| Objective Microcystin-LR(MCLR)is a biologically active cyclic heptapeptide compound that has been shown to be carcinogenic,but its carcinogenic mechanism is not fully understood.Resveratrol(Res)is a polyphenolic compound synthesized by plants against foreign pathogens.It is widely found in many plants and has special functions in the chemoprevention and treatment of cancer.This study was to investigate the effects of long-term low-dose MCLR exposure on the proliferation and malignant transformation of human hepatocyte cell line WRL68 and the intervention of Res,to study the expression difference of long non-coding RNA H19 in MCLR-induced malignant transformation with the intervention of Res,explore the molecular biological mechanism of Res affecting the proliferation of MCLR malignant cells.Methods1.Construction of MCLR-induced hepatic malignant transformation modelWRL68 cells was continuously infected with 10 nmol/L MCLR,and the same generation control group was established,and treated with PBS containing 0.01%DMSO.The cells were passaged every 3 days,and the cells were always exposed to 10 nmol/L of MCLR and cultured until the 25th generation.Morphological changes of cells in different transformation periods were observed.MTT assay was used to detect the growth viability of the cells;the soft agar assay was used to detect the colony forming ability of the cells,and the tumor formation ability of the cells was tested by nude mouse tumorigenicity assay.Flow cytometry was used to detect cell cycle and apoptosis in the P25 normal control group and MCLR treated group.2.Observing the effect of resveratrol on the proliferation of MCLR-transformed cells and the expression of lncRNA H19Routinely cultured WRL68 cells,six groups were set:normal control group,normal control+25 nmol/L Res intervention group,normal control +100 nmol/L Res intervention group,MCLR treated group,MCLR treated+25 nmol/L Res intervention group,MCLR treated+100 nmol/L Res intervention group.The cells in each MCLR exposure group were always treated with 10 nmol/L MCLR,while other normal control groups were treated with PBS containing 0.01%DMSO.For the Res intervention group,different concentrations(25 nmol/L and 100 nmol/L)of resveratrol were added after 4 hours of MCLR treated and passaged every 3 days,continuously cultured until the 25th generation.Dynamic monitoring cell growth change under MCLR treated and resveratrol intervention:MTT assay was used to detect cell proliferation ability;soft agar assay was used to evaluate cell anchorage-independent growth ability.qRT-PCR was used to dynamically monitor the changes of lncRNA H19 in cells exposed to MCLR by Res intervention.3.Construction of lncRNA H19 stable high expression cell lineConstruction of lncRNA H19 overexpressed and negative control lentiviral expression vector by Gateway technology.Packing lentivirus and infecting human liver cell line WRL68,screening stable transfected cell lines with puromycin.The green fluorescence of the cells was observed by fluorescence microscopy to detect the transfection effect,and the transfected cell lines were identified by qRT-PCR.4.Observing the effect of long-term low-dose MCLR exposure on the proliferation of H19-overexpressing cells and the intervention of res veratrolConventionally cultured WRL68 tranfected cells with stably overexpressed lncRNA H19 and corresponding transfected negative control cells.Six groups were set:negative control group,negative control+MCLR treated group,MCLR treated negative control+100 nmol/L Res intervention group,H19 overexpressed group,MCLR treated H19 group,MCLR treated H19+100 nmol/L Res intervention group.MCLR treated and resveratrol intervention are consistent with previous experiments.MTT assay was used to detect changes in cell proliferation ability.Results1.Long-term low-dose MCLR exposure induces malignant transformation of human liver cell line WRL68(1)Observing the morphology of cells under an inverted light microscope,the cells grown in a single layer,arranged in order,and the structure of the nucleus and cytoplasm was clear in the normal control group.However in the MCLR treated group,the cell boundary was not smooth,even with folds,the boundary between the nucleus and the cytoplasm was not clear,and the number of nucleoli was significantly increased,showing the infiltrating growth.(2)MTT assay results showed that when cultured to the 15th generation,the proliferation of cells in the MCLR treated group was faster than that in the normal control group.The proliferation rate of the 25th generation cells in the MCLR treated group was significantly faster than in the normal control group(P<0.05).(3)The results of 25th generation cell cycle experiments showed that the proportion of MCLR-treated cells in G0-G1 phase was lower than that in the same-generation normal control group(P<0.05).The proportion of G2-M phase in MCLR-treated group was higher than that in the same-generation normal control group(P<0.05).(4)The results of 25th generation cell apoptosis experiments showed that the apoptosis rate of MCLR treated cells was lower than that of the normal control group(P<0.05).(5)The results of soft agar experiment showed that no cell colonies were found in the normal control group when the 15th generation cells were cultured,while cell colonies were formed on the soft agar in the MCLR treated group.Compared with the normal control group and P15 MCLR treated group,the cell colonies of P25 MCLR group increased significantly and the volume also increased.The difference was statistically significant(P<0.05).(6)The results of tumor formation in nude mice showed that no tumors were found in nude mice inoculated with P15 normal control group and MCLR-treated group in the head and neck;no tumors were found in nude mice inoculated with P25 normal control cells while tumors were observed in nude mice inoculated with P25 MCLR treated group.Pathological section observation showed that the tumor tissue cells were disordered and the ratio of nucleoplasm was increased,which was a hepatocellular carcinoma with low degree of differentiation.2.Effect of resveratrol on the proliferation of MCLR-transformed cells and the expression of lncRNA H19(1)The MTT assay results showed that when cultured to the 25th generation,there was no significant change in cell proliferation rate in the normal control+25nM Res and the cell proliferation rate of normal control+100nM Res group was slower than that of normal control group(P<0.05).There was no significant change in cell proliferation rate in the MCLR+25nM Res group and the cell proliferation rate of MCLR+100nM Res group was significantly slower than MCLR treated group(P<0.05).(2)Soft agar experiment results show that when cultured to the 25th generation,the clone formation rate of MCLR treated group and MCLR treated+100nM Res group was higher than that of the corresponding uninfected group(P<0.05).The clone formation rate of MCLR treated+100nM Res group was lower than that of the MCLR treated group(P<0.01).(3)The results of qRT-PCR showed that the relative expression of H19 in the MCLR group was higher than that in the normal control group from the 10th generation.The relative expression of H19 in the MCLR+100nM Res group was lower than that of the MCLR in the same generation(P<0.05).The relative expression of H19 in the 15th generation,20th generation and 25th generation of MCLR group was significantly higher than that in the normal control group(P<0.05)and the relative expression of H19 increased gradually with the increase of the infected algebra(P<0.05)The relative expression of H19 in the 15th,20th and 25th generations of MCLR+100nM Res group was significantly lower than that of the same generation MCLR group(P<0.05);The relative expression of H19 in the 15th,20th and 25th generations of normal control+100nM Res group was significantly lower than that of the same generation normal control group(P<0.05).3.Construction of IncRNA H19 stable high expression cell lineAfter digestion with the recombinant plasmid,a fragment of about 2346 bp was observed by agarose gel electrophoresis,which was consistent with the expected results.The positive plasmids after electrophoresis were sequenced,and the sequencing results were consistent with the design.After lentivirus infection of target cells,almost all cells were fluorescent after 2 weeks of 2?g/ml puromycin selection,and the expression level of lncRNA H19 in H19 stably transfected cells was significantly higher than that of negative control group(P<0.05).4.Effects of long-term low-dose MCLR exposure on the proliferation of H19-overexpressing cells and the intervention of resveratrolThe results of MTT assay showed that the proliferation rate of cells in H19 high expression group was faster than that in negative control group;After the MCLR treated and resveratrol intervention in negative control group and H19 high expression group,the results of the MTT experiment showed that the proliferation rate of H19+MCLR group was faster than that of negative control+MCLR group(P<0.05);the proliferation rate of negative control+MCLR+100nM Res group was slower than that of negative control+MCLR group,the proliferation rate of H19+MCLR+100nM Res group was slower than that of H19+MCLR group(P<0.05).The proliferation rate of H19+MCLR+100nM Res group was faster than that of NC+MCLR+100nM Res group(P<0.05).Conclusions1.Long-term low-dose MCLR exposure can induce malignant transformation of human hepatocytes WRL68 with rapid cell proliferation,cell cycle G2-M arrested,and apoptosis rate decreased.Resveratrol can inhibit the proliferation and malignant transformation of cells induced by MCLR.2.The expression of lncRNA H19 was gradually increased during MCLR-induced WRL68 cell malignant transformation.Res intervention could down-regulate the expression of IncRNA H19.Overexpression of lncRNA H19 can reverse the inhibitory effect of Res on the proliferation of MCLR malignant transformed cells,suggesting that Res may inhibit MCLR-induced malignant growth by down regulating the expression level of lncRNA H19. |
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