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Targeting protein arginine methyltransferase 5 inhibits human hepatocellular carcinoma growth via the downregulation of beta-catenin.

Zhang B, Dong S, Li Z, Lu L, Zhang S, Chen X, Cen X, Wu Y - J Transl Med (2015)

Bottom Line: Knockdown of PRMT5 significantly reduced the proliferation of HCC cells, but did not affect the growth of normal liver cells.Silencing PRMT5 significantly down-regulated the expression of β-catenin and the downstream effector Cyclin D1 in HCC cells.Meanwhile, AMI-1 decreased the expression levels of symmetric dimethylation of H4 (H4R3me2s), a histone mark of PRMT5.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Basic Medical Sciences, Lanzhou University; Key Lab of Preclinical Study for New Drugs of Gansu Province, No 199, Dongang West Road, Lanzhou, 730000, Gansu, China. zhangbl@lzu.edu.cn.

ABSTRACT

Background: Protein arginine methyltransferase 5 (PRMT5), a type II PRMT, is highly expressed in some tumors, but its role in hepatocellular carcinoma (HCC) is still unknown.

Methods: PRMT5 level in HCC specimens was determined by immunohistochemical staining and the association with clinicopathologic features was evaluated. PRMT5 was inhibited by AMI-1 (a small molecule inhibitor of PRMTs) or small interference RNA (siRNA). The proliferation of HCC cells was tested by Cell Counting Kit-8, cell migration was evaluated by Transwell assay and cell cycle and apoptosis were analyzed by flow cytometry. The effect of AMI-1 on HCC in vivo was examined by mouse xenograft model.

Results: PRMT5 expression was markedly upregulated in HCC tissues, and correlated inversely with overall patient survival. Knockdown of PRMT5 significantly reduced the proliferation of HCC cells, but did not affect the growth of normal liver cells. Furthermore, β-catenin was identified as a target of PRMT5. Silencing PRMT5 significantly down-regulated the expression of β-catenin and the downstream effector Cyclin D1 in HCC cells. AMI-1 strongly inhibited HCC growth in vivo, increased the ratio of Bax/Bcl-2, and led to apoptosis and loss of migratory activity in several HCC cells. Meanwhile, AMI-1 decreased the expression levels of symmetric dimethylation of H4 (H4R3me2s), a histone mark of PRMT5.

Conclusions: PRMT5 plays an important role in HCC. PRMT5 may be a promising target for HCC therapy.

No MeSH data available.


Related in: MedlinePlus

Upregulation of PRMT5 in HCC tissues and its association with poor survival. A Immunohistochemical staining of PRMT5 in HCC tissues and corresponding NATs. Positive cells were stained brown. Magnification, (a, c) ×50; (b, d) ×100; scale bar 50 μm. B PRMT5 scores based on the nuclear or cytoplasmic levels of expression in 54 HCC patients, compared with matched normal tissues. C Association between PRMT5 expression and the survival of HCC patients
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Fig1: Upregulation of PRMT5 in HCC tissues and its association with poor survival. A Immunohistochemical staining of PRMT5 in HCC tissues and corresponding NATs. Positive cells were stained brown. Magnification, (a, c) ×50; (b, d) ×100; scale bar 50 μm. B PRMT5 scores based on the nuclear or cytoplasmic levels of expression in 54 HCC patients, compared with matched normal tissues. C Association between PRMT5 expression and the survival of HCC patients

Mentions: To evaluate the clinical significance of PRMT5 in HCC development, we analyzed PRMT5 level and its distribution in HCC tissues by immunohistochemical (IHC) staining. The array included the cancer tissue samples and corresponding normal adjacent tissues (NATs) from 54 HCC patients, which consisted of 48 males and 6 females, with a medium age of 53 years (range 38–72 years). Among the patients 31 (57.4 %) died of tumor related to causes, and 23 (42.6 %) were still alive as of the last follow-up (Table 1). PRMT5 levels in the tissues array specimens were assessed based on the staining density scores. We found that in cancer tissues, 31 cases (57.4 %) exhibited strong immunopositivity, 18 cases (33.3 %) exhibited moderate immunopositivity, and 5 cases (9.3 %) exhibited no or weak immunopositivity. In contrast, the majority of normal tissues (51.9 %) exhibited no or weak PRMT5 expression (Table 2). Statistical analysis revealed that PRMT5 level significantly increased in cancer tissues compared with the corresponding NATs (Fig. 1A, B). In addition, cytoplasmic PRMT5 level, but not nuclear PRMT5 level, had a trend to be negatively correlated with the survival rate of the patients with HCC (Fig. 1C). As negative control, PRMT5 antibody could not detect positive staining (Additional file 1: Figure 1). Notably, no significant association was found between PRMT5 expression in HCC and tumor size or TNM stage (P > 0.05, Table 1). Univariate Cox regression analyses showed that PRMT5 expression in the cytoplasm was significantly correlated with overall survival (Table 3). Furthermore, multivariate Cox regression analysis confirmed that cytoplasmic PRMT5 expression could serve as independent predictor of the overall survival of patients with HCC (Table 3). In addition, we performed Western blot analysis to determine of PRMT5 protein level in several HCC cell lines. As shown in Additional file 1: Figure 2, PRMT5 was expressed in normal liver HL-7702 cells and highly expressed in Bel-7704, HepG2 and CBRH-7919 HCC cell lines. Taken together, these results indicate that PRMT5 overexpressed is tightly linked to HCC proliferation and development.Table 1


Targeting protein arginine methyltransferase 5 inhibits human hepatocellular carcinoma growth via the downregulation of beta-catenin.

Zhang B, Dong S, Li Z, Lu L, Zhang S, Chen X, Cen X, Wu Y - J Transl Med (2015)

Upregulation of PRMT5 in HCC tissues and its association with poor survival. A Immunohistochemical staining of PRMT5 in HCC tissues and corresponding NATs. Positive cells were stained brown. Magnification, (a, c) ×50; (b, d) ×100; scale bar 50 μm. B PRMT5 scores based on the nuclear or cytoplasmic levels of expression in 54 HCC patients, compared with matched normal tissues. C Association between PRMT5 expression and the survival of HCC patients
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4635578&req=5

Fig1: Upregulation of PRMT5 in HCC tissues and its association with poor survival. A Immunohistochemical staining of PRMT5 in HCC tissues and corresponding NATs. Positive cells were stained brown. Magnification, (a, c) ×50; (b, d) ×100; scale bar 50 μm. B PRMT5 scores based on the nuclear or cytoplasmic levels of expression in 54 HCC patients, compared with matched normal tissues. C Association between PRMT5 expression and the survival of HCC patients
Mentions: To evaluate the clinical significance of PRMT5 in HCC development, we analyzed PRMT5 level and its distribution in HCC tissues by immunohistochemical (IHC) staining. The array included the cancer tissue samples and corresponding normal adjacent tissues (NATs) from 54 HCC patients, which consisted of 48 males and 6 females, with a medium age of 53 years (range 38–72 years). Among the patients 31 (57.4 %) died of tumor related to causes, and 23 (42.6 %) were still alive as of the last follow-up (Table 1). PRMT5 levels in the tissues array specimens were assessed based on the staining density scores. We found that in cancer tissues, 31 cases (57.4 %) exhibited strong immunopositivity, 18 cases (33.3 %) exhibited moderate immunopositivity, and 5 cases (9.3 %) exhibited no or weak immunopositivity. In contrast, the majority of normal tissues (51.9 %) exhibited no or weak PRMT5 expression (Table 2). Statistical analysis revealed that PRMT5 level significantly increased in cancer tissues compared with the corresponding NATs (Fig. 1A, B). In addition, cytoplasmic PRMT5 level, but not nuclear PRMT5 level, had a trend to be negatively correlated with the survival rate of the patients with HCC (Fig. 1C). As negative control, PRMT5 antibody could not detect positive staining (Additional file 1: Figure 1). Notably, no significant association was found between PRMT5 expression in HCC and tumor size or TNM stage (P > 0.05, Table 1). Univariate Cox regression analyses showed that PRMT5 expression in the cytoplasm was significantly correlated with overall survival (Table 3). Furthermore, multivariate Cox regression analysis confirmed that cytoplasmic PRMT5 expression could serve as independent predictor of the overall survival of patients with HCC (Table 3). In addition, we performed Western blot analysis to determine of PRMT5 protein level in several HCC cell lines. As shown in Additional file 1: Figure 2, PRMT5 was expressed in normal liver HL-7702 cells and highly expressed in Bel-7704, HepG2 and CBRH-7919 HCC cell lines. Taken together, these results indicate that PRMT5 overexpressed is tightly linked to HCC proliferation and development.Table 1

Bottom Line: Knockdown of PRMT5 significantly reduced the proliferation of HCC cells, but did not affect the growth of normal liver cells.Silencing PRMT5 significantly down-regulated the expression of β-catenin and the downstream effector Cyclin D1 in HCC cells.Meanwhile, AMI-1 decreased the expression levels of symmetric dimethylation of H4 (H4R3me2s), a histone mark of PRMT5.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Basic Medical Sciences, Lanzhou University; Key Lab of Preclinical Study for New Drugs of Gansu Province, No 199, Dongang West Road, Lanzhou, 730000, Gansu, China. zhangbl@lzu.edu.cn.

ABSTRACT

Background: Protein arginine methyltransferase 5 (PRMT5), a type II PRMT, is highly expressed in some tumors, but its role in hepatocellular carcinoma (HCC) is still unknown.

Methods: PRMT5 level in HCC specimens was determined by immunohistochemical staining and the association with clinicopathologic features was evaluated. PRMT5 was inhibited by AMI-1 (a small molecule inhibitor of PRMTs) or small interference RNA (siRNA). The proliferation of HCC cells was tested by Cell Counting Kit-8, cell migration was evaluated by Transwell assay and cell cycle and apoptosis were analyzed by flow cytometry. The effect of AMI-1 on HCC in vivo was examined by mouse xenograft model.

Results: PRMT5 expression was markedly upregulated in HCC tissues, and correlated inversely with overall patient survival. Knockdown of PRMT5 significantly reduced the proliferation of HCC cells, but did not affect the growth of normal liver cells. Furthermore, β-catenin was identified as a target of PRMT5. Silencing PRMT5 significantly down-regulated the expression of β-catenin and the downstream effector Cyclin D1 in HCC cells. AMI-1 strongly inhibited HCC growth in vivo, increased the ratio of Bax/Bcl-2, and led to apoptosis and loss of migratory activity in several HCC cells. Meanwhile, AMI-1 decreased the expression levels of symmetric dimethylation of H4 (H4R3me2s), a histone mark of PRMT5.

Conclusions: PRMT5 plays an important role in HCC. PRMT5 may be a promising target for HCC therapy.

No MeSH data available.


Related in: MedlinePlus