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Effects of Ras homolog gene family, member C gene silencing combined with rapamycin on hepatocellular carcinoma cell growth.

Xie SL, Zhu MG, Chen GF, Wang GY, Lv GY - Mol Med Rep (2015)

Bottom Line: No significant difference was found between the RNAi + RAPA and the normal HL7702 group.The number of silver nitrate‑stained particles was reduced in the RNAi + RAPA group compared with that in the other groups.No significant difference was found between the RNAi + RAPA and HL7702 groups.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China.

ABSTRACT
The aim of the present study was to investigate the combined effects of inhibiting the Ras homolog gene family, member C (RhoC)/Rho kinase and phosphoinositide 3 kinase/Akt/mammalian target of rapamycin (mTOR) pathways on hepatocellular carcinoma cell growth. The RhoC gene was silenced by RNA interference (RNAi) and mTOR was inhibited by rapamycin (RAPA). Subsequently, an MTT assay for cell growth detection, western blot analysis for gene expression analysis, silver nitrate staining for cell proliferation, Wright's staining for analysis of the apoptotic rate analysis, soft agar clonogenic assay for the determination of cell growth characteristics and a Transwell assay for cell migration were performed. RhoC expression in hepatoma cell lines was lower than that in the HL7702 normal human liver cell line. The level of cell proliferation in the RNAi + RAPA group was lower than that in the RNAi, RAPA and Scramble groups. The levels of cyclin‑dependent kinase 2 in the RNAi + RAPA group were lower than those in the other groups, while the levels of P16 in the RNAi + RAPA group were higher than those in the other experimental groups. No significant difference was found between the RNAi + RAPA and the normal HL7702 group. The number of silver nitrate‑stained particles was reduced in the RNAi + RAPA group compared with that in the other groups. No significant difference was found between the RNAi + RAPA and HL7702 groups. Wright's staining for apoptosis demonstrated that apoptosis in the Scramble group was rare, while the RAPA and RNAi groups contained a large number of apoptotic cells, which displayed nuclear condensation, fragmentation, deepened staining, as well as a wrinkled membrane. B‑cell lymphoma‑2 (Bcl‑2) expression in the RNAi + RAPA group was lower than that in the other groups, while the gene expression of Bcl‑2‑associated X protein in the RNAi + RAPA group was increased compared with that in the other groups. No cell colony formation was observed in the soft agar cloning experiment in the RNAi + RAPA and HL7702 group, while in the other groups, visible cell clones appeared. In the Transwell assay the number of migrated cells in the RNAi + RAPA group was lower than that in the other groups. The gene expression of matrix metalloproteinase (MMP)2, MMP‑9 and vascular endothelial growth factor in the RNAi + RAPA group was lower than that in the other experimental groups. In conclusion, RhoC gene silencing combined with RAPA was able to significantly inhibit the growth of hepatocellular carcinoma cells.

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Cell migration and expression of migration-associated genes. (A) Representative microscopic images (magnification, ×100) and statistical evaluation of migrated cells. Transmembrane cells in the RNAi + RAPA and HL7702 groups were rare and sparsely distributed, while in the Scramble group were greater in quantity and widely distributed. (B) Western blot analysis of migration-associated proteins. MMP2/9 and VEGF protein expression were decreased in the RNAi + RAPA group to the levels of HL7702. Values are expressed as the mean ± standard deviation, **P<0.01. RAPA, rapamycin; RNAi, RNA interference; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor.
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f6-mmr-12-04-5077: Cell migration and expression of migration-associated genes. (A) Representative microscopic images (magnification, ×100) and statistical evaluation of migrated cells. Transmembrane cells in the RNAi + RAPA and HL7702 groups were rare and sparsely distributed, while in the Scramble group were greater in quantity and widely distributed. (B) Western blot analysis of migration-associated proteins. MMP2/9 and VEGF protein expression were decreased in the RNAi + RAPA group to the levels of HL7702. Values are expressed as the mean ± standard deviation, **P<0.01. RAPA, rapamycin; RNAi, RNA interference; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor.

Mentions: The Transwell assay showed that cells which migrated across the membrane in the RNAi, RAPA, RNAi + RAPA and HL7702 groups were rare and scattered, while in the Scramble group, transmembrane cells were greater in number and dense in distribution. Statistical analysis revealed that the number of transmembrane cells in the RNAi + RAPA group was significantly lower than that in the RNAi, RAPA and Scramble groups (35.33±3.51 vs. 65.33±6.51, 60.67±8.50 and 161.67±14.50; P<0.01), and no significant difference was found when compared with the HL7702 group (35.33±3.51 vs. 25.67±5.86; P>0.05) (Fig. 6A). Detection of cell migration-associated genes revealed that matrix metal-loproteinase (MMP)-2 expression in the RNAi + RAPA group was significantly lower than that in the RNAi, RAPA and Scramble groups (0.04±0.02 vs. 0.21±0.03, 0.20±0.04 and 0.67±0.04; P<0.01), and the difference was not significant compared with that in the HL7702 group (0.04±0.02 vs. 0.03±0.01; P>0.05). MMP-9 expression in the RNAi + RAPA group was markedly decreased compared with that in the RNAi, RAPA and Scramble groups (0.06±0.02 vs. 0.23±0.02, 0.19±0.04 and 0.62±0.03; P<.0.01), and no significant difference was observed when compared with that in the HL7702 group (0.06±0.02 vs. 0.03±0.01; P>0.05). VEGF expression in the RNAi + RAPA group was also significantly lower than that in the RNAi, RAPA and Scramble groups (0.07±0.02 vs. 0.24±0.04, 0.20±0.06 and 0.62±0.06; P<0.01); while there was no significant difference compared with that in the HL7702 group (0.07±0.02 vs. 0.04±0.02; P>0.05) (Fig. 6B). These results demonstrated that RhoC gene silencing combined with RAPA treatment exerted marked migration-inhibitory effects on hepatocellular carcinoma cells.


Effects of Ras homolog gene family, member C gene silencing combined with rapamycin on hepatocellular carcinoma cell growth.

Xie SL, Zhu MG, Chen GF, Wang GY, Lv GY - Mol Med Rep (2015)

Cell migration and expression of migration-associated genes. (A) Representative microscopic images (magnification, ×100) and statistical evaluation of migrated cells. Transmembrane cells in the RNAi + RAPA and HL7702 groups were rare and sparsely distributed, while in the Scramble group were greater in quantity and widely distributed. (B) Western blot analysis of migration-associated proteins. MMP2/9 and VEGF protein expression were decreased in the RNAi + RAPA group to the levels of HL7702. Values are expressed as the mean ± standard deviation, **P<0.01. RAPA, rapamycin; RNAi, RNA interference; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581818&req=5

f6-mmr-12-04-5077: Cell migration and expression of migration-associated genes. (A) Representative microscopic images (magnification, ×100) and statistical evaluation of migrated cells. Transmembrane cells in the RNAi + RAPA and HL7702 groups were rare and sparsely distributed, while in the Scramble group were greater in quantity and widely distributed. (B) Western blot analysis of migration-associated proteins. MMP2/9 and VEGF protein expression were decreased in the RNAi + RAPA group to the levels of HL7702. Values are expressed as the mean ± standard deviation, **P<0.01. RAPA, rapamycin; RNAi, RNA interference; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor.
Mentions: The Transwell assay showed that cells which migrated across the membrane in the RNAi, RAPA, RNAi + RAPA and HL7702 groups were rare and scattered, while in the Scramble group, transmembrane cells were greater in number and dense in distribution. Statistical analysis revealed that the number of transmembrane cells in the RNAi + RAPA group was significantly lower than that in the RNAi, RAPA and Scramble groups (35.33±3.51 vs. 65.33±6.51, 60.67±8.50 and 161.67±14.50; P<0.01), and no significant difference was found when compared with the HL7702 group (35.33±3.51 vs. 25.67±5.86; P>0.05) (Fig. 6A). Detection of cell migration-associated genes revealed that matrix metal-loproteinase (MMP)-2 expression in the RNAi + RAPA group was significantly lower than that in the RNAi, RAPA and Scramble groups (0.04±0.02 vs. 0.21±0.03, 0.20±0.04 and 0.67±0.04; P<0.01), and the difference was not significant compared with that in the HL7702 group (0.04±0.02 vs. 0.03±0.01; P>0.05). MMP-9 expression in the RNAi + RAPA group was markedly decreased compared with that in the RNAi, RAPA and Scramble groups (0.06±0.02 vs. 0.23±0.02, 0.19±0.04 and 0.62±0.03; P<.0.01), and no significant difference was observed when compared with that in the HL7702 group (0.06±0.02 vs. 0.03±0.01; P>0.05). VEGF expression in the RNAi + RAPA group was also significantly lower than that in the RNAi, RAPA and Scramble groups (0.07±0.02 vs. 0.24±0.04, 0.20±0.06 and 0.62±0.06; P<0.01); while there was no significant difference compared with that in the HL7702 group (0.07±0.02 vs. 0.04±0.02; P>0.05) (Fig. 6B). These results demonstrated that RhoC gene silencing combined with RAPA treatment exerted marked migration-inhibitory effects on hepatocellular carcinoma cells.

Bottom Line: No significant difference was found between the RNAi + RAPA and the normal HL7702 group.The number of silver nitrate‑stained particles was reduced in the RNAi + RAPA group compared with that in the other groups.No significant difference was found between the RNAi + RAPA and HL7702 groups.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China.

ABSTRACT
The aim of the present study was to investigate the combined effects of inhibiting the Ras homolog gene family, member C (RhoC)/Rho kinase and phosphoinositide 3 kinase/Akt/mammalian target of rapamycin (mTOR) pathways on hepatocellular carcinoma cell growth. The RhoC gene was silenced by RNA interference (RNAi) and mTOR was inhibited by rapamycin (RAPA). Subsequently, an MTT assay for cell growth detection, western blot analysis for gene expression analysis, silver nitrate staining for cell proliferation, Wright's staining for analysis of the apoptotic rate analysis, soft agar clonogenic assay for the determination of cell growth characteristics and a Transwell assay for cell migration were performed. RhoC expression in hepatoma cell lines was lower than that in the HL7702 normal human liver cell line. The level of cell proliferation in the RNAi + RAPA group was lower than that in the RNAi, RAPA and Scramble groups. The levels of cyclin‑dependent kinase 2 in the RNAi + RAPA group were lower than those in the other groups, while the levels of P16 in the RNAi + RAPA group were higher than those in the other experimental groups. No significant difference was found between the RNAi + RAPA and the normal HL7702 group. The number of silver nitrate‑stained particles was reduced in the RNAi + RAPA group compared with that in the other groups. No significant difference was found between the RNAi + RAPA and HL7702 groups. Wright's staining for apoptosis demonstrated that apoptosis in the Scramble group was rare, while the RAPA and RNAi groups contained a large number of apoptotic cells, which displayed nuclear condensation, fragmentation, deepened staining, as well as a wrinkled membrane. B‑cell lymphoma‑2 (Bcl‑2) expression in the RNAi + RAPA group was lower than that in the other groups, while the gene expression of Bcl‑2‑associated X protein in the RNAi + RAPA group was increased compared with that in the other groups. No cell colony formation was observed in the soft agar cloning experiment in the RNAi + RAPA and HL7702 group, while in the other groups, visible cell clones appeared. In the Transwell assay the number of migrated cells in the RNAi + RAPA group was lower than that in the other groups. The gene expression of matrix metalloproteinase (MMP)2, MMP‑9 and vascular endothelial growth factor in the RNAi + RAPA group was lower than that in the other experimental groups. In conclusion, RhoC gene silencing combined with RAPA was able to significantly inhibit the growth of hepatocellular carcinoma cells.

Show MeSH
Related in: MedlinePlus