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MicroRNA-192 suppresses liver metastasis of colon cancer.

Geng L, Chaudhuri A, Talmon G, Wisecarver JL, Are C, Brattain M, Wang J - Oncogene (2013)

Bottom Line: Alterations associated with the metastatic phenotype in the primary tumors include increased apoptosis, decreased proliferation and angiogenesis.Finally, studies performed on human colonic adenocarcinoma show that expression of miR-192 is significantly reduced in neoplastic cells as compared with normal colonic epithelium.Importantly, there is a significant decrease in miR-192 expression in stage IV tumors when compared with stage I or II lesions.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, Omaha, NE, USA.

ABSTRACT
Metastasis causes most deaths from colon cancer yet mechanistic understanding and therapeutic options remain limited. Here we show that expression of microRNA (miR)-192 is inversely correlated with metastatic potential of colon cancer cells. Ectopic expression of miR-192 sensitizes colon cancer cells to growth factor deprivation stress-induced apoptosis, whereas inhibition of miR-192 confers resistance. Overexpression of miR-192 inhibits metastatic colonization to the liver in an orthotopic mouse model of colon cancer. Alterations associated with the metastatic phenotype in the primary tumors include increased apoptosis, decreased proliferation and angiogenesis. Further studies indicate that miR-192 downregulates expression of Bcl-2, Zeb2 and VEGFA in vitro and in vivo, which is responsible for enhanced apoptosis, increased expression of E-cadherin and decreased angiogenesis in vivo, respectively. Finally, studies performed on human colonic adenocarcinoma show that expression of miR-192 is significantly reduced in neoplastic cells as compared with normal colonic epithelium. Importantly, there is a significant decrease in miR-192 expression in stage IV tumors when compared with stage I or II lesions. These findings indicate that miR-192 has an important role in colon cancer development and progression. Our studies underscore the clinical relevance and prognostic significance of miR-192 expression in colon cancer. Therefore, a major implication of our studies is that restoration of miR-192 expression or antagonism of its target genes (Bcl-2, Zeb2 or VEGFA) may have considerable therapeutic potential for anti-metastatic therapy in patients with colon cancer.

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miR-192 directly or indirectly regulates expression of Bcl2, Zeb2 and VEGFAA, C and E, Bcl2 mRNA and protein (A), Zeb2 mRNA (C) and VEGFA protein (E) were determined in HCT116 control and miR-192-expressing cells (left panels). Luciferase constructs were co-transfected into HCT116 cells with β-gal expression vector. Luciferase activity was normalized to β-gal activity. The data are presented as mean ± SE of triplicate experiments (right panels). * P < 0.001 (A), * P < 0.01 (C). B, Bcl2 was ectopically expressed in HCT116 control and miR-192-expressing cells. Western blot analysis of cleaved caspase 3 was performed. D, Expression of E-cadherin was determined in HCT116 control and miR-192-expressing cells (upper panel). Expression of E-cadherin was determined in HCT116 control (Scr) and Zeb2-knocked down (shZeb2) cells (lower panel).
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Figure 4: miR-192 directly or indirectly regulates expression of Bcl2, Zeb2 and VEGFAA, C and E, Bcl2 mRNA and protein (A), Zeb2 mRNA (C) and VEGFA protein (E) were determined in HCT116 control and miR-192-expressing cells (left panels). Luciferase constructs were co-transfected into HCT116 cells with β-gal expression vector. Luciferase activity was normalized to β-gal activity. The data are presented as mean ± SE of triplicate experiments (right panels). * P < 0.001 (A), * P < 0.01 (C). B, Bcl2 was ectopically expressed in HCT116 control and miR-192-expressing cells. Western blot analysis of cleaved caspase 3 was performed. D, Expression of E-cadherin was determined in HCT116 control and miR-192-expressing cells (upper panel). Expression of E-cadherin was determined in HCT116 control (Scr) and Zeb2-knocked down (shZeb2) cells (lower panel).

Mentions: The ability of miR-192 to impede multiple steps of the cancer metastasis cascade might be attributed to its ability to regulate expression of genes involved in diverse aspects of metastatic dissemination. To identify effectors of miR-192, we used several algorithms that predict the mRNA targets of miRNAs – TargetScan (26), PicTar (27) and miRanda-mirSVR (28). Based on the representation of miR-192 sites in their 3′ UTRs and their implicated role in tumor progression, we chose the top ten target genes to perform further studies. Three of the ten genes studied (Bcl-2, Zeb2 and VEGFA) showed reduced expression in HCT116 cells expressing miR-192 as compared to vector control cells (Fig. 4A, 4C & 4E, left panels). To determine whether they are direct targets of miR-192, we cloned the 3′ UTRs of Bcl-2, Zeb2 or VEGFA containing the potential miR-192 recognition element into a luciferase construct described above. Reporter assays revealed that miR-192 repressed the UTRs of Bcl-2 and Zeb2, but not of VEGFA (Fig. 4A, * P < 0.001, 4C, * P < 0.01 & 4E, right panels,). This indicates that expression of Bcl-2 and Zeb2 are directly regulated by miR-192, whereas VEGFA could be an indirect target of miR-192.


MicroRNA-192 suppresses liver metastasis of colon cancer.

Geng L, Chaudhuri A, Talmon G, Wisecarver JL, Are C, Brattain M, Wang J - Oncogene (2013)

miR-192 directly or indirectly regulates expression of Bcl2, Zeb2 and VEGFAA, C and E, Bcl2 mRNA and protein (A), Zeb2 mRNA (C) and VEGFA protein (E) were determined in HCT116 control and miR-192-expressing cells (left panels). Luciferase constructs were co-transfected into HCT116 cells with β-gal expression vector. Luciferase activity was normalized to β-gal activity. The data are presented as mean ± SE of triplicate experiments (right panels). * P < 0.001 (A), * P < 0.01 (C). B, Bcl2 was ectopically expressed in HCT116 control and miR-192-expressing cells. Western blot analysis of cleaved caspase 3 was performed. D, Expression of E-cadherin was determined in HCT116 control and miR-192-expressing cells (upper panel). Expression of E-cadherin was determined in HCT116 control (Scr) and Zeb2-knocked down (shZeb2) cells (lower panel).
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getmorefigures.php?uid=PMC4016997&req=5

Figure 4: miR-192 directly or indirectly regulates expression of Bcl2, Zeb2 and VEGFAA, C and E, Bcl2 mRNA and protein (A), Zeb2 mRNA (C) and VEGFA protein (E) were determined in HCT116 control and miR-192-expressing cells (left panels). Luciferase constructs were co-transfected into HCT116 cells with β-gal expression vector. Luciferase activity was normalized to β-gal activity. The data are presented as mean ± SE of triplicate experiments (right panels). * P < 0.001 (A), * P < 0.01 (C). B, Bcl2 was ectopically expressed in HCT116 control and miR-192-expressing cells. Western blot analysis of cleaved caspase 3 was performed. D, Expression of E-cadherin was determined in HCT116 control and miR-192-expressing cells (upper panel). Expression of E-cadherin was determined in HCT116 control (Scr) and Zeb2-knocked down (shZeb2) cells (lower panel).
Mentions: The ability of miR-192 to impede multiple steps of the cancer metastasis cascade might be attributed to its ability to regulate expression of genes involved in diverse aspects of metastatic dissemination. To identify effectors of miR-192, we used several algorithms that predict the mRNA targets of miRNAs – TargetScan (26), PicTar (27) and miRanda-mirSVR (28). Based on the representation of miR-192 sites in their 3′ UTRs and their implicated role in tumor progression, we chose the top ten target genes to perform further studies. Three of the ten genes studied (Bcl-2, Zeb2 and VEGFA) showed reduced expression in HCT116 cells expressing miR-192 as compared to vector control cells (Fig. 4A, 4C & 4E, left panels). To determine whether they are direct targets of miR-192, we cloned the 3′ UTRs of Bcl-2, Zeb2 or VEGFA containing the potential miR-192 recognition element into a luciferase construct described above. Reporter assays revealed that miR-192 repressed the UTRs of Bcl-2 and Zeb2, but not of VEGFA (Fig. 4A, * P < 0.001, 4C, * P < 0.01 & 4E, right panels,). This indicates that expression of Bcl-2 and Zeb2 are directly regulated by miR-192, whereas VEGFA could be an indirect target of miR-192.

Bottom Line: Alterations associated with the metastatic phenotype in the primary tumors include increased apoptosis, decreased proliferation and angiogenesis.Finally, studies performed on human colonic adenocarcinoma show that expression of miR-192 is significantly reduced in neoplastic cells as compared with normal colonic epithelium.Importantly, there is a significant decrease in miR-192 expression in stage IV tumors when compared with stage I or II lesions.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, Omaha, NE, USA.

ABSTRACT
Metastasis causes most deaths from colon cancer yet mechanistic understanding and therapeutic options remain limited. Here we show that expression of microRNA (miR)-192 is inversely correlated with metastatic potential of colon cancer cells. Ectopic expression of miR-192 sensitizes colon cancer cells to growth factor deprivation stress-induced apoptosis, whereas inhibition of miR-192 confers resistance. Overexpression of miR-192 inhibits metastatic colonization to the liver in an orthotopic mouse model of colon cancer. Alterations associated with the metastatic phenotype in the primary tumors include increased apoptosis, decreased proliferation and angiogenesis. Further studies indicate that miR-192 downregulates expression of Bcl-2, Zeb2 and VEGFA in vitro and in vivo, which is responsible for enhanced apoptosis, increased expression of E-cadherin and decreased angiogenesis in vivo, respectively. Finally, studies performed on human colonic adenocarcinoma show that expression of miR-192 is significantly reduced in neoplastic cells as compared with normal colonic epithelium. Importantly, there is a significant decrease in miR-192 expression in stage IV tumors when compared with stage I or II lesions. These findings indicate that miR-192 has an important role in colon cancer development and progression. Our studies underscore the clinical relevance and prognostic significance of miR-192 expression in colon cancer. Therefore, a major implication of our studies is that restoration of miR-192 expression or antagonism of its target genes (Bcl-2, Zeb2 or VEGFA) may have considerable therapeutic potential for anti-metastatic therapy in patients with colon cancer.

Show MeSH
Related in: MedlinePlus