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Microvascular invasion in hepatocellular carcinoma overexpression promotes cell proliferation and inhibits cell apoptosis of hepatocellular carcinoma via inhibiting miR-199a expression.

Shi Y, Song Q, Yu S, Hu D, Zhuang X - Onco Targets Ther (2015)

Bottom Line: MVIH expression was significantly increased and miR-199a expression was significantly decreased in tumor tissue and HCC cells. si-MVIH inhibited HCC cell viability and promoted cell apoptosis, but this effect was reversed by miR-199a inhibitor.Luciferase reporter assay and RNA immunoprecipitation experiment showed that miR-199a had a direct binding ability to MVIH RNA.MVIH promoted cell growth and inhibited cell apoptosis of HCC via inhibiting miR-199a expression.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Affiliated Hospital of Xuzhou Medical College, Xuzhou, People's Republic of China.

ABSTRACT

Objectives: Long non-coding RNA (lncRNA) associated with microvascular invasion in hepatocellular carcinoma (MVIH) has been recently reported to act as a predictor for the poor recurrence-free survival of hepatocellular carcinoma (HCC) after hepatectomy. However, the biological role of MVIH in the tumorigenesis of HCC is still unclear.

Methods: In the study reported here, MVIH expression levels were detected by real-time polymerase chain reaction (PCR) in tumor tissue of HCC patients and in HCC cells, including SMMC7721 and HepG2 cells. Cell viability and apoptosis were determined by MTT and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) methods, respectively. The model of transplantation tumor of HepG2 cells in nude mice was used to evaluate the effects of MVIH and miR-199a on HCC in vivo.

Results: MVIH expression was significantly increased and miR-199a expression was significantly decreased in tumor tissue and HCC cells. si-MVIH inhibited HCC cell viability and promoted cell apoptosis, but this effect was reversed by miR-199a inhibitor. Luciferase reporter assay and RNA immunoprecipitation experiment showed that miR-199a had a direct binding ability to MVIH RNA. In nude mice with transplantation, the tumor volume was reduced by si-MVIH, and miR-199a inhibitor canceled this decrease.

Conclusion: MVIH promoted cell growth and inhibited cell apoptosis of HCC via inhibiting miR-199a expression.

No MeSH data available.


Related in: MedlinePlus

miR-199a targets 3′UTR of microvascular invasion in hepatocellular carcinoma (MVIH) in hepatocellular carcinoma cells. Luciferase reporter plasmid, wild-type luciferase reporter plasmid (p-MIR-MVIH-WT) or mutant luciferase reporter plasmid (pMIR-MVIH-Mut), was co-transfected with miR-199a mimic into SMMC7721 or HepG2 cells; the relative luciferase activity was detected by luciferase reporter assay (A). HepG2 cells lysate was treated with antibody Ago2 or immunoglobulin (Ig) G, and then the RNA immunoprecipitation was performed to detect the relative MVIH and miR-199a enrichment (B). Co-transfection of miR-199a mimic with pcDNA-MVIH or pcDNA-NC into SMMC7721 or HepG2 cells; the concentrations of pcDNA-MVIH were 1 μg, 2 μg, and 4 μg; the relative miR-199a level was quantified by real-time polymerase chain reaction (C). The protein expression of FZD7 was detected by Western blot (D).Note: **P<0.01, versus the group treated with negative control (NC; A) or anti-IgG (B) or transfection of miR-199a with empty pcDNA (C).Abbreviations: IP, immunoprecipitation; RIP, RNA immunoprecipitation; WB, western blot.
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f3-ott-8-2303: miR-199a targets 3′UTR of microvascular invasion in hepatocellular carcinoma (MVIH) in hepatocellular carcinoma cells. Luciferase reporter plasmid, wild-type luciferase reporter plasmid (p-MIR-MVIH-WT) or mutant luciferase reporter plasmid (pMIR-MVIH-Mut), was co-transfected with miR-199a mimic into SMMC7721 or HepG2 cells; the relative luciferase activity was detected by luciferase reporter assay (A). HepG2 cells lysate was treated with antibody Ago2 or immunoglobulin (Ig) G, and then the RNA immunoprecipitation was performed to detect the relative MVIH and miR-199a enrichment (B). Co-transfection of miR-199a mimic with pcDNA-MVIH or pcDNA-NC into SMMC7721 or HepG2 cells; the concentrations of pcDNA-MVIH were 1 μg, 2 μg, and 4 μg; the relative miR-199a level was quantified by real-time polymerase chain reaction (C). The protein expression of FZD7 was detected by Western blot (D).Note: **P<0.01, versus the group treated with negative control (NC; A) or anti-IgG (B) or transfection of miR-199a with empty pcDNA (C).Abbreviations: IP, immunoprecipitation; RIP, RNA immunoprecipitation; WB, western blot.

Mentions: Bioinformatics analysis revealed MVIH contains one conserved target site of miR-199a. To confirm this prediction, the wild-type sequence of MVIH (MVIH-WT) or its mutant sequence (MVIH-Mut) was sub-cloned into the pMIR luciferase reporter and then co-transfected with miR-199a mimic into SMMC7721 or HepG2 cells. The relative luciferase activity of pMIR-UCA1-WT was significantly reduced after the transfection of miR-199a mimic. However, no effect was observed in pMIR-UCA1-Mut (Figure 3A). The RIP experiments was used to confirm whether both MVIH and miR-199a might be in the RISC complex. The results showed that MVIH and miR-199a were enriched to more than 100 times in Ago2 pellets relative to control IgG immunoprecipitates (Figure 3B), which demonstrated that MVIH is present in Ago-2-containing RISC through association with miR-199a. In miR-199a mimic co-transfected with pcDNA-MVIH (or pcDNA-NC) in SMMC7721 or HepG2 cells, the miR-199a expression level was significantly inhibited by pcDNA-MVIH in a dose-dependent manner (Figure 3C). However, FZD7, the target gene of miR-199a, was upregulated by pcDNA-MVIH (Figure 3D).


Microvascular invasion in hepatocellular carcinoma overexpression promotes cell proliferation and inhibits cell apoptosis of hepatocellular carcinoma via inhibiting miR-199a expression.

Shi Y, Song Q, Yu S, Hu D, Zhuang X - Onco Targets Ther (2015)

miR-199a targets 3′UTR of microvascular invasion in hepatocellular carcinoma (MVIH) in hepatocellular carcinoma cells. Luciferase reporter plasmid, wild-type luciferase reporter plasmid (p-MIR-MVIH-WT) or mutant luciferase reporter plasmid (pMIR-MVIH-Mut), was co-transfected with miR-199a mimic into SMMC7721 or HepG2 cells; the relative luciferase activity was detected by luciferase reporter assay (A). HepG2 cells lysate was treated with antibody Ago2 or immunoglobulin (Ig) G, and then the RNA immunoprecipitation was performed to detect the relative MVIH and miR-199a enrichment (B). Co-transfection of miR-199a mimic with pcDNA-MVIH or pcDNA-NC into SMMC7721 or HepG2 cells; the concentrations of pcDNA-MVIH were 1 μg, 2 μg, and 4 μg; the relative miR-199a level was quantified by real-time polymerase chain reaction (C). The protein expression of FZD7 was detected by Western blot (D).Note: **P<0.01, versus the group treated with negative control (NC; A) or anti-IgG (B) or transfection of miR-199a with empty pcDNA (C).Abbreviations: IP, immunoprecipitation; RIP, RNA immunoprecipitation; WB, western blot.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556036&req=5

f3-ott-8-2303: miR-199a targets 3′UTR of microvascular invasion in hepatocellular carcinoma (MVIH) in hepatocellular carcinoma cells. Luciferase reporter plasmid, wild-type luciferase reporter plasmid (p-MIR-MVIH-WT) or mutant luciferase reporter plasmid (pMIR-MVIH-Mut), was co-transfected with miR-199a mimic into SMMC7721 or HepG2 cells; the relative luciferase activity was detected by luciferase reporter assay (A). HepG2 cells lysate was treated with antibody Ago2 or immunoglobulin (Ig) G, and then the RNA immunoprecipitation was performed to detect the relative MVIH and miR-199a enrichment (B). Co-transfection of miR-199a mimic with pcDNA-MVIH or pcDNA-NC into SMMC7721 or HepG2 cells; the concentrations of pcDNA-MVIH were 1 μg, 2 μg, and 4 μg; the relative miR-199a level was quantified by real-time polymerase chain reaction (C). The protein expression of FZD7 was detected by Western blot (D).Note: **P<0.01, versus the group treated with negative control (NC; A) or anti-IgG (B) or transfection of miR-199a with empty pcDNA (C).Abbreviations: IP, immunoprecipitation; RIP, RNA immunoprecipitation; WB, western blot.
Mentions: Bioinformatics analysis revealed MVIH contains one conserved target site of miR-199a. To confirm this prediction, the wild-type sequence of MVIH (MVIH-WT) or its mutant sequence (MVIH-Mut) was sub-cloned into the pMIR luciferase reporter and then co-transfected with miR-199a mimic into SMMC7721 or HepG2 cells. The relative luciferase activity of pMIR-UCA1-WT was significantly reduced after the transfection of miR-199a mimic. However, no effect was observed in pMIR-UCA1-Mut (Figure 3A). The RIP experiments was used to confirm whether both MVIH and miR-199a might be in the RISC complex. The results showed that MVIH and miR-199a were enriched to more than 100 times in Ago2 pellets relative to control IgG immunoprecipitates (Figure 3B), which demonstrated that MVIH is present in Ago-2-containing RISC through association with miR-199a. In miR-199a mimic co-transfected with pcDNA-MVIH (or pcDNA-NC) in SMMC7721 or HepG2 cells, the miR-199a expression level was significantly inhibited by pcDNA-MVIH in a dose-dependent manner (Figure 3C). However, FZD7, the target gene of miR-199a, was upregulated by pcDNA-MVIH (Figure 3D).

Bottom Line: MVIH expression was significantly increased and miR-199a expression was significantly decreased in tumor tissue and HCC cells. si-MVIH inhibited HCC cell viability and promoted cell apoptosis, but this effect was reversed by miR-199a inhibitor.Luciferase reporter assay and RNA immunoprecipitation experiment showed that miR-199a had a direct binding ability to MVIH RNA.MVIH promoted cell growth and inhibited cell apoptosis of HCC via inhibiting miR-199a expression.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Affiliated Hospital of Xuzhou Medical College, Xuzhou, People's Republic of China.

ABSTRACT

Objectives: Long non-coding RNA (lncRNA) associated with microvascular invasion in hepatocellular carcinoma (MVIH) has been recently reported to act as a predictor for the poor recurrence-free survival of hepatocellular carcinoma (HCC) after hepatectomy. However, the biological role of MVIH in the tumorigenesis of HCC is still unclear.

Methods: In the study reported here, MVIH expression levels were detected by real-time polymerase chain reaction (PCR) in tumor tissue of HCC patients and in HCC cells, including SMMC7721 and HepG2 cells. Cell viability and apoptosis were determined by MTT and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) methods, respectively. The model of transplantation tumor of HepG2 cells in nude mice was used to evaluate the effects of MVIH and miR-199a on HCC in vivo.

Results: MVIH expression was significantly increased and miR-199a expression was significantly decreased in tumor tissue and HCC cells. si-MVIH inhibited HCC cell viability and promoted cell apoptosis, but this effect was reversed by miR-199a inhibitor. Luciferase reporter assay and RNA immunoprecipitation experiment showed that miR-199a had a direct binding ability to MVIH RNA. In nude mice with transplantation, the tumor volume was reduced by si-MVIH, and miR-199a inhibitor canceled this decrease.

Conclusion: MVIH promoted cell growth and inhibited cell apoptosis of HCC via inhibiting miR-199a expression.

No MeSH data available.


Related in: MedlinePlus