Journal of Jilin University Medicine Edition ›› 2016, Vol. 42 ›› Issue (03): 481-485.doi: 10.13481/j.1671-587x.20160312

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Establishment of tongue squamous cell carcinoma TSCCA cell model with silencing α-catulin gene

HAN Chunyao, ZHANG Bin, MA Xue, LIU Mingyuan, XUE Zhongyuan, HAO Lijing, GE Shuqing   

  1. Department of Stomatology, First Affiliated Hospital, Liaoning Medical College, Jinzhou 121001, China
  • Received:2015-11-23 Published:2016-06-17

Abstract:

Objective: To design and construct the expression vectors of α-catulin-shRNA,and to establish the TSCCA cell model with silencing α-catulin gene of tongue squamous cell carcinoma (TSCC) and observe its silencing efficiency,and to provide foundation for gene treatment of TSCC. Methods: The TSCCA cells were divided into transfection groups (transfected with sh-catu1,sh-catu2,sh-catu3, and sh-catu4),negative control group (transfected with sh-nc),and blank control group(untreated).According to the mRNA sequence of α-catulin obtained from GenBank, the expressing vectors of α-catulin-shRNA plasmids were designed and compounded.After the vectors were identified by DNA sequencing,the expressing vectors were transfected into the TSCCA cells cultured in vitro after mediated with liposome.The expression of green fluorescence protein (GFP) in TSCCA cells was observed by fluorescence microscope;the relative expression levels and silencing efficiency of α-catulin mRNA and protein were detected by RT-PCR and Western blotting methods. Results: The DNA sequencing results showed the plasmid expression vectors containing shRNA against α-catulin gene (sh-catu1,sh-catu2,sh-catu3,and sh-catu4) were constructed correctly.After transfection,the expressing vectors were expressed successfully in the TSCCA cells.The RT-PCR results showed that compared with blank control group,the relative expression levels of α-catulin mRNA in TSCCA cells in ttansfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05).Compared with negative control group,the relative expression levels of α-catulin mRNA in TSCCA cells in transfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05).The Western blotting results showed that compared with blank control group,the relative expression levels of α-catulin protein in TSCCA cells in transfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05).Compared with negative control group,the relative expression levels of α-catulin protein in TSCCA cells in transfection groups were decreased (P<0.05) and the silencing efficiencies were increased (P<0.05). Conclusion: The recombinant plasmid vectors are constructed successfully,which could silence the α-catulin expression in TSCCA cells.This study will provide a new cell model for further study of the role of α-catulin in the pathogenesis of TSCC.

Key words: carcinoma,squamous cell, tongue neoplasms, α-catulin gene, TSCCA cell, cell transfection

CLC Number: 

  • R739.86