The effects of interfering in COX-2 gene expression on malignant proliferation of the human lung adenocarcinoma A2 cell in vitro

Objective:We explored the interfering effect of COX-2 gene expression and the influence on the malignant proliferation of A2 cells by RNAi after quenching COX-2 in vitro. Methods:COX-2 was selected as the subject. Three COX-2 siRNA expression vectors with human U6 promoter were constructed and three vectors and the vacant vector (pEGFP) were transfected into A2 cells with lipofectamine respectively. The cell strains transfected were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A2 cells after silencing COX-2 were studied by cell growth curve and clonogenic assay in vitro. Results: The three siRNA and U6 promoter cloned into pEGFP plasmid were validated by PCR, restriction endonucleases identification, DNA sequencing and BLAST alignment. The cell strains transfected were named as A2-3, A2-7, A2-10 and A2-p respectively. Green fluorescence was seen in A2-p cells and not in A2-3, A2-7 and A2-10 cells in 24, 48 and 72 h after transfected. The results of RT-PCR and Western blot showed the three siRNA expression vectors produced effects and the expression of COX-2 was inhibited in different extent. In contrast to A2 cells, the levels of COX-2 mRNA of A2-3, A2-7 and A2-10 cells reduced 15.6%, 20.4% and 64.2% respectively; the levels of COX-2 protein of A2-3, A2-7 and A2-10 cells reduced 23.7%, 36.7% and 60.2% respectively. The results of cell growth curve and clonogenic assay showed the growth of A2-10 cell slowed and the colonial formation rate reduced but the growth of A2-3 and A2-7 cells had not obvious changes in contrast to the controls (A2 and A2-p). Conclusion: Silencing the COX-2 gene in vitro by RNAi technique can significantly inhibit the malignant proliferation of A2 cells.

The effects of the same target on malignant proliferation of human lung cancer cells with different expression levels of COX-2 protein

Objective: The aim of the study was to explore the effects of the same target (si-10) on lung cancer cells with different expression levels of cyclooxygenase-2 (COX-2) protein by RNAi and malignant proliferation of these cells. Methods: COX-2 was selected as the target and one siRNA expression vector with the best effect was selected and thought as the subject from three COX-2 siRNA expression vectors with human U6 promoter. The siRNA expression vector (psi-10) and the vacant vector (pEGFP) were transfected into these cells with different COX-2 expression states (801D, A549 and LTEP-A2) with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve and clonogenic assay. Results: The siRNA and U6 promoter were validated by PCR, restriction endonucleases identification and DNA sequencing and BLAST alignment and cloned into the pEGFP vector. The cell strains transfected that 801D was used as maternal line were named as 801D-p and 801D-10 respectively. The cell strains transfected that A549 was used as maternal line were named as A549-p and A549-10 respectively. The cell strains transfected that LTEP-A2 was used as maternal line were named as LTEP-A2-p and LTEP-A2-10 respectively. These cells transfected pEGFP (801D-p, A549-p and LTEP-A2-p) had the expression of GFP and 801D-10, A549-10 and LTEP-A2-10 cells had not in 24, 48 and 72 hours after transfected. The results of RT-PCR and Western blot showed the siRNA expression vector produced marked effects in two cells (A549 and LTEP-A2) expressing COX-2 and the expression of COX-2 was inhibited. But the inhibited effects were differ- ent and the expression of COX-2 was more inhibited obviously in LTEP-A2 cells than in A549 cells though the expression of COX-2 was also inhibited obviously in A549 cells. In contract to their maternal line, the levels of COX-2 mRNA of LTEP-A2-10 and A549-10 cells reduced 64.2% and 61.2% respectively; the levels of COX-2 protein reduced 60.2% and 56.2% respectively. But the levels of COX-2 mRNA and protein had not change in 801D cells not expressing COX-2. The results of cell growth curve and clonogenic assay showed the growth of LTEP-A2-10 cells slowed and the clonal formation rate reduced and the size of the colonies became small; the growth of A549-10 cells showed slow and more obviously in the cell growth curve especially. But the growth of 801D-10 cells had not obvious change. Conclusion: The si-10 target of COX-2 has different inhibition effects on lung cancer cells with different COX-2 expression levels and the different inhibition effects have different effects on cells malignant proliferation.

CLONAL PROLIFERATION AND LONG-TERM CULTURE OF MALIGNANT LYMPHOMA CELLS UNDER SERUM-FREE CULTURE CONDITIONS

We developed a serum-free culture system that promoted the growth of B cell colonies in peripheral blood, bone marrow, lymph nodes and cerebrospinal fluid (CSF) from 7 out of 8 patients with non-Hodgkin's lymphomas of B cell type. The culture cells were pretreated with or without galactose oxi-dase (GO) prior to plating. Colony growth was best supported with BCGF. A moderate increment was observed with rIL-3, as well as rIL-1β and even to a lesser degree, by rlL-2, while B cell stimulating factor-2 (rBCSF-2) and rlL-1β did not show significant activity. rGM-CSF and rG-CSF had little effect, while rM-CSF enhanced the formation of lymphoma colonies. The cells from different patients had different requirements for Staphylococcus aureus protein A and GO pretreatment. It reflected the differences in activation and differentiation status and surface properties of lymphoma cells from different patients. The cells from CSF of one patient were successfully maintained in serum-free culture medium supplemented with 10% BCGF or 5% PHA-LCM for more than 4 months. The long-term culture cells were EBV negative, phenotypically consistent with B cells and gene rearrangements for JH, Kappa and myc. This serum-free culture system allowed extensive analysis of the growth requirements for clonogenic precursors.

COX-2 silencing inhibits cell proliferation in A549 cell

Objective: The aim of this study was to explore the effects on malignant proliferation of A549 cell by silencing cy-clooxygenase (COX)-2. Methods: In the present study, we constructed three siRNA vectors producing small interference RNA. The siRNA vectors and the vacant vectors were transfected into A549 cell with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve, clonogenic assay and xenograft assays. Results: The siRNA expression vectors produced marked effects in A549 cell but the inhibited effects were different. The effect of psi-10 was best and the mRNA and protein levels of COX-2 reduced 61.2% and 56.2% respectively in A549-si10 cell in contrast to the control. The growth of A549 cell slowed and the colony formation rate reduced after silencing COX-2. In xenograft assays, the growth speeds of tumor became slow and the numbers of tumor reduced after silencing COX-2. Conclusion: The si10 target of COX-2 has the best silencing effect in A549 cell and the best inhibition effect on malignant proliferation of A549 cell in vivo and in vitro.

miR-504 knockout regulates tumor cell proliferation and immune cell infiltration to accelerate oral cancer development

miR-504 plays a pivotal role in the progression of oral cancer.However,the underlying mechanism remains elusive in vivo.Here,we find that miR-504 is significantly down-regulated in oral cancer patients.We generate miR-504 knockout mice(miR-504^(-/-))using CRISPR/Cas9 technology to investigate its impact on the malignant progression of oral cancer under exposure to 4-Nitroquinoline N-oxide(4NQO).We show that the deletion of miR-504 does not affect phenotypic characteristics,body weight,reproductive performance,and survival in mice,but results in changes in the blood physiological and biochemical indexes of the mice.Moreover,with 4NQO treatment,miR-504^(-/-)mice exhibit more pronounced pathological changes char-acteristic of oral cancer.RNA sequencing shows that the differentially expressed genes observed in samples from miR-504^(-/-)mice with oral cancer are involved in regulating cell metabolism,cytokine acti-vation,and lipid metabolism-related pathways.Additionally,these differentially expressed genes are significantly enriched in lipid metabolism pathways that influence immune cell infiltration within the tumor microenvironment,thereby accelerating tumor development progression.Collectively,our results suggest that knockout of miR-504 accelerates malignant progression in 4NQO-induced oral cancer by regulating tumor cell proliferation and lipid metabolism,affecting immune cell infiltration.

EXPRESSION OF PROLIFERATING CELL NUCLEAR ANTIGEN IN CUTANEOUS MALIGNANT LYMPHOMAS AND LYMPHOID INFILTRATES

Paraffin-embedded tissue of skin biopsy specimens taken retrospectively from 24 patients with cutaneous malignant lymphomas (CML) and 8 patients with cutaneous lymphoid infiltrates (CLI) and other dennatoses were studied retrospectively with PCIO immunostaining. The results show a statistical significant difference among PCIO indices for cutaneous genuine histiocytic lymphoma (CGHL), cutaneous germinal center cell-derived lymphomas (CGCCL), cutaneous peripheral T-cell lymphomas (CPTL), non-mycosis fungoides (MF) and Sezary's syndrome (SS), and MF when compared with those for CLI. There is a linear relationship between PCIO index and square root of PCIO density, both of which seem to have a parallel relationship to the severity of malignancy in CML. The nuclear volume of the positive tumor cell or lymphocyte with PCIO immunostaining may be also useful in differentiating CML from CLI.