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. 2016 May 31;7(26):40266–40284. doi: 10.18632/oncotarget.9733

Figure 4. JARID2 Promotes Metastasis of Hepatocellular Carcinoma by inhibiting PTEN Expression through H3K27 Trimethylation.

Figure 4

(A) Western blot analysis showed, compared with control cells, a decrease in H3K27me3 in HCCLM3shJARID2-1 and MHCC97-HshJARID2-1 cells, but an increase in H3K27me3 in HepG2JARID2 cells. Histones H3 and β-actin were used as loading controls. (B) PTEN expression was upregulated in HCC cells with JARID2 knockdown and downregulated in HCC cells with JARID2 overexpression. The expression levels of PTEN was measured in HCCLM3, MHCC97-H cells with JARID2 knockdown, HepG2 cells with JARID2 overexpression and their corresponding control by qPCR (B1), semiquantitative RT-PCR (B2), western blot (B3). (C) The expression level of JARID2 exhibited an inverse trend with that of PTEN in HCC samples. The expression of JARID2 and PTEN was detected in 30 paired HCC tissues and ANLTs. (C1) The relative level of JARID2 mRNA expression was plotted against the relative level of PTEN mRNA expression. (C2) Western blot detected the expression of JARID2 and PTEN protein in the same HCC tissues and ANLTs. (D) JARID2 was associated with increased H3K27me3 at PTEN promoter region. Quantitative chromatin immunoprecipitation (qChIP) was used to assess H3K27me3 at PTEN promoters region in HCCLM3, MHCC97-H cells with shJARID2 knockdown, HepG2 cells with JARID2 overexpression and their corresponding control vector. qChIP was performed using the indicated antibodies. Purified rabbit IgG was a negative control. The percentage fold difference of shJARID2/control, Vector/JARID2 was calculated as the difference in percentage of the qChIP in HCCLM3, MHCC97-H cells transfected with shJARID2 relative to cells transfected with control vector, in HepG2 cells transfected with control vector relative to cells transfected with JARID2 vector respectively. Each bar represents the mean ± SEM for triplicate experiments. (E, F) Gain- and loss-off function studied with PTEN expression vector, shPTENs or its control showed PTEN is a critical downstream effector in JARID2-promoted invasion and metastasis in HCC. HCC cells were transfected with shRNAs for PTEN or PTEN expression vector to inhibit or restore the PTEN expression. Wound-healing assays (E) and transwell assays (F) were determined in HCCLM3, MHCC97-H, HepG2 cells. **, P < 0.01; ***, P < 0.001.