All procedures adhered to the approved medical ethics practices and the Human Ethics Review Board of the Second Hospital of Shandong University, China [Approval no.: KYLL-2014 (LW) P-001]. Clinical specimens were collected from patients at the Second Hospital of Shandong University from October 2014 to December 2016. Histological classifications and clinical staging were based on the classification system by the International Federation of Gynecology and Obstetrics (International Federation of Gynecology and Obstetrics Cancer Committee; FIGO, 2009) (29). A tumor size of 4 cm was chosen as a ‘watershed’ value according to previous studies (30–32). The human cervical cancer cell lines, Caski and HeLa, and normal cervical cell, CRL-2614, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO₂. Transfection was performed with Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocols.

Total RNA was isolated from tissue and cells using TRIzol reagent (Invitrogen), according to manufacturer's protocols. Total RNA (1 µg) was reverse-transcribed into cDNA using PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China), and qPCR was conducted using SYBR-Green dye mix (Invitrogen). Thermocycling conditions for RT-qPCR were as follows: 95°C for 10 min, 40 cycles of 95°C for 20 sec, 55°C for 30 sec, and 72°C for 20 sec. The relative expression level of Trio was calculated using 2^−ΔΔCq, and the expression level of Trio mRNA was normalized to that of GAPDH. The Trio sense and antisense primers were designed as follows: 5′-AGATTCTCTGTCGTTAAG-3′, and 5′-TTCTAAGACCAGGATGTA-3′, respectively. The GAPDH sense and antisense primers were: 5′-CTCAAGATCATCAGCAAT-3′ and 5′-CGATACCAAAGTTGTCAT-3′, respectively.

The guide RNA sequences targeting the human Trio gene were designed using an online sgRNA design tool at https://crispr.mit.edu/. Guide sequences with high scores for on-target activity and minimal predicted off-target activity were selected. Table I displays the three sets of oligonucleotides. Primer oligonucleotides were annealed under 88°C for 2 min, 65°C for 10 min, 37°C for 10 min, and 25°C for 5 min. The annealing primer was then purified and cloned into the PX330 vector (Addgene, Cambridge, MA, USA). The CRISPR/Cas9 backbone and CRISPR/Cas9-gRNA plasmids were separately transfected into cells using a lipidosome.

For the invasion assay, a Transwell chamber placed into a 24-well plate was coated with 30 µl of Matrigel and incubated for 40 min at 37°C. In the Transwell assay, the cells were trypsinized and seeded in chambers at a density of 5×10⁵ cells/well and were cultured in serum-free medium, while 500 µl of 10% FBS-RPMI-1640 were added to the lower chamber. After 24 h, the cells on the upper surface were removed, and the migrated cells were fixed with 100% methanol for 30 min. The cells on the bottom surface of the membrane were stained with eosin for 20 min. The cell images were obtained under a phase-contrast microscope.

Cells were seeded in 6-well plates. When the cells reached 90–100% confluence, the cell monolayers were wounded by scraping with a micropipette tip. The spreading of wound closure was observed after 48 h. Images were captured using a phase-contrast microscope (Olympus, Tokyo, Japan) either immediately or 48 h after wounding. All experiments were repeated thrice.

Western blot analysis was performed using anti-Trio (Rabbit, Polyclonal, 1:1,000; Abnova, Taipei, Taiwan), anti-RhoA (Rabbit, monoclonal, 1:1,000, cat. no. 2117; Cell Signaling Technology, Beverly, MA, USA), anti-Rock1 (Mouse, monoclonal, 1:800, cat. no. sc-365628), anti-Rock2 (Mouse, monoclonal, 1:800, cat. no. sc-398519) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-LIMK1 (Rabbit, Polyclonal, 1:1,000, cat. no. 3842) antibodies, p-LIMK1 (Thr508) (Rabbit, Polyclonal, 1:1,000, cat. no. 3841) (both from Cell Signaling Technology), with rabbit anti-β-actin (Mouse, monoclonal, 1:5,000; Bioworld, Nanjing, China) were used as a loading control. Band intensities of the western blotting were analyzed using Image Analysis Software v2.0 (Thermo Fisher Scientific Inc.).

RhoA activity was assessed using a Rho Activation Assay Biochem kit™ (Cytoskeleton, Inc., Denver, CO, USA) according to the manufacturer's protocol.

All values were expressed as the mean ± SD of three individual experiments. Statistical analyses were performed using Student's t-test. When appropriate, the Mann-Whitney U-test was used to compare the two groups. P<0.05 were considered to indicate a statistically significant result. SPSS software was used for statistical analyses.

To ascertain Trio expression in cervical cancer, we performed real-time PCR on 50 clinical specimens from cervical cancer patients. The expression levels of Trio in the tumor tissues for all 50 samples were significantly higher than that in the matching adjacent tissue (Fig. 1A). Furthermore, we determined the protein level of Trio in randomly selected cancer and adjacent normal tissues (n=3). The protein level was also increased compared to the normal tissues (Fig. 1B). The expression of Trio was also determined in cervical cancer cell lines (Caski and HeLa) and in a normal cervical cell line (Crl-2614) (Fig. 1C), where the results obtained from the two analyses were in agreement with the data obtained from real-time PCR, indicating the significant increase of Trio levels in cervical cancer tissues and cell lines compared with normal cervical tissue and a cell line.

We analyzed the expression level of Trio to determine its clinicopathological significance. Notably, Trio levels were significantly increased in patients with lymph node involvement (P=0.005) (Table II). Therefore, Trio expression associates with lymph node metastasis and is a potential diagnostic marker for cervical cancer.

We used Caski cells to examine the effects of Trio gene knockdown by CRISPR/Cas9. We transfected Caski cells with the control, CRISPR+Cas9+gRNA empty, CRISPR+Cas9+Trio-1, CRISPR+Cas9+Trio-2, and CRISPR+Cas9+Trio-3 and cultured them for two days. Fig. 2A revealed the transfection results as determined by western blotting, which confirmed the reduced expression of Trio by CRISPR+Cas9-gRNA1 in comparison with the control at 48 h. CRISPR+Cas9+Trio-1 exhibited effective knockdown of Trio gene expression at 48 h (Fig. 2A and C). Significantly decreased Trio expression was also demonstrated in the HeLa cell line (Fig. 2B and C) as determined using western blotting. Therefore, we obtained an effective gRNA for Trio.

Migration assay through wound-healing revealed that the migration ability of Caski and HeLa cells transfected with the control was significantly higher in cervical cancer cells than that of cells transfected with knocked down Trio-1 (Fig. 3).

Invasion assay using the Transwell method revealed that the invasiveness of Caski and HeLa cells transfected with CRISPR+Cas9+Trio-1 was significantly lower than that of cells transfected with the control (Fig. 4). Trio silencing inhibited cervical cancer cell migration and invasion via inactivation of the RhoA/Rock signaling pathway.

Rho-GTPase proteins participate in cell motility and actin cytoskeleton reorganization (18). RhoA is a member of the Rho-GTPase proteins and acts as a switch between the active GTP-bound form and inactive GDP-bound form. The activated RhoA-GTPase is associated with invasive cancers and tumor metastasis (30,31). To determine whether the inactivation of the Rho-GTPase pathway mediated the inhibitory effect of Trio on cervical cancer cell migration and invasion, we examined the expression level of the active RhoA-GTPase under Trio knockdown by western blotting. The active form of GTP-bound RhoA was downregulated after Trio decreased. We assessed the protein levels of ROCK1, ROCK2 and p-LIMK1, a protein directly downstream of ROCK, after transient transfection of CRISPR+Cas9+gRNA empty and CRISPR+Cas9+Trio-1 for 48 h. Knockdown of Trio significantly inhibited the protein expression of activated RhoA, ROCK1, ROCK2 and p-LIMK1 compared with the control (Fig. 5), indicating that Trio may mediate the metastatic ability of cervical cancer cells through inactivation of the RhoA-GTPase pathway.