Limits...
GNAI1 Suppresses Tumor Cell Migration and Invasion and is Post-Transcriptionally Regulated by Mir-320a/c/d in Hepatocellular Carcinoma.

Yao J, Liang LH, Zhang Y, Ding J, Tian Q, Li JJ, He XH - Cancer Biol Med (2012)

Bottom Line: The GNAI1 protein was significantly downregulated in HCC samples without changes in its mRNA levels.GNAI1 is downregulated in HCC and inhibits the migration and invasion of HCC cells.Regulation of GNAI1 by miR-320a/c/d indicates new therapeutic avenues for targeting HCC metastasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China ; State Key Laboratory for Diagnosis and Treatment for Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.

ABSTRACT

Objective: To explore the role and regulation of guanine nucleotide-binding protein G(i), α-1 subunit (GNAI1) in hepatocellular carcinoma (HCC).

Methods: Expression of GNAI1 in HCC samples was determined by qRT-PCR and immunohistochemical (IHC) staining. Huh-7 and SNU-387 cells stably expressing GNAI1 were established by the infection of lentivirus transducing unit containing GNAI1. siRNA against GNAI1 was transfected into SMMC-7721 cells to knock down the GNAI1 expression in HCC cells. Mir-320a/c/d mimics were transfected into SMMC-7721 and SK-Hep-1 cells and the expression of GNAI1 was determined by Western blot. The migration and invasion of Huh-7, SNU-387, SK-Hep-1 and SMMC-7721 cells were investigated by Transwell assays.

Results: The GNAI1 protein was significantly downregulated in HCC samples without changes in its mRNA levels. GNAI1 could inhibit the migration and invasion of HCC cells in vitro. Further investigations indicated that GNAI1 was a target of miR-320a/c/d in HCC cells. Transwell assays demonstrated that these microRNAs could promote the migratory ability and invasivesess of HCC cells in vitro.

Conclusions: GNAI1 is downregulated in HCC and inhibits the migration and invasion of HCC cells. This study is the first to investigate the role of GNAI1 in cancer. Regulation of GNAI1 by miR-320a/c/d indicates new therapeutic avenues for targeting HCC metastasis.

No MeSH data available.


Related in: MedlinePlus

A set of miRNAs can downregulate the GNAI1 protein level by binding to its 3’ UTR. A: Schematic diagram of the predicted binding sites of the GNAI1-regulating miRNAs in the 3’ UTR of GNAI1. Several miRNAs shared the same target sites; B: Relative luciferase activity analyzed following the co-transfection of a luciferase reporter plasmid containing the WT GNAI1 3’ UTR and GNAI1-regulating miRNAs. Normalized to the Renilla luciferase activity, the data are shown as mean±SEM; C: Western blot assays of the endogenous GNAI1 protein levels in SMMC-7721 cells transfected with GNAI1-regulating miRNAs or negative control. The intensity was normalized to GAPDH, and the ratios indicate the relative expression levels compared with the negative control; D: Sequences of WT and MT (shadowed and underlined) binding sites; E: Relative luciferase activity in cells co-transfected with GNAI1-regulating miRNAs, and a luciferase reporter plasmid containing the WT or MT 3’UTR. The data are presented as mean±SEM, along with the P values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643671&req=5

f3: A set of miRNAs can downregulate the GNAI1 protein level by binding to its 3’ UTR. A: Schematic diagram of the predicted binding sites of the GNAI1-regulating miRNAs in the 3’ UTR of GNAI1. Several miRNAs shared the same target sites; B: Relative luciferase activity analyzed following the co-transfection of a luciferase reporter plasmid containing the WT GNAI1 3’ UTR and GNAI1-regulating miRNAs. Normalized to the Renilla luciferase activity, the data are shown as mean±SEM; C: Western blot assays of the endogenous GNAI1 protein levels in SMMC-7721 cells transfected with GNAI1-regulating miRNAs or negative control. The intensity was normalized to GAPDH, and the ratios indicate the relative expression levels compared with the negative control; D: Sequences of WT and MT (shadowed and underlined) binding sites; E: Relative luciferase activity in cells co-transfected with GNAI1-regulating miRNAs, and a luciferase reporter plasmid containing the WT or MT 3’UTR. The data are presented as mean±SEM, along with the P values.

Mentions: Our previous experimental data has shown that the GNAI1 expression varies at the mRNA and protein levels, which may involve post-transcriptional regulation. Because microRNAs could down-regulate the protein levels of target genes without necessarily changing their mRNA expression, we examined whether miRNA was involved in the regulation of GNAI1. Using two internet-based miRNA target-prediction algorithms (TargetScan and PicTar), we identified nine potential binding sites (Figure 3A) for nine miRNAs in the 3’ UTR of GNAI1. We co-transfected the HEK293T cells with individual miRNA mimics and luciferase reporter plasmids to determine whether the predicted miRNAs were bound directly to the GNAI1 3’ UTR. The full-length wild-type GNAI1 3’ UTR was directly inserted into the region immediately downstream of a luciferase reporter gene. As shown in Figure 3B, all the predicted miRNAs, except miR-186, significantly reduced the luciferase activity. Furthermore, Western blot assays revealed that miR-320a/c/d and miR-9* dramatically down-regulated the endogenous GNAI1 protein level (Figure 3C), which indicates that these four miRNAs may directly target GNAI1 in HCC cells. To clarify whether these four miRNAs could bind directly to the 3’UTR of GNAI1, we analyzed the binding sites predicted by the algorithms and mutated the binding site sequences (Figure 3D). Reporter assays revealed that in the cells transfected with 3’ UTRs that contained mutations in the miR-320a/c/d or miR-9* binding site 1 but not site 2, the level of luciferase activity remained similar to that of the control cells (Figure 3E). This analysis indicated that these binding sites were authentic.


GNAI1 Suppresses Tumor Cell Migration and Invasion and is Post-Transcriptionally Regulated by Mir-320a/c/d in Hepatocellular Carcinoma.

Yao J, Liang LH, Zhang Y, Ding J, Tian Q, Li JJ, He XH - Cancer Biol Med (2012)

A set of miRNAs can downregulate the GNAI1 protein level by binding to its 3’ UTR. A: Schematic diagram of the predicted binding sites of the GNAI1-regulating miRNAs in the 3’ UTR of GNAI1. Several miRNAs shared the same target sites; B: Relative luciferase activity analyzed following the co-transfection of a luciferase reporter plasmid containing the WT GNAI1 3’ UTR and GNAI1-regulating miRNAs. Normalized to the Renilla luciferase activity, the data are shown as mean±SEM; C: Western blot assays of the endogenous GNAI1 protein levels in SMMC-7721 cells transfected with GNAI1-regulating miRNAs or negative control. The intensity was normalized to GAPDH, and the ratios indicate the relative expression levels compared with the negative control; D: Sequences of WT and MT (shadowed and underlined) binding sites; E: Relative luciferase activity in cells co-transfected with GNAI1-regulating miRNAs, and a luciferase reporter plasmid containing the WT or MT 3’UTR. The data are presented as mean±SEM, along with the P values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643671&req=5

f3: A set of miRNAs can downregulate the GNAI1 protein level by binding to its 3’ UTR. A: Schematic diagram of the predicted binding sites of the GNAI1-regulating miRNAs in the 3’ UTR of GNAI1. Several miRNAs shared the same target sites; B: Relative luciferase activity analyzed following the co-transfection of a luciferase reporter plasmid containing the WT GNAI1 3’ UTR and GNAI1-regulating miRNAs. Normalized to the Renilla luciferase activity, the data are shown as mean±SEM; C: Western blot assays of the endogenous GNAI1 protein levels in SMMC-7721 cells transfected with GNAI1-regulating miRNAs or negative control. The intensity was normalized to GAPDH, and the ratios indicate the relative expression levels compared with the negative control; D: Sequences of WT and MT (shadowed and underlined) binding sites; E: Relative luciferase activity in cells co-transfected with GNAI1-regulating miRNAs, and a luciferase reporter plasmid containing the WT or MT 3’UTR. The data are presented as mean±SEM, along with the P values.
Mentions: Our previous experimental data has shown that the GNAI1 expression varies at the mRNA and protein levels, which may involve post-transcriptional regulation. Because microRNAs could down-regulate the protein levels of target genes without necessarily changing their mRNA expression, we examined whether miRNA was involved in the regulation of GNAI1. Using two internet-based miRNA target-prediction algorithms (TargetScan and PicTar), we identified nine potential binding sites (Figure 3A) for nine miRNAs in the 3’ UTR of GNAI1. We co-transfected the HEK293T cells with individual miRNA mimics and luciferase reporter plasmids to determine whether the predicted miRNAs were bound directly to the GNAI1 3’ UTR. The full-length wild-type GNAI1 3’ UTR was directly inserted into the region immediately downstream of a luciferase reporter gene. As shown in Figure 3B, all the predicted miRNAs, except miR-186, significantly reduced the luciferase activity. Furthermore, Western blot assays revealed that miR-320a/c/d and miR-9* dramatically down-regulated the endogenous GNAI1 protein level (Figure 3C), which indicates that these four miRNAs may directly target GNAI1 in HCC cells. To clarify whether these four miRNAs could bind directly to the 3’UTR of GNAI1, we analyzed the binding sites predicted by the algorithms and mutated the binding site sequences (Figure 3D). Reporter assays revealed that in the cells transfected with 3’ UTRs that contained mutations in the miR-320a/c/d or miR-9* binding site 1 but not site 2, the level of luciferase activity remained similar to that of the control cells (Figure 3E). This analysis indicated that these binding sites were authentic.

Bottom Line: The GNAI1 protein was significantly downregulated in HCC samples without changes in its mRNA levels.GNAI1 is downregulated in HCC and inhibits the migration and invasion of HCC cells.Regulation of GNAI1 by miR-320a/c/d indicates new therapeutic avenues for targeting HCC metastasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China ; State Key Laboratory for Diagnosis and Treatment for Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.

ABSTRACT

Objective: To explore the role and regulation of guanine nucleotide-binding protein G(i), α-1 subunit (GNAI1) in hepatocellular carcinoma (HCC).

Methods: Expression of GNAI1 in HCC samples was determined by qRT-PCR and immunohistochemical (IHC) staining. Huh-7 and SNU-387 cells stably expressing GNAI1 were established by the infection of lentivirus transducing unit containing GNAI1. siRNA against GNAI1 was transfected into SMMC-7721 cells to knock down the GNAI1 expression in HCC cells. Mir-320a/c/d mimics were transfected into SMMC-7721 and SK-Hep-1 cells and the expression of GNAI1 was determined by Western blot. The migration and invasion of Huh-7, SNU-387, SK-Hep-1 and SMMC-7721 cells were investigated by Transwell assays.

Results: The GNAI1 protein was significantly downregulated in HCC samples without changes in its mRNA levels. GNAI1 could inhibit the migration and invasion of HCC cells in vitro. Further investigations indicated that GNAI1 was a target of miR-320a/c/d in HCC cells. Transwell assays demonstrated that these microRNAs could promote the migratory ability and invasivesess of HCC cells in vitro.

Conclusions: GNAI1 is downregulated in HCC and inhibits the migration and invasion of HCC cells. This study is the first to investigate the role of GNAI1 in cancer. Regulation of GNAI1 by miR-320a/c/d indicates new therapeutic avenues for targeting HCC metastasis.

No MeSH data available.


Related in: MedlinePlus