Objective: To screen the effective target sequences of laryngeal carcinoma related gene LCRG1 using RNAi.
Methods: PCR site mutation method was used to reconstruct pSuper vector. Five pairs of siRNA sequences designed by siRNA software were annealed and inserted into the reconstructed pSuper vector. The reconstructed pSuper 362,398,432,789,903,and pSuper vectors were transfected into Hela cell lines and selected with the appropriate drugs to get resistant and pool cells, respectively. The colonies were identified by RT-PCR or real-time RT-PCR analysis. The silence effects were observed by cloning formation analysis.
Results: pSuper vector was reconstructed to restore Bgl II restriction enzyme sites using PCR mutation. The RT-PCR or real-time RT-PCR Results of pool clones showed 362, 398, and 432 pool clones all had better effects of LCRG1 gene-silence, especially 362 pool clones. The expression level of LCRG1 mRNA of selected 362 group anti-puromycin clones A2 and A5 was decreased. The Results of clone forming efficiency revealed that the cellular proliferation in A2 of 362 group was significantly higher than that of the vector and control Hela cells (P<0.05).
Conclusion: The reconstructed pSuper vector is successfully constructed. The 362 group has better gene silence and has 2 effective 362 group anti-clones, suggesting that methodology has important values in studYing the function and molecular mechanism of LCRG1.