Melanoma is a malignant tumor that originates from melanocytes, commonly occurs on the skin surface, and has the highest mortality rate of superficial tumors.1,2 Highly invasive metastasis is one of the important characteristics of melanoma, and is a main reason for the poor prognosis. Ras homology C (RhoC) is considered a molecular switch that controls tumor metastasis because it is involved in tumor invasion and metastasis through multiple mechanisms. The results from preliminary experiments indicate that RhoC or its downstream effectorspecific inhibitors are used to control tumor metastasis.3,4 RNA interference (RNAi), a specific post-transcriptional gene silencing,5 can control various cell activities by manipulating the gene expression.6,7 However, little research has been done about the effects of RNAi targeting RhoC on the invasion and metastasis of melanoma.
In this study, a lentivirus vector targeting the RhoC gene of melanoma cells was constructed, and its silencing effects on the RhoC gene were determined in order to provide a basis for further investigating the mechanism of RhoC in the invasion and metastasis of melanoma.
Target point design and shRNA plasmid construction of RNAi targeting the RhoC gene
Based on the human RhoC gene data (Ras homolog gene family, member C, NM_175744.4), three pairs of oligonucleotide sequences and a pair of negative control sequences were designed and synthesized by GenePharma Technology Co., Ltd. (Shanghai, China) with RNAi designer 2.0 software. Based on the number from the start sites of the target sequence to the start sites of transcription, they were named RhoC336, RhoC453, and RhoC680. Their sequences are shown in Table 1. After the synthesized oligos underwent annealing and pGPU6/ GFP/Neo underwent linearizing by digestion, they were used to construct the pGPU6/GFP/Neo-shRNA plasmid followed by transformation into the E. coli competent cell DH5α (Tiangen Biotech, Co., Ltd., Beijing, China). After kanamycin resistance screening, positive clones were selected. Positive recombinant plasmid sequences were identified by Shanghai Invitrogen Biotechnology Co., Ltd. (Shanghai, China).
Screening for the optimal RNAi target sequence
Human melanoma A375 cells (Shanghai Institute of Cellular Biology, Chinese Academy, Shanghai, China) were incubated in DMEM, 10% FBS, and 1% P/S in an atmosphere of 95% humidity and 5% CO2 at 37°C. When A375 cell density reached 50%-70%, the three shRNA plasmids were respectively transfected into A375 cells using a LipofectamineTM 2000 kit (Invitrogen, USA). The level of RhoC mRNA was determined with real-time PCR 72 hours after transfection. At the same time, normal cell and negative vector control groups were set. Total RNA was extracted with Trizol reagent (Invitrogen), and then synthesized into cDNA. The RhoC primers were 5′-ACAGCAGGGCAGGAAGACTA-3′ and 5 ′ - TTCATCTTGGCCAGCTCTCT-3′. The internal reference GAPDH primers were 5 ′ - GAGTCAACGGATTTGGTCGT-3′ and 5′-TTGATTTTGGAGGGATCTCG-3′. PCR was performed with SYBR Green PCR Master mix kit (ABI, USA). PCR conditions were as follows: 95°C for 5 minutes, 94°C for 20 seconds, 58°C for 20 seconds, 72°C for 20 seconds, 72°C for 5 minutes, 55°C for 10 seconds, 40 cycles; then 0.5°C for 10 seconds, 80 cycles. The average Ct values of RhoC and GAPDH were calculated with the data analysis software of the PCR instrument. The relative levels of RhoC and GAPDH mRNA were indicated by 2−ΔΔCt (RQ). The level of RhoC protein 72 hours after transfection was determined by Western blotting. The primary antibody of GAPDH was mouse anti-GAPDH antibody (Promab, USA, 1:1000 dilution) and the secondary antibody was HRP-labeled goat anti-mouse IgG (Zymed, USA, 1:50 000 dilution). The primary antibody of RhoC was goat anti-RhoC antibody (Santa Cruz, USA, 1:400 dilution), and the secondary antibody was HRP-labeled rabbit anti-goat IgG (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China, 1:40 000 dilution). The integrated optical density (IOD) of the target band was analyzed with software and then obtained the relative level of RhoC protein.
Construction of lentivirus vector with the optimal interference target
A pair of oligos were synthesized according to the optimal interference target, and its sequence was as follows: S: 5′-TGCTGTCAATGTCCGCAATATAGTTCGTTTTGGC CACTGACTGACGAACTATAGCGGACATTGA-3′; A: 5′-CCTGTCAATGTCCGCTATAGTTCGTCAGTCAGTG GCCAAAACGAACTATATTGCGGACATTGAC-3′.
After annealing and linearization, pcDNA6.2-GW/EmGFPmiR was linked to the oligo, and then transformed into E. coli followed by incubation in Luria-Bertani broth containing spectinomycin resistance overnight. Positive clones were used for identification. The target fragment underwent PCR amplification, and then its two ends were linked to restriction enzyme cutting sites respectively. PCR products of the target gene and lentivirus vector were respectively digested with Asc I and Pme I. The target fragment was obtained by electrophoretic separation, and then transformed into competent cells followed by incubation in Luria-Bertani broth containing ampicillin resistance overnight. When single colonial morphology appeared, PCR amplification, identification by restriction endonuclease digestion, and sequencing were performed.
Lentivirus package and titer determination
293T cells were incubated using a conventional method. Lentivirus expression vector and lentivirus package vectors pHelper1.0, pHelper2.0, and pVSVG (Invitrogen) were co-transfected into 293T cells. Lentivirus supernatant was collected 24 and 48 hours after co-transfection respectively for concentration by centrifugation. The titer of lentivirus was determined by the hole-by-dilution titer assay.
Lentivirus with optimal interference target sequence infection of A375 cells
A375 cells were incubated using a conventional method. When the cell density reached 50%-70%, cells might be used for infection. In an infection pre-experiment, the MOI (multiplicity of infection) value gradients were 0, 1, 5, 10, and 20, and then lentivirus solution of corresponding doses were respectively added followed by addition of 5 μg/ml polybrene. At the same time, the control group (absence of polybrene) was set. Green fluorescent protein (GFP) expression per well was observed 72 hours after lentivirus infection to determine the optimal MOI value. A375 cells were then infected with the optimal lentivirus solution.
Identification of pLenti6.3-EGFP-453 interference with RhoC of human melanoma A375 cells
Normal cells served as the normal control group (group A), lentivirus-negative cells infected by lentivirus served as the negative lentivirus control group (group B), and pLenti6.3-EGFP-453-transfected cells served as the lentivirus group (group C). The levels of RhoC mRNA and protein after infection were determined by real-time PCR and Western blotting respectively as previously described.
All data were analyzed with SPSS 15.0 software (SPSS Inc., IL, USA). Measurement data were expressed as mean ± standard deviation (SD). Analysis of variance was used for comparisons between multiple samples. Least significant difference (LSD) or Dunnett T3 test were used for comparisons between two samples. Statistical significance was established at P <0.05.
Sequencing of pGPU6/GFP/Neo shRNA-positive recombinant plasmids
Three RhoC shRNA interference sequences were all successfully inserted into pGPU6/GFP/Neo plasmids, which were entirely consistent with the designed sequence without base deletions or mismatch.
Effects of the three pGPU6/GFP/Neo-shRNA plasmids on RhoC mRNA and protein expression in melanoma A375 cells
The level of RhoC mRNA and protein was indicated with RQ (2−ΔΔCt) and IOD values respectively. The three shRNA interference plasmids could all inhibit RhoC mRNA and protein expression. Compared to the normal and negative control groups, the levels of RhoC mRNA and protein in the three pGPU6/GFP/Neo-shRNA groups were significantly decreased. In the pGPU6/GFP/Neo-shRNA453 group, the levels of RhoC mRNA and protein were significantly lower than that in both control groups and the other pGPU6/GFP/ Neo-shRNA groups (P <0.05, Table 2 and Figure 1).
Sequencing of pcDNA6.2-GW/EmGFP-453
The optimal interference sequence shRNA453 was successfully inserted into the pcDNA6.2-GW/EmGFP-miR plasmid, which was entirely consistent with the designed sequence without base deletions or mismatch (Figure 2).
Identification of recombinant vector pLenti6.3-EGFP-453
Recombinant vector pLenti6.3-EGFP-453 was successfully digested into two bands. The electrophoretic bands were consistent with those expected, suggesting the constructed recombinant vector was correct (Figure 3).
Sequencing of the pLenti6.3-EGFP-453 lentivirus vector
The interference sequence 453 was successfully inserted to construct pLenti6.3-EGFP-453, which was entirely consistent with the designed sequence without base deletions or mismatch (Figure 4).
Determination of lentivirus titer and optimal MOI value of pLenti6.3-EGFP-453 for infection of A375 cells
The titer of lentivirus was determined with the hole-bydilution titer assay, and the pLenti6.3-EGFP-453 lentivirus titer was 1.0×107 TU/ml. GFP expression was observed under a fluorescence microscope 72 hours after lentivirus infection. At MOI values ≤10, green fluorescence was enhanced with increasing values. When the MOI value was >10, the MOI value-dependent enhancement of green fluorescence was not marked. The results suggested that human melanoma A375 cells could be infected by the pLenti6.3-EGFP-453 lentivirus, and that the optimal MOI value was 10. Addition of polybrene (5 μg/ml) markedly promoted infection (Figure 5).
Interference effects of pLenti6.3-EGFP-453 on the expression of RhoC mRNA and protein of human melanoma A375 cells
pLenti6.3-EGFP-453 could significantly inhibit RhoC mRNA and protein expression. Compared to the normal and negative lentivirus groups, RhoC mRNA and protein were significantly decreased in the pLenti6.3-EGFP-453 lentivirus group (P <0.05). Compared to the normal group, RhoC mRNA and protein were also decreased in the negative lentivirus group, but the differences were not statistical significant (P >0.05, Table 3 and Figure 6).
Invasive metastasis is one of the important characteristics of malignant tumors, and is a main factor of poor prognosis. Invasive metastasis is multifactorial, multistage, and multistep complex process, and is associated with a variety of adhesion molecules, matrix metalloproteinases, cytokines, signal transduction, and related gene alterations. RhoC, a primary member of the RhoC subfamily,8 interacts with downstream effector molecules and is involved in tumor malignant transformation, invasive metastasis, and angiogenesis via a variety of signal transduction pathways. RhoC gene overexpression controls the expression of many tumor metastasis-related genes, leading to tumor metastasis. The level of RhoC may be a useful clinical indicator for evaluating tumor invasion, lymphatic metastasis, and prognosis.9 Many studies have indicated that RhoC protein expression increased in a variety of tumors, especially in highly invasive tumors, and is positively correlated with tumor stage and metastasis.10–13 The aforementioned findings suggest that RhoC and its downstream target molecules may become important targets to inhibit tumor metastasis.
It is universally acknowledged that RNAi technology can achieve fast and effective gene silencing. RNAi can regulate or knockdown gene expression to control a variety of cellular activities. In the preparation of RNAi, the interference sequence, delivery system, and transfection conditions have been shown to be strongly associated with the effects of RNAi.14–17 In order to obtain satisfactory silencing, in this study, three interference sequences targeting the RhoC gene were designed and constructed, and then these successfully constructed plasmids were respectively transfected into human melanoma A375 cells. Real-time PCR indicated that the three plasmids all could inhibit the expression of RhoC mRNA, and pGPU6/ GFP/Neo-shRNA453 exhibited the strongest inhibitory effect. The results of Western blotting were similar to those of real-time PCR. The three plasmids also could inhibit the expression of RhoC protein, and pGPU6/ GFP/Neo-shRNA453 exhibited the strongest inhibitory effect. The results suggest that the constructed plasmid vectors have RNAi effects because they can inhibit the target gene (RhoC) mRNA and protein expression, which is similar to the findings in other malignancies research; our results were consistent with the studies on other cancer. For example, Pille and his colleagues constructed the stable RhoC-siRNA MDA-MB cell lines which RhoC expression was significantly suppressed by using RNAi interference technology.18 Meanwhile, the results suggested that interference effects are related to the target sequences, and RhoC453 is the optimal interference sequence. Achievement of the RNAi effect needs efficient transfection and stable expression of the exogenous gene. In order to further improve the interference effects, the optimal interference sequence was used to construct a more stable and effective lentivirus vector with a thirdgeneration lentivirus vector system in this study. As a novel gene vector,19 Lentiviral vectors are becoming a new tool of choice for gene delivery. Currently, the advantages of lentiviral vectors include high transfection efficiency, transfecting almost all cells, avoiding host immune response as no reproduced HIV was found in the in vitro studies. Nowadays, the lentiviral vectors system which was from the first generation to the third is more and more stable and safe such as that used in the RNAi technology. We also use lentiviral transfection in our study. Human melanoma A375 cells were infected with the lentivirus vector to obtain a stable interference cell line. We optimized the MOI value by using lentivirus with different MOI values to infect the A375 cells. Since the vector expresses GFP, infected A375 cells also express GFP which can be used to observe transfection efficiency. Our results indicated that the optimal MOI value was 10 for the infection of A375 cells by pLenti6.3-EGFP-453 because at MOI values ≤10 tested, green fluorescence was enhanced with increasing values, and when the MOI value was >10 the MOI valuedependent enhancement of green fluorescence was not marked. Polybrene, a type of enhancer, can effectively improve transfection efficiency for a variety of cells.20 We observed the fluorescent cell number and fluorescent intensity in the presence and absence of polybrene, and found that the transfection efficiency was markedly enhanced by polybrene demonstrating that polybrene can upregulate the transfection efficiency of the lentivirus vector for human melanoma A375 cells. Based on these results, the optimal MOI value for pLenti6.3-EGFP-453 lentivirus infection of A375 cells is 10, and polybrene markedly promotes infection.
Since the interference effects of pLenti6.3-EGFP-453 are different from those before lentivirus packaging, we again determined the interference effects on target gene. Our results indicated that the inhibition rates of RhoC mRNA and protein were increased from 65.16% and 64.95% to 77.35% and 73.59% respectively demonstrating that the lentivirus-packaged interference vector can further enhance RNAi effects, and pLenti6.3-EGFP-453 can effectively infect human melanoma A375 cells and inhibit target gene expression. The lentivirus vector targeting the RhoC gene successfully constructed in this study will provide a basis for further investigating the role of RhoC in the invasion and metastasis of melanoma, and for exploring new therapeutic methods against melanoma.
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Keywords:© 2014 Chinese Medical Association
human melanoma cell; Ras homology C; RNA interference; lentivirus vector