| Objective:（1）long-noncoding RNA H19 was knocked down at the cell level to study the effects of H19 on HL60 cell（acute myeloid leukemia cell）on biological function（proliferation,apoptosis,invasion and migration）,so as to provide some theoretical basis for the exploration of targeted therapy for some types of acute leukemia.（2）We studied the possible downstream effectors of H19 and explored the methylation of CpG island in target gene promoter,and elaborated the preliminary mechanism of H19 on HL60 cells.Methods:（1）Using RNAi interference technique,the pGV248-H19-RNAi-GFP and Puro lentivirus interference vectors are constructed and transfected into HL60 cells（2）After lentivirus successfully transfected HL60 and achieved high transfection rate,the effects of knocking down H19 on HL60 proliferation were detected by CCK-8,colony formation assay and flow cytometry respectively.（3）The benzimidazole fluorescent dyes（bisbenzimide,Hoechst 33258）staining was used to observe the morphology of apoptosis and cell apoptosis rate was detected by flow cytometry and Western blot was applied for detecting the expression of apoptosis related proteins Caspase-3,Caspase-8 and Caspase-9 in cell lines,The study of apoptosis by these experiments.（4）The influence of H19 on the invasion and migration of HL60 was detected by Transwell experiment,The OD value of transmembrane cells was used for measuring the reflected transmembrane cell number,and in order to verify the effect on invasion and m(5ig)r aSticorne.ening downstream target genes that may be regulated by H19 throughning downstream target genes that may be regulated by H19 through literature retrieval,and then detection of the expression of related target genes and potential downstream effector molecules by RT-qPCR at the cell level after knocking down H19,and then Western blot was applied to detect the protein expression of target gene and downstream effector.Finally,pyrosequencing was used to detect the methylation of CpG island in the promoter region of the target gene after H19 knocking down at the cell level,and then comparison of the methylation rate of the target genes in the experimental group and the control group to explain the preliminary mechanism of the impact of knocking-down H19 on the biological function of HL60 cells.Results:（1）The GV248-H19-RNAi lentiviral vector was successfully constructed and transfected into HL60 cell line.RT-qPCR assay showed that the expression of H19 in HL60 cells was down regulated,which was statistically significant,it could provide a basis for subsequent experiments.（2）After successful transfection of HL60 cell with lentivirus and high transfection rate,The effects of knocking down H19 on the proliferation of HL60 cell were detected by CCK-8,clonogenic and flow cytometry.The results suggest that knocking down of H19 can inhibit the proliferation of HL60 cells and lead to G0/G1 phase block and decrease of S phase in cell cycle.（3）Morphologic of benzimidazole fluorescent dye to observe the apoptosis of HL60 cells after H19 knocking down shows that Some cell nucleus are dense and some are broken into fragments,the apoptotic bodies are also produced,and the morphologic changes of apoptotic cells appear.Flow cytometry shows a significant increase in the rate of apoptosis in HL60 cells.The reault of Western blotting is that the expression of apoptosis related proteins Caspase-3,Caspase-8 and Caspase-9 were up-regulated in HL60 cell lines after transfection.（4）After knocking down H19 on HL60 cells,Tanswell assay detected the invasion and migration ability.The results showed that there was no significant difference in the number of penetrating cells.The difference was not statistically significant.Knocking down H19 had no significant effect on the invasion and migration of HL60 cells.（5）By reading related literature,we speculate that the 1 target genes that H19 may regulate:RB gene,which is based on existing research as tumor suppressor gene.（6）After knocking down H19,RT-qPCT detected the possible target gene RB up regulation.The possible downstream effector DNMT1gene、DNMT3Agene、DNMT3B gene was down regulated.（7）After knocking down H19,the expression of related proteins was detected by Western-blotting,and the DNMT1 protein、DNMT3A protein、DNMT3B protein was downregulated in HL60 cells,RB protein were upregulated.The result is consistent with RT-qPCT.（8）After H19 knocking down,the methylation rate of CpG island in the promoter region of the RB gene decreased in HL60 cells.Conclusion:（1）Knocking down H19 in vitro can inhibit the proliferation of HL60 cells,blocks in G0/G1 phase and S phase declines,and also promote cell apoptosis.It may induce apoptosis through activating Caspase apoptotic protein related pathways.（2）Tanswell test was applied for detecting the the effect of knocking down H19on the invasion and migration of HL60 cells.The results showed that there was no significant difference in the number of membrane cells,and the difference was not statistically significant.The knocking down of H19 had no significant effect on the invasion and migration of HL60 cells.（3）By searching relevant literature,we have predicted RB gene was the target genes that may be regulated by H19.This gene is a tumor suppressor gene based on relevant literature.（4）After H19 knocking down,the methylation rate of CpG island in the promoter region of the RB gene decreased in HL60 cells.（5）The possible mechanism of the effect of H19 on the biological function of HL60 cells is that inducing the low methylation rate of the CpG island of the promoter region of RB gene by affecting the expression of the downstream effector molecule DNMT1、DNMT3A、DNMT3B,thereby regulates the high expression of RB gene and then affects the biological function of HL60 cells. |
