Effect Of Silencing PARG In Human Colon Carcinoma Lovo Cells On The Ability Of HUVEC Angiogenesis

Objective: To initially investigate the influence of silencing poly-(ADP-ribose)glycohydrolase(PARG)in colon carcinoma Lovo cells on the ability of HUVEC angiogenesis and its possible mechanisms.Methods: PARG-shRNA lentivirus vectors were transfected into colon carcinoma Lovo cells, then Lovo cells with silenced PARG were perpetually selected. Untreated Lovo cells served as blank control, Lovo cells that were treated with lentivirus empty vectors served as empty-vector control and Lovo cells that were treated with PARG-shRNA lentivirus vectors served as experimental control. The expression of PARG mRNA of each group was detected by Reverse Transcriptase-PCR and Real Time-PCR. The expressions of PARG, poly-(ADP-ribose)polymerases(PARP), p38, p-p38, ERK, p-ERK, NF-κB, p-IκBα, VEGF, b-FGF, ICAM-1 and MMP-9 of each group were detected by Western Blotting. Regarding the influence of silencing PARG in colon carcinoma Lovo cells on the ability of HUVEC migration, the latter was observed by Transwell migration assay. The supernatant of each group of Lovo cells was collected and added into HUVEC culture medium separately. CCK-8 was used to check the ability of HUVEC proliferation. Moreover, to further investigate the relationship between p38, ERK and NF-κB in Lovo cells, the ERK inhibitor(U0126)and p38 inhibitor(SB203580)were used to treat Lovo cells apartly. Meanwhile, the changes of NF-κB and p-IκBαexpressions were detected by Western Blotting. The NF-κB inhibitor(PDTC)was used to treat Lovo cells. The changes of PARP, p38, p-p38, ERK, p-ERK expressions were detected by Western Blotting.Results: 1. Both RT-PCR and Western Blotting results showed that the expressions of PARG in the experimental group were obviously lower than that in the control group(P<0.05). These illustrated that PARG was silenced in Lovo cells.2. The results of migration assay showed that the quantity of migratory HUVEC through the microporous membrane was decreased in the experimental group than that in the control group(P<0.05)and migratory inhibition rate was 55.23%. The results of CCK-8 showed that the proliferation of HUVEC in the experimental group was obviously lower than that in the control group(P<0.05), and the inhibition rates were reduced in pace with the continuous decrease in the concentration of Lovo cell supernatant.3. The expressions of PARP, p38, p-p38, ERK, p-ERK, NF-κB, p-IκBα, VEGF, b-FGF, ICAM-1 and MMP-9 in the experimental group were all obviously lower than that in the control group(P<0.05).4. The expressions of NF-κB and p-IκBαwere decreased after Lovo cells were treated by U0126 and SB203580 separately(P<0.05). The expression of PARP was obviously lower than that in the blank control group(P<0.05), and the expressions of p38, p-p38, ERK and p-ERK were not changed after Lovo cells were treated by PDTC.Conclusion: 1. The silencing PARG in Lovo cells can inhibit the ability of HUVEC migration significantly, and the ability of HUVEC prolifiration also. The inhibition rate was reduced in pace with the continuous decrease in the concentration of Lovo cell supernatant. PARG in Lovo cell probably plays an important role in promoting angiogenesis.2. The silencing PARG in Lovo cells can decrease the expressions of PARP, p38, p-p38, ERK, p-ERK, NF-κB and p-IκBαsynchronously. It suggests that the silencing PARG in Lovo cell inhibiting the ability of angiogenesis maybe related to p38, ERK and NF-κB pathways. The activity of NF-κB was decreased after p38 and ERK pathways had been inhibited in Lovo cells. It demonstrates that p38 and ERK pathways can regulate the activity of NF-κB in Lovo cells.3. The silencing PARG in Lovo cells can decrease the expressions of VEGF, b-FGF, ICAM-1 and MMP-9 simultaneously. These indicate that silencing PARG down-regulates the activity of NF-κB and finally decreases the expressions of NF-κB-dependent angiogenic factors like VEGF, b-FGF, ICAM-1 and MMP-9 by inhibiting the activity of PARP and the phosphorylation of p38 and ERK in Lovo cells. It plays a role in inhibiting the ability of angiogenesis.