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Lentiviral-Mediated Short Hairpin RNA Knockdown of MTDH Inhibits Cell Growth and Induces Apoptosis by Regulating the PTEN/AKT Pathway in Hepatocellular Carcinoma.

Li WF, Ou Q, Dai H, Liu CA - Int J Mol Sci (2015)

Bottom Line: We found that MTDH protein levels are higher in most HCC cancerous tissues compared with their matched adjacent non-tumor tissues.Additionally, the MTDH mRNA was also higher inHCC tissues compared to their matched adjacent non-tumor tissues.All these results indicated that MTDH protein levels in most HCC tissues are higher than non-tumor tissues, and knockdown of MTDH inhibited growth and induced apoptosis in HCC cells through the activation of PTEN.Therefore, MTDH might be an effective targeted therapy gene for HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatibiliary Surgery, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China. wen_fang79@yahoo.com.

ABSTRACT
The activation of oncogenes and the loss of tumor suppressor genes are believed to play critical roles in the pathogenesis of human hepatocellular carcinoma (HCC). Metaherin(MTDH), also called astrocyte elevated gene-1 (AEG-1), is frequently amplified in a variety of cancers, but the roles of MTDH with regard to growth and apoptosis in HCC have not yet been studied. In the present study, we first analyzed the expression of MTDH in HCC samples. We found that MTDH protein levels are higher in most HCC cancerous tissues compared with their matched adjacent non-tumor tissues. Additionally, the MTDH mRNA was also higher inHCC tissues compared to their matched adjacent non-tumor tissues. Knockdown of the endogenous MTDH using small interfering RNA further showed that deficiency of MTDH suppressed cell growth and caused apoptosis in HCC cells. Knockdown MTDH promoted PTEN and p53 expression in HCC cells and inhibited AKT phosphorylation. Knockdown MTDH also inhibited tumor growth in vivo. All these results indicated that MTDH protein levels in most HCC tissues are higher than non-tumor tissues, and knockdown of MTDH inhibited growth and induced apoptosis in HCC cells through the activation of PTEN.Therefore, MTDH might be an effective targeted therapy gene for HCC.

No MeSH data available.


Related in: MedlinePlus

shRNA silencing of MTDH expression effectively reduces MTDH mRNA and protein expression in HepG2 cells. (A) Stably transfected HepG2 cells on fluorescence microscopy (200×); (B) q-PCR quatification of MTDH mRNA, and products were observed with 1.5% agarose gel containing 0.5 μg/mL ethidium bromide with an ultraviolet illuminator; (C) Western blot to test MTDH protein expression.
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ijms-16-19419-f002: shRNA silencing of MTDH expression effectively reduces MTDH mRNA and protein expression in HepG2 cells. (A) Stably transfected HepG2 cells on fluorescence microscopy (200×); (B) q-PCR quatification of MTDH mRNA, and products were observed with 1.5% agarose gel containing 0.5 μg/mL ethidium bromide with an ultraviolet illuminator; (C) Western blot to test MTDH protein expression.

Mentions: To further explore the molecular mechanism of MTDH-mediated pathological functions in HCC, we examined knockdown MTDH protein expression in HepG2 cells. Cells were infected by a lentivirus which contained MTDH shRNA (LV-GFP-MTDH-shRNA) or an empty vector (LV-GFP-NC-shRNA) (Figure 2A). Uninfected HepG2 cells were included as the negative control. The infected efficiency was calculated by dividing fluorescent HepG2 cell number and total HepG2 cells with fluorescence microscopy. The infected efficiencies were 93.2% and 92.8% in the LV-GFP-MTDH-shRNA and LV-GFP-NC-shRNA groups, respectively. Following infection, the cells were collected for mRNA and protein expression analysis. As indicated in Figure 2B, apparently, compared with the LV-GFP-NC-shRNA cells and uninfected negative control cells, the mRNA and protein expression levels of MTDH in the LV-GFP-MTDH-shRNA cells was decreased. Based on the RT-PCR results, the mRNA level of MTDH was reduced by 89.6% in the LV-GFP-MTDH-shRNA cells, while the protein level of MTDH was significantly reduced by 73.8% in the LV-GFP-MTDH-shRNA cells vs. that in the LV-GFP-NC-shRNA and control cells as detected by Western blot.


Lentiviral-Mediated Short Hairpin RNA Knockdown of MTDH Inhibits Cell Growth and Induces Apoptosis by Regulating the PTEN/AKT Pathway in Hepatocellular Carcinoma.

Li WF, Ou Q, Dai H, Liu CA - Int J Mol Sci (2015)

shRNA silencing of MTDH expression effectively reduces MTDH mRNA and protein expression in HepG2 cells. (A) Stably transfected HepG2 cells on fluorescence microscopy (200×); (B) q-PCR quatification of MTDH mRNA, and products were observed with 1.5% agarose gel containing 0.5 μg/mL ethidium bromide with an ultraviolet illuminator; (C) Western blot to test MTDH protein expression.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581304&req=5

ijms-16-19419-f002: shRNA silencing of MTDH expression effectively reduces MTDH mRNA and protein expression in HepG2 cells. (A) Stably transfected HepG2 cells on fluorescence microscopy (200×); (B) q-PCR quatification of MTDH mRNA, and products were observed with 1.5% agarose gel containing 0.5 μg/mL ethidium bromide with an ultraviolet illuminator; (C) Western blot to test MTDH protein expression.
Mentions: To further explore the molecular mechanism of MTDH-mediated pathological functions in HCC, we examined knockdown MTDH protein expression in HepG2 cells. Cells were infected by a lentivirus which contained MTDH shRNA (LV-GFP-MTDH-shRNA) or an empty vector (LV-GFP-NC-shRNA) (Figure 2A). Uninfected HepG2 cells were included as the negative control. The infected efficiency was calculated by dividing fluorescent HepG2 cell number and total HepG2 cells with fluorescence microscopy. The infected efficiencies were 93.2% and 92.8% in the LV-GFP-MTDH-shRNA and LV-GFP-NC-shRNA groups, respectively. Following infection, the cells were collected for mRNA and protein expression analysis. As indicated in Figure 2B, apparently, compared with the LV-GFP-NC-shRNA cells and uninfected negative control cells, the mRNA and protein expression levels of MTDH in the LV-GFP-MTDH-shRNA cells was decreased. Based on the RT-PCR results, the mRNA level of MTDH was reduced by 89.6% in the LV-GFP-MTDH-shRNA cells, while the protein level of MTDH was significantly reduced by 73.8% in the LV-GFP-MTDH-shRNA cells vs. that in the LV-GFP-NC-shRNA and control cells as detected by Western blot.

Bottom Line: We found that MTDH protein levels are higher in most HCC cancerous tissues compared with their matched adjacent non-tumor tissues.Additionally, the MTDH mRNA was also higher inHCC tissues compared to their matched adjacent non-tumor tissues.All these results indicated that MTDH protein levels in most HCC tissues are higher than non-tumor tissues, and knockdown of MTDH inhibited growth and induced apoptosis in HCC cells through the activation of PTEN.Therefore, MTDH might be an effective targeted therapy gene for HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatibiliary Surgery, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China. wen_fang79@yahoo.com.

ABSTRACT
The activation of oncogenes and the loss of tumor suppressor genes are believed to play critical roles in the pathogenesis of human hepatocellular carcinoma (HCC). Metaherin(MTDH), also called astrocyte elevated gene-1 (AEG-1), is frequently amplified in a variety of cancers, but the roles of MTDH with regard to growth and apoptosis in HCC have not yet been studied. In the present study, we first analyzed the expression of MTDH in HCC samples. We found that MTDH protein levels are higher in most HCC cancerous tissues compared with their matched adjacent non-tumor tissues. Additionally, the MTDH mRNA was also higher inHCC tissues compared to their matched adjacent non-tumor tissues. Knockdown of the endogenous MTDH using small interfering RNA further showed that deficiency of MTDH suppressed cell growth and caused apoptosis in HCC cells. Knockdown MTDH promoted PTEN and p53 expression in HCC cells and inhibited AKT phosphorylation. Knockdown MTDH also inhibited tumor growth in vivo. All these results indicated that MTDH protein levels in most HCC tissues are higher than non-tumor tissues, and knockdown of MTDH inhibited growth and induced apoptosis in HCC cells through the activation of PTEN.Therefore, MTDH might be an effective targeted therapy gene for HCC.

No MeSH data available.


Related in: MedlinePlus