BACKGROUNDMelanoma has the highest mortality among all superficial malignant tumors. The poor prognosis is due to its high metastasis rate and the lack of therapeutic targets. As a molecular switch that controls tumor metastasis, Ras homology C (RhoC) has been correlated with tumor progression, especially tumor invasion and metastasis. However, little research has been done about the effects of RNA interference (RNAi) targeting RhoC on the invasion and metastasis of melanoma. In this study, we constructed an RNAi lentivirus vector targeting the RhoC gene of melanoma cells and identified its silencing effects on the RhoC gene.

METHODSBased on the RhoC gene encoding information, three pGPU6/GFP/Neo-short hairpin (shRNA) plasmids were constructed. After detecting their silencing effects on the RhoC gene of A375 cells, the most effective pGPU6/GFP/Neo-shRNA plasmid was packed with lentivirus to construct the recombinant pLenti6.3-EGFP-453 targeting RhoC. The lentivirus vector was used to infect A375 cells, and then the expression of RhoC mRNA and protein were determined with real-time PCR and Western blotting.

RESULTSThe plasmids pGPU6/GFP/Neo-shRNA 336, pGPU6/GFP/Neo-shRNA 453, and pGPU6/GFP/Neo-shRNA 680 were constructed. After they were transfected into A375 cells, the expressions of RhoC mRNA and protein were 1.47 ± 0.26, 1.13 ± 0.16, 1.39 ± 0.11 and 70.98 ± 9.21, 50.67 ± 6.06, 65.77 ± 4.06, respectively. pGPU6/GFP/Neo-shRNA 453 was the most effective sequence, and was used to successfully construct the pLenti6.3-EGFP-453 lentiviral vector targeting RhoC. pLenti6.3-EGFP-453 was used to infect A375 cells. The expression of RhoC mRNA and protein were 1.05 ± 0.05 and 62.04 ± 15.86 in the lentivirus group, 4.21 ± 0.24 and 220.86 ± 24.07 in the negative lentivirus control group, and 4.63 ± 0.32 and 257.39 ± 12.30 in the normal control group respectively with the difference between the lentivirus group and the control groups being statistically significant (P < 0.05).

CONCLUSIONThe successfully constructed pLenti6.3-EGFP-453 vector targeting the RhoC can effectively infect human melanoma A375 cells in vitro, and significantly inhibit the RhoC mRNA and protein expression.

Cell Line, Tumor ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; Melanoma ; genetics ; therapy ; RNA Interference ; physiology ; rho GTP-Binding Proteins ; genetics ; metabolism ; rhoC GTP-Binding Protein

OBJECTIVETo detect the expression of RhoC protein in human primary hepatocellular carcinoma (HCC) and pericancerous liver (PCL) tissues and its relation to HCC prognosis.

METHODSImmunohistochemical method was used to detect the expression of RhoC protein in HCC, PCL of 43 patients. Thirty-six patients were followed up after they received radical resection.

RESULTThe expression of RhoC protein in HCC was significantly higher than that in PCL. Rhoc expression was increased in cases with poor differentiation, portal vascular invasion, unintact envelope and multiple masses. The survival analysis showed that HCC with lower RhoC protein expression had better clinical outcomes.

CONCLUSIONRhoC protein expression is correlated with infiltration and metastasis in HCC. RhoC protein can be used as a prognostic indicator for HCC.

Adult ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Male ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Prognosis ; rho GTP-Binding Proteins ; biosynthesis ; genetics ; rhoC GTP-Binding Protein

OBJECTIVETo investigate the expressions of RhoC and osteopontin (OPN) protein in esophageal squamous carcinoma (ESC) and their association with the biological behavior of ESC.

METHODSThe expressions of RhoC and OPN protein were detected in 80 ESC cases by immunohistochemistry.

RESULTSThe positive expression rate of RhoC was 66.25% in these ESC cases. The rate was significantly higher in cases with lymph node metastasis than in those without (r(s)=-2.115, P<0.05), but RhoC expression was not associated with the tumor diameter, differentiation or TNM grade (P>0.05). The RhoC-positive patients had significantly shorter survival time than the negative patients (P<0.001). All the 80 ESC patients were positive for OPN expression, and OPN expression levels were correlated with the differentiation (chi(2)=10.766, P<0.05) and lymph node metastasis of the tumor (r(s)=-2.289, P<0.05), but not with the tumor diameter or TNM grade (P>0.05). Higher expression level of OPN was closely related to shorter survival time of the patients (P<0.05). A positive correlation was found between RhoC protein and OPN expressions (r(s)= 0.408, P<0.001) in these cases.

CONCLUSIONThe expressions of RhoC and OPN protein are closely related to lymph node metastasis of ESC and the patients survival time, and therefore may serve the purpose of prognostic evaluation of ESC.

Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; biosynthesis ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Osteopontin ; biosynthesis ; Prognosis ; Survival Analysis ; rho GTP-Binding Proteins ; biosynthesis ; rhoC GTP-Binding Protein

OBJECTIVETo investigate the expressions of RhoC, CD44v6 and ICAM-1 in gastric cancer and their correlations.

METHODSSABC immunohistochemistry was used to detect the expressions of RhoC, CD44v6 and ICAM-1 in the specimens from 40 cases with gastric cancer. Their correlations were reviewed by clinicopathological data.

RESULTSThe expression of RhoC, CD44v6 and ICAM-1 were not correlated with tumor differentiation and invasion depth (P> 0.05), but significantly correlated with lymph metastasis and pTNM stage (P< 0.05). There was a significant correlation between the expression of RhoC and the expression of CD44v6 or ICAM-1 (r=0.355, P=0.006; r=0.354, P=0.003) respectively. If RhoC was positive-expressed, and either of CD44v6 or ICAM-1 was positive-expressed, the sensitivity and the specificity for predicting the lymphatic metastasis was 93.75%, 62.5% respectively.

CONCLUSIONSThe positive expressions of RhoC, CD44v6 and ICAM-1 are useful biological markers for predicting the metastatic potential of gastric cancer. The combined detection of three markers is a useful method for predicting lymphatic metastasis of gastric cancer.

Adult ; Aged ; Humans ; Hyaluronan Receptors ; metabolism ; Immunohistochemistry ; Intercellular Adhesion Molecule-1 ; metabolism ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Staging ; Stomach Neoplasms ; metabolism ; pathology ; rho GTP-Binding Proteins ; metabolism ; rhoC GTP-Binding Protein

Female ; Humans ; Lymphatic Metastasis ; Male ; Microdissection ; Neoplasm Invasiveness ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms ; genetics ; pathology ; rho GTP-Binding Proteins ; genetics ; rhoC GTP-Binding Protein

OBJECTIVETo investigate the expression of RhoA, RhoC and their effector ROCK-1 in four ovarian cancer cell lines in vitro and their correlation with invasiveness.

METHODSExpression of RhoA, RhoC and ROCK-1 mRNA and protein in four ovarian cancer cell lines SW626, Skov-3, A2780 and Caov-3 was detected by RT-PCR and Western blot assay. Invasion assay was done in Boyden chamber.

RESULTSThe expression levels of RhoA, RhoC and ROCK-1 mRNA and protein varied in the four different cell lines examined. The expression level of RhoC, but not RhoA and ROCK-1, was significantly correlated with the invasive capability of these cells in vitro (r = 0.95, P < 0.01). Expression of RhoA at the level of transcription was not correlated with that at the translation level. The expression of RhoA and RhoC did not correlate with that of ROCK-1.

CONCLUSIONExpression level of RhoC may serve as an independent parameter in evaluating metastasis and become a new target in inhibiting ovarian cancer metastasis.

Cell Line, Tumor ; Cell Movement ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Intracellular Signaling Peptides and Proteins ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Phenotype ; Protein Biosynthesis ; Protein-Serine-Threonine Kinases ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transcription, Genetic ; rho GTP-Binding Proteins ; biosynthesis ; genetics ; rho-Associated Kinases ; rhoA GTP-Binding Protein ; biosynthesis ; genetics ; rhoC GTP-Binding Protein

Actins ; chemistry ; Amides ; therapeutic use ; Animals ; Apoptosis ; drug effects ; Cytoskeleton ; drug effects ; Enzyme Inhibitors ; therapeutic use ; Female ; Liver Neoplasms, Experimental ; drug therapy ; pathology ; Mice ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Pyridines ; therapeutic use ; ras Proteins ; analysis ; rho-Associated Kinases ; analysis ; antagonists & inhibitors ; rhoA GTP-Binding Protein ; analysis ; rhoC GTP-Binding Protein

BACKGROUND AND OBJECTIVEPhosphatase of regenerating liver-3 (PRL-3) is a newly identified protein-tyrosine phosphatase, which belongs to phosphatase of regenerating liver family, and plays a role in promoting tumor metastasis; Ras homologue C (RhoC) belongs to Rho subfamily of small-molecule G protein superfamily. However, the mechanisms of PRL-3 and RhoC are unknown. The aim of this study is to investigate the expressions of PRL-3 and RhoC proteins and their correlation to invasion and metastasis of non-small cell lung cancer (NSCLC), which may provide experiment evidence of the mechanism of PRL-3 in tumorigenesis and tumor-development.

METHODSImmunohistochemical staining was used to detect the expressions of PRL-3 and RhoC in NSCLC in 92 cases, and statistical methods were used to analyse statistical significances of their expressions in different groups and their correlation.

RESULTSThe positive rates of PRL-3 and RhoC expressions in NSCLC were 69.6% (64/92) and 73.9% (68/92), respectively, and the expressions of PRL-3 and RhoC were closely correlated with TNM stage and lymphatic metastasis and pleural metastasis (P < 0.01), and they were correlated with each other (r = 0.754, P < 0.001).

CONCLUSIONThe expressions of PRL-3 and RhoC are higher in the higher TNM stage and lymphatic metastasis and pleural metastasis cases, and closely correlate with each other in NSCLC, which suggests that PRL-3 and RhoC might be in the same signal pathway and PRL-3 might promote the distant metastasis of cancer cell by RhoC and downstream factors.

Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; chemistry ; pathology ; Female ; Humans ; Immunohistochemistry ; Lung Neoplasms ; chemistry ; pathology ; Male ; Middle Aged ; Neoplasm Metastasis ; Neoplasm Proteins ; analysis ; genetics ; physiology ; Neoplasm Staging ; Protein Tyrosine Phosphatases ; analysis ; genetics ; physiology ; rho GTP-Binding Proteins ; analysis ; genetics ; physiology ; rhoC GTP-Binding Protein

OBJECTIVETo detect the expression of RhoC and Rho kinase 1 (ROCK-1) in prostate carcinoma, and explore the possible mechanism of RhoC/ROCK-1 in the pathogenesis of prostate carcinoma.

METHODSTissue specimens from 73 patients with prostate carcinoma and corresponding paracancerous tissues were obtained by prostate cancer biopsy or radical prostatectomy. The expression of RhoC/ROCK-1 mRNA was detected by RT-PCR. Western blot and immunohistochemistry were performed to dertect the expression of RhoC/ROCK-1 protein. Eukaryotic expression plasmids of RhoC were constructed and transfected into PC-3M-2B4 cells. p-MAPK and p-Akt were detected by Western bolt.

RESULTSThe expression levels of RhoC and ROCK-1 mRNA in the prostate carcinomas were significantly higher than those in corresponding paracancerous tissues [72.6% (53/73) vs. 34.2% (25/73); 68.5% (50/73) vs. 38.4% (28/73), P < 0.01], respectively. The results indicated that RhoC/ROCK-1 mRNA expression had no significant correlation with Gleason grade. However, the expression of RhoC/ROCK-1 mRNA showed a significant positive correlation with distant metastasis. The RhoC/ROCK-1 protein expression in prostate cancer was also higher than corresponding paracancerous tissues, and showed a significant positive correlation with p-MAPK and p-Akt expression levels. In addition, p-MAPK and p-Akt expression levels were up-regulated in the transcripts.

CONCLUSIONExpression levels of RhoC and ROCK-1 in prostate carcinoma are higher than those in corresponding paracancerous tissues, showing a significant positive correlation with distant metastasis. RhoC/ROCK-1 may be involved in the development, invasion and metastasis of prostate carcinoma.

Bone Neoplasms ; metabolism ; secondary ; Cell Line, Tumor ; Humans ; Male ; Mitogen-Activated Protein Kinases ; metabolism ; Neoplasm Grading ; Neoplasm Staging ; Phosphorylation ; Prostatectomy ; Prostatic Neoplasms ; metabolism ; pathology ; surgery ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; metabolism ; Transfection ; Up-Regulation ; rho GTP-Binding Proteins ; genetics ; metabolism ; rho-Associated Kinases ; genetics ; metabolism ; rhoC GTP-Binding Protein

OBJECTIVERho, a ras homologous gene, encodes a group of GTP-binding proteins. Our previous study suggested that one member of the Rho gene family, RhoC, was related to the progression of human hepatocellular carcinoma (HCC). This study is to elucidate correlation of Rho overexpression with invasion and metastasis of HCC.

METHODSThe expression level of RhoC mRNA and protein in 25 cases of HCC and adjacent non-cancerous liver tissue was examined by RT-PCR and Western blot. Mutation of RhoC gene was examined by PCR-SSCP.

RESULTSThe expression of RhoC mRNA and protein was found in all HCC and adjacent non-cancerous liver tissue. The expression level of RhoC mRNA and protein was significantly higher in tumor tissue than in adjacent non-cancerous liver tissue (1.8 +/- 1.1 vs 1.0 +/- 0.7; 33 992 +/- 10 384 vs 17 342 +/- 9998, P < 0.01). The degree of RhoC overexpression was even more marked in metastatic lesions than in primary tumors (P < 0.01). Overexpression of the rhoC gene was significantly correlated with such clinic-pathological findings as cell differentiation, portal vein invasion, number of primary tumor nodules and metastatic lesions (P < 0.05). Mutation of RhoC gene was found in none of the HCC specimens examined.

CONCLUSIONOverexpression of RhoC gene may play an important role in carcinogenesis and progression of HCC.

Adult ; Aged ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Differentiation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Liver ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Invasiveness ; Portal Vein ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; rho GTP-Binding Proteins ; biosynthesis ; genetics ; rhoC GTP-Binding Protein