Construction and identification of an RNA interference lentivirus vector targeting the Ras homology C gene of melanoma cells.
BACKGROUND
Melanoma 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.


METHODS
Based 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.


RESULTS
The 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).


CONCLUSIONS
The 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.
Department of Plastic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China.
Qiying
Q
Wang
Department of Plastic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China.
Ximei
X
Wang
Department of Plastic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China.
Xiaomei
X
Zhai
Department of Plastic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China.
Jianwen
J
Zhang
Department of Plastic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China.
Minjing
M
Chen
Department of Plastic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China. Email: liulinbo@zzu.edu.cn.
Linbo
L
Liu
D045744:Cell Line, Tumor; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008545:Melanoma / Q000235:genetics* / Q000628:therapy; D034622:RNA Interference / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000075024:rhoC GTP-Binding Protein
C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D000075024:rhoC GTP-Binding Protein