| Objective:To study the expression of mPGES-1 gene and clinicopathological features in hepatocellular carcinoma (HCC).To investigate the expression of mPGES-1 in HepG2 cells treated by MK886,the mPGES-1 inhibitors,and observe the impact of MK886 on cell proliferation,adherence,migration and invasion abilities.The lentiviral vector-mediated RNA interference targeting mPGES-1 gene was constructed and transfected into HepG2 cells. To observe the inhibitory effect of RNA interference(RNAi) on mPGES-1 gene expression and protein level of HepG2 cells,as well as its effect on the biology of HepG2 cells and to investigate its significance in the progression,metastasis and invasion.Methods:(1)The expressions of mPGES-1 were determined by reverse transcription-polymerase chain reaction(RT-PCR) and Western Blot in 60 HCC samples,40 para-carcinoma tissue samples,23 far-carcinoma tissue samples and 5 normal liver tissue samples.The correlation of mPGES-1 to clinicopathologic parameters of HCC was also analyzed.(2)The RNA and protein of HepG2 cells was extracted after cultured with MK886 in different concentration for 48h,respectively.The RT-PCR and Western Blot was used to detect mPGES-1 mRNA and protein respectively;The morphology of HepG2 cells was observed under inverted microscope under different concentration of chemotherapeutics;The growth inhibitory rate of HepG2 cell was assessed by MTT assay;The adhesive abilities of HepG2 cells interfered by MK886 in different concentration for 2h was also detected by MTT assay;The migration and invasion abilities of HepG2 cells interfered by MK886 in different concentration for 24h was investigated by transwell technique.(3)Four designed DNA sequences targeting mPGES-1 with small hairpin structure were cloned into the transcripted carrier pGCSIL-GFP in order to construct recombinants.The most effective reorganization plasmid screened by Western blot, pHelper 1. 0 and pHelper 2. 0 were transfected into the 293T cells.After sueeessful construction of the recombinant lentiviruses,they were injected into HepG2 cells which the expression of the mPGES-1 were examined by RT-PCR and Western Blot. The effects of LV- mPGES-1-shRNA on proliferation,apoptosis,adherence,migration and invasion of HepG2cells were detected by MTT assays,transmission electron microscopy and Transwell technique.Results:1.The expression of mPGES-1 mRNA(semi-quantitative,mPGES-1/GAPDH) and protein(semi-quantitative,mPGES-1/β-actin)were0.805±0.466,0.471±0.246,0.457±0.172,0.076±0.009 and 0.320±0.153,0.205±0.171,0.204±0.098,0.029±0.005 in HCCs,para-carcinoma tissues,far-carcinoma tissues and normal liver tissues respectively.MPGES-1 was significantly higher in HCCs than normol tissues.There was statistical significance among each group(α′=0.00714).2.The expression of mPGES-1 in HCCs was associated with histological grades,the related factors to the tumor invasion and metastasis(P<0.01),not associated with sex,tumor size and histological types(P>0.05),which was increased following histological grade increasing(P<0.01).Furthermore,mPGES-1 expression level was higher in the capsule invasion and metastasis tumor than primary locus(P<0.01).3.In HepG2 cells, the expression of mPGES-1 mRNA and protein decreased in a dose-dependent after cultured with MK886 for 48h.4.The proliferation of HepG2 cells was inhibited in a dose and time-dependent manner after exposed to MK886.The IC50 was 41.42μmol/L in HepG2 cells after exposed to MK886 for 48h.The cell morphology became shrinkage and smaller,the cytoplasm was vacuolated and the nuclear pyknosis or fragmentation.5.The rate of adhesion cells observed a dose-dependent in experimental groups were 85.3%±1.3%,70.5%±1.5%,45.8%±2.4%,respectively,less than that in control group100.0%±0%(F=626.313,P=0.000).6.The migration cells was (92.47±1.90),(62.63±1.96),(37.33±0.83) respectively in the experimental groups after 24h,lower than that of the control group (128.93±2.60)(F=1253.805,P<0.01).The invasion assay revealed that the invading cells were(41.67±1.30),(25.47±1.30),(13.93±1.66) in the experimental groups,in contrast to (55.67±2.08) in control group after 24h.The differerce between these groups was significan(tF=372.615,P<0.01).The number of migration and invasion of HepG2 cells were observed a dose-dependent in MK886 groups.7. PCR and DNA sequencing demonstrated that the inserted sequences were correct.The most effective reorganization plasmid screened by Western blot was KD3,KD4 which their silence efficiency of mPGES-1 gene was at least 80%.The titer of lentivirus was equal 9x108TU/ mL.And the vector KD4 was selected to conduct successor experiment.8. Compared with non-transfected cells and empty vector transfected cells,the expression of mPGES-1 mRNA and protein decreased significantly(P<0.01).9. We found nucleus electron density increased, nuclear fragmention of apoptotic cell and apoptotic body by transmission electron microscopy,all of which was most significant in the transfected cells.And the proliferation,adherence,migration and invasion abilities of HepG2 cells were also reduced(P<0.01).Conclusion:1. The expression of mPGES-1 was very low in normal liver tissues,but much higher in HCCs.The mPGES-1 may be a new target in hepatocellular carcinoma (HCC) treatment.2. MK886,a mPGES-1 inhibitor, reduced mPGES-1 mRNA and protein expression dose-dependently in HepG2 cells. The proliferation,adherence,migration and invasion abilities was also reduced following dose-dependently.The down-regulation of mPGES-1 gene expression indicated the proficiency on degrading the invasion and metastasis process of HCC.3. The siRNA expression vectors targeting mPGES-1 were successfully constructed.The expression of mPGES-1 gene was inhibited effectively in HepG2 cells transfected by vector KD4,which laid a basis for its application in the experimental treatment of hepatocellular carcinoma. 4. The proliferation,adherence,migration and invasion abilities of HepG2 cells were inordinately inhibited after transfecting with LV-mPGES-1-shRNA,inducing cell apoptosis meanwhile.And the mechanism of inhibition tumor growth,metastasis were related with the down-regulation of mPGES-1.
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