Effects of hsa-circ-0001862 on Phenotype of Tongue Squamous Cell Carcinoma Cells

[Objectives]To investigate the molecular mechanism of hsa_circ_0001862 on the proliferation,migration,invasion and apoptosis of tongue squamous cell carcinoma Tca-8113 cells.[Methods]hsa_circ_0001862 plasmid was constructed,and the interaction relationship between hsa_circ_0001862 and miR-23a-3p was verified by dual luciferase reporter gene and qRT-PCR.CCK8 assay,colony formation assay,scratch assay and Transwell assay were used to detect the proliferation,migration and invasion ability of Tca-8113 cells.Western blot was used to detect the expression level of apoptosis-related protein molecules.The effects of hsa_circ_0001862 on the apoptosis of Tca-8113 cells was detected.[Results]hsa_circ_0001862 and miR-23a-3p could interact,and their expression was negatively correlated in tongue squamous cell carcinoma cells.In Tca-8113 cells,hsa_circ_0001862 inhibited cell proliferation,migration,and invasion(P<0.01),and promoted cell apoptosis(P<0.01).[Conclusions]The hsa_circ_0001862 interacts with miR-23a-3p,and hsa_circ_0001862 plays an inhibitory role in the development of tongue squamous cell carcinoma.The hsa_circ_0001862 may be a new biomarker and target for the treatment of tongue squamous cell carcinoma.

POLE2 Regulates Apoptosis of Oral Squamous Cell Carcinoma Cells through the PI3K/AKT Signaling Pathway

Objective Oral squamous cell carcinoma(OSCC)is the most common malignant tumor of the head and neck,but its occurrence and progression mechanisms remain unclear.In addition-there is a lack of effective targeting drugs.The second major subunit of DNA polymerase(POLE2)catalyzes the prolongation of new strand replication and modifies exonuclease domain activity.Our previous study found that POLE2 was associated with OSCC progression,but the mechanism remains unclear.Methods The expression of POLE2 in OSCC tissues was detected using immunological assays.Mann-Whitney U analysis was used to investigate the relationship between POLE2 gene expression and tumor classification and prognosis of OSCC.POLE2 expression was inhibited in OSCC cells,and the effects of gene and protein expression were detected using RT-PCR and Western blotting.The POLE2 knockout model was constructed by transfecting a lentiviral vector.Cell proliferation,apoptosis,and migration were detected using various assays including colony formation,MTT,flow cytometry,wound healing assay,Transwell assay,and the Human Apoptosis Antibody Array.The animal model of OSCC was established by subcutaneous injection of transfected HN6 into 4-week-old female nude mice.After 30 days,tumors were removed under anesthesia and tumor weight and dimension were recorded.Tumor cell proliferation was analyzed using Ki67 staining.Results POLE2 gene levels were significantly higher in the OSCC tissues than in the normal tissues.In addition,POLE2 gene levels were statistically correlated with tumor classification and prognosis.Silencing POLE2 inhibited the proliferation of oral cancer cells and promoted apoptosis in vitro.Animal experiments also supported a positive correlation between POLE2 and OSCC tumor formation.We further demonstrated that POLE2 could upregulate the expression of apoptosis-related proteins such as caspase-3,CD40,CD40L,DR6,Fas,IGFBP-6,p21,and SMAC.In addition,POLE2 regulated OSCC development by inhibiting the PI3K/AKT signaling pathway.Conclusion POLE2 is closely related to the progression of OSCC.Thus,POLE2 may be a potential target for OSCC treatment.

Targeting LncRNA LLNLR-299G3.1 with antisense oligonucleotide inhibits malignancy of esophageal squamous cell carcinoma cells in vitro and in vivo

Accumulating evidence has indicated that long non-coding RNAs(lncRNAs)play critical roles in the development and progression of cancers,including esophageal squamous cell carcinoma(ESCC).However,the mechanisms of lncRNAs in ESCC are still incompletely understood and therapeutic attempts for in vivo targeting cancer-associated lncRNA remain a challenge.By RNA-sequencing analysis,we identified that LLNLR-299G3.1 was a novel ESCC-associated lncRNA.LLNLR-299G3.1 was up-regulated in ESCC tissues and cells and promoted ESCC cell proliferation and invasion.Silencing of LLNLR-299G3.1 with ASO(antisense oligonucleotide)resulted in opposite effects.Mechanistically,LLNLR-299G3.1 bound to cancerassociated RNA binding proteins and regulated the expression of cancer-related genes,including OSM,TNFRSF4,HRH3,and SSTR3.ChIRP-seq(chromatin isolation by RNA purification and sequencing)revealed that these genes contained enriched chromatin binding sites for LLNLR-299G3.1.Rescue experiments confirmed that the effects of LLNLR-299G3.1 on ESCC cell proliferation were dependent on interaction with HRH3 and TNFRSF4.Therapeutically,intravenous delivery of placental chondroitin sulfate A binding peptide-coated nanoparticles containing antisense oligonucleotide(pICSA-BP-ANPs)strongly inhibited ESCC tumor growth and significantly improved animal survival in vivo.Overall,our results suggest that LLNLR-299G3.1 promotes ESCC malignancy through regulating gene-chromatin interactions and targeting ESCC by pICSA-BP-ANPs may be an effective strategy for the treatment of lncRNA-associated ESCC.

DAPT Enhances the Apoptosis of Human Tongue Carcinoma Cells

Aim To investigate the effect of DAPT （γ-secretase inhibitor） on the growth of human tongue carcinoma cells and to determine the molecular mechanism to enable the potential application of DAPT to the treatment of tongue carcinoma. Methodology Human tongue carcinoma Tca8113 cells were cultured with DAPT. Cell growth was determined using Indigotic Reduction method. The cell cycle and apoptosis were analyzed by flow cytometry. Real-time PCR and Immuno-Fluorescence （IF） were employed to determine the intracellular expression levels. Results DAPT inhibited the growth of human tongue carcinoma Tca8113 cells by inducing G0-G1 cell cycle arrest and apoptosis, The mRNA levels of Hairy/Enhancer of Split-1 （Hes-1）, a target of Notch activation, were reduced by DAPT in a dose-dependent manner. Coincident with this observation, DAPT induced a dose-dependent promotion of constitutive Caspase-3 in Tca8113 cells. Conclusion DAPT may have a therapeutic value for human tongue carcinoma. Moreover, the effects of DAPT in tumor inhibition may arise partly via the modulation of Notch- 1 and Caspase-3.

Manganese antagonizes iron blocking mitochondrial aconitase expression in human prostate carcinoma cells

Aim： To investigate the possible role of manganese in the regulation of mitochondrial aconitase （mACON） activity human prostate carcinoma cell line PC-3 cells. Methods： The mACON enzymatic activities of human prostate carcinoma cell line PC-3 cells were determined using a reduced nicotinamide adenine dinucleotide-coupled assay. Immunoblot and transient gene expression assays were used to study gene expression of the mACON. The putative response element for gene expression was identified using reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays. Results： In vitro study revealed that manganese chloride （MnCI2） treatment for 16 h inhibited the enzymatic activity of mACON, which induced the inhibition of citrate utility and cell proliferation of PC- 3 cells. Although results from transient gene expression assays showed that MnCI2 treatment upregulated gene translation by approximately 5-fold through the iron response element pathway, immunoblot and reporter assays showed that MnCl2 treatments inhibited protein and gene expression of mACON. This effect was reversed by cotreatment with ferric ammonium citrate. Additional reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays suggested that a putative metal response element in the promoter of the mACON gene was involved in the regulation of MnCh on the gene expression of mACON. Conclusion： These findings suggest that manganese acts as an antagonist of iron, disrupting the enzymatic activity and gene expression of mACON and citrate metabolism in the prostate.

Vascular Endothelial Growth Factorl65-regulated Nasopharyngeal Carcinoma Cell Lines Invasion and Migration Involve Expression and Activation of Matrix Metalloproteinase-2

The effect of vascular endothelial growth factor （VEGF） overexpression on matrix metalloproteinase-2 （MMP-2） in nasopharyngeal carcinoma （NPC） cells in vitro and the possible mechanism involved were investigated, and the correlation between the expression of VEGF and MMP-2 in NPC evaluated. The NPC cells were transfected with PAd-trackVEGF165 plasmid. The expression levels of VEGF and MMP-2 mRNA and protein in NPC cells were detected by semi-quantitative RT-PCR and Western blot respectively. It was found that the expression of VEGF and MMP-2 mRNA and protein was significantly increased in NPC cells after transfection of VEGF 165. It was concluded that the expression of VEGF was correlated to the in vitro invasion of NPC cells, and the induction of MMP-2 by VEGF was a key process of NPC cell invasion.

Effects of Meloxicam on Vascular Endothelial Growth Factor and Angiopoietin-2 Expression in Colon Carcinoma Cell Line HT-29

To investigate the effect of meloxicam, a selected NSAIDs, on cell growth, expression of VEGF and angiopointin-2 （Ang-2） protein in HT-29 cell line, cultured HT-29 cells were treated with meloxicam of various concentrations for various lengths of time. The proliferation of HT-29 was detected by cell counting kit-8 （CCK-8）, the cell cycle was determined by flow cytometer and the levels of VEGF and Ang-2 protein in supernatants were examined by enzyme linked immunosorbent assay （ELISA）. The mRNA expressions of VEGF and Ang-2 in cultured HT-29 were determined by real-time quantitative reverse-transcription polymerase chain reaction. Our results showed that treatment of meloxicam of different concentrations and for various lengths of time had a cytotoxicic effect on the cell proliferation of HT-29 cells in a concentration-dependant and time-dependant manner. Cell cycle analysis showed that the cells were mainly blocked in G0/G1 phase. The VEGF and Ang-2 protein levels in supernatants of the culture medium were decreased gradually in a concentration-dependent or time-dependent fashion. The mRNA expression of cox-2, VEGF and Ang-2 showed a gradual and concentration-dependent reduction. It is concluded that meloxicam can reduce the expression of VEGF and Ang-2 at the protein and mRNA level in colon carcinoma cell line.

STUDY ON THE EXPRESSION OF CYCLOOXYGENASE-2 IN HEPATOCELLULAR CARCINOMA CELL LINES AND ON THE GROWTH INHIBITION EFFECT OF NS-398

Objective： To investigate the expression of cyclooxygenase -2 （COX-2） in hepatocellular carcinoma cell lines and to explore the effect of NS-398, a selective inhibitor for COX-2, on HepG-2 cell line. Methods： lmmunohistochemistry and RT-PCR were used to investigate COX-2 expression in 6 HCC cell lines. MTT and Flowcytometry were used to evaluate the effect of the selective inhibitor of COX-2, NS-398, on HepG-2 cell lines. Results： All six HCC cell lines showed COX-2 expression at protein level. Five out of 6 cell lines showed COX-2 expression at mRNA level. NS-398 could suppress the growth of HepG-2 cell line, in a time and dose dependant manner. Conclusion： NS-398, a selective inhibitor of COX-2, showed inhibition effect on HepG-2 HCC cell line. The efficacy of inhibition was time and dose dependent, providing a new evidence for chemoprovention of hepatocellular carcinorma with COX-2 selective inhibitors.

The Effect of Nano-apatite on the Expression of Telomerase Gene of Human Hepatocellular Carcinoma Cells

To investigate the effect of nano-apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the hybridization in situ method to detect the expression of the telomerase gene of human hepatocellular carcinoma cells treated with the nano-apatite for 4 h at 37 ℃ . The hybridization in situ showed that the cytoplasm of the positive cells was stained in nigger- brown. The positive cell rate of the control group was 88.49% , the cisplatin group was 25.6% , the nano-apatite group was 63.6% . The activity of telomerase gene was both obviously dedined comparing with the control group and the difference had significance （ p 〈 0. 05, p 〈 0.01 ）. The nanoapatite obviously inhabit the expression of the telomerase gene of human hepatocellular carcinoma cells.

Hypoxia-induced enhancement of cell invasiveness in SMMC7721 hepatocellular carcinoma cells

Objective To explore the effects of hypoxia(1% O2)on the ability of cell invasiveness and expression of KAI1/CD82 in SMMC7721 hepatocellular carcinoma cells.Methods SMMC7721 hepatocellular carcinoma cells were cultured by hypoxia(1% O2)in vitro,and the ability of cell invasiveness was analyzed by cell invasion assay.Immunohistochemistry staining technique was used to evaluate the protein expression of KAI1/CD82.Results Cell invasion assay revealed that hypoxia enhanced the ability of invasiveness of hepatocellular carcinoma cells.In addition,KAI1/CD82 protein expression was positive in cultured SMMC7721 hepatocellular carcinoma cells,and it was located diffusedly in the cytoplasm and on the membrane.KAI1/CD82 protein expression was down-regulated when mediated by hypoxia;at the same time,it showed a time-effect relationship.Conclusion Hypoxia can enhance invasiveness of hepatocellular carcinoma cells.The down-regulation of KAI1/CD82 expression may play a certain role in those courses.

Apoptosis of Human Pancreatic Carcinoma Cells Induced By All-Trans Retinoic Acid and Interferon

Objective： To investigate the apoptosis of human pancreatic carcinoma PC3 cells induced by the combination of all-trans retinoic acid （ATRA） with interferon alpha （IFN-α）. Methods： PC3 cells were treated with ATRA and IFN-α. The inhibitory rate of PC3 cell proliferation was detected using MTT method. Cellular apoptosis was determined with flow cytometry. The percentage of PC3 cell apoptosis was assayed using TUNEL methods. Results： ATRA and IFN-α could inhibit cellular proliferation and induces cellular apoptosis of PC3 cells. The inhibitory effect was stronger when the ATRA and IFN-α were combined as a therapy. Conclusion： ATRA inhibits the proliferation of PC3 cells and induce the apoptosis of PC3 cells. The combination of IFN-α with ATRA may enhance these effects on PC3 cells.

Construction,identification and biological feature study of human hepatocellular carcinoma cells stably expressing HBeAg

Objective: To construct a HepG2 cell line which stably expressing Hepatitis B e antigen(HBeAg)and investigate the effects of HBeAg on the proliferation,migration and invasion of HepG2 cells. Methods: The lentivirus carrying HBeAg gene was constructed and packaged. HepG2 cells were infected with the lentivirus and screened with puromycin to obtain HepG2 cells which stably expressing HBeAg(HepG2-HBeAg cells).The expression levels of HBeAg mRNA and protein were detected by RT-qPCR and Western blot,respectively. The content of HBeAg secretion in cell supernatant in both HepG2-HBeAg cells and control cells(HepG2-NC cells and HepG2 cells)were detected by IFMA assay. Furthermore,CCK-8 proliferation assay,colony formation assay and transwell migration and invasion assays were conducted to compare the abilities of cell proliferation,migration and invasion,respectively. Results: The expression of HBeAg in the HepG2-HBeAg cell was significantly higher than those in HepG2 cells and HepG2-NC cells. Secreted HBeAg in the supernatant of HepG2-HBeAg cells was 26. 33±2. 13 PEIU/mL but was undetectable in supernatant of control cells. The proliferation,migration and invasion were all significantly lower in HepG2-HBeAg cells compared to control cells(P<0. 01). Conclusion: A HepG2 cell line which stably expressing HBeAg was constructed successfully,and the over-expression of HBeAg could attenuate the proliferation,migration and invasion of hepatocellular carcinoma cells.

THE SENSITIVITY OF EHRLICH ASCITIC CARCINOMA CELLS TO THE EMPERIPOLESIS OF THYMUS LYMPHOCYTES

In mixed lymphocyte-tumor all culture in vitro, the ability of thymus lymphocytes adhering to tumor cells was found to be essentially consistent with its emperipolesis index, though not synchronous. Tumor cells at different progressive stages and in the mixed cultures with or without Con A stimulation also varied in the sensitivity to lymphocyte adhesion and emperipolesis. Tumor adhesiveness anl emperipolesis of theymus lymphocytes and their PNA-, PNA+ subgroups were shown to be different significantly from splenic lymphocytes. Thymosin exhibited certain promotive effect on lymphocyte-tumor adhesion and emperipolesis.

Cyclin L2, a novel RNA polymerase Ⅱ-associated cyclin, is involved in pre-mRNA splicing and induces apoptosis of human hepatocellular carcinoma cells

We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.

SELECTIVE TOXICITY OF RHODAMINE 123 ON CARCINOMA CELL IN VITRO

The selective toxicity of the mitochondria-specific cationic fluorescent dye rhodamine 123 (Rh-123) on He La cells in culture was studied. In this report, we demonstrate that with continuous exposure, Rh-123 markedly inhibited the growth of HeLa cells but had little effect on normal human kidney fibroblasts. With continuous exposure to Rh-123, the growth rate, colony forming ability, anl mitotic index of HeLa cells were decreased. The mechanism of toxicity of Rh-123 on HeLa cells was investigated by EM and enzyme cytochemistry stain. The mitochondria of carcinoma cells were the main targets for the inhibitory action of Rh-123, since they selectively accumulated the dye. At the dosage of Rh-123 which was toxic to HeLa cells, the structure and function of mitochondria were disrupted, as the mitochondria-related enzymes, i.e., ATPase, LDH and SDH were inhibited. The possible mechanism of the action of Rh-123 on HeLa cells is briefly discussed.

INTERACTIONS BETWEEN THE HUMAN GASTRIC CARCINOMA CELL AND THE HUMAN VASCULAR ENDOTHELIAL CELL

Objective To definite the interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the establishment and maintenance of the tumor vascular system and the tumor hematogenous metastasis.Methods We prepared the conditioned mediums of each cell so as to study the effect of the conditioned medium on itself or others by MTT colorimetry. The comprehensive effect of interactions between two cells was determined by stratified transfilter co culture or direct contact co culture.Results The conditioned medium of human gastric carcinoma cell can stimulate the proliferation of the human vascular endothelial cell, but the CM of HVEC can inhibit the growth of HGCC. Both kinds of cells can inhibit the growth of itself. The ultimate comprehensive effect of the interactions between two kinds of cells was increase of total cell numbers.Conclusion There exist the complicated interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the tumor angiogenesis and the tumor hematogenous metastasis. The ultimate comprehensive effect of the interactions is increase of total cells numbers and tumor volume.

Induced Differentiation of Epithelioid Carcinoma Cell Lines: Evidence for Tumor Cell Quantal Mitosis

The effects of growth factors and calcium concentrations present in different culture media on induction of terminal differentiation were investigated for four different epidermoid carcinoma cell lines, Hela, KB, A431, and SCC-25, and their responses determined relative to those elicited by normal human keratinocytes subjected to these culture conditions. Differentiation status was determined cyto-chemically by a validated keratin protein staining method, and by autoradiographic analyses. Growth and differentiation promoting factors that influenced the direction of integrated control of growth and differentiation in normal human keratinocytes were found to be effective for some cell lines but not others. The factors examined were 1) high density arrest in serum-free and serum-containing media, 2) media shifts from high density culture in serum-containing media to low density growth factor-depleted or supplemented serum-free medium, and 3) the concentration of calcium in the media. The extent and degree of differentiation achieved varied among different cell lines depend on the presence or absence of serum, EGF and insulin protein growth factors. Certain growth media appear to sponsor keratin protein, cyto-chemically-detected differentiation, and evidence of quantal mitotic division in low density HeLa cell and SCC25 cell cultures. Epidermoid carcinoma cell lines retain limited capacity to commit to early stages of cell differentiation.

The effect of interleukin 6 on the growth of LNCaP and PC-3 prostatic carcinoma cells

Objective To investigate the effect of IL-6 on prostatic carcinoma cell lines, and differential effects on androgen-dependent and androgen-independent prostatic carcinoma cells. Methods The IL-6 producing capacities of LNCaP and PC-3 cells were determined, and effects of exogenous IL-6 and anti-IL - 6 antibodies on LNCaP and PC - 3 cells were examined. Results LNCaP produced a very small amount of IL-6, but PC-3 produced more, the concentraion of IL-6 being 190 pg/48 h per ml(1 × 106). The exogenous IL-6 inhibited LNCaP growth significantly,but had no obvious effect on PC -3 cells. Anti-IL-6 antibodies lowered PC-3 cells growth rate but had neutral effect on LNCaP. Conclusion PC-3 cells produces IL-6 massively in autocrine manner. IL-6 could be antagonized by anti-IL-6 antibodies,resulting in slowing PC-3 cells growth, and LNCaP cells growth could be inhibited by exogenous IL-6.7 refs,2 tabs.