TRIM44 promotes proliferation and metastasis in non-small cell lung cancer via mTOR signaling pathway

Tripartite motif-containing protein 44 (TRIM44) was recently identified as a potential therapeutic target in several types of malignancy, but its effect on the clinical course of malignancy and its underlying regulatory mechanism remain largely unknown. The present study shows that upregulation of TRIM44 is associated with poor differentiation, advanced pTNM stage, adenocarcinoma subtype, lymph node metastasis and, most importantly, unfavorable survival in patients with non-small cell lung cancer (NSCLC). TRIM44 knockdown inhibited the invasion and migration of human NSCLC cells, which was concurrent with downregulation of mesenchymal markers and upregulation of epithelial markers. Overexpression of TRIM44 induced the epithelial-to-mesenchymal transition (EMT) and increased the metastatic potential of lung cancer cells. Additionally, TRIM44 induced cell proliferation in vitro and tumor growth in vivo by accelerating G1/S transition via upregulation of cyclins and CDKs. TRIM44-induced mTOR signaling, EMT, and cyclin/CDK upregulation were reversed by treatment with a mammalian target of rapamycin (mTOR) inhibitor. These results provide a model for the relationship between TRIM44 expression and lung cancer progression, and open up new avenues for the prognosis and therapy of lung cancer.

For siRNA delivery, double-stranded RNA oligonucleotides were transfected using Lipo2000 (Invitrogen). A549 or NCI-H520 (grown to 30-50% confluence in six-well plates) were transfected with siRNAs (75 pmol per well) using 5 µL of Lipo2000 according to the manufacturer's instructions and then harvested 72 hours later for analysis.

Establishment of stable cell lines
To establish stable TRIM44 knockdown cell lines, the Lenti-shRNA vector system (pGCSIL-GFP) was constructed, packed, and purified by GeneChem (Shanghai, China) and manipulated according to the manufacturer's protocol. A TRIM44 short hairpin sequence (5ʹ-TAGGATTGTCCTGACTCACTA-3ʹ) was chosen. Briefly, shTRIM44 and shControl lentiviruses were used to infect A549 cells for 3 days. Stable clones were then selected with puromycin (1 μg/mL) and the knockdown efficiency determined by western blotting.

Wound healing assay
Cells were seeded in six-well plates and cultured in RPMI 1640 or DMEM containing 10% FBS until confluent. The cells were then scratched with a sterile 10 μL pipette tip to create artificial wounds. Phase-contrast images of the wound healing process were obtained digitally at 0 and 24 h after wounding using an inverted Olympus I × 50 microscope fitted with a 10× objective lens. Eight images per treatment were analyzed to determine average parameters with respect to the position of the migrating cells at the wound edges. Lines were digitally drawn using Image-Pro Plus software (Media Cybernetics, Rockville, USA).

Western blot analysis
Frozen tissue samples or lung cancer cells were homogenized in RIPA buffer containing a 1% protease inhibitor mixture. The mixture was centrifuged at 12 000 g for 15 min at 4°C and the supernatant obtained. Total protein was quantified using the Bradford method (Thermo Scientific, Waltham, MA, USA). Briefly, 30 µg of protein extract was separated by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore Company, Billerica, MA, USA). The membranes were blocked with 2% bovine serum albumin (BSA) at 37°C for 1 h and incubated with the primary antibody overnight at 4°C. After washing, the membrane was incubated with a horseradish peroxidase-labeled secondary antibody for 1 h at room temperature and then washed again. The blots were stained using Super ECL Reagent kit (HaiGene, Harbin, China; M2301) and imaged using the FluorChem™ HD2 System (Proteinsimple, CA, USA). The antibodies used for western blotting are listed in Supplementary Table S2. The experiment was repeated three times.

RNA preparation and reverse transcription
Immediately following resection, fresh tissues were stored at -80°C until RNA extraction. Total RNA was extracted from fresh frozen samples or lung cells using Trizol reagent (Invitrogen, Carlsbad, CA), following confirmation by pathologists that the tumor samples contained at least 75% tumor cells. RNA quality and concentration were measured using a NanoDrop 2000 spectrophotometer (Thermo, Wilmington, DE, USA). Reverse transcription was performed using 2.0 μg total RNA in a 14.5-μL reaction mixture containing 2 × Power Taq PCR Master Mix kit (PR1702, BioTeke, Beijing, China), according to the manufacturer's instructions.