S Phase Research Articles

Dual Delivery of HNF4α and Cisplatin by Mesoporous Silica Nanoparticles Inhibits Cancer Pluripotency and Tumorigenicity in Hepatoma-Derived CD133-Expressing Stem Cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly malignancies characterized by high rate of recurrence. Tumor recurrence is often attributed to the presence of a subpopulation of cells with stem cell properties, referred to as cancer stem cells (CSCs). Traditionally, cancer therapies target the entire bulk of tumor cells; however, they are poorly effective against CSCs, characterized by higher drug resistance. Therefore, approaches targeting CSCs may be required in addition to conventional chemotherapy to prevent tumor recurrence. In this study, we investigated an approach to target HCC by combining the conventional chemotherapeutic drug, cisplatin, to target the bulk of tumor cells, and differentiation therapy by delivering the gene encoding HNF4α, an important regulator of hepatocyte differentiation, to target CSCs. We used the Huh7 cell line as an in vitro model of HCC, which is characterized by a high proportion of CD133-expressing CSCs. By using flow cytometry, we separated CD133+ and CD133- Huh7 cell subpopulations and have shown that the former has highly pronounced in vivo tumorigenic capacity in contrast to the latter, which could not generate tumors in vivo. For the dual delivery of HNF4α-encoding plasmid and cisplatin, we used polyethyleneimine-modified mesoporous silica nanoparticles (PMSNs) as the nanocarriers. Here, we show that the treatment of CD133-expressing Huh7 cells with HNF4α-loaded PMSNs can suppress their proliferation rate, decrease the proportion of CSCs, downregulate stemness-associated genes, and increase the expression of mature hepatocyte-associated genes. At the same time, the treatment of Huh7 with PMSNs loaded with both HNF4α-encoding plasmid and cisplatin could block them in the S-phase of the cell cycle and cause apoptosis. In addition, dually loaded PMSNs were the most efficient formulation in suppressing tumor growth in vivo. To summarize, in this study, we tested the nanoparticle-based delivery system as both chemotherapy and gene-based therapy agents, which has great potential for development of effective treatment of HCC.

Construction of short hairpin RNA interfering milk fat globule-epidermal growth factor 8 gene lentiviral vector and its effect on inhibition of breast cancer cells

Objective To construct the lentiviral vector of milk fat globule-epidermal growth factor 8 (MFG-E8) short hairpin RNA (shRNA), and stably transfected the HCC1973 cell line to detect its interference efficiency and influence in breast cancer cell HCC1973. Methods The knockout gene fragment was synthesized and inserted into the shRNA lentiviral vector PLKO.1 with a cherry protein reporter. The constructed recombinant plasmids were transfected into 293T cells respectively. The lentiviruses were transfected into HCC1973 cells and stably transfected PLKO.1-MFG-E8 shRNA recombinant lentiviruses were obtained. The expression of MFG-E8 was detected by real-time PCR and Western blotting. The cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay and Flow cytometry was used to detect the cell cycle and apoptosis, Transwell was used to detect the invasion and migration of cells. Statisticalanalysis of data using the statistical product and service solutions 17.0 software. Results The constructed vector of PLKO.1-MFG-E8 shRNA significantly reduced the expression of MFG-E8 gene and protein in breast cancer cell HCC1973, the lentivirus titer was 3×108 PFU/ml. The proliferation of breast cancer cells was significantly suppressed (t=4.426, P<0.05), G0/G1 phase cells increased significantly with a decrease of cells in S phase (t=2.264, P<0.05), the ability of cell invasion and migration was significantly reduced (t=4.802, P<0.05), after the lentiviral infection. Conclusion The expression of MFG-E8 gene and protein by shRNA interference can inhibit the malignant biological behavior of breast cancer cells effectively, which indicates that MFG-E8 is involved in the invasion, metastasis and apoptosis of breast cancer during the process of breast carcinogenesis. Key words: Milk fat globule-epidermal growth factor 8 short hairpin RNA; Cell cycle; Cell proliferation; Cell poptosis; Invasion and metastasis

Effect of silencing troponin I3 gene expression on biological property of rat embryonic H9C2 cardiomyocytes

Objective To investigate the effect of silencing troponin I3 (Tnni3) gene expression on biological property of rat embryonic H9C2 cardiomyocytes. Methods The rat embryonic H9C2 cardiomyocytes were cultured and divided into 2 groups: control group transfected with negative control small interfering RNA (NC-siRNA group) and experimental group transfected with Tnni3 small interfering RNA (Tnni3-siRNA group). At 48 h, 72 h after transfection, the cells were collected, and real time quantitative polymerase chain reaction (qPCR)was used to detect the mRNA expressions of Tnni3 and Caspase-3, and Western blot was used to detect the protein expressions of Tnni3, Cyclin A1 and Cyclin B1.Annexin V-fluorescein isothiocyanate(FITC) apoptosis detection kit was used to analyze cell apoptosis.Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) solution and cell cycle was detected by flow cytometry. Results At 48 h post-transfection with Tnni3-siRNA, H9C2 cells exhibited a significant decrease in Tnni3 mRNA (0.27±0.05 vs. 1.00±0.00) and protein (0.18±0.03 vs. 1.00±0.00) compared with those transfected with NC-siRNA, and the differences were statistically significant (t=25.26, 47.40, all P<0.01). Apoptotic cells were observed in the NC-siRNA group and the Tnni3-siRNA group.At 72 h post-transfection, the percentage of apoptotic cells significantly increased in H9C2 cells transfected with Tnni3-siRNA [(11.30±1.85)% vs. (0.33±0.15)%] compared with those transfected with NC-siRNA, an increased expression of Caspase-3 mRNA was also observed in Tnni3-siRNA-transfected H9C2 cells (1.39±0.13 vs. 1.00±0.00), and the differences were statistically significant (t=10.24, 5.19, all P<0.01). Compared with NC-siRNA-transfected H9C2 cells, a time-dependent reduction in cell proliferation was observed in Tnni3-siRNA-transfected H9C2 cells (48 h: 0.32±0.06 vs. 0.46±0.03; 72 h: 0.31±0.01 vs. 0.63±0.04; 96 h: 0.36±0.01 vs 0.75±0.04), and the differences were statistically significant (t=3.62, 13.45, 16.39, all P<0.01). At 72 h post-transfection with Tnni3-siRNA, the percentage of G1 phase, S phase and G2 phase cells was (71.25±3.82)%, (18.28±2.78)% and (9.94±1.09)%, respectively.There was a significant increase in the proportion of G2 phase cells [(9.94±1.09)% vs. (4.54±0.99)%] in H9C2 cells transfected with Tnni3-siRNA compared with those transfected with NC-siRNA, an increased expression of Cyclin A1 protein (1.89±0.09 vs.1.00±0.00) and a decreased expression of Cyclin B1 protein (0.47±0.06 vs.1.00±0.00) were observed in Tnni3-siRNA-transfected H9C2 cells, respectively, and the differences were statistically significant (t=6.35, 17.12, 15.32, all P<0.01). Conclusions Silencing Tnni3 gene expression in rat embryonic H9C2 cardiomyocytes can induce cell apoptosis, suppress cell proliferation, and led to G2 cell cycle arrest. Key words: Troponin I3 gene; Cell apoptosis; Cell proliferation; Cell cycle

Effect of apoptosis and proliferation of endometrial cancer HEC-1B cell by siRNA down-regulating expression of Ezrin

Objective To observe the changes of cell cycle, apoptosis and proliferation of endometrial cancer cells after the expression and down-regulation of Ezrin in endometrial cancer cells and to explore whether Ezrin may be a candidate gene for targeted therapy. Methods Endometrial cancer cells were from Shanghai Institute of Cell Research, of Chinese Academy of Medical Sciences in February 2017 and divided into blank control group and siEzrin group according to the intervention methods.Western blot and qRT-PCR was used to detect the expression of Ezrin protein and mRNA in endometrial cell lines.Small interfering RNA(siRNA) was used to transfect HEC-1B cell and down-regulate Ezrin.Cell cycle and apoptosis were detected by flow cytometry.MTT assay was used to detect multiplication. Results Western blot showed that Ezrin protein was expressed in Ishikawa(31.742±5.832)、HEC-1A(16.326±3.135)、HEC-1B(17.636±4.426) and KLE(14.862±5.109) and qRT-PCR showed that mRNA was expressed in Ishikawa (2.513±0.725), HEC-1A (1.655±0.692), HEC-1B (3.237±0.411) and KLE (0.962±0.235) cell lines, and expressed highest in HEC-1B cells (F=6.173, P<0.05; F=7.042, P<0.05). Flow cytometry assay showed that compared with blank control group less cells stayed in G1 phase and G2 phase, more stayed in S phase (t=3.118, P<0.05; t=5.435, P<0.05; t=3.332, P<0.05). The apoptotic rate of HEC-1B cells increased from (9.84±2.37)% to (17.64±5.96)%(t=8.963, P<0.01) after Ezrin was down-regulated.MTT assay showed that the proliferation of HEC-1B cells in 72 h and 96 h siEzrin transfection group was lower than that in blank control group (t=3.209, P<0.05; t=3.726, P<0.05). Conclusion Down-regulating of Ezrin may promote more endometrial cancer cells stay in S phase and promote apoptosis, inhibit proliferation, Ezrin may become target candidate gene in target therapy. Key words: Endometrial cancer; Ezrin protein; Cell cycle; Apoptosis; Proliferation

PD-L2 generates a signal through the PD-1 co-receptor distinct from that of PD-L1

Abstract Immune checkpoint blockade of the PD-1/PD-L1/PD-L2 signalling axis yields potent anti-tumor responses by disinhibiting T cell effector function. Mouse and human data demonstrating an inhibitory role for PD-L1 has resulted in FDA approval of anti-PD-L1 antibodies; however, PD-L2 remains sparsely studied despite being the higher affinity of the two ligands for PD-1. Recent findings demonstrate that PD-L2 is widely expressed by human malignancies. PD-L1 and PD-L2 have also been shown to induce distinct conformational changes on engaging human PD-1. We utilized a Jurkat T cell based bioluminescent assay of T cell activation and PD-1 inhibition to determine the unique mechanism of action of human PD-L2. In this system, human PD-L2 generates a solely co-inhibitory signal, but at reduced inhibitory potential relative to PD-L1. We find that unlike murine PD-L2, human PD-L2 appears to have a dedicated co-inhibitory function as we find no significant engagement of the human homolog of its murine co-stimulatory partner RGMb. Reverse phase protein array analysis of human PD-L1 and PD-L2 engagement of PD-1 on Jurkat T cells revealed significant differences in downstream T cell signalling generated by each ligand. We also find that human PD-L1 and PD-L2 have disparate impacts on proliferation wherein PD-L2 supports a preferential arrest of T cells in the S-phase of the cell cycle. We observed that combination blockade of PD-L1 and PD-L2 improves on the blockade of PD-L1 alone resulting in increased production of IL-2 and IFNγ in primary human mixed lymphocyte reactions. Our findings address critical gaps in our knowledge about the basic function of PD-L2 and will inform future attempts to manipulate the PD-1 co-inhibitory circuit.

In yeast cells arrested at the early S-phase by hydroxyurea, rRNA gene promoters and chromatin are poised for transcription while rRNA synthesis is compromised

Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase that is used as a chemotherapeutic agent to treat a number of chronic diseases. Addition of HU to cell cultures causes reduction of the dNTP cellular pool below levels that are required for DNA replication. This trigger dividing cells to arrest in early S-phase of the cell cycle. Cell division hinges on ribosome biogenesis, which is tightly regulated by rRNA synthesis. Remarkably, HU represses the expression of some genes the products of which are required for rRNA maturation. To gain more information on the cellular response to HU, we employed the yeast Saccharomyces cerevisiae as model organism and analyzed the changing aspects of closed to open forms of rRNA gene chromatin during cell cycle arrest, the arrangement of RNA polymerase-I (RNAPI) on the open genes, the presence of RNAPI transcription-factors, transcription and rRNA maturation.The rRNA gene chromatin structure was analyzed by psoralen crosslinking and the distribution of RNAPI was investigated by chromatin endogenous cleavage. In HU arrested cells nearly all rRNA genes were in the open form of chromatin, but only a portion of them was engaged with RNAPI. Analyses by chromatin immuno-precipitation confirmed that the overall formation of transcription pre-initiation complexes remained unchanged, suggesting that the onset of rRNA gene activation was not significantly affected by HU. Moreover, the in vitro transcription run-on assay indicated that RNAPI retained most of its transcription elongation activity. However, in HU treated cells, we found that: (1) RNAPI accumulated next to the 5′-end of rRNA genes; (2) considerably less rRNA filaments were observed in electron micrographs of rDNA transcription units; and (3) rRNA maturation was compromised.It is established that HU inhibition of ribonucleotide reductase holds back DNA replication. This study indicates a hitherto unexplored cellular response to HU, namely altered rRNA synthesis, which could participate to hamper cell division.

Anthecotulide Sesquiterpene Lactone Exhibits Selective Anticancer Effects in Human Malignant Melanoma Cells by Activating Apoptotic and Autophagic Pathways, S-Phase Cell Cycle Arrest, Caspase Activation, and Inhibition of NF-κB Signalling Pathway.

BackgroundWe examined the anticancer potential of anthecotulide against SK-MEL-24 malignant melanoma cells. The apoptotic and autophagic effects of anthecotulide were also investigated.Material/MethodsThe cell viability of SK-MEL-24 human malignant melanoma cells was evaluated by WST-1 assay. Fluorescence microscopy using acridine orange and ethidium bromide staining, as well as Western blot analysis, were used to study apoptotic effects induced by anthecotulide. Autophagy was assessed by Western blot analysis and fluorescence microscopy. Effects of anthecotulide on cell cycle progression were analyzed by flow cytometry.ResultsThe results revealed that anthecotulide exerts significant growth-inhibitory effects on SK-MEL-24 cells. The IC50 of anthecotulide against the SK-MEL-24 cells was found to be 10 μM. However, the anticancer effects against the normal cells were minimal (IC50; 100 μM). Investigation of the underlying mechanism revealed that anthecotulide prompts apoptotic cell death of the SK-MEL-24 cells, which was linked with increased expression of Bax and decreased expression of Bcl-2. It also triggered concentration-dependent activation of caspase 3 and 9. Anthecotulide induced autophagy in the SK-MEL-24 cells, which was associated with upregulation of LC3 II and Beclin-1 expression. Anthecotulide also halted the SK-MEL-24 cells at S-phase of the cell cycle and downregulated the expression of Cyclin B1. However, the expression of p27 was upregulated.ConclusionsThese results indicate anthecotulide is a potent lead molecule for the treatment of melanoma. In vivo and other related experiments are warranted to further assess this promising drug candidate.

Relationship between microRNA-95 and proliferation, apoptosis of gastric cancer cells and underlying mechanism

Objective To investigate the role of microRNA (miRNA, miR)-95 in the proliferation and apoptosis of gastric cancer cells and the possible mechanism. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect the expression of miR-95 in tumor tissues and gastric mucosal tissues from 70 patients with gastric cancer. The patient’s clinicopathological features were recorded and the relationship between miR-95 and these clinicopathologic features was analyzed. The human gastric cancer cell line BGC823 was transfected with anti-miR-95 and the cell viability, cell cycle and apoptosis rate were examined before and after transfection. Simultaneously, Real-time PCR and Western blotting were used to detect the expression of proliferation- and apoptosis-related genes proliferating cell nuclear antigen (PCNA), Cyclin D1, p21, Survivin, Livin and Bad. Results The miR-95 level in gastric cancer was significantly higher than that in gastric mucosal tissues (t=-3.399, P<0.05). Its expression was related to the degree of tumor differentiation and lymph node metastasis. The level of miR-95 in poorly differentiated tumors and in tumors with lymph node metastasis was significantly higher than that in well-differentiated tumors and without lymph node metastasis (t=-3.021, P<0.05; t=3.348, P<0.05). In gastric cancer cell lines AGS, SGC7901, MKN45 and BGC823, and gastric epithelial cell line GES-1, the miR-95 expression was higher in gastric cancer cell lines than GES-1, and highest in BGC823 cells (F=5.998, P<0.05). After transfection of anti-miR-95, the activity of the cells significantly decreased, and the ratio of G0/G1 phase increased (F=39.758, P<0.01), but the ratio of G2/M (F=10.349, P<0.05) and S phases decreased (F=15.118, P<0.01); apoptosis rate increased significantly after anti-miR-95 transfection (F=25.182, P<0.01). The mRNA and protein expression levels of PCNA, Cyclin D1 and Survivin significantly decreased, and those of p21 and Bad increased (P<0.05). Conclusion The up-regulated expression of miR-95 in gastric cancer is associated with tumor differentiation and lymph node metastasis, which may be involved in the progression of gastric cancer by regulating the expression of some proliferative and apoptotic genes. Key words: Gastric cancer; MicroRNA-95; Proliferation; Apoptosis; Clinicopathological features

Effect of dehydrocostus lactone on the proliferation of chronic myeloid leukemia K562 cells and its mechanism

Objective To explore the effect of dehydrocostus lactone on the proliferation of chronic myeloid leukemia cell line K562 and its mechanism. Methods K562 cells in logarithmic growth phase were treated with different concentrations of dehydrocostus lactone. Cell morphology was observed by Wright-Giemsa staining. Cell proliferation was detected by CCK-8 method. Cell cycle, apoptosis and the expressions of cell surface differentiation antigen CD14 and CD11b were detected by flow cytometry. JAK-STAT pathway and the expressions of apoptosis and cell cycle related proteins were detected by Western blot. Results Compared with the control group, the proliferation of K562 cells could be inhibited after treatment with different concentrations (4.0, 6.0, 8.0, 10.0 and 12.0 μmol/L) of dehydrocostus lactone for 24 h, and the difference was statistically significant (F = 109.510, P < 0.05). After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h, the apoptosis rates of K562 cells were (16.1±3.8)% and (29.6±4.3)%, which were higher than that of the control group [(3.1±0.5)%] (F = 83.255, P < 0.05). After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h, the proportion of G2/M phase cells in K562 cells were (17.0±3.2)% and (28.8±3.9)%, which were higher than that of the control group [(9.1±2.3)%] (F = 161.598, P < 0.05); the proportion of S phase cells in K562 cells were (48.1±3.9)% and (61.0±5.4)%, which were higher than that of the control group [(39.6±3.6)%] (F = 192.356, P < 0.05). After treatment with 2.5 and 5.0 μmol/L dehydrocostus lactone for 72 h, the expression rates of CD14 in K562 cells were (28.6±3.9)% and (41.1±4.4)%, which were higher than that in the control group [(3.1±0.5)%] (F = 132.811, P < 0.05); the expression rates of CD11b in K562 cells were (42.4±5.0)% and (61.2±5.7)%, which were higher than that in the control group [(4.2±1.1)%] (F = 179.553, P < 0.05). Dehydrocostus lactone could decrease the expressions of JAK2, STAT5, cyclin E, CDK2, cyclin A, CDC25C, cyclin B1, CDK1 and bcl-2 proteins, and up-regulate the expressions of p21 and bax proteins. Conclusion Dehydrocostus lactone can inhibit the proliferation of chronic myeloid leukemia K562 cells, which may be achieved by cell cycle arrest, induction of apoptosis and differentiation. Key words: Dehydrocostus lactone; Cell cycle; Apoptosis; Differentiation; Leukemia

Effect of microRNA-153 targeting phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway on proliferation in acute lymphoblastic leukemia cell

Objective To investigate the effect of microRNA (miRNA, miR)-153 on the proliferation and apoptosis of in human acute lymphoblastic leukemia (ALL) cell line Jurkat cells by phosphatidylinositol 3 kinase (PI3K)/serine-threonine protein kinase (Akt) pathway. Methods After miR-153 mimics (miR-153 mimics group) and miR-153 inhibitor (miR-153 inhibitor group) were transfected into Jurkat cells, cell proliferation, cycle and apoptosis. was detected; the expression of miR-153, PI3K and Akt mRNA and protein was measured; whether PI3K was the target gene of miR-153. was confirmed. Results Compared with the negative control group, the expression of miR-153 in the miR-153 mimics group was significantly increased by approximately 10-fold (t=39.596, P<0.01), and the expression of miR-153 was significantly decreased in the miR-153 inhibitor group (t=14.651, P<0.01). The activity of the cells in the miR-153 mimics group was significantly decreased (t=10.931, P<0.01), and the activity of the miR-153 inhibitor group was significantly increased (t=7.640, P<0.01). The expression of PI3K total protein, PI3K mRNA and p-Akt in the miR-153 mimics group was significantly decreased (t=7.161, P<0.01; t=6.684, P<0.01; t=10.769, P<0.01), and the expression of PI3K total protein, PI3K mRNA and p-Akt in the miR-153 inhibitor group was significantly increased (t=3.998, P<0.05; t=8.902, P<0.01; t=7.078, P<0.01). The proportion of G0/G1 phase cells increased significantly, while the S phase and G2/M phasecells decreased significantly (t=3.786, P<0.05; t=3.012, P<0.05; t=4.948, P<0.01) in the miR-153 mimics group; the G0/G1 phase cells decreased significantly, and the proportion of cells in S phase and G2/M phase increased significantly (t=5.356, P<0.01; t=3.055, P<0.05; t=3.893, P<0.05) in the miR-153 inhibitor group. The apoptosis rate of the miR-153 mimics group was significantly increased (t=4.673, P<0.05), while the apoptotic rate of the miR-153 inhibitor group was significant decline (t=5.618, P<0.01). Compared to the empty plasmid group, the relative luciferase activity of 3’untranslated regions (3’UTR) plasmid group was significantly reduced (t=7.925, P<0.01). Conclusion MiR-153 can directly target PI3K/Akt signaling pathway and regulate Jurkat cell proliferation and apoptosis. Key words: Acute lymphoblastic leukemia; MicroRNA-153; Phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway

Effect of alanine-glyoxylate aminotransferase 2-like 1 on proliferation, apoptosis and cell-division cycle of human hepatoma cell lines 97H and HCCLM3

Objective To investigate the effect of alanine-glyoxylate aminotransferase 2-like 1 (AGXT2L1) on the proliferation, apoptosis and cell-division cycle of hepatoma cells. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression of AGXT2L1 in 97H, HCCLM3 and LO2 cells. The siRNA targeting AGXT2L1 was designed, synthetized and transfected into 97H and HCCLM3 cells to construct AGXT2L1 knockdown group, and the efficiency of knockdown was validated by RT-qPCR. The cell proliferation of each group at 0, 12, 24, 48, 72 and 96 h was tested by cell counting kit-8 (CCK-8) assay. Cell cycle assay was applied to detect the distribution of cells in G1, S and G2 phases. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis rate of cells in each group. Results The expression of AGXT2L1 in 97H and HCCLM3 cells was significantly higher than that in normal liver cells (1 350.04±91.36, 1 710.64±92.34, t97H=25.574, P<0.01; tHCCLM3=32.068, P<0.01). The AGXT2L1 expression level in 97H and HCCLM3 cells was decreased significantly after transfection with AGXT2L1-siRNA (0.19±0.04 vs. 1.10±0.04, t97H =-28.722, P<0.01; 0.20±0.01 vs. 0.93±0.09, tHCCLM3=-13.887, P<0.01). The CCK-8 assay showed that proliferation rate of 97H and HCCLM3 cells decreased significantly after knocking down AGXT2L1 (0.71±0.01 vs. 0.95±0.12, t97H=-31.243, P<0.01; 0.67±0.13 vs. 0.87±0.17, tHCCLM3=-17.083, P<0.01). After transfection of AGXT2L1-siRAN into cells, the cell cycle assay showed that the ratio of S phase cells was lower than that in the control group [(13.45±1.20)% vs. (18.73±0.28)%, t97H=-7.451, P<0.01; (15.26±0.52)% vs. (20.45±1.08)%, tHCCLM3=-7.483, P<0.01], and the ratio of G2 phase cells was lower than that in the control group [(16.90±0.54)% vs. (19.99±0.54)%, t97H=-6.992, P<0.01; (16.23±0.28)% vs. (23.36±0.39)%, tHCCLM3=-25.720, P<0.01]. The knockdown of AGXT2L1 could inhibit the transformation of 97H and HCCLM3 cells from G1 to S phase and inhibit the proliferation of human hepatoma cells. Compared with the control group, after the knockdown of AGXT2L1, the apoptosis rate of 97H and HCCLM3 cells was increased [(13.06±1.86)% vs. (1.06±0.75)%, t97H=14.673, P<0.01; (14.41±2.21)% vs. (0.80±0.32)%, tHCCLM3=14.947, P<0.01]. Conclusion The knockdown of AGXT2L1 can inhibit the proliferation of 97H and HCCLM3 cells and promote apoptosis. Key words: Carcinoma, hepatocellular; Alanine-glyoxylate aminotransferase 2-like 1; Proliferation; Apoptosis

Effects of tanshinone II A on proliferation, apoptosis and cell cycle distribution of osteoblasts MC3T3-E1

Objective Objective to investigate the effects of tanshinone Ⅱ A on proliferation, apoptosis and cell cycle distribution of osteoblasts MC3T3-E1. Methods The osteoblasts MC3T3-E1 were divided into four groups: blank control group, low concentration group (5×10-3 mg/L), medium concentration group (5×10-2 mg/L) and high concentration group (5×10-1 mg/L). The control group was treated with routine cell culture medium without any drugs, while the low concentration group, the middle concentration group and the high concentration group were treated with tanshinone Ⅱ A to culture the cells, methyl thiazol tetrazolium (MTT) assay was used to detect the proliferation of thyroid cancer cells, and flow cytometry was used to detect the cell cycle and apoptosis. Results The proliferation rate of osteoblasts MC3T3-E1 in low, middle and high concentration group was significantly higher than that in blank control group at different time points, and that in high concentration group was significantly higher than that in low concentration group at different time points (F=2.140, 2.130, 3.120, P< 0.05). The apoptosis rate of osteoblasts MC3T3-E1 in low, middle and high concentration groups was significantly lower than that in blank control group at different time points, and that in high concentration group was significantly lower than that in low concentration group at different time points (F=2.390, 2.990, 3.020, P< 0.05). The proportion of cells in G0/G1 phase in low, middle and high concentration group was significantly higher than that in blank control group at different time, and the proportion of cells in S phase and G2/M phase was significantly lower than that in blank control group, andthe proportion of cells in G2/M phase was significantly lower than that in low concentration group and medium concentration group (F=2.310, 2.700, 2.340, 2.170, 4.920, 4.420, 2.840, 2.120, 4.180, P<0.05). Conclusion Tanshinone Ⅱ A can promote the proliferation of osteoblasts MC3T3-E1, inhibit the apoptosis of osteoblasts MC3T3-E1, and significantly improve the cycle distribution of osteoblasts MC3T3-E1. Key words: Tanshinone Ⅱ A; Osteoblast MC3T3-E1; Proliferation; Apoptosis

Cell Cycle Analysis Using In Vivo Staining of DNA-Synthesizing Cells.

The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters.In the current step-by-step protocol, we present` two approaches optimized for in vivo study of the cell cycle and the limitations that such approaches imply: (1) determination of the cell flow rate into the G2-phase by dual EdU/BrdU DNA-labelling method and (2) determination of the outflow of DNA-labelled cells arising from the mitosis.

Effects of resveratrol on proliferation and invasion of lung cancer 95D cells

Objective To study the effects of resveratrol on the proliferation and invasion capacity of human lung adenocarcinoma 95D cells. Methods Human lung adenocarcinoma 95D cells were treated with 0, 10, 20, 40, 80 μmol/L resveratrol and treated with 0 μmol/L as the control group. The proliferation level of 95D cells was measured by methyl thiazolyl tetrazolium (MTT). Cell cycle and apoptosis were detected by flow cytometry. Cell adhesion rate was determined by in vitro adhesion test. Cell invasiveness was measured by Transwell chamber. The expressions of matrix metalloproteinase-2 (MMP-2) and tissue inhibitior of metalloproteinase-2 (TIMP-2) were determined by fluorescent immunocytochemistry. Results When the 95D cells were treated with resveratrol for 72 h, the cell proliferation rates in groups treated with 0, 10, 20, 40, 80 μmol/L resveratrol were 100%, (82.23±0.33)%, (62.45±0.27)%, (49.89±0.43)%, (45.11±0.35)% respectively, with a significant difference (F=87.830, P=0.002). The proliferation rates of 95D cells in the 10, 20, 40, 80 μmol/L resveratrol groups were significantly inhibited compared with the control group (P=0.017, P<0.001, P<0.001, P<0.001). When the 95D cells were treated for 48 h, the apoptosis rates of cells in each group were 0, (34.90±0.91)%, (41.33±0.13)%, (45.47±0.87)%, (59.46±0.59)% respectively, with a significant difference (F=21.032, P=0.002). The apoptosis rates of 95D cells in the 10, 20, 40, 80 μmol/L resveratrol groups were significantly increased compared with the control group (P=0.001, P<0.001, P<0.001, P<0.001). When the 95D cells were treated for 48 h, the S phase cell percentages in each group were (18.12±0.62)%, (38.33±0.62)%, (54.15±0.74)%, (44.85±0.82)%, (50.01±0.35)% respectively, with a significant difference (F=104.156, P=0.001). The S phase cell percentages in the 10, 20, 40, 80 μmol/L resveratrol groups were significantly higher compared with the control group (P=0.001, P<0.001, P<0.001, P<0.001). When the 95D cells were treated for 24 h, the cell adhesion rates in each group were 100%, (87.41±0.02)%, (84.32±0.03)%, (68.23±0.04)%, (63.01±0.02)% respectively, with a significant difference (F=13.760, P<0.001). The cell adhesion rates in the 20, 40, 80 μmol/L resveratrol groups were significantly inhibited compared with the control group (P=0.035, P<0.001, P<0.001), while the 10 μmol/L resveratrol group had no significant difference compared with the control group (P=0.058). When the 95D cells were treated for 24 h, the cell invasion rates in each group were 100%, (97.01±0.03)%, (74.89±0.07)%, (34.07±0.03)%, (14.65±0.02)% respectively, with a significant difference (F=39.382, P=0.001). The cell invasion rates in the 20, 40, 80 μmol/L groups were significantly inhibited compared with the control group (P=0.012, P<0.001, P<0.001), while the 10 μmol/L resveratrol group had no significant difference compared with the control group (P=0.881). When the 95D cells were treated for 48 h, MMP-2 protein expression was decreased in the 20 mol/L group, while TIMP-2 protein expression was increased compared with the control group. Conclusion Resveratrol can inhibit the proliferation of human lung adenocarcinoma 95D cells and has effect of anti-tumor cell invasiveness, and its mechanism may involve two-way regulation of MMP-2/TIMP-2 expression. Key words: Lung neoplasms; Cell proliferation; Cell adhesion; Neoplasm invasiveness; Resveratrol

Effect of over-expression of microRNA-637 on biological behaviors of thyroid cancer cells

Objective To observe the effect of over-expression of microRNA (miRNA, miR)-637 on the biological behaviors of thyroid cancer cell line TPC-1. Methods The over-expressed miR-637 plasmids (experimental group) and blank plasmid (negative control group) were transferred into TPC-1 cells respectively by lentivirus transfection. Cell proliferation was observed by cell counting kit-8 (CCK-8), cell apoptosis rate and cycle distribution were analyzed by flow cytometry, and scratch test and Transwell invasion test were used to evaluate the cell proliferation, cell migration and invasiveness. Results The growth curve of TPC-1 cells in the experimental group was relatively slow, and the cell growth rate was significantly lower than that in the negative control group (t=5.840, P=0.028). As compared with the negative control group [(12.50±1.71)%, the apoptosis rate of TPC-1 cells in the experimental group [(20.75±0.93)%] was significantly increased (t=19.674, P=0.003). The ratio of cell apoptosis was significantly higher in the experimental group than that in the negative control group. As compared with the experimental group, the percentage of cells in S phase was decreased to 23.35% after 48 h and that in G2/M phase decreased to 17.58%. Scratch test and Transwell invasion test showed that the number of migrating cells in the experimental group decreased significantly as compared with the negative control group (t=-26.186, P=0.001). Conclusion The over-expression of miR-637 can reduce the proliferation, apoptosis, migration and invasion of TPC-1 cells. Key words: Thyroid cancer; MicroRNA-637; Proliferation; Apoptosis; Migration; Invasion

Biosynthesis of histone messenger RNA employs a specific 3' end endonuclease.

Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo β-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target.

Construction of prokaryotic expression vector with human esophageal cancer related gene 4 and the effect of recombinant protein on the cell cycle of EC-18 in esophageal carcinoma

Objective To construct the prokaryotic expression vector of human esophageal cancer related gene 4 (ECRG4), to purify the recombinant ECRG4 protein and to verify the biological function of the recombinant ECRG4 protein. Methods DNA recombination technology was utilized to construct the ECRG4 protein prokaryotic expression vector. The recombinant ECRG4 protein was purified with the transformation of escherichia coli. Then the purity of the recombinant ECRG4 protein was examined by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Furthermore, esophageal cancer EC-18 cell line was treated by recombinant ECRG4 protein (10 μg/ml) for phosphate buffer (PBS) 48 h respectively, and the tumor cell cycle change was examined by using flow cytometry. Results SDS-PAGE analysis showed that the high purity of recombinant ECRG4 protein was obtained and the ECRG4 protein prokaryotic expression vector was successfully constructed. The cell proportion of G1 phase in PBS control group was lower than that in the recombinant ECRG4 protein group [(60.4±2.1) % vs. (71.6±1.8) %; t = 25.695, P = 0.002]. The cell proportion of S phase in PBS control group was higher than that in the recombinant ECRG4 protein group [(24.6±1.4) % vs. (16.5±1.0) %; t = 36.905, P = 0.001]. The cell proportion of G2/M phase in PBS control group was higher than that in the recombinant ECRG4 protein group[(15.0±1.1) % vs. (11.9±0.8) %; t = 6.471, P = 0.023], which indicated that the recombinant ECRG4 protein could induce the G1 phase arrest of EC-18 cells. Conclusion The ECRG4 protein prokaryotic expression vector is successfully constructed. And the recombinant ECRG4 protein has an active biological function in esophageal carcinoma. Key words: Esophageal cancer related gene 4; Recombinant protein; Cell cycle

HERC2 Facilitates BLM and WRN Helicase Complex Interaction with RPA to Suppress G-Quadruplex DNA.

BLM and WRN are RecQ DNA helicasesessential for genomic stability. Here, we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacted with BLM, WRN, and replication protein A (RPA) complexes during the S-phase of the cell cycle. Depletion of HERC2 dissociated RPA from BLM and WRN complexes and significantly increased G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4-suppressing function. In vitro, HERC2 released RPA onto single-stranded DNA (ssDNA) rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 inhibited ubiquitination of RPA2, caused RPA accumulation in the helicase complexes, and increased G4, indicating an essential role for E3 activity in the suppression of G4. Both depletion of HERC2 and inactivation of E3 sensitized cells to the G4-interacting compounds telomestatin and pyridostatin. Overall, these results indicate that HERC2 is a master regulator of G4 suppression that affects the sensitivity of cells to G4 stabilizers. Given that HERC2 expression is frequently reduced in many types of cancers, G4 accumulation as a result of HERC2 deficiency may provide a therapeutic target for G4 stabilizers.Significance: HERC2 is revealed as a master regulator of G-quadruplex, a DNA secondary structure that triggers genomic instability and may serve as a potential molecular target in cancer therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6371/F1.large.jpg Cancer Res; 78(22); 6371-85. ©2018 AACR.

Effects of parthenolide on proliferation inhibiting and cell cycle of human renal cancer 786-O cells

Objective To explore the effects of parthenolide on growth inhibition, cell aoptosis and molecular mechanism of human 786-O kidney cancer cells. Methods The inhibitory effect of parthenolide on human786-O cells was determined by MTT assay.The cell apoptosis and the cell cycle were analyzed by flow cytometry.Bax and the gene change of Bax and Bcl-2 were analyzed by fluorescence quantitative PCR, Bax and Bcl-2 family proteins were analyzed by Westerm blot. Results Parthenolide inhibited the proliferation of 786-O cells in a dose-dependent and time-dependent manner.As showed by flow cytometry, parthenolide treatment of the 786-O cells led to significant G1-phase cell cycle arrest in a dose-dependent manner and a decrease in cell population in the S-phase, whereas the population of cells in G2/M phase did not chang significantly.Flow cytometIy also showed a dose-dependent increase in 786-O cells apoptosis by parthenolide treatment.Westem blot analysis indicated a down-regulation of phosphorylated CyclinDl in a dose-dependent manner in 786-O cells by parthenolide treatment.In the gene and protein level, parthenolide treatment to 786-O cell lines resulted a significant reduction in Bcl-2 expression but an increase in Bax protein in a dose-dependent manner. Conclusions Parthenolide treatment can 1ead to a significant dose-dependent and time-dependent inhibition in the growth and an increase in apoptosis of human 786-O Kidney cancer cells.The mechanism of action of parthenolide may be related to inhibition of CyclinDl activation and regulation of Bcl-2 and Bax expression in human 786-O Kidney cancer cells. Key words: Kidney Neoplasms; Tanacetum parthenium; Cell Proliferation; Apoptosis; Cell Cycle

Study on proliferation effects of low intensity pulsed ultrasound on human bone marrow mesenchymal stem cells in vitro

Objective To investigate the effect of low intensity pulsed ultrasound (LIPUS) on the proliferation of human bone marrow mesenchymal stem cells (hBMSCs) and the effect of Cyclin D1 on the proliferation of hBMSCs induced by LIPUS. Methods The cells were inoculated on the a 12-well culture culture plate at a cell concentration of 2.0×104 cells/ml, LIPUS with radiation intensity of 0, 30, 40, 50, 60 and 80 mW/cm2 was used to stimulate for 5 min, the morphological changes of the cells were observed at 0, 24, 72 and 96 h, cell counting was performed with a Scepter 2.0 handheld cell counter, the cell cycle and value-added index were measured by flow cytometry, the cell proliferation was detected by cell counting kit-8 (CCK-8). The expression of Cyclin D1 was detected by Western blotting. The expression of Cyclin D1 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Results Cell counts were found except for the 30 mW/cm2 (P=0.871) group that the number of cells in 40 W/cm2 (P=0.006), 50 W/cm2 (P=0.001), 60 W/cm2 (P=0.001), 80 W/cm2 (P=0.004) group was significantly higher than that in control group. Cell proliferation assay was found that the proliferation of 30 mW/cm2 (P=0.042), 40 W/cm2 (P=0.012), 50 W/cm2 (P=0.001), 60 W/cm2 (P=0.001), 80 W/cm2 (P=0.046) cells was significantly higher than that of the control group. Flow cytometry detection found that the cell proliferation index of LIPUS group was significantly higher than that of control group (P=0.001). Western blotting and RT-PCR showed that there was no significant difference between the 30 mW/cm2 group and the control group. The expression of Cyclin D1 was significantly increased in 40 W/cm2 (P=0.002), 50 W/cm2 (P=0.004), 60 W/cm2 (P=0.023), 80 W/cm2 (P=0.012) groups. The expression of Cycling D1 mRNA was significantly different from that of control group (P=0.001). Conclusion LIPUS can significantly promote the proliferation of hBMSCs, and promote the proliferation of hBMSCs from G1 phase to S phase by up-regulation of Cyclin D1 expression. Key words: Low intensity pulsed ultrasound; Human bone marrow mesenchymal stem cells; Proliferation; Cyclin D1