The Study On Microcystins And Its Relationship With Primary Hepatocellular Carcinoma

[Objective]To determine the effects on BRL-3A cellular proliferation and DNA damage induced by MC-LR with XRCC1 knocked down, then further to explore the role of XRCC1 in BRL-3A cells. Through the epidemiology survey in Fuzhou city of Fujian Province, to explore the relationship among the types of drinking water, miRNAs polymorphism of inflammatory pathway and primary hepatocellular carcinoma.[Methods]1 RNAi was used to silence the expression of XRCC1 of BRL-3A cells, and the expression changes of XRCC1 was detected by qRT-PCR for12h, 24h and 48h after transfection.2 XRCC1 knock-down BRL-3A cells, normal BRL-3A cells and missense siRNA-transfected BRL-3A cells were exposured respectively to different doses(0, 2, 6 and 10μg/ml) of MC-LR for 4h, 8h and 16h, and CCK-8 kit was used to determine the cellular proliferation.3 XRCC1 knock-down BRL-3A cells, normal BRL-3A cells and missense siRNA-transfected BRL-3A cells were exposured respectively to different doses(0, 2, 6 and 10μg/ml) of MC-LR for 4h, 8h and 16h, and qRT-PCR was used to determine the expression of PARP-1..4 XRCC1 knock-down BRL-3A cells, normal BRL-3A cells and missense siRNA-transfected BRL-3A cells were exposured respectively to different doses(0, 2, 6 and 10μg/ml) of MC-LR for 16h, and comet assay was used to determine the levels of DNA damage, which induced by MC-LR.5 Designing case-control study, 226 cases and 235 controls were recuited, and surveyed by questionare. Using logistic regression models, influence factors of primary liver cancer were evaluated, OR and 95%CI and additive interaction effect were compute. 6 Designing a two-stage molecular epidemiology study, peripheral blood of cases and controls were collected, then their DNA were extracted, and MALDI-TOF-MS was used to evaluate SNPs in the miRNAs of inflammatory pathway.[Results]1 We use transient transfection technology suppress the expression of XRCC1 successfully, and the inhibition efficiency after transfected in12h, 24h and 48h were 34.5%,64.4% and 62.9%, respectively.2 The differences among three groups were not significant at three time points when expoursed to the dose of 2μg/ml. Expoursed to 6μg/ml MC-LR for 16h, the difference between siRNA-XRCC1 group and normal control group was significant.And expoursed to 10μg/ml MC-LR for 8h or 16h, the difference between siRNA-XRCC1 group and two control groups was significant.3 Compared with the two control groups, the expression of PARP-1 of siRNA-XRCC1 group was enhanced by MC-LR(2μg/ml for 4h); Compared with siRNA-XRCC1(2μg/ml for 4h), the levels of the expression of PARP-1 of siRNA-XRCC1(2μg/ml for 8h) and siRNA-XRCC1 (2μg/ml for 16h) were reduced; The level of expression of PARP-1 of siRNA-XRCC1(10μg/ml for 4 h) is lower than siRNA-XRCC1(2μg/ml for 4 h).4 Compared with each 0μg/ml group, the Tail Moment decreased significantly. The siRNA-XRCC1 group compared with either normal group or missense siRNA group, the differences were significant, which show that XRCC1 knocked down cells were sensitive for MC-LR. And positive control explained that the comet assay was successful.5 Multivariate logistic regression analysis show that hepatitis B (OR=164.799) and drinking ditch pond water (OR=8.750) were risk factors of liver cancer, and drinking spring water (OR=0.136), drinking deep well water (OR=0.028), drinking tea (more than once a day) (OR=0.143) and eating pickled food (more than once a week) (OR1=0.031, OR2=0.034, OR3=0.012) were protective factors.6 Under codominant model, carriers of rs2168709 GG genotype had a statistically significantly increased risk for liver cancer (OR=1.782, 1.033-3.074); carriers of rs11807848 TC genotype had a statistically significantly increased risk for liver cancer (OR=1.622, 1.088-2.418). Under dominant model, carriers of rs11807848 TC or CC genotype had a statistically significantly increased risk for liver cancer (OR=1.665, 1.138-2.436). Under recessive model, carriers of rs2168709 CC genotype had a statistically significantly increased risk for liver cancer (OR=1.786, 1.044-3.053); carriers of rs17175796 GG genotype had a statistically significantly increased risk for liver cancer (OR=1.621, 1.002-2.624).[Conclusion]1 MC-LR can inhibited the proliferation of XRCC1 knocked-down BRL-3A; MC-LR regulated the expression of PARP-1 when the XRCC1 knocked-down BRL-3A cells expoursed to low dose for short time, on the contrary, reduced the expression of PARP-1 expoursed to high dose or a long time; MC-LR can induced DNA damage.2 Hepatitis B and drinking ditch pond water were the risk factors of liver cancer and drinking spring water, drinking deep well water, drinking tea (more than once a day) and eating pickled food (more than once a week) were protective factors.