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ABSTRACT Objectives: We examined the expression of Lnc-ZFAS1 in osteosarcoma and comprehensively evaluated its effects on osteosarcoma in vitro and vivo. Moreover, we revealed the regulatory mechanism between Lnc-ZFAS1 and miR-520b/miR-520e-mediated RHOC and provided a novel clue for ameliorating osteosarcoma. Method: The expression of Long non-coding RNA Zinc Finger Antisense 1 (LncRNA ZFAS1) osteosarcoma tissues and normal tissues in the TCGA database was analyzed. Then, LncRNA ZFAS1 expression was further verified in clinical samples and osteosarcoma cell lines (U2OS and KHOS), as well as the human osteoblast cell line hFOB1.19 by qRT-PCR. Thereafter, LncRNA ZFAS1 was overexpressed or silenced to explore its effects on cell proliferation, apoptosis, migration, invasion, and Epithelial-Mesenchymal Transition (EMT). The fundamental mechanism through which Lnc-ZFAS1 affects osteosarcoma progression was further investigated and verified. Results: We found that LncRNA ZFAS1 was upregulated in osteosarcoma, and Lnc-ZFAS1 overexpression facilitated osteosarcoma cells proliferation, migration, invasion and EMT, while Lnc-ZFAS1 silence exerted reverse influence. Mechanistically, Lnc-ZFAS1 functionally acted as a sponger of microRNA-520b (miR-520b) and micro-RNA-520e (miR-520e) to up-regulate Ras Homologue C (RHOC). In addition, depleted Lnc-ZFAS1 restrained osteosarcoma cells proliferation, migration, and invasion, which could be rescued by RHOC overexpression. Lnc-ZFAS1 was upregulated in osteosarcoma and Lnc-ZFAS1 could exert promoted impact upon osteosarcoma cells proliferation, migration, invasion, and EMT in vitro. Conclusions: Lnc-ZFAS1 acted sponger of miR-520b and miR-520e to promote RHOC, indicating that Lnc-ZFAS1/miR-520b/RHOC and Lnc-ZFAS1/miR-520e/RHOC axes might serve as potential therapeutic strategies against osteosarcoma.
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Objective To investigate the effect of RhoC expression in vascular endothelial cells on the proliferation and invasion of myeloma RPMI8226 cells and its possible mechanism. Methods RhoC shRNA lentivirus vector was constructed and transfected into myeloma vascular endothelial cells (MVECs) and human umbilical vein endothelial cells (HUVECs). The effects of conditioned medium on the proliferation, cell cycle and invasion of RPMI8226 cells were detected by CCK-8 test, flow cytometry and Transwell test. The expression of CDK, CyclinD1, AKT, PI3K, MMP2 and MMP9 were detected by Western blot. Results The expression of RhoC in MVECs and HUVECs were downregulated. The proliferation and invasion of RPMI8226 cells in RhoC shRNA group were lower than those in negative control group, and the cell cycle was blocked in G0/G1 phase (P < 0.05). The expressions of CDK, CyclinD1, AKT, PI3K, MMP2 and MMP9 in RhoC shRNA group were lower than those in negative control group (P < 0.05). Conclusion The expression of RhoC in MVECs can regulate the proliferation and invasion of myeloma RPMI8226 cells, and the mechanism may be related to the participation of CDK, CyclinD1, AKT, PI3K, MMP2 and MMP9.
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Objective To investigate the effects of RhoC on biological behavior in the laryngeal squamous carcinoma.Methods By delivering exogenous gene into Hep2 laryngeal squamous carcinoma cell line,we alternatively repressed and strengthened the expression of RhoC.We tested the apoptosis of Hep2 tumor cell line with TUNEL,visualized tumor cells shape by staining cell skeleton with Alexa fluor phalloidin,measured the mRNA of CSC marker ALDH1A1 with QPCR.Results After repressing the expression of RhoC in Hep2 cell line,the apoptosis of cancer cells was elevated,the expression of CSC marker ALDH1A1 was significantly decreased.RhoC impacted the shapes of Hep2.Conclusion RhoC havd a positive role in LSCC metastasis.RhoC is a promising target of anti-metastases in LSC.
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Objective To investigate the effect of miR-106b on the apoptosis and proliferation of nasopharyngeal carcinoma (NPC ) cells. Methods We analyzed differences in miRNA expression in nasopharyngeal carcinoma and adjacent normal tissues with miRNA microarray.Taq Man miRNA detection kit and Real-time fluorescence quantitative PCR were used to detect the expressions of miR-106 and RhoC mRNA in nasopharyngeal carcinoma and adjacent tissues.The miR-106b and target gene binding sites were predicted with miRnada.The target gene was verified by double luciferase.Western blot was used to detect the expression of RhoC regulated by miR-106b.Annexin and TUNEL were used to detect the effect of miR-106b on the apoptosis of nasopharyngeal carcinoma cells;the effect of miR-106b on the proliferation of nasopharyngeal carcinoma cells was detected by MTT assay.Results miRNA microarray analysis showed that the expression of miR-106b was lower in NPC tissues than in adjacent normal tissues.The results of RT-PCR showed that the expression of miR-106b in nasopharyngeal carcinoma was decreased (P <0.05)while the expression of RhoC was increased in nasopharyngeal carcinoma (P <0.05).The expressions of miR-106b and RhoC in NPC were negatively correlated (r =-0.5866, P <0.001).The results of luciferase reporter assay showed that the activity of luciferase in miR-106b group was lower than that in empty plasmid group (P < 0.05 ).The results of Western blot showed that miR-106b could decrease the expression of RhoC in NPC tissues (P <0.05).Annexin V-PI and TUNEL showed that the apoptosis ofnasopharyngeal carcinoma cells was significantly higher in miR-106 group than in empty plasmid group (P <0.05). MTT results showed that the proliferation of nasopharyngeal carcinoma cells in miR-106b group was lower than that in empty plasmid group (P <0.05).Conclusion miR-106b may induce the apoptosis of nasopharyngeal carcinoma cells and inhibit the proliferation of nasopharyngeal carcinoma cells by down-regulating the expression of RhoC.
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Objective To investigate the effect of miR-106b on the apoptosis and proliferation of nasopharyngeal carcinoma (NPC ) cells. Methods We analyzed differences in miRNA expression in nasopharyngeal carcinoma and adjacent normal tissues with miRNA microarray.Taq Man miRNA detection kit and Real-time fluorescence quantitative PCR were used to detect the expressions of miR-106 and RhoC mRNA in nasopharyngeal carcinoma and adjacent tissues.The miR-106b and target gene binding sites were predicted with miRnada.The target gene was verified by double luciferase.Western blot was used to detect the expression of RhoC regulated by miR-106b.Annexin and TUNEL were used to detect the effect of miR-106b on the apoptosis of nasopharyngeal carcinoma cells;the effect of miR-106b on the proliferation of nasopharyngeal carcinoma cells was detected by MTT assay.Results miRNA microarray analysis showed that the expression of miR-106b was lower in NPC tissues than in adjacent normal tissues.The results of RT-PCR showed that the expression of miR-106b in nasopharyngeal carcinoma was decreased (P <0.05)while the expression of RhoC was increased in nasopharyngeal carcinoma (P <0.05).The expressions of miR-106b and RhoC in NPC were negatively correlated (r =-0.5866, P <0.001).The results of luciferase reporter assay showed that the activity of luciferase in miR-106b group was lower than that in empty plasmid group (P < 0.05 ).The results of Western blot showed that miR-106b could decrease the expression of RhoC in NPC tissues (P <0.05).Annexin V-PI and TUNEL showed that the apoptosis ofnasopharyngeal carcinoma cells was significantly higher in miR-106 group than in empty plasmid group (P <0.05). MTT results showed that the proliferation of nasopharyngeal carcinoma cells in miR-106b group was lower than that in empty plasmid group (P <0.05).Conclusion miR-106b may induce the apoptosis of nasopharyngeal carcinoma cells and inhibit the proliferation of nasopharyngeal carcinoma cells by down-regulating the expression of RhoC.
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Objective:To investigate the effect of heavy ion beam on the invasion and metastasis associated gene RhoC expression in tongue squamous cell carcinoma Tca8113 cells. Methods:Real-time PCR and Western-blot were used to detect the expression of RhoC mRNA and RhoC protein respectively after Tca8113 cells were irradiated by heavy ion beams of 12 C6+. Results:The expression levels of RhoC mRNA in the experimental group were lower than that in the control group(P<0. 05) except for the levels at 12 h after 2 Gy and 4 Gy 12 C6+ irradiation. In addition, the expression levels of RhoC protein increased in a dose-dependent manner at 12 h af-ter irradiation, reaching a peak at 4 Gy, and subsequently decreased with the increase of irradiation dose. The expression level of RhoC protein was consistent with that of RhoC mRNA. Conclusion:Heavy ion beam of 12 C6+ may inhibit RhoC gene expression in Tca8113 cells.
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Aim To investigate the effect of neferine on proliferation and invasion of human hepatocellular car-cinoma. Methods HepG2 and Bel-7402 cells were cultured in vitro with different concentrations of nefer-ine, then cell proliferation was observed by CCK-8 as-say; cell invasion was observed by transwell invasion assay; the protein expression of RhoA, RhoC and ROCK was detected by Western blot. Results CCK-8 results showed that neferine could significantly inhibit cell proliferation in a dose-dependent manner compared with the control group. Transwell invasion assay showed that cell invasion was significantly decreased with neferine 3 μmol · L-1 . Western blot results showed that RhoA, RhoC and ROCK protein expres-sion was decreased when neferine was co-incubated with hepatocellular carcinoma cells. Conclusion Nef-erine can inhibit proliferation and invasion of HepG2 and Bel-7402 cells, which is mediated mainly by the inhibition of RhoA, RhoC and ROCK protein expres-sion.
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Objective To investigate the expression and clinical significance of RhoC protein in breast cancer.Methods Expression of RhoC protein was detected by immunohistochemlstry in 112 cases of breast cancer,20 cases of adjscent non-cancerous normal tissues and 15 cases of normal mammary gland tissues.The relation between expression of RhoC protein and clinicopathological characteristics was analyzed.Expression of CD34 in breast cancer tissues was detected.Microvessel density(MVD)was calculated and its correlation with RhoC protein Was analyzed.Results RhoC had a positive expression rate of 40.2%(45/112)in breast cancer,while it was not expressed in adjacent non-cancerous tissues or normal mammary gland tissues.Rhoc expression in breast tissues was related to lymph node metastasis and pathological stages(P<0.05).MVD value in patients with positive RhoC expression was significantly higher than that in patients with negative RhoC expression(P<0.05).Conclusions The expression of RhoC protein is related to lymphatic metastasis,pathological stages and MVD value.The upregulation of RhoC protein expression may promote the metastasis and invasion of breast cancer.
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Objective To investigate the expression of RhoC mRNA in the gastric carcinoma (GC) and paracarcinoma gastric mucosa tissues (PGMT) and their relationship with clinical pathological features.Methods RhoC mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) in 23 cases of primary gastric carcinoma and the paracarcinoma gastric mucosa tissues.Results The opacity density of RhoC mRNA in 23 cases of GC was 1.40±0.23,higher than that of PGMT (0.36±0.15)(P<0.01).In addition.RhoC mRNA in gastric carcinoma tissues were positively related to invasion depth,TNM stage and lymph node metastasis (P<0.01),Conclusion The overexpression of RhoC mRNA in GC may be closely related to the carcinogenesis,development,invasion and metastasis of gastric carcinoma,which is a frefered biomarker for early diagnosis.
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This study investigated the effect of RhoC GTPase on the proliferation and metastasis of cervical cancer cells, SiHa cells, in vitro. RhoC siRNA was introduced into SiHa cells to silence the RhoC gene. The mRNA and protein expression of RhoC, before and after RhoC siRNA transfection,was examined by RT-PCR and Western blotting, respectively. The proliferation and apoptosis of SiHa cells were examined by MTT assay and flow cytometry (FACS), respectively. Adhesive rate was evaluated by Matrigel adhesive assay, and the invasive capability and migration capability were assessed by transwell invasive assay and migration assay, respectively. The results showed that after the RhoC siRNA transfection, the mRNA and protein expression of RhoC was down-regulated in SiHa cells. The down-regulation of RhoC GTPase did not affect the cell proliferation and apoptosis (P>0.05), but it did suppress SiHa cells' adhesion to matrigel (P<0.01), the invasive capability (P<0.01) and the migration capability (P<0.01). It was concluded that RhoC obviously promotes the adhesion, invasion and migration of SiHa cells in vitro, but not proliferation and apoptosis, suggesting that RhoC plays an important rote in the progression in cervical cancer.
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Objective To study the expressions of RhoC and MMP-2 proteins in epithelial ovarian cancer and their clinical significances in order to provid the basis for assessing the biological behaviour of epithelial ovarian cancer.Methods Immunohistochemistry was used to detect the expressions of RhoC and MMP-2 proteins in 10 cases of normal ovarian tissues,12 cases of epithelial benign ovarian tumor and 91 cases of epithelial ovarian cancer,and the relationship with the clinical features was analyzed.Results The RhoC and MMP-2 proteins positive expression rates in ovarian benign tumor and ovarian normal tissues were 0 respectively; the positive expression rates of RhoC and MMP-2 proteins in epithelial ovarian cancer were 37.3% and 68.1%.The expressions of RhoC and MMP-2 proteins in ovarian cancer were significantly higher than those in ovarian benign tumor and ovarian normal tissues.The positive expression rates of RhoC and MMP-2 in moderately and poorly differentiated cancer were significantly higher than those in well differentiated cancer(P
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Objective To construct a RhoC-siRNA expression vector, and study its role on the biological behaviors of hepatoma carcinoma cells. Methods RhoC-siRNA gene was synthesized and cloned into the expression vector pSilencer2.1. The constructed RhoC-siRNA expression vector was stably transfected into hepatoma carcinoma cell line SK-Hep1. The inhibitory effect of RhoC-siRNA on the expression of RhoC in transfected cells was detected by Western blotting. The morphous, growth velocity and the ability of cell adhesion, cell migration, cell invasion before and after transfection was observed. Results Enzyme digestion and DNA sequencing confirmed that the RhoC specific siRNA expression vector was constructed successfully. After transfection, RhoC expression was inhibited by 60%, and no marked difference was observed in cellular morphous and growth curve, while the ability of cell adhesion, cell migration, and cell invasion were markedly decreased. Conclusion The RhoC-siRNA expression vector can effectively suppress RhoC expression in transfected hepatoma carcinoma cells. Although having no effect on the morphous and growth velocity of hepatoma carcinoma cells, it decreases the potentiality of cell invasion and cell metastasis, which may provide a novel applicable strategy for gene therapy on hepatocellular carcinoma.
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Objective To study the expression of RhoC mRNA in human primary hepatocellular carcinoma(PHCC) and paracarcinoma liver(PCL) tissues .Method Reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expression of RhoC mRNA in the PHCC and PCL tissue of 30 patients with PHCC. Results The opacity density (OD)of RhoC mRNA expression in PHCC tissues was significantly higher than that in PCL tissures(P
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Objective To study the influence of pcDNA3.1-RhoC on the expression of endogenous angiogenic factors in HCC cells.Methods The reconstructed plasmid pcDNA3.1-RhoC was transfected into HepG2 cells, and expression of VEGF and bFGF was detected with the RT-PCR and immunohistochemical stain. HepG2 cells transfected with pcDNA3.1-RhoC or pcDNA3.1 were implanted into nude mice to observe the tumor occurrence rate.Results HepG2 cells transfected with pcDNA3.1/RhoC showed higher expression of RhoC . The expression of RhoC enhanced the expression of VEGF and bFGF(P
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Objective To establish an experimental model for exploring the role of RhoC gene in the invasiveness and metastasis of hepatocellular carcinoma.Method The RhoC gene was digested with restricted enzyme Hind III and XbaI,and direct cloned to pcDNA3.1.The recombinant vector (pcDNA3.1 RhoC) and the vector alone (pcDNA3.1) were transfected into HEPG2 cells with LIPFECTAMINETMReagent.After selected with hygromycin,resistant cloneies was obtained.The transcription and translation of RhoC gene were analysed with the reverse transcription PCR and immunohistochemical stain.Results The recombinant vectort (pc DNA3.1 Rhoc) express steadily in HerpG2 cells.Conclusions The modified tumor cells(HEPG2 RhoC) could be used to study the effect of RhoC protein on the invasiveness and metastasis of hepatocellular carcinoma.