higher than that in adjacent tissues (1.02±0.59, t=8.086, P<0.01); The expression of lncRNA GACAT3 in PLC/PRF/5 (1.92±0.21, t=7.074, P<0.01), Hep3B (2.42±0.25, t=9.327, P<0.01) and HepG2 cells(2.96±0.31, t=10.582, P<0.01) were significantly higher than that in normal liver cells THLE-3 (0.97±0.10). Compared with the silencing control group (1.03±0.11), the expression of lncRNA GACAT3 in the GACAT3 group (0.16±0.02) was significantly decreased (t=13.478, P<0.01). Cell proliferation was significantly reduced at 48 h and 72 h (P<0.01). Scratch healing rate and migratory and invasive ability were significantly decreased (P<0.01). The expression of Wnt/β-catenin signaling pathway protein β-catenin (0.15±0.02) and its downstream proteins c-Myc (0.22±0.02) and Cyclin D1 (0.21±0.02) were significantly lower than that of the silencing control group(0.81±0.08, t=13.863; 0.65±0.07, t=9.731; 0.78±0.08, t=10.230; P all<0.01). The expression of p-β-catenin protein (0.75±0.08) was significantly higher than that of the silencing control group (0.27±0.03, t=11.972, P<0.01). Conclusion LncRNA GACAT3 is highly expressed in hepatocellular carcinoma. Silencing LncRNA GACAT3 can inhibit the proliferation, migration and invasion of hepatocellular carcinoma cells, and its mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway. Key words: Long chain non coding RNA gastric cancer-associated transcript 3; Hepatocellular carcinoma; HepG2; Wnt/β-catenin signaling pathway

The expression of microRNA-532-5p in glioma and mechanism research

Objective To explore the expression of microRNA(miRNA, miR)-532-5p in primary glioma and to detect the effects of miR-532-5p overexpression on proliferation, migration and invasion in U251 glioma cell line. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was conducted to measure the expression of miR-532-5p in 30 cases of glioma tissues and traumatic brain specimens. We then analyzed the relationship of the level of miR-532-5p and the clinical pathological data of glioma patients. After transfection with miR-532-5p, the effects of on proliferation, migration and invasion were detected using methyl thiazol tetrazolium (MTT) assay, wound healing assay and transwell assay. Results The relative expression of miR-532-5p was significantly lower in glioma tissues(0.381±0.310) than in normal brain tissues (1.040±0.470, P<0.01). The level of miR-532-5p was significantly associated with Karnofsky (KPS) scores and seizures. Expression of miR-532-5p in U251 cell line (0.280±0.080) were significantly higher than those in Normal Human Astrocyte (NHA) cell line (1.021±0.069, P<0.01). The proliferation [(59.5±9.5)%, P<0.01], migration [(55.4±4.2)%, P<0.05] an invasion [(45.7±6.3)%, P<0.01] of U251 cell with miR-532-5p mimic transfection were significantly lower than those in miR-532-5p negative control after transfection. Conclusion The expression of miR-532-5p were decreased in patients with glioma. The low level of miR-532-5p was significantly associated with the KPS scores and seizures. It was shown that miR-532-5p was associated with proliferation, migration and invasion in U251 cell line and high expression of miR-532-5p was significantly associated with the KPS scores and seizures Overexpression of miR-532-5p could inhibit the capability of proliferation, migration and invasion. Key words: MicroRNA-532-5p; Glioma; Migration; Invasion; Pathological parameters

Effect of microRNA-451 on proliferation and migration of colorectal carcinoma via inhibition the expression of macrophage migration inhibitory factor

Objective To explore effect of microRNA (miRNA, miR)-451 on proliferation and migration of colorectal carcinoma via macrophage migration inhibitory factor (MIF). Methods miR-451 mimics and miR-451 NC were transfected into colorectal carcinoma cell by liposome Lipofectamine™3000. The expression of miR-451 was detected by real-time quantitative polymerase chain reaction (Real-time PCR). Cell viability was measured by methyl thiazol tetrazolium (MTT) assay. Cell clone ability was detected by cell clone formation test. Cell cycle was detected by flow cytometry. Cell migration ability was detected by Transwell method. The expression of MIF protein and mRNA was measured by Western blotting and Real-time PCR. Luciferase reporter analysis was performed. Results The expression of miR-451 in miR-451 mimics group (2.46±0.5) was higher than that in miR-451 NC group (1.00±0.11, t=52.374, P<0.05). Cell viability in miR-451 mimics group (0.37±0.03) was lower than that in miR-451 NC group (0.58±0.06, t=30.672, P<0.05). Cell clone number in miR-451 mimics group (41.56±4.17) was lower than that in miR-451 NC group (132.37±11.29, t=73.927, P<0.05). The G1 period in miR-451 mimics group was longer than that in miR-451 NC group (t=4.093, P<0.05), and the G2 period in miR-451 mimics group was shorter than that in miR-451 NC group (t=6.349, P<0.05). The number of cell invasion in miR-451 mimics group (56.59±5.66) was less than that in the miR-451NC group (184.73±18.47, t=64.992, P<0.05). The fluorescence intensity in miR-451 mimics+ pGL4 MIF 3’untranslated regions (3’UTR)-Wt group was lower than other transfection groups (F=2 875.12, P<0.05). The expression of MIF mRNA in miR-451 mimics (0.35±0.03) group was lower than that in miR-451 NC group (1.00±0.10, t=61.001, P<0.05). The western blot results showed that expression amount of MIF protein in mir-451 mimics group (0.23±0.02) was lower than that in miR-451 NC group (0.92±0.10, t=66.293, P<0.05). Conclusion Over-expression of miR-451 could inhibit colorectal carcinoma cell proliferation and migration by targeting down-regulation expression of MIF. Key words: Carcinoma carcinoma; MicroRNA-451; Macrophage migration inhibitory factor; Proliferation; Migration

Impact and mechanism of Loganin on epithelial mesenchymal transformation of gastric cancer cells

Objective To explore impact and molecular mechanism of Loganin on epithelial mesenchymal transformation of gastric cancer cells. Methods Human gastric cancer cell lines AGS, BGC823 and SGC7901 were cultured routinely. The effect of Loganin on cells activity was tested by utilizing methyl thiazol tetrazolium (MTT) assay. Also Wound assay and Transwell assay were employed to detect the effect of Loganin on migration and invasion in BGC823 cells. Furthermore, real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to examine the variations of associated genes and phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt) signal pathway in BGC823 cells prior and post Loganin interventions. Results Results of MTT assay showed that the activity of BGC823 was lowest among AGS (0.748±0.051), BGC823 (0.581±0.042) and SGC7901 (0.412±0.037) (F=44.101, P<0.01); the expression level of PCNA mRNA and protein declined gradually with the increase dose of Loganin (F=36.298, 47.942, P<0.01). Compared with the control group, the ability of migration and invasion in BGC 823 cells decreased, and this changes were particularly distinct in high dose Loganin group (200 mg/L), which were more significant than in low dose group (50 mg/L) (migration: F=87.750, P<0.01; invasion: F=85.749, P<0.01). Results of qPCR and Western blotting showed that expressions of migration, invasion associated genes in BGC823 cells changes significantly compared with the control group, especially in high dose Loganin group (P<0.01). Results of qPCR and Western blotting illustrated that EMT related gene in BGC823 cells changes significantly compared with the control group, especially in high dose Loganin group (P<0.01). The activity of PI3K-Akt signal pathway declined significantly post Loganin intervention, and expression of p-PI3K, p-Akt decreased significantly, especially in high dose Loganin group (F=38.590, 63.563; P<0.01). Conclusion Loganin may inhibit EMT in gastric cancer cells via regulating PI3K-Akt signal pathway. Key words: Gastric cancer; Loganin; Invasion; Epithelial mesenchymal transformation; Phosphatidylinositol 3 kinase-protein kinase B signal pathways

Effect of silencing Kinesin family member 21B expression in hepatoma cells BEL-7404 on cell biological behavior

Objective To detect the differential expression of Kinesin family member 21B (KIF21B) gene in hepatocellular carcinoma (HCC) tissues and normal tissues and its influence on cell. Methods The Cancer Genome Atlas (TCGA) database was used to screen the differential expression of KIF21B gene, the expression level of KIF21B gene in hepatocellular carcinoma cell lines was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), and the short hairpin RNA (shRNA) targeting KIF21B gene was constructed to infect BEL-7404 cells. The expression of KIF21B mRNA in transfected hepatoma cells was detected by real-time quantitative polymerase chain reaction (Real-time PCR), and the expression of KIF21B protein was detected by Western blotting. The effect of KIF21B gene depletion on cell proliferation was detected by Celigo. KIF21B gene subtraction was detected by methyl thiazol tetrazolium (MTT) assay. The effect on cell proliferation was detected by flow cytometric fluorescence sorting (FACS). The effect of KIF21B gene depletion on apoptosis was detected by colony formation assay. The effect of KIF21B gene subtraction on cell clone formation ability was detected by colony formation assay. The measurement data were expressed as mean±standard deviation (Mean±SD), and t-test was used for comparison between groups. Results Compared with normal tissues, the expression of KIF21B gene was up-regulated in 31 HCC tissues, unchanged in 14 and down-regulated in 5. The expression level of KIF21B in 4 HCC cell lines was higher than that in normal Liver tissue cells (P<0.05). ShRNA targeting KIF21B effectively silenced the expression of KIF21B, and MTT assay showed that the growth rate of bel-7404 cells was significantly inhibited after KIF21B reduction (P<0.05). Apoptosis was significantly increased (P<0.05). The clonal formation ability of KIF21B cells was significantly decreased (25.3±3.7 vs. 418.2±26.5, P<0.05). Conclusion After KIF21B gene silencing, the apoptosis of hepatocellular carcinoma cells increased, the proliferation and cloning ability decreased significantly. Key words: Hepatoma cells; Kinesin family member 21B; Apoptosis; Proliferation

Effects of FAM96B on the proliferation, apoptosis, invasion and metastasis of hepatocellular carcinoma HepG2 cells

Objective To investigate the effects of family with sequence similarity 96 member B (FAM96B) on the proliferation, apoptosis, invasion and metastasis of hepatocellular carcinoma (HCC) HepG2 cells. Methods FAM96B mRNA and protein were examined respectively by real-time fluorogenic quantitative polymerase chain reaction (Real-time PCR) and Western blotting in L02 and HepG2 cells. Moreover, the eukaryotic expression vector of FAM96B was constructed and transfected into HepG2 cells. The cell proliferation of each group at 0, 24, 48, 72 and 96 h was tested by methyl thiazol tetrazolium (MTT). Then the apoptosis, invasion and metastasis were tested respectively by Flow cytometry, wounding healing assay and Transwell. Meanwhile, the subcutaneous tumorigenic model of nude mice were established by 10 male and aged 4-5 weeks BALB/c-nu/nu mice. The model mice were randomized into control group and FAM96B group, 5 mice in each group. And which were injected with 0.3 ml 1×1010 pcDNA3.1-HepG2 cells and pcDNA3.1-FAM96B HepG2 cells. The weight and volume of tumors were measured every three days. All the mice were executed on the 30th day. Statistical analysis was performed using SPSS 21.0. The measurement data were expressed as mean±standard deviation (Mean±SD). The mean comparison between the two groups was performed by t test. Results The FAM96B mRNA expression was decreased in HepG2 cells than in L02 cells (t=25.343, P<0.01). Meanwhile, The FAM96B protein expression was decreased in HepG2 cells than in L02 cells. Compared with control group, the proliferative ability of FAM96B group was significantly reduced (P<0.01). The apoptosis rate in FAM96B group was increased in FAM96B group than in control group [(8.61±1.05)% vs. (47.34±4.82)%, t=23.544, P<0.01]. Compared with control group, the migration ability of FAM96B group was obviously reduced [(222.33±32.78) μm vs. (284.13±46.18) μm, t=4.227, P<0.01], and the invasion ability was obviously reduced [(45.47±4.05) vs. (101.13±5.54), t=31.413, P<0.01]. Moreover, in the subcutaneous tumorigenic model of nude mice, the tumour volume and weight were all decreased in FAM96B group than in control group (tvolume=0.610, tweight=7.186, P all<0.05). Conclusion FAM96B in hepatocellular carcinoma HepG2 cells is low expression, up-regulated its expression levels can inhibit the proliferation, invasion, metastasis and induce apoptosis of HepG2 cells. And up-regulated FAM96B expression levels can inhibit growth of tumors in the subcutaneous tumorigenic model of nude mice. Key words: Family with sequence similarity 96 member B; Hepatocellular carcinoma; HepG2 cells; Proliferation; Apoptosis; Invasion and metastasis

Effect of epigallocatechin gallate on Th1/Th2 immune balance in patients with plaque-type psoriasis

Objective To evaluate the effect of epigallocatechin gallate (EGCG) on T helper cell 1 (Th1) and Th2 in psoriasis patients. Methods A total of 33 patients with plaque-type psoriasis vulgaris were enrolled, and peripheral blood mononuclear cells (PBMC) were isolated and cultured. The appropriate concentration of EGCG was determined by methyl thiazol tetrazolium (MTT) assay. PBMC at exponential growth phase were divided into 2 groups to be treated with EGCG (EGCG group) or not (control group) for 24 hours. Flow cytometry was performed to determine proportions of Th1 and Th2 cells, enzyme-linked immunosorbent assay (ELISA) to detect levels of Th1 (interleukin[IL]-2, interferon[IFN]-γ) and Th2 cytokines (IL-4, IL-10) in the cell culture supernatant, and real-time quantitative RCR (qRT-PCR) to determine the mRNA expression of T-bet (a Th1 transcription factor) and GATA3 (a Th2 transcription factor) . Statistical analysis was carried out by using t test. Results According to the MTT assay results, EGCG at a non-toxic concentration of 60 μmol/L was chosen for subsequent experiments. Compared with the control group, the EGCG group showed significantly decreased number of Th1 cells (t = 3.43, P = 0.026) , increased number of Th2 cells (t = 6.68, P = 0.026) , and decreased Th1/Th2 ratio (P < 0.05) . The levels of IL-2 and IFN-γ in the culture supernatant of PBMC were both significantly lower in the EGCG group (824.45 ± 101.21 ng/L, 1 623.62 ± 185.56 ng/L respectively) than in the control group (1 568.32 ± 196.45 ng/L, 3 287.63 ± 235.54 ng/L respectively) , while the levels of IL-4 and IL-10 were significantly higher in the EGCG group (389.48 ± 46.63 ng/L, 285.95 ± 53.28 ng/L respectively) than in the control group (225.38 ± 26.92 ng/L, 165.46 ± 32.25 ng/L respectively) . Compared with the control group, the EGCG group showed significantly decreased T-bet mRNA expression (t = 11.99, P < 0.001) , but increased GATA3 mRNA expression (t = 18.62, P < 0.001) . Conclusion EGCG can reduce the number of Th1 cells, inhibit the production of Th1 cytokines and transcription factors, and increase the number of Th2 cells and the production of Th2 cytokines and transcription factors, followed by the modulation of Th1/Th2 immune imbalance. Key words: Psoriasis; Th1 cells; Th2 cells; Interferon-gamma; Interleukins; Transcription factor; Epigallocatechin gallate

Comparison of breast cancer cell culture models and drug sensitivity study

Objective A three-dimensional (3D) culture model of human breast cancer MCF-7 cells was constructed and compared with a two-dimensional (2D) culture model to observe its drug sensitivity to doxorubicin. Methods A 2D culture model was established by using a planar culture plate, and a 3D culture model was established using an ultra-low adsorption culture plate. The morphology and growth of MCF-7 cells in the two culture models were observed under microscope. Two culture models were determined by immunohistochemistry. Expression of B cell lymphoma/leukemia-2 (bcl-2), proliferation cell nuclear antigen (Ki-67) and Permeability glycoprotein (P-gp) in cells; cell proliferation was detected by methyl thiazol tetrazolium (MTT) assay, and drug sensitivity of MCF-7 cells to doxorubicin under different culture conditions was compared; reverse transcriptase-polymerase chain reaction (RT-PCR) The gene expression levels of P-gp and CD44 were detected, and the protein expression levels of P-gp and CD44 were detected by Western blotting. Differences between groups were analyzed and compared using independent sample t-test. Results In the 3D model, MCF-7 cells aggregated and rapidly formed typical multicellular spheres; while in 2D culture, cells dispersed in adherent growth. The positive expression of bcl-2 and P-gp in the 3D group (0.159±0.127, 0.115±0.621) was significantly higher than that in the 2D group (0.144±0.151, 0.058±0.374, t=2.288, 2.500, P<0.05), Ki-67 expression. (0.034±0.175) was lower than 2D group (0.069±0.351, t=2.832, P<0.05). The proliferation inhibition rate of doxorubicin in each concentration of MCF-7 cells in 3D group (42.75±2.24, 49.21±4.80, 50.64±3.93, 51.40±4.13, 59.21±4.07, 60.64±3.08, 61.40±2.57) was significantly lower than that of 2D group. (50.37±4.02, 73.53±5.67, 64.10±3.89, 65.53±3.80, 74.10±4.14, 83.53±3.17, 84.10±3.15, t=3.701, 7.319, 5.443, 5.632, 5.734, 11.589, 12.487, P<0.05) And the proliferation inhibition rate of the two groups of cells increased significantly with the increase of drug concentration. The expression levels of P-gp and CD44 in the 3D group (2.085±0.235, 1.115±0.741) were significantly higher than those in the 2D group (1.408±0.158, 0.708±1.175, t=5.339, 4.795, P<0.05), protein level (0.432±). 0.197, 0.759±0.356) was also higher than the 2D group (0.154±0.094, 0.305±0.103, t=2.851, 2.737, P<0.05). Conclusion The tumor characteristics of MCF-7 cells in 3D culture were significantly different from those in 2D culture, and the drug sensitivity of the 3D group was higher, which provided an experimental basis for in vitro drug testing and drug resistance related mechanisms. Key words: Breast cancer; Three dimensional cell culture; Doxorubicin; Drug sensitivity

The expression and mechanism of trihistidine nucleotide binding protein 1 in esophageal carcinoma

Objective To study the expression and mechanism of trihistidine nucleotide binding protein 1 in esophageal carcinoma. Methods The Eca-109 esophageal cancer cell line was cultured in laboratory. The Hint1 vector was expressed by pLVX-IRES-ZsCreen1 lentivirus. After 293T cells were purified and concentrated, the Eca-109 cells were infected to obtain Eca-109 Hint1 group cells. To detect the expression of green fluorescent protein (CFP) in two groups, to detect whether the transfection was successful or not, to detect the expression of Hint1 protein by Western blotting method, detection of invasiveness in different groups of cells by Transwell invasion assay, to detect the cell cycle by flow cytometry, and to detect the cell growth by methyl thiazol tetrazolium (MTT) method. Results The average expression rate of fluorescent protein in Eca-109 Hint1 group was (68.26±3.12)% (t=8.250, P<0.01). These results indicated that the transfection of human esophageal cancer cells infected with Hint1 vector virus was successful. The expression of Hint1 in Eca-109 Hint1 group was (1.86±0.02), and that of Hint1 in Eca-109 group was (0.61±0.01) by Western blot assay (t=5.529, P<0.05). The invasiveness of Eca-109Hint1+ group was (49.36±3.02), while that of Eca-109 group was (114.11±10.01). The invasiveness of Eca-109Hint1+ group was significantly lower than that of Eca-109 group (t=8.988, P<0.05). The cell cycle of Eca-109 group was (50.31±0.41), and that of Eca-109 Hint1 group was (41.24±0.62). On the third day of culture, the absorbance of cells in Eca-109 Hint1 group was (0.82±0.01) and that in Eca-109 group was (0.57±0.02). The results showed that the absorbance value of Eca-109 Hint1 group was significantly higher than that of Eca-109 group (t=6.527, P<0.05). Conclusion The expression of tri-histidine nucleotides binding protein-1 in esophageal carcinoma resulted in the increase of cell cycle and cell viability, which could inhibit the growth of esophageal cancer cells. Key words: Trihistidine nucleotide binding protein 1; Esophageal carcinoma; Cell expression and inhibition

Impact and mechanism of PMs-3 to epithelial mesenchymal transformation of non-small cell lung cancer cell line A549

Objective To explore Impact and mechanism of PMs-3 to epithelial mesenchymal transformation (EMT) of non-small cell lung cancer (NSCLC) cell line A549. Methods Nanocomposite structure synthetized by our team was utilized on human NSCLC cell line A549 and human normal pulmonary epithelial cell line BEAS-2B with concentrations of 0, 20, 40, 60, 80, 100, 120 nmol/L for 48 h. Methyl thiazol tetrazolium (MTT) assay was employed to detect the effect of PMs-3 on A549 cells. Wound assay and Transwell chamber assay were used to test the migration and invasion of PMs-3 on A549 cells. EMT related genes were detected with real time PCR (RT-qPCR) and Western blotting. Results Results of MTT assay detected that PMs-3 could inhibit activity of A549 cells significantly, and inhibition rate was (3.203±1.222)% (0 nmol/L), (11.958±4.000)% (20 nmol/L), (25.348±7.710)% (40 nmol/L), (34.775±9.155)% (60 nmol/L), (41.163±10.988)% (80 nmol/L), (50.430±9.743)% (100 nmol/L), (51.895±6.161)% (120 nmol/L), which showed a dose-dependent manner (F=35.451, P 0.05). Conclusion PMs-3 could inhibit NSCLC cells via regulating EMT. Key words: Non-small cell lung cancer; PMs-3; Epithelial mesenchymal transformation; Drug treatment

Effect of peroxiredoxin 1 on apoptosis and autophagy of human gastric cancer cells and possible mechanism

Objective To investigate the effect and molecular mechanism of peroxiredoxin 1 (PRDX1) in human gastric cancer cell lines. Methods The expression of PRDX1 was detected in gastric cancer cell lines SGC7901, AGS and the normal gastric mucosa cell line GES-1 with real-time quantitative polymerase chain reaction (Real-time PCR). Small interfering RNA to PRDX1 was synthetized for the transfection with lentivirus vector of LV-PRDX1-small interfering RNA (siRNA), which was transfected into AGS. Methyl thiazol tetrazolium (MTT) assay was used to test activity of cells. Flow cytometry (FCM) was used to detect the apoptosis rate and mitochondrial trans-membrane potential. Real-time PCR and Western blotting were used to detect the expression of mRNAs and proteins associated with apoptosis and autophagy. Results The expression of PRDX1 mRNA (1.049±0.124, 1.597±0.103) and proteins (0.873±0.135, 1.083±0.204) in SGC7901, AGS cells was significantly higher than that in GES-1 cells (mRNA: 0.451±0.060, and protein: 0.305±0.022), and the difference was significant (F=200.162, 48.432, P 0.05), and the expression of Beclin-1 and LC-3Ⅱ/LC-3Ⅰ increased significantly (F=30.332, 24.260, P<0.01). Conclusion PRDX1 was up-regulated in gastric cancer cells, and inhibition of PRDX1 can regulate apoptosis and autophagy via regulating apoptosis and autophagy associated genes. Key words: Gastric cancer; Peroxiredoxin1; Autophagy; Target gene

Expression and biological function of long chain non coding RNA-HOX transcript antisense RNA in colorectal cancer

Objective To evaluate the long intervening noncoding RNA (LincRNA)-HOX transcript antisense RNA (HOTAIR) expression in colorectal cancer and its biology function of proliferation and metastasis in human colorectal cancer cell line LoVo. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was applied to analysis the expression of HOTARI in paired colorectal cancer tissues and para-cancer normal colorectal tissues. And the HOTAIR was knocked down by small interfering RNA (siRNA) in LoVo cells. Methyl thiazol tetrazolium (MTT), wound healing and transwell assay were applied to evaluated the cell proliferation and invasion ability. Results HOTAIR expression were 7.96±4.15 and 4.47±2.70 for cancer tissue and para-cancer normal colorectal tissue respectively with statistical differenc (t=4.520, P 0.05). Conclusion HOTAIR was high expressed in human colorectal cancer tissue and down-regulation of HOTAIR in colorectal cancer can promote cell migration/invasion. Key words: HOX transcript antisense RNA; Colorectal cancer; Proliferation; Invasion; Migration

Effect of macrophage capping protein expression on proliferation and migration of prostate cancer cells

Objective To investigate the expression and explore the biological function of macrophage capping protein (CapG) in prostate cancer (PCa). Methods Immunohistochemistry was used to detect the expression of CapG in 115 cases of prostate cancer and 42 cases of adjacent normal prostate in the Second Affiliated Hospital of Wenzhou Medical University from January 2013 to June 2018. The correlation between CapG expression and clinicopathological features in PCa was evaluated by statistical methods. The expression of CapG in 3 prostate cancer tissues was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting. CapG expression was knocked down by small interfering RNA (siRNA), and the effect of CapG on proliferation and migration of human prostate cancer cells was detected by methyl thiazol tetrazolium (MTT) (5 g/L) and Transwell chamber experiments. Results The positive rates of CapG expression in prostate cancer and adjacent normal prostate tissues were 55.6% and 16.7%, respectively, and the difference was statistically significant (χ2=18.879, P<0.05); CapG expression was significantly correlated with prostate specific antigen (PSA) level, Gleason score, T stage and lymph node metastasis of prostate cancer (P<0.05). Down-regulation of CapG significantly inhibited the proliferation and migration of PC3 and DU145 cells (P<0.05). Conclusion CapG is highly expressed in prostate cancer and affects the proliferation and migration of prostate cancer cells. Key words: Prostate cancer; Macrophage capping protein; Proliferation; Metastasis

Knockdown of mediator complex subunit 19 suppresses the proliferation in prostate cancer LNCaP cells

Objective To study the role of mediator complex subunit 19 (Med 19) in prostate cancer by analyzing the effects of lentivirus-mediated suppression of Med 19 expression on LNCaP prostate cancer cells in vitro and explore the mechanism of proliferation. Methods The lentivirus vectors containing small interfering RNA (siRNA) targeting Med 19 gene were constructed and transfected to LNCaP cells. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting analysis were used to detect the Med 19 expression in the siRNA group (LNCaP-Med 19-siRNA cells)and the control group (LNCaP-Med 19-scRNA cells) at 72 h after the transfection. The influence of Med 19 on the proliferation of prostate cancer cells were assessed using methyl thiazol tetrazolium (MTT), Soft Agar Assay. Cell cycle was analyzed with flow cytometry assay. Results Med 19 relative mRNA level and Med19 protein expressing in the siRNA group were significantly inhibited (t=-69.1413, P<0.01). Compared with the control group, the siRNA group displayed a significant cell proliferation defect by MTT and the number of colonies (8.61±0.51) was significant decreased (t=-11.948, P<0.01; t=-98.775, P<0.01). The siRNA group displayed an increased proportion of cells (67.29±1.70)% in the G0/G1 phase compared with the control group [(56.23±1.46)%, t=-21.906, P<0.01]. Conclusion Knockdown of Med 19 gene increased the number of human prostate cancer LNCaP cells in G0/G1 phase, resulting in significantly inhibited proliferation and growth of prostate cancer. Key words: Mediator complex subunit 19; Prostate carcinoma; Proliferation