| Objective Hepatocellular carcinoma is the most common cancer in Guangxi province. Water contamination expecially microcystics pollution has attracted wide attention because of its correlation with the progress of hepatocellular carcinoma, and the abnormal expression of long non-coding RNA has been found to be related with cancer. Microcystin-LR is one of the cancerogens in the world, so we determin to establish the malignant transformation model of WRL68 cell induced by exposured in long-low-dose Microcystin-LR and investigate the expression of Long non-coding RNAs in the process of malignant transformation. The dynamic expression of these LncRNAs will provide scientific basis for the further research of MC-LR carcinogenic mechanism.Methods WRL68 cells were exposured in MC-LR with the concentration of lOnmol/L at all the time, and including a control group with 0.01%concentration DMSO of PBS, passaged every two days until the twenty fifth.The cells that were treated and passaged for 5, 10, 15, 20, and 25 passages were labeled P5, P10, P15, P20 and P25, respectively. The characteristics of cellular morphology in different passages were observed and malignant transformation of WRL68 cells were identified through serum resistance assay,soft agar assay and nude mice tumorigenesis assay. Then the malignant transformation model of WRL68 cells was established. RNAs from different passages of the model and the corresponding control groups were extracted and reverse transcriptased into cDNA. Quantitative real-time PCR was used to determine the relative expression of LncRNA H19, HOTAIR, HULC, MEG3 and AFAP1-AS1.Results1. The result of cell morphology showed that when compared with the parallel control group, the boundaries between the nucleus and cytoplasm in P25 MC-LR treated cells were indistinct, the numbers of nucleolus were obviously increased, the boundaries between cells were not smooth, but fold,and showed invasive growth.2. The result of soft agar assay showed that the colonies appared in P15 MC-LR treated cells and the colonies were significantly more and bigger in P25 MC-LR treated cells than the corresponding control cells and P15 MC-LR treated cells (P<0.05).3. The results of MTT and serum dependence assay showed that P25 MC-LR treated cells grew faster outstandingly than control group in 10% FBS concentration, and P25 MC-LR treated cells were grew faster than the corresponding control cells in low FBS concentration, such as 0.5% and 1%FBS concentration, which means P25 MC-LR treated cells has the abality to survive in low FBS concentration (P<0.05).4. The result of tumorigenicity in nude mice showed that tumors were appeared when injected with P25 MC-LR treated cells, and no tumors were appeared when injected with P15 MC-LR treated cells and control cells.Pathology examination results show that the tumor tissue was with high degree of malignant transformation, and the arrangement of cells was disordered and nucleoplasm ratio was increased.5. The expression of H19, HOTAIR and HULC in different passages showed that H19, HOTAIR, HULC in cells treated with MC-LR were all up-regulated with the treatment time prolonged (P<0.05). And compared with the corresponding control cells (1.00±0.07), the expression of H19 in P25 MC-LR treated cells was statistically up-regulated to (2.23±0.29), and the expression of HOTAIR was statistically up-regulated to (3.18±0.18) when compared with the corresponding control cells (1.03±0.11), and compared with the corresponding control cells (1.00±0.16), the expression of HULC in P25 MC-LR treated cells was significantly up-regulated to (2.26±0.17).6. The expression of MEG3 and AFAP1-AS1 in different passages showed that MEG3 and APAP1-AS1 in P10 MC-LR treated cells were all up-regulated and the expression of MEG3 was statistically up-regulated to (1.311±0.09) when compared with the corresponding control cells (1.02±0.06), and the expression of AFAP1-AS1 was statistically up-regulated to (1.38±0.09) when compared with the corresponding control cells (1.05±0.04). And the expression of MEG3 and AFAP1-AS1 were down-regulated in P20 MC-LR treated cells. The expression of MEG3 and AFAP1-AS1 were statistically down-regulated to (0.34±0.05) and (0.50 ±0.08), when compared with the corresponding control cells.7. The results of linear correlation showed the expression of H19 was positively correlated with HOTAIR (r=0.90, P<0.05) and also positively correlated with HULC (r=0.94, P<0.01) , and the expression of HOTAIR also positively correlated with HULC (r=0.95, P<0.01), and the expression of MEG3 also positively correlated with AFAP1 -AS1 (r=0.99, P<0.01) 。Conclusion1. Hepatocyte exposured in low-dose MC-LR for a long time could induce malignant transformation.2. The expression of H19, HOTAIR, HULC were continuously up-regulated during the malignant transformation, and the expression of MEG3 and AFAP1-AS1 were up-regulated in the early stage of malignant transformation and down-regulation in the stage of malignant transformation, which means LncRNAs like H19, HOTAIR might be involved in the biology effect of microcystin-LR-induced carcinogenesis. |
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