Inhibitory effect of shRNA targeting GPR48 on invasion and metastasis of human cervical carcinoma cell line HeLa.

BACKGROUND AND OBJECTIVE

GPR48 regulates invasion and metastasis of cancer cells by linking Gs protein to stimulate intracellular signaling pathway and affect actin filament aggregation and the activity of matrix metalloproteinase. This study was to explore the effects of GPR48 short hairpin RNA (shRNA) on GPR48 expression, and invasion and metastasis of cervical carcinoma cell line HeLa.

METHODS

Plasmids containing two different sequences of human GPR48 mRNA coding region were constructed and transfected into HeLa cells. HeLa cell clones with stable small interfering RNA (siRNA) expression were screened by neomycin resistance. The expression of GPR48 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. In vitro invasive activity was measured by Boyden chamber experiment. GPR48 shRNA-transfected or untransfected HeLa cells were inoculated subcutaneously into nude mice to observe tumor growth and lung metastasis.

RESULTS

Compared with that in negative shRNA-transfected cells, GPR48 mRNA expression in GPR48 shRNA-transfected cells was downregulated by more than 80% and the number of HeLa cells infiltrated Boyden chamber membrane was decreased (94.4 +/- 15.7 vs. 28.3 +/- 1.5 and 17.6 +/- 1.5, p < 0.01). The depletion of endogenous GPR48 suppressed in vivo metastasis: the number of lung metastases was significantly larger in negative shRNA group than in GPR48 shRNA groups (7.3 +/- 1.8 vs. 1.3 +/- 0.3 and 1.5 +/- 0.4, p < 0.01).

CONCLUSION

GPR48 shRNA can inhibit in vitro invasion and in vivo metastasis of HeLa cells efficiently.