Long non-coding RNA Myd88 promotes growth and metastasis in hepatocellular carcinoma via regulating Myd88 expression through H3K27 modification

Abstract

Enhanced Myd88 expression has been found in various parenchymal tumors especially in hepatocellular carcinoma with little mechanism of its upregulation known. A lot of long non-coding RNAs are reported to regulate the protein-coding genes which have location association through various mechanisms. In our study we confirmed a new long non-coding RNA Myd88 aberrant upregulated in HCC located upstream of Myd88 and verified a positive regulation relationship between them indicating that Lnc-Myd88 might participate in the enhanced expression of Myd88 in HCC. The gain- and loss-of-function analysis revealed that Lnc-Myd88 could promote the proliferation and metastasis of HCC both in vitro and in vivo. In addition, ChIP assays demonstrated that Lnc-Myd88 might increase Myd88 expression through enhancing H3K27Ac in the promoter of Myd88 gene, thus resulting in the activation of both NF-κB and PI3K/AKT signal pathways. In conclusion, we proposed that Lnc-Myd88 might serve as a novel diagnosis and therapeutic target for HCC.

Figure 1

Lnc-Myd88 is upregulated with a high correlation with Myd88 in hepatocellular carcinoma tissues and correlated with poor prognosis. (a) Ectopic expression of Lnc-Myd88 in HCC tumor tissues and corresponding adjacent normal liver tissues were detected by quantitative real-time PCR normalized to GAPDH (N=110, P<0.001). (b) Enhanced expression of Myd88 in HCC tissues compared with the adjacent normal liver tissues were detected by quantitative real-time PCR normalized to GAPDH (N=110, P<0.001). (c) A positive correlation between expression levels of Myd88 and Lnc-Myd88 determined by Pearson analysis (N=110, r²=0.5665,  P<0.001). (d and e) Relative expression level of Myd88 protein in HCC tissues and adjacent tissues were tested by western blotting and immunohistochemical assays (original magnification × 200). (f) According to the median value, patients were divided into two groups according to Lnc-Myd88 expression in HCC tissues. The log-rank test was used to calculated the overall survival and recurrence-free survival of patients

Figure 2

Lnc-Myd88 promotes HCC cells proliferation in vitro. (a) The transfection efficiency of ectopic expression and gene silencing of Lnc-Myd88 in SMMC-7721 and Huh7 cells was determined by qRT-PCR. (b) Proliferation ability was detected by CCK8 assay, overexpression of Lnc-Myd88 promoted SMMC-7721 cells proliferation, whereas knockdown of Lnc-Myd88 inhibited Huh7 cells proliferation. (c) Colony formation assays were performed on differently treated HCC cells for 2 weeks, representative graphs are shown. (d) EdU immunofluorescence staining confirmed the function of Lnc-Myd88 on HCC cells proliferation. Original magnification × 200. Stable overexpression of Lnc-Myd88 increased the proliferation of SMMC-7721 cells while knockdown of Lnc-Myd88 decreased the proliferation of Huh7 cells. All experiments were performed in triplicate and presented as the mean±S.E.M. (*P<0.05, **P<0.01)

Figure 3

Lnc-Myd88 regulates hepatoma cells cell cycle, cell apoptosis and cell migration in vitro. (a) Cell-cycle analysis of SMMC-7721 cells stable overexpressing Lnc-Myd88 and Huh7 cells stable silenced Lnc-Myd88 expression was conducted by flow cytometry. The distribution of the cell cycle was shown in the graphs. (b) Cells were cultured with complete medium with 0.5 mM peroxide overnight, and cell apoptosis rate was detected by flow cytometry using the Annexin V-APC/7-AAD staining kit. The bar graph shows the percentage of apoptotic cells. (c) Invasion and migration assay of Lnc-Myd88 overexpressed and silenced cells. The bar graph shows the number of cells migrated or invaded through the membrane. Original magnification × 200. All experiments were performed in triplicate and presented as the mean±S.E.M. (*P<0.05, **P<0.01)

Figure 4

Lnc-Myd88 enhances tumor growth and metastasis in vivo. (a–c) BAB/c nude mice (6 weeks of age) were subcutaneous transplantated with SMMC-7721 cells, Lv-Lnc-Myd88-SMMC-7721 cells (1 × 10⁷) in the right groin and Lv-NC SMMC-7721 (1 × 10⁷) cells in the left groin (5 mice in each group). The volume of tumors was calculated every 5 days after transplantation and mice were killed 30 days after implantation. Lnc-Myd88 strengthen the tumor growth of SMMC-7721 cells in nude mice. The volume of each tumor was calculated as the length × width² × 0.5. (d) Myd88 expression level in the tumor samples determined by IHC. Original magnification × 200. (e) In the tail vein xenograft model, mice (8 in each group) were injected with Huh7 cells (1 × 10⁷ suspended in 200 μl PBS) through the tail vein and killed 5 weeks later, lung metastasis were investigated in each group respectively by an in vivo fluorescence imaging system. (f) Compared with the Lnc-Myd88 knockdown group (three mice presented lung colonization), six mice presented lung colonization with more and larger tumors in the control group. (g) All the results of lung colonization were validated by the histological examination (H&E). Original magnification × 200 (*P<0.05, **P<0.01)

Figure 5

Lnc-Myd88 induces an upregulation of Myd88 by enhanced acetylation of the promoter of Myd88 and then activates NF-κB and PI3K/AKT signal pathways. (a) Enrichment prediction of H3K4m3, H3K27Ac and H3K27m3 in the promoter region of Myd88 through ENCODE database. (b) The enrichment of H3K4m3, H3K27Ac and H3K4m3 in the promoter region of Myd88 in HCC patients. (c) The alteration of the enrichment of H3K27Ac in the promoter region of Myd88 in HCC cells treated with Lnc-Myd88. (d and e) The alteration of mRNA and protein level of Myd88 in HCC cells induced by Lnc-Myd88. GAPDH was used as a loading control. (f and g) The levels of NF-κB, p-NF-κB, AKT, p-AKT and GAPDH were examined by western blotting in SMMC-7721 and Huh7 cells treated with Lnc-Myd88. The experiments were performed in triplicate; the data are expressed as the mean±S.E.M. (*P<0.05, **P<0.01)