Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry.

The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (P<0.05). No significant difference in RhoA mRNA and protein expression was detected (P>0.05), but the expression of RhoA membrane protein decreased compared with the negative control group and blank control groups (P<0.05). Cyclin D1 expression decreased, whereas p21 expression significantly increased and the relative expression of ERK protein in the experimental group did not significantly different that in the control group (P>0.05). However, the phosphorylation level of ERK was significantly reduced (P<0.05). The cell cycles of TSCC CAL-27 and SCC-4 were altered in G1/S, cell proliferation activity was inhibited, and apoptosis was induced (P<0.05).

Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.