lncRNA KIAA0125 functions as a tumor suppressor modulating growth and metastasis of colorectal cancer via Wnt/β-catenin pathway

Patient samples

Twenty-five pairs of human colorectal carcinoma tissues and adjacent normal colon tissues were collected from China to Japan union hospital of Jilin University. For each patient, at least three samples were collected from different parts of cancer tissues. The patients were diagnosed with CRC based on the histopathological detection and divided into early stage (I/II) group (n = 13) and late stage (III/IV) group (n = 12). All the patients did not undergo preoperative therapy prior to the surgical resection and volunteered to participate in the research by written consent form. This study was approved by the ethic committee of Jilin University and all the procedures were performed according to the approved protocol.

Real-time quantitative polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the CRC tissues or cells with TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocols. After concentration measurement, the RNA was reverse transcribed into complementary DNA (cDNA) and analyzed by qRT-PCR. The targeted gene expression was normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and calculated using method. The primer sequences were used as follows: KIAA0125: 5′-TGGCAAAGGCAAGTGAC-3′ and 5′-GGCAGAAGGATGAACCC-3′; GAPDH: 5′-TGACAACTTTGGTATCGTGGAAGG-3′ and 5’-AGGCAGGGATGATGTTCTGGAGAG-3′; β-catenin 5′-GGCAGCAACAGTCTTACCT-3′ and β-catenin 5′-CATACAGGACTTGGGAGGT-3′.

Cell culture and cell transfection

FHC, HCT116, LS174T, SW480, SW620, and LOVO cell lines were obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics and maintained at 37°C in a humidified incubator with 5% CO₂.

si-KIAA0125 (5′-GGAGCAAAUGGAGAGGGAA-3′) or scramble small interfering RNA (siRNA) (5′-GGAAAUAGGGAGAGGCGAA-3′) was transiently transfected into cells using Lipofectamine RNAi Max (Invitrogen) according to the manufacturer's instructions. For the lncRNA-KIAA0125 overexpressing construct, the full-length lncRNA-KIAA0125 was cloned into pcDNA3.1 vector (Invitrogen) between HindIII and BamHI sites. The primer sequences are as follows: forward, 5′-CCCAAGCTTAGGGTTGTCTGGATGGGCAG-3′ and reverse, 5′-CGCGGATCCTTAGCCTACAAATTTAATGTAA-3′. pcDNA3.1-KIAA0125 or pcDNA3.1 empty vector was delivered into cells using Lipofectamine 2000 (Invitrogen) transfection reagent according to manufacturer's protocol.

Cell counting kit-8 (CCK-8) cell proliferation assay

The CCK-8 assay was used to define HCT116 or SW480 cells viability under KIAA0125 knockdown or overexpression. The cells were seeded into 96-well plates and cultured for the indicated time points. Ten microliters of CCK-8 reagent was added into each well and incubated for another 2 h. The optical density of solution was detected at 450 nm.

The scratch assay

HCT116 and SW480 cells were seeded into a 24-well plate and reached 80–90% density after 24 h of growth. A linear wound was scratched across the center of the well using a pipette tip. The wound closure rate was measured after 36 h.

Transwell assay

The invasion and migration of HCT116 and SW480 cells was detected using matrigel-coated or non-coated chambers with a pore size of 0.8 μM. The cells were seeded into upper chamber in DMEM with 1% FBS and DMEM with 10% FBS in the lower chamber was added as chemoattractant. After 36 h incubation, cells in the upper chamber were removed and cells in the lower side were fixed with 4% paraformaldehyde and stained by 1% crystal violet in phosphate-buffered saline.

Western blotting

The cellular proteins were extracted in 2× Laemmli buffer supplemented with protease inhibitor. Nuclear and cytoplasmic proteins were separated using cytoplasmic extract buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 60 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.075% NP40, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol, pH 7.6) and nuclear extract buffer (20 mM Tris-HCl, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM PMSF, and 25% glycerol, pH 8.0). The proteins were analyzed by gel electrophoresis system and transferred to the polyvinylidene fluoride membrane. After blocking in 3% bovine serum albumin, the membrane was incubated with primary antibodies at 4° overnight followed by incubation with peroxidase-conjugated secondary antibody for one hour at room temperature. The protein signals were detected and the intensity of the bands was quantified using Image Lab Software (Bio Rad, China).

Statistical analysis

All data are expressed as mean ± standard error mean (SEM). The difference between two groups was compared using Student's t test and the differences among multiple groups were analyzed using one-way analysis of variance. A P < 0.05 was considered as statistically significant (*P < 0.05 and **P < 0.01).

The expression of KIAA0125 is downregulated in CRC tissues and cells

LncRNA KIAA0125 was found differentially expressed in many cancers (Yao et al., 2017; Diniz et al., 2018; Hornung et al., 2018). From our previous RNA-sequencing bioinformatic analysis, lncRNA-KIAA0125 was differentially expressed in several carcinomas (data unpublished). To understand the role of KIAA0125 in the CRC, we examined the expression of KIAA0125 in both CRC tissues and CRC cells. The 25 pairs of tumor and paratumor tissues were analyzed by qRT-PCR and the expression level of KIAA0125 was normalized by GAPDH. As shown in Figure 1A, the expression of KIAA0125 was significantly downregulated in CRC tissues when compared with adjacent normal tissues. Besides, the relative expression of KIAA0125 in each patient showed different levels of decrease as indicated in Figure 1B. Moreover, we classified the CRC tissues into two groups (Stage I/II as early stage and Stage III/IV as late stage). The expression levels of KIAA0125 in Stage III/IV (n = 12) were much lower than in Stage I/II (n = 13), which suggested that the expression of KIAA0125 correlated with the clinical cancer development (Figure 1C). Next, we analyzed the expression of KIAA0125 in CRC cell lines (HCT116, LS174T, SW480, SW620, and LOVO) and human normal colon epithelial cell line fetal human cell (FHC). The expression levels of KIAA0125 in CRC cells were also lower than in FHC cells (Figure 1D). Collectively, the above results demonstrated that KIAA0125 was significantly downregulated in CRC tissues and cells.

Overexpression of KIAA0125 suppresses cell proliferation, migration, and invasion in CRC

To elucidate the mechanism of KIAA0125 in cell proliferation, migration, and invasion, we transfected KIAA0125 expressing plasmid or empty vector into HCT116 and SW480 cells, which were chosen for further study because of their relatively low expression levels of KIAA0125 (Figure 2A). For cell proliferation, KIAA0125 overexpression led to growth retardation of HCT116 and SW480 cells, which was confirmed by CCK-8 assays (Figure 2B). Since cell motility and consequent invasion have been recognized as the major features during cancer progression (Reya et al., 2001), we further investigated the functions of KIAA0125 in cell migration and invasion. In wound healing assays, the wound closure rates in HCT116 and SW480 cells were delayed by KIAA0125 overexpression (Figures 2C and 2D). Moreover, the property of invasion was determined by transwell assays. The results indicated that KIAA0125 overexpression suppressed their invasive potential (Figures 2E and 2F). Overall, these data indicated that KIAA0125 acted as a tumor suppressor gene that suppressed cell proliferation, migration, and invasion in CRC cells.

Knockdown of KIAA0125 promotes cell proliferation, migration, and invasion in CRC

To further confirm the functions of KIAA0125 in cell proliferation, migration, and invasion, we used siRNA to knockdown endogenous KIAA0125. The pre-designed siRNA effectively silenced KIAA0125 in HCT116 and SW480 cells (Figure 3A). After siRNA transfection for 72 h, the cell viability was increased in HCT116 and SW480 cells when compared with scramble siRNA transfection groups, which suggested that KIAA0125 silencing promoted cell proliferation (Figure 3B). Next, we examined the potential of cell migration and invasion under KIAA0125 knockdown using transwell assays with or without matrigel coating, respectively. As shown in Figures 3C, 3D, and 3E, KIAA0125 silencing increased the migratory and invasive ability of HCT116 and SW480 cells. In summary, KIAA0125 was directly involved in the regulation of cell proliferation, migration, and invasion in CRC cells.

KIAA0125 inhibits EMT through Wnt/β-catenin signaling in CRC

EMT has been demonstrated as a key program and been activated during cancer development (Mani et al., 2008). Among several signaling pathways, Wnt signaling pathway has been shown to directly contribute to EMT (Gasior et al., 2017). Thus, we first examined the expression of several EMT-related proteins under the condition of ectopic KIAA0125 expression in HCT116 and SW480 cells. As shown in Figures 4A and 4B, the epithelial marker, E-cadherin, was increased and mesenchymal markers, N-cadherin and vimentin, were decreased after KIAA0125 overexpression. In addition, the expression of matrix metalloproteinase-2, which represented the cell invasive potential was decreased after KIAA0125 overexpression. Next, we explored the effects of KIAA0125 overexpression on β-catenin, which is the key regulator in Wnt canonical pathway (MacDonald et al., 2009). The overexpression of KIAA0125 suppressed β-catenin at both messenger RNA (mRNA) level (Figure 4C) and protein level (Figures 4D and 4E). We then analyzed myc and cyclin D1, which are the targets of β-catenin. The levels of protein expression were also decreased after KIAA0125 overexpression. Taken together, the above data suggested that KIAA0125 exerted functions by regulating Wnt/β-catenin signaling in CRC cells.