Effect Of PARG On Tumor Lymphangiogenesis And Its Molecular Mechanisms In Murine Colon Carcinoma CT26 Cells

PART ONE STUDY ON THE EFFECT OF PARG ON TUMOR LYMPHANGIOGENESIS IN MURINE COLON CARCINOMA CT26 CELLS IN VITROObjectiveThe aim was to study the effect of PARG on tumor lymphangio- genesis in murine colon carcinoma CT26 cells in vitro.MethodsLentivirus vector-mediated PARG-shRNA(LV-PARG-shRNA) and Lentivirus vector-mediated PARG-cDNA ( LV-PARG-cDNA ) were transfected into CT26 cells, respectively. The expression of PARG mRNA was measured by the means of Real-time PCR. Western blot analysis was used to detect PARG; BALB/c mice with benign lymphangiomas in their abdominal cavities were induced by injection of IFA (incomplete Freund's adjuvant) and mechanically disrupted to obtain LECs. Expression of Podoplanin tested by immunofluorescence cytochemistry was used to confirm LECs. The effects of CT26-PARG-shRNA cells and CT26-PARG-cDNA cells on LECs migration were performed by the Transwell method, respectively. The influences of CT26-PARG-shRNA cells and CT26-PARG-cDNA cells on the capability of LECs to form lymphatic vessel-like structures were detected by the lymphatic vessel formation assay in vitro, respectively. The CT26 cells without transfection and CT26 cells transfected with LV empty vectors were used as control.ResultsWestern blot and Real-time PCR showed that PARG expression in CT26 cells transfected with LV-PARG-shRNA was significantly decreased than that in CT26 cells without transfection and CT26 cells transfected with LV empty vectors (P<0.01), while PARG expression in CT26 cells transfected with LV-PARG-cDNA was significantly enhanced than that in CT26 cells without transfection and CT26 cells transfected with LV empty vectors(P<0.01). PARG expression in CT26 cells without transfection and CT26 cells transfected with LV empty vectors had no significant difference (P>0.05).We successfully performed the animal model. The expressions of Podoplanin were positive in isolated LECs, showing LECs were obtained. The LECs migration in CT26-PARG-shRNA cells group was lower than that in CT26 cells without transfection and CT26 cells transfected with LV empty vectors group (P<0.01), whereas the LECs migration in CT26-PARG-cDNA cells group was higher than that in CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group (P<0.01). The LECs migration in CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group had no significant difference (P>0.05).The lymphatic vessel-like structures in CT26- PARG-shRNA cells group were significantly less than that in CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group (P<0.01). The lymphatic vessel-like structures in CT26- PARG-cDNA cells group were significantly more than that in CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group (P<0.01). The lymphatic vessel-like structures in CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group had no significant difference (P>0.05).ConclusionWe successfully obtained cell lines stably expressing PARG-shRNA (CT26-PARG-shRNA) and PARG-cDNA (CT26-PARG-cDNA), respectively. PARG silencing inhibited the migration of LECs and the formation of lymphatic vessel-like structures, while PARG over-expression enhanced the migration of LECs and the formation of lymphatic vessel-like structures. PARG probably plays an important role in promoting tumor lymphangiogenesis. PART TWO STUDY ON THE EFFECT OF PARG ON TUMOR LYMPHANGIOGENESIS IN MURINE COLON CARCINOMA CT26 CELLS IN VIVOObjectiveThe aim was to determine the effect of PARG on the tumor lymphangiogenesis of BALB/C tumor xenografts in vivo.MethodsCT26-PARG-cDNA and CT26-PARG-shRNA were injected subcutaneously in the left axillary region of BALB/C mice, respectively. CT26 cells without transfection and CT26 cells transfected with LV empty vectors served as control. The growth of the tumor xenografts was observed and 35 days later, the mice were sacrificed. The tumor xenografts were excised and weighed. Lymphangiogenesis was investigated by the tissue immunohistochemistry assay and LVD (lymphatic vessel density) was counted.Results1. A significant decrease of tumor weight was observed in the tumor xenografts of CT26-PARG-shRNA cells group compared to CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group( P<0.01). The tumor weight of CT26-PARG-cDNA cells group was significantly higher than that in CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group (P<0.01). The tumor weight of CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group had no significant difference (P>0.05).2. LVD in the CT26- PARG-shRNA cells group was drastically less than that in CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group(P<0.01). The LVD in CT26- PARG-cDNA cells group was significantly more than that in CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group(P<0.01). LVD in CT26 cells without transfection group and CT26 cells transfected with LV empty vectors group had no significant difference (P>0.05).ConclusionPARG silencing may inhibit tumor lymphangiogenesis, and PARG over-expression may promote tumor lymphangiogenesis in BALB/C mouse in vivo. PARG may regulate tumor lymphangiogenesis. PARG probably plays an important role in tumor metastasis. PART THREE EFFECT OF PARG INHIBITION ON EXPRESSION VEGF-C IN MURINE COLON CARCINOMA CT26 CELLSObjectiveThe objective was to investigate the role of PARG inhibition in the regulation of VEGF-C expression in colon carcinoma CT26 cells.MethodsCT26 cells were treated with or without the PARG inhibitor Gallotannin (GLTN). The expressions of PARG, NF-κBp65 and VEGF-C proteins in CT26 cells were measured by Western blot analysis; while VEGF-C mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). VEGF-C secreted by murine colon carcinoma CT26 cells was measured by enzyme-linked immunosorbent assay (ELISA).ResultsThe protein expression of PARG, NF-κBp65, and VEGF-C were reduced in GLTN-treated CT26 cells (P<0.01, P<0.05). VEGF-C mRNA in GLTN-treated CT26 cells were significantly weaker than that of GLTN-untreated cells (P<0.01), and moreover, the VEGF-C in GLTN-treated CT26 cells culture supernatants were significantly lower than that in GLTN-untreated CT26 cells culture supernatant (P < 0.05).ConclusionHere, we provide evidence for the first time that PARG inhibition dramatically reduces the expression of VEGF-C protein, and it is probably related with NF-κB signalling pathways. PART FOUR STUDY ON THE MECHANISMS OF PARG REGULATE TO THE VEGF-C EXPRESSION IN MURINE COLON CARCINOMA CT26 CELLSObjectiveThe aim was to explore the possible molecular mechanisms of the effect of PARG on the VEGF-C expression in mouse colon carcinoma CT26 cellsMethodsThe expressions of PARG, p-ERK, p-p38, NF-κBp65 and VEGF-C were detected by Western blot in tumor xenografts. The expression of VEGF-C mRNA in tumor xenografts was assessed by Real-time PCR. The tumor xenografts of CT26 cells without transfection and the tumor xenografts of CT26 cells transfected with LV empty vectors served as control. Western blot analysis was also used to detect PARG, p-ERK, p-p38, NF-κBp65 and VEGF-C protein expressions in CT26-PARG-shRNA cell lines and in CT26- PARG-cDNA cell lines. VEGF-C secreted by different CT26 cells was measured by enzyme-linked immunosorbent assay (ELISA). The CT26 cells transfected with LV empty vectors and CT26 cells without transfection were used as control. In order to further highlight the relationship between ERK, p38, NF-κB and VEGF-C and the relationship between NF-κB and VEGF-C, Western blot was used to investigate the effects of ERK inhibitor (U126) and p38 inhibitor (SB203580) on the expression of NF-κBp65 in CT26 cells transfected with LV-PARG-cDNA. The effects of ERK inhibitor (U126), p38 inhibitor (SB203580) and NF-κB inhibitor (PDTC) on the expression of VEGF-C in CT26 cells transfected with LV-PARG-cDNA were measured by Western blot and ELISA.Results1. The expressions of PARG, p-ERK, p-p38, NF-κBp65 and VEGF-C were significantly reduced in tumor xenografts of CT26-PARG-shRNA cells group and obviously enhanced in tumor xenografts of CT26-PARG-cDNA cells group, when compared to control. The expressions of PARG, p-ERK, p-p38, NF-κBp65 and VEGF-C in tumor xenografts of CT26 cells without transfection and tumor xenografts of CT26 cells transfected with LV empty vectors had no significant difference (P>0.05). The expressions of p-ERK, p-p38, NF-κBp65 and VEGF-C were significantly reduced in CT26-PARG-shRNA cell lines and obviously enhanced in CT26- PARG-cDNA cell lines, when compared with control. The expressions of p-ERK, p-p38, NF-κBp65 and VEGF-C in CT26 cells without transfection and CT26 cells transfected with LV empty vectors had no significant difference (P>0.05). Enhancements of NF-κBp65 and VEGF-C induced by PARG-cDNA were blocked by U126 and SB203580. VEGF-C over-expression induced by PARG-cDNA was inhibited by PDTC.2. The VEGF-C mRNA level was obviously decreased in tumor xenografts of CT26-PARG-shRNA group than that in tumor xenografts of CT26 cells without transfection group and tumor xenografts of CT26 cells transfected with LV empty vectors group (P<0.01). Compared to control, The VEGF-C mRNA level was significantly enhanced in tumor xenografts of CT26-PARG-cDNA. The VEGF-C mRNA level in tumor xenografts of CT26 cells without transfection group and tumor xenografts of CT26 cells transfected with LV empty vectors group had no significant difference (P>0.05).ConclusionPARG silencing in CT26 cells reduced the expressions of p-ERK, p-p38, NF-κBp65, VEGF-C and decreased the secretion of VEGF-C; PARG over-expression in CT26 cells enhanced the expressions of p-ERK, p-p38, NF-κBp65, VEGF-C and up-regulated the secretion of VEGF-C. ERK inhibitor and p38 inhibitor inhibited the expressions of NF-κBp65 and VEGF-C in CT26 cells transfected with LV-PARG-cDNA. NF-κB inhibitor suppressed the VEGF-C expression in CT26 cells transfected with LV-PARG-cDNA. These results suggested that PARG regulated the expression of VEGF-C by ERK, p38 and NF-κB signalling pathways in murine colon carcinoma CT26 cells. PARG plays an important role in tumor lymphangiogenesis in Murine Colon Carcinoma CT26 cells.