Silencing of tripartite motif (TRIM) 29 inhibits proliferation and invasion and increases chemosensitivity to cisplatin in human lung squamous cancer NCI-H520 cells

Abstract

Background: TRIM29 belongs to the tripartite motif (TRIM) protein family. It has been reported to be a tumor suppressor or have oncogenic function in many cancer types. The aim of this study was to investigate whether downregulation of TRIM29 by small interfering ribonucleic acid (siRNA) could inhibit cell proliferation and invasion and increase chemosensitivity to cisplatin in human lung squamous cancer NCI-H520 cells in vitro.

Methods: We transformed TRIM29 siRNA into NCI-H520 cells. Real time reverse transcriptase polymerase chain reaction and Western blotting assay were employed to determine TRIM29 messenger (m)RNA and protein expressions. MTT assay was used to determine the cell proliferation. Transwell invasion assay was used to determine the cell invasion. An Annexin V-propidium iodide (AnnV/PI) staining apoptosis test was used for detecting apoptosis.

Results: TRIM29 siRNA could specifically and efficiently suppress TRIM29 expression at both mRNA and protein levels. Silencing of the TRIM29 by siRNA in NCI-H520 cells inhibited cell proliferation and invasion in vitro. TRIM29 knockdown resulted in chemosensitivity enhancement in NCI-H520 cells.

Conclusion: Downregulation of TRIM29 can lead to potent antitumor activity and chemosensitizing effect in human lung squamous cancer NCI-H520 cells.

Keywords: Chemosensitivity; TRIM29; lung cancer; siRNA.

Figure 1

Specific downregulation of tripartite motif (TRIM)29 messenger ribonucleic acid (mRNA) and protein expression by TRIM29 small interfering (si)RNA. Cells were transfected with TRIM29 siRNA or scrambled siRNA (siCONTROL) for 48 hours. (a) The relative mRNA level of TRIM29 was quantified by real-time polymerase chain reaction analysis, and 18S was used as an internal standard. (b) The relative TRIM29 protein level was determined by Western blotting, and data was normalized using β-actin as an internal standard. Compared with TRIM29 siRNA1 and 2, siRNA3 mostly silenced TRIM29 expression. P < 0.01 versus siCONTROL.

Figure 2

RNA interference (RNAi)-mediated downregulation of tripartite motif (TRIM)29 reduces NCI-H520 cell proliferation. Cells treated with TRIM29 small interfering (si)RNA, scrambled control siRNA or with medium alone were plated on a 96-well plate. Cells were evaluated for proliferation at 24-hour intervals by MTT assay. Data were from three independent experiments. P < 0.01 versus siCONTROL. , NCI-H520; , siCONTROL; , TRIM29 siRNA.

Figure 3

Downregulation of tripartite motif (TRIM)29 inhibits invasion of NCI-H520 cells. (a) NCI-H520, siCONTROL and TRIM29 small interfering ribonucleic acid (siRNA) cells were seeded in the upper chamber in medium supplemented with five percent fetal bovine serum (FBS. After 24 hours, cells were fixed, stained, and counted (bar shows 100 μm). (b) The number of invasive cells in the TRIM29 siRNA treated group were significantly lower than in the NCI-H520 and siCONTROL groups (P < 0.01).

Figure 4

Small interfering ribonucleic acid (siRNA) for tripartite motif (TRIM29) induces apoptosis and enhances chemosensitivity. (a and c) TRIM29 inhibition by siRNA induced apoptosis in NCI-H520 cells. (b and d) After treating with 5 μg/mL cisplatin, cell apoptosis (including early and late apoptosis) was further enhanced. Combining TRIM29 inhibition with cisplatin increased the incidence of apoptosis. After 48 hours transfecting with siRNA, cells were treated with 5 μg/mL cisplatin for 24 hours, and then cells were double stained with Annexin V-FITC and propidium iodide (PI) followed by FACS analysis. FACS analysis scatter-grams of Annexin V/PI staining display four different cell populations marked as: double negative (unstained) cells showing live cell population (lower left), Annexin V positive and PI negative stained cells showing early apoptosis (lower right), Annexin V/PI double-stained cells showing late apoptosis (upper right), and, finally, PI positive and Annexin V negative stained cells showing dead cells (upper left). P < 0.01 versus siCONTROL.