The Function And Mechanism Research Of ARHGEF7 Promoting The Invasion And Metastasis Of Hepatocellular Carcinoma

Research BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a high rate of metastasis, which has a serious threat to the Chinese people. Currently the long-term prognosis of HCC is very poor because of the high rate of recurrence and metastasis. Hence, further study on the invasion and metastasis of HCC will be helpful to develop novel therapeutic strategies and it is of great significance to improve the overall treatment outcome of HCC.ARHGEF7 is an important member of the guanine nucleotide exchange factor family. Recent studies have found that ARHGEF7 can affect cell movement, proliferation and apoptosis by regulating intercellular adhesion and pseudopodia formation, thereby inducing vascular permeability changes, and ultimately lead to tumor formation. Further studies showed that ARHGEF7 protein level was significantly higher in the gastric cancer cells than that in the adjacent and normal tissues. ARHGEF7 can promote breast cancer growth and metastasis through the activation of Yap/Taz. In addition, the protein level of ARHGEF7 was significantly higher in renal cell carcinoma than that in HEK293 cells, suggesting that ARHGEF7 may play an important role in tumorigenesis.Our preliminary experimental study showed elevated ARHGEF7 mRNA and protein expression level in the HCC tissues compared with the adjacent and normal tissues, and it was closely related to multiple tumor nodules, Edmondson-Steiner classification, and venous invasion, suggesting that ARHGEF7 may be involved in the invasion and metastasis of HCC. However, the underlying mechanisms are not clear. Based on the previous results, in this study, we investigated ARHGEF7 expression in multiple hepatocellular carcinoma cell lines with different invasion and metastasis ability. We also investigated the role and the molecular mechanisms of ARHGEF7 in the regulation of HCC invasion and metastasis through a series of in vitro and in vivo methodsPurpose and significanceThe aim of this study was to investigate the correlations of ARHGEF7 expression with clinicopathologic characteristics and prognosis of HCC. In addition, RNA interference (RNAi) mediated by lentiviral vectors (which induce strong down-regulation of ARHGEF7 in HCCLM3 cell lines) was used to analyze the role of ARHGEF7 in the pathogenesis of HCC. Our findings may uncover the mechanisms of ARHGEF7 in the regulation of HCC invasion and metastasis, supplying potential therapeutic targets for HCC treatment.Methods and Results1. High expression of ARHGEF7 is significantly associated with clinicopathologic characteristics and prognosis of HCCInitially, ARHGEF7 mRNA level was examined in 30 HCC samples and adjacent normal liver tissues (ANLT) as well as 5 normal liver tissues (NL) by using QRT-PCR assay. Our results showed that ARHGEF7 mRNA was highly expressed in HCC tissues than that in ANLT and NL (P<0.01). Next, we tested the protein level of ARHGEF7 in the same batch of tissue samples and found that ARHGEF7 protein level was significantly elevated in HCC tissues compared with ANLT and NL. Spearman’s rank correlation analysis indicated a positive correlation between the mRNA and protein level of ARHGEF7 (r=0.843, P<0.01). Furthermore, ARHGEF7 mRNA and protein level was compared between HCC tissues with different clinicopathologic characteristics. The results showed that he ARHGEF7 mRNA and protein expression levels were closely associated with the clinicopathologic characteristics of HCC such as capsule formation, number of nodules, TNM stage, Edmondson Steiner grade and venous infiltration and so on (P< 0.05).2. High expression of ARHGEF7 in HCC cell lines is closely correlated with the invasion and metastasis of HCC.To confirm the results obtained from the fresh HCC tissues, ARHGEF7 mRNA level was detected in six hepatocellular carcinoma cell lines (HepG2, SMMC-7721, Huh-7, MHCC-97L, MHCC-97H, HCCLM3) with different invasion and metastasis ability by QRT-PCR, and a normal hepatic cell line (HL-7702) was used as control. Our results showed that ARHGEF7 mRNA level was highly expressed in six HCC cell lines than that in HL-7702, and it was significantly related to the invasion and metastasis ability. We further tested the protein level of ARHGEF and found that ARHGEF7 expression was significantly elevated in hepatocellular carcinoma cell lines compared with normal hepatic cell line, and the difference was statistically significant (P<0.05).3. High expression of ARHGEF7 in Training cohort and its relationship with clinicopathologic characteristics and prognosis of HCC.Next, we measured ARHGEF7 expression in Training cohort by immunohistochemistry in 92 cases of HCC paraffin section samples and analyzed its relationship with clinicopathologic characteristics of HCC. The results showed that ARHGEG was mainly distributed in the cytoplasm, and its high expression was correlated to cirrhosis, multiple tumor nodules, TNM stage, and Edmondson Steiner grade (P<0.05). The median overall survival time in patients with high expression of ARHGEF7 was significantly lower than that in patients with low expression (P=0.001). Likewise, the median disease-free survival time in patients with high expression of ARHGEF7 was shorter than that in patients with low expression (P=0.003). We further established the Cox proportional hazard regression model by using the clinical follow-up data for overall survival time and tumor-free survival time. Single factor Cox regression analysis on overtime survival time showed that liver cirrhosis and no liver cirrhosis, capsule formation and no capsule formation, number of nodules, TNM stage, Edmondson Steiner grade, venous infiltration, and ARHGEF7 expression level had significant influence on prognosis of HCC. Multi-factor Cox regression analysis on overtime survival time showed that liver cirrhosis, tumor diameter, Edmondson Steiner grade, venous infiltration, and ARHGEF7 expression level were independent risk factors for HCC prognosis. Single factor Cox regression analysis on tumor-free survival time showed that liver cirrhosis and no liver cirrhosis, capsule formation and no capsule formation, number of nodules, Edmondson Steiner grade, and ARHGEF7 expression level were closely associated with the prognosis of HCC. Multi-factor Cox regression analysis on tumor-free survival time revealed that tumor diameter and ARHGEF7 were independent risk factors for HCC prognosis.4. High expression of ARHGEF7 in Validation cohort and its relationship with clinicopathologic characteristics and prognosis of HCC.In order to verify the previous results, we measured ARHGEF7 expression in Validation cohort by immunohistochemistry in 65 cases of HCC paraffin section samples and analyzed its relationship with clinicopathologic characteristics of HCC. We found that ARHGEG was mainly distributed in the cytoplasm, and its high expression was related to capsule formation and no capsule formation, multiple tumor nodules, TNM stage, and Edmondson Steiner grade and venous infiltration (P<0.05). The median overall survival time in patients with high expression of ARHGEF7 was significantly shorter than that in patients with low expression (P=0.009). Likewise, the median tumor-free survival time in patients with high expression of ARHGEF7 was lower than that in patients with low expression, and the tumor free survival rate with high expression of ARHGEF7 was also lower than that in patients with low expression (P=0.003). We further established the Cox proportional hazard regression model by using the clinical follow-up data for overall survival time and tumor-free survival time. Single factor Cox regression analysis on overtime survival time showed that capsule formation and no capsule formation, number of nodules, Edmondson Steiner grade, venous infiltration, and ARHGEF7 expression level were correlated to the prognosis of HCC. Multi-factor Cox regression analysis on overtime survival time showed that number of nodules, TNM stage, venous infiltration, and ARHGEF7 expression level were independent risk factors for HCC prognosis. Single factor Cox regression analysis on tumor-free survival time revealed that capsule formation and no capsule formation, number of tumor nodules, Edmondson Steiner grade, venous infiltration, and ARHGEF7 expression level were closely associated with the prognosis of HCC. Multi-factor Cox regression analysis on tumor-free survival time showed that number of nodules, TNM stage, venous infiltration, and ARHGEF7 expression level were independent risk factors for HCC prognosis.5. Inhibition of ARHGEF7 by sh-RNA mediated by lentivirus in HCCLM3 cell linesSubsequently, we chose HCCLM3 cell lines with high invasive potential as the experimental cells for the study of HCC invasion and metastasis. Using lentivirus infection, we constructed HCCLM3ARHGEF7 cell lines (KD group) and control HCCLM3vector cell lines (NC group).72h cells after infection, the transfection efficiency was greater than 90% with no large area of cell death. We further examined the expression of ARHGEF7 in HCCLM3 cells. As shown by QRT-PCR, the ARHGEF7 RNAi led to a substantial down-regulation of ARHGEF7 mRNA compared with NC group, and the efficiency of knockdown was up to 83.3% (P<0.05). Western blotting results also showed that ARHGEF7 expression was significantly decreased in KD group compared with NC group (P<0.05). The results indicated that lentivirus can effectively infect target cells and inhibit the expression of ARHGEF7 in HCCLM3 cells, providing a foundation for further research on the function and mechanism of this gene.6. In vitro inhibition of ARHGEF7 can inhibit the invasion and migration of hepatocellular carcinoma cells and affect its adhesion ability.We then examined the ability of ARHGEF7 on HCC migration and movement by using wound healing assay and Transwell assay, respectively. The wound healing assay showed that no significant difference in 8 h average-mobility was found between NC and KD group (P>0.05), while significant difference in 24 h average-mobility was observed between the two groups (P<0.05), indicating the decreased HCC migration and movement by inhibition of ARHGEF7 expression. In addition, Transwell assay revealed that the number of cells in KD group that migrated to the lower chamber was significantly decreased compared with NC group (P< 0.05). Furthermore, the effects of ARHGEF7 inhibition on hepatocellular carcinoma morphology were analyzed in KD and NC group by ilmmunofluorescence. We found that the actin cytoskeleton in KD group was obviously changed relative to NC group. Considering the correlation between ARHGEF7 and cell adhesion, we then observed whether ARHGEF7 could regulate the adhesion ability of HCC cells by homogeneous and heterogeneous adhesion experiments. The results showed that the homogeneous adhesion ability in KD group was significantly enhanced than that in NC group (P<0.05), while the heterogeneous adhesion ability in KD group was obviously weakened compared with NC group (P<0.05). These results indicate that inhibition of ARHGEF7 expression can affect the adhesion ability of HCC cells and inhibit HCC invasion and metastasis in vitro.7. In vitro inhibition of ARHGEF7 expression does not affect the proliferation and apoptosis of hepatocellular carcinoma cells.Next, we used MTT assays to examine the effects of ARHGEF7 on the proliferation of hepatocellular carcinoma cells. The results showed that there was no significant difference in cell count between KD group and NC group at day 7 (P>0.05). Then we used colonogenic assays to test the effects of ARHGEF7 on colony formation of HCC cells in vitro and found that ARHGEF7 did not promote the colony formation of HCC cells. There is no significant difference in the colony forming number between KD and NC group (P>0.05). In order to further confirm the effects of ARHGEF7 on the viability of HCC cells, we detected the cell cycle between the two groups. Our results showed that the percentage of cells in G2/M-phase was increased in KD group compared with NC group(P<0.05), whereas it decreased in S-phage in KD group (P<0.05) with no obvious change in G1-phage between the two groups (P>0.05). These findings indicate that ARHGEF7 can not promote the viability of HCC cells in vitro. Furthermore, cell apoptosis was detected by Flow cytometry between NC and KD group. Compared with NC group, no obvious cell apoptosis was found in KC group (P>0.05). Taken together, these results suggest that ARHGEF7 does not affect the proliferation and apoptosis of HCC cells.8. Inhibition of ARHGEF7 expression can inhibit the proliferation and metastasis of hepatocellular carcinoma cells in nude mice.Subsequently, we established HCCLM3 xenografted tumor model to investigate the effects of ARHGEF7 expression on the invasion and metastasis of hepatocellular carcinoma cells. The cells of KD and NC group were injected into the subcutaneous of nude mice, and the the growth and pulmonary metastasis of orthotopic implant tumor was analyzed between the two groups. The results showed that the average volume of tumors of KD group was significantly less than that of NC group (P<0.05). A further study was performed to examine the expression of ARHGEF7 with hematoxylin and eosin (H&E) staining and immunohistochemistry, and found that ARHGEF7 expression was significantly increased in tumors of NC group compared with KD group, further confirming the efficacy of RNAi mediated by lentivirus. More importantly, we performed serial sections of lung tissue and the H&E staining was used to detect the pulmonary metastasis of tumors from two groups. Our results showed that only one of the five nude mice in KD group was found to have lung metastasis of HCC cells, while four of the five nude mice in NC group were observed to have lung metastasis of HCC cells, and this difference was statistically significant (P<0.05). The above findings indicate that Inhibition of ARHGEF7 expression can significantly inhibit the proliferation and metastasis of hepatocellular carcinoma cells in nude mice.9. In vivo inhibition of ARHGEF7 expression in nude mice can inhibit the proliferation and metastasis of hepatocellular carcinoma cells.The subcutaneous tumor tissues of the two groups were transplanted into the liver of 10 nude mice, and the growth and metastasis of liver tumor was compared between two groups. Our results showed that 7 of the 10 nude mice in KD group formed liver tumor, whereas all the nude mice in NC group formed liver tumor, and the average tumor volume of KD group was significantly lower than NC group (P<0.05). A further study was performed to examine the expression of ARHGEF7 in liver tumor tissues with hematoxylin and eosin (H&E) staining and immunohistochemistry, and found that ARHGEF7 expression was significantly decreased in tumors of KD group compared with NC group, further confirming the efficacy of RNAi mediated by lentivirus. More importantly, only two of the ten nude mice in KD group were found to have tumor nodules within the liver tissue (i.e. intrahepatic metastases) outside the planting area, while eight of the ten nude mice in NC group were found to have tumor nodules within the liver tissue (i.e. intrahepatic metastases) outside the planting area, and the difference was statistically significant (P<0.05). We then performed serial sections of lung tissue and the pulmonary metastasis of tumors from two groups was detected by H & E staining. Only one nude mouse in KD group were found to have lung metastasis of HCC cells, while five nude mice in NC group were found to have lung metastasis of HCC cells, and this difference was statistically significant (P<0.05). In summary, these results suggest that in vivo inhibition of ARHGEF7 expression in nude mice can inhibit the proliferation and metastasis of hepatocellular carcinoma cells.Conclusions1. The mRNA and protein level of ARHGEF7 were significantly elevated in the HCC tissues and cell lines.2. High expression of ARHGEF7 in HCC tissue was closely associated with the clinicopathologic characteristics of HCC including liver cirrhosis, capsule formation, number of nodules, TNM stage, Edmondson Steiner grade and venous infiltration, and it was the independent risk factor for the prognosis of HCC patients.3. In vitro inhibition of ARHGEF7 can inhibit the invasion and migration of hepatocellular carcinoma cells and affect its adhesion ability, but did not affect the proliferation and metastasis of hepatocellular carcinoma cells.4. In vivo inhibition of ARHGEF7 expression can inhibit the proliferation and metastasis of hepatocellular carcinoma cells.5. ARHGEF7 can be used as a novel molecular marker for evaluating the prognosis of HCC patients and the molecular intervention therapy for the recurrence and metastasis of liver cancer.