Down-regulation of lncRNA DNAJC3-AS1 inhibits colon cancer via regulating miR-214-3p/LIVIN axis

ABSTRACT Long non-coding RNAs (lncRNAs) play a key role in the development and metastasis of cancer. However, the biological role and clinical significance of lncRNA DNAJC3-AS1 in the development of colon cancer is still unknown. In this study, the effects of DNAJC3-AS1 on cell proliferation, migration, and invasion were evaluated by MTT assay, wound-healing assay, and transwell assay, respectively. The relationship between DNAJC3-AS1, miR-214-3p and LIVIN was predicted by the online software and confirmed by dual-luciferase reporter assay. We found that the down-regulation of DNAJC3-AS1 inhibited the proliferation of colon cancer cells and induced growth arrest. Down-regulation of DNAJC3-AS1 also inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of colon cancer cells. Moreover, miR-214-3p can bind to DNAJC3-AS1, and knockdown of DNAJC3-AS1 increased miR-214-3p expression in colon cancer cells. LIVIN was identified as a target of miR-214-3p. The up-regulation of miR-214-3p inhibited the protein expression of LIVIN and suppressed the activation of the NF-κB signaling pathway. Besides, down-regulation of DNAJC3-AS1 reduced cell viability, invasion, and EMT of colon cancer cells, while miR-214-3p inhibitor could reverse these effects. The expression of LIVIN and the activation of the NF-κB signaling pathway were suppressed by down-regulating DNAJC3-AS1, while these effects could be restored by miR-214-3p inhibitor. These findings suggested that DNAJC3-AS1 may promote colon cancer progression by regulating the miR-214-3p/LIVIN axis. DNAJC3-AS1 may serve as a new biomarker and therapeutic target for colon cancer, stimulating new research directions and treatment options.


Introduction
Colon cancer is one of the malignant cancers with the highest morbidity and mortality in the world, and it seriously threatens people's health and life [1]. The incidence of colon cancer varies worldwide. The rate of colon cancer is higher in developed countries than in non-industrialized countries, and in men than in women [2]. Besides, the incidence of colon cancer continues to rise in adults under the age of 50 and increases with age [2]. However, the advances in cancer diagnostics and treatments have not significantly extended the overall survival of patients with colon cancer [3]. The main reason is that current treatments lack effective biomarkers [4].
LncRNAs are non-coding RNAs with a transcript longer than 200 nucleotides and have more cell-type specificity compared with mRNAs [5]. Previous studies have demonstrated that lncRNAs participate in the regulation of various biological processes in cells [6][7][8]. MicroRNA (miRNA) is a class of endogenous single-stranded non-coding RNA molecules with a length of about 22 bp [9]. miRNAs can also participate in various biological processes such as cell proliferation, differentiation, apoptosis, and angiogenesis through regulating downstream target genes [10][11][12]. miRNAs are closely related to the occurrence and development of many cancers, including colon cancer [13]. The hypothesis of competitive endogenous RNA (ceRNA) was proposed in 2011 [14], suggesting a new regulatory mechanism of miRNAs and lncRNAs. The ceRNA hypothesis indicates that some endogenous RNAs contain certain miRNA binding sites, and they can regulate the expression of miRNAs target mRNAs through regulating miRNA expression [14].
Recent research indicates that lncRNA DNAJC3-AS1 can promote the progression of osteosarcoma [18]. However, its role in colon cancer has not been reported. Bioinformatic prediction indicated that DNAJC3-AS1 contains miR-214-3p binding sites. Many studies show that miR-214-3p could inhibit colon cancer [19][20][21]. Moreover, we predicted that miR-214-3p could bind to LIVIN. Researchers found that over-expressing LIVIN can activate the NF-κB signaling pathway and promote the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of colon cancer cells [22]. In contrast, inhibiting the expression of LIVIN can inhibit the malignant phenotype of colorectal cells [22].
The aim of the current study is to investigate the role of DNAJC3-AS1 on the progression of colorectal cancer and reveal the possible mechanisms. Our results demonstrate that the down-regulation of DNAJC3-AS1 suppressed the proliferation, migration, and invasion of colorectal cells. Further, DNAJC3-AS1 functions as a sponge to regulate the miR-241-3p level, thus, affect the malignant activity of colorectal cells via the miR-214-3p/LIVIN axis.

MTT assay
Cell proliferation of colon cancer cells was detected by MTT assay according to the manufacturer's instruction. Cells were cultured in the 96well plate and were detected at 24, 48, 72 and 96 hours after transfection. MTT (KeyGEN BioTECH, China) was put into the medium and incubated at 37°C for 5 hours. The absorbance (OD) was measured using EXL-800 Microplate Reader (BIOTEK, USA) at 570 nm.

5-ethynyl-2ʹ-deoxyuridine (EdU)
Cell proliferation was assayed using the Click-iT EdU Imaging Kit (KeyGEN BioTECH, China) according to the manufacturer's instructions, and then stained with Hoechst 33,342 (Invitrogen, USA) for 15 min. These results were from three independent experiments.

Flow-cytometry analysis
For cell cycle analysis, we used the Cell Cycle Kit (Beyotime, China). After transfection, cells were was harvested and fixed with 70% ethanol at 4°C for 2 h. Then, cells were centrifuged at 1000 g for 5 min to collect these cells. Next, these cells were incubated with 25 μl of propidium iodide (PI) liquid and 10 μl RNase A at 37°C for 30 min. NovoCyte Flow Cytometer (ACEA Biosciences, China) was used to measure the cell cycle.

Wound-healing assay
After transfection, the monolayer cell was scratched with a 200 μl pipette tip, and the medium was replaced with a serum-free medium. Each scratch wound was recorded with a microscope at the same position at 0 h and 24 h, respectively. The experiments were conducted in triplicate.

Transwell assay
The capacity of cell invasion was assessed by transwell assay. First, the transwell chamber was covered with 40 μl Matrigel (BD, USA). The colon cancer cells were seeded on the upper chamber and incubated with serum-free culture medium. Next, 800 μl medium was added in the lower chamber with 30% serum. After a 48-h culture, the colon cancer cells in the upper chamber were washed twice. The lower cells were fixed with 4% paraformaldehyde (Aladdin, China) for 25 min and stained with 0.4% crystal violet stain (Amresco, USA) for 5 min. Five randomly microscopic fields were selected for analysis.

Immunofluorescence (IF)
Cells were seeded on the coverslips and cultured for proper cell density. The cells were blocked with FBS after fixing by 4% paraformaldehyde Table 1. Primer sequences used for qPCR.

Statistical analysis
GraphPad Prism 8 was used to analyze experimental data. The differences between 2 groups were analyzed by the T-test. Multiple groups were compared by one-way ANOVA. Data were presented as mean ± standard deviation (SD) from at least three independent experiments. The P-value <0.05 was considered statistically significant.

DNAJC3-AS1 may involve in the development of colon cancer
We measured the expression of DNAJC3-AS1 in five colon cancer cells (SW620, SW480, HT29, Caco-2, and LoVo). The highest level of DNAJC3-AS1 was found in LoVo cells, followed by Caco-2 cells (Figure 1(a)). Further experiments showed that the level of DNAJC3-AS1 was significantly down-regulated in LoVo cells after transfection with si-DNAJC3-AS1 (Figure 1(b)). Consistently, the level of DNAJC3-AS1 was significantly down-regulated in Caco-2 cells after transfection with si-DNAJC3-AS1 (Figure 1(c)). Next, the MTT assay indicated that the downregulation of DNAJC3-AS1 could inhibit the proliferation of LoVo cells (Figure 1(d)) and Caco-2 cells (Figure 1(e)). Flow-cytometry results showed that the percentage of cells in the G1 phase was markedly increased in LoVo cells (Figure 1(f)) and Caco-2 cells (Figure 1(g)) after transfection with si-DNAJC3-AS1. Down-regulation of DNAJC3-AS1 could decrease the S and G2 phases in LoVo cells (Figure 1(f)) and Caco-2 cells (Figure 1(g)). Additionally, the percentage of EdU + cells was significantly decreased in LoVo cells (Figure 1(h,i)) and Caco-2 cells (Figure 1(j,  k)) by down-regulating DNAJC3-AS1. The Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/) was used to analyze the differences of DNAJC3-AS1 in colon cancer patients and the effect of DNAJC3-AS1 on overall survival. The expression of DNAJC3-AS1 was markedly up-regulated in colon tumor tissues compared to normal colon tissues (Figure 1(l)). Kalpen-Meier survival plot showed that the patients with high DNAJC3-AS1 had shorter overall survival than those with low DNAJC3-AS1 (Figure 1(m)). Together, our results suggested that the down-regulation of DNAJC3-AS1 could inhibit the proliferation of colon cancer cells, and DNAJC3-AS1 may play a vital role in colon cancer development.

DNAJC3-AS1 affected the cell migration, invasion, and EMT of colon cancer cells
Tumor metastasis is one of the most important reasons for the failure of colon cancer treatment [24]. We performed the wound-healing assay to detect the migration ability of colon cancer cells.
The migration rate of LoVo cells transfected with si-DNAJC3-AS1 was lower than that of LoVo cells transfected with si-NC (Figure 2(a,b)). Downregulation of DNAJC3-AS1 could decrease the migrate rate of Caco-2 cells (Figure 2(c,d)).
Transwell assay results indicated that the downregulation of DNAJC3-AS1 could decrease the invasion cell number of LoVo cells (Figure 2(e,f)) and Caco-2 cells (Figure 2(h,i)), suggesting that colon cancer cells with si-DNAJC3-AS1 had weaker invasion ability compared with colon cancer cells with si-NC. Western blot results showed that the expression of E-cadherin was enhanced by down-regulating DNAJC3-AS1 in LoVo cells and Caco-2 cells (Figure 2(i)). The levels of N-cadherin were decreased by down-regulating DNAJC3-AS1 in LoVo cells and Caco-2 cells (Figure 2(i)). Besides, immunofluorescence was used to determine the levels of E-cadherin in colon cancer cells. The knockdown of DNAJC3-AS1 could increase the expression level of E-cadherin in LoVo cells (Figure 2(j)) and Caco-2 cells (Figure 2(k)). These data revealed that the down-regulation of DNAJC3-AS1 could inhibit the migration, invasion, and EMT of colon cancer cells.

DNAJC3-AS1/miR-214-3p axis affected the cell proliferation, invasion, and EMT of colon cancer cells
MTT assay results revealed that down-regulation of DNAJC3-AS1 could inhibit the proliferation of LoVo cells, while down-regulation of miR-214-3p could increase the cell proliferation ( Figure 5(a)). The invasion cell numbers of LoVo cells were reduced by si-DNAJC3-AS1, while this effect was abrogated by down-regulating miR-214-3p ( Figure 5(b,c)). Besides, the protein expression of E-cadherin was increased by si-DNAJC3-AS1, while this increase could be reversed by a miR-214-3p inhibitor ( Figure  5(d)). The expression of LIVIN was reduced by si-DNAJC3-AS1, while this effect was attenuated by a miR-214-3p inhibitor ( Figure 5(e)). In addition, down-regulation of DNAJC3-AS1 could also reduce the phosphorylation of IκB and p65, while this effect was abrogated by a miR-214-3p inhibitor ( figure 5(f,  g)). These suggested that down-regulation of DNAJC3-AS1 could inhibit the cell proliferation, invasion and EMT of colon cancer cells through regulating miR-214-3p, and affect LIVIN expression, NF-κBp65 signaling through regulating miR-214-3p.

Discussion
In cancer biology, the earliest evidence was the difference in gene expression between tumor tissues and normal tissues [25]. Previous studies indicated that lncRNA expression levels in tumors are significantly increased or decreased compared to normal tissues [25][26][27]. The basic hallmark capabilities of malignant transformation of cancer are: sustaining proliferation; evading growth suppressors; activating invasion and metastasis; inducing angiogenesis and resisting cell death [28]. It has been reported that some lncRNAs can affect cell proliferation [29], and participate in the complex interaction of cancer cells [30]. In addition, lncRNAs also play a role in tumor angiogenesis [30]. Therefore, lncRNAs can be used as novel biomarkers for diagnosis, prognosis, and treatment.
In this study, we found that lncRNA DNAJC3-AS1 expression was higher in colon tumor tissues than normal colon tissues. The overall survival of patients with the low expression levels of DNAJC3-AS1 was longer than that of patients with a strong expression level of DNAJC3-AS1. Moreover, the down-regulation of DNAJC3-AS1 inhibited the proliferation, migration, and invasion of colon cancer cells, and induced growth arrest in vitro. These findings indicated that DNAJC3-AS1 may play a direct role in colon cancer progression.
Inspired by the 'competitive endogenous RNA' regulatory network [14], we searched potential interactions with miRNAs. We used bioinformatics analysis and dual-luciferase reporter assays to verify the predicted binding capacity of miR-214-3p on DNAJC3-AS1. As expected, we found that miR-214-3p could complement base pairing with DNAJC3-AS1 and induce translational suppression of the RLuc-DNAJC3-AS1 reporter gene. qPCR results demonstrated that the expression of miR-124-3p was negatively correlated with the expression of DNAJC3-AS1 in colon cancer cells. Previous research has shown that miR-214 is a colon cancer inhibitor [31]. miR-214 inhibits colon cancer by down-regulating Wnt signaling in colon cancer [20]. Besides, miR-214 sensitizes colon cancer cells to 5-fluorouracil (5-FU) by targeting Hsp27 to overcome chemotherapy resistance [21]. Our results showed that knockdown of DNAJC3-AS1 inhibited the proliferation, invasion, and EMT of colon cancer cells, while these effects could be eliminated by miR-214-3p inhibitor. The data suggested that the DNAJC3-AS1/miR-214-3p axis may be related to the pathogenesis of colon cancer.
In order to study the mechanism of DNAJC3-AS1 /miR-214-3p axis in colon cancer, we selected the predicted target gene LIVIN of miR-214-3p for further research. LIVIN is a member of the inhibitor of apoptosis proteins (IAPs) [32]. The expression of LIVIN is rarely found in normal tissues but is abundant in tumor tissues [32]. Overexpression of LIVIN protein has been reported in leukemia, hepatocellular carcinoma, and melanoma, colorectal cancer [33]. In addition, overexpression of LIVIN is associated with the high risk of bladder cancer recurrence [34]. Mountains studies have suggested that LIVIN could regulate the proliferation, migration, and invasion of cancer cells and act as an oncogene in various tumor cells [35][36][37]. More importantly, our team has shown that forced over-expression of LIVIN significantly promoted the proliferation, migration, invasion and EMT activities of colorectal cancer cells, on the contrary, siRNA-mediated knockdown of LIVIN had opposite effects [22]. In the present study, the down-regulation of DNAJC3-AS1 resulted in increased miR-214-3p level and reduced LIVIN expression. These results suggesting that DNAJC3-AS1 may affect the biological behavior of colorectal cancer cells through the regulation of LIVIN expression mediated by miR-214-3p.
The NF-kB pathway is considered to play pivotal roles in proliferation, apoptosis, invasion and other physiological processes of cancer cells [38,39]. The activation of the NF-kB pathway is tightly controlled by the IkB proteins, which inhibit the DNA binding activity and prevent the nuclear uptake of NF-kB complexes [40]. Mehrotra et al [41]. have proved that the IAPs contributes to tumor progression by the activation of the NF-kB pathway. Moreover, we have shown that over-expression of LIVIN promoted the nuclear transfer of NF-kB, and the NF-κB inhibitor BAY 11-7028 or P65 siRNA restores LIVIN-overexpression induced invasion and EMT of colorectal cells [22]. In this study, the downregulation of DNAJC3-AS1 resulted in the reduction of LIVIN level, accompanied by the inactivation of NF-kB, as evidenced by the reduced p-IkB and p-P65 levels. In addition, these effects were obviously restored by the miR-214-3p inhibitor. These results indicate that the DNAJC3-AS1/miR-214-3p axis affects the activation of the NF-kB pathway via the regulation of LIVIN expression, which further controls the malignant phenotype of colorectal cancer cells.

Conclusion
In summary, DNAJC3-AS1 was involved in cell proliferation, migration, invasion, and EMT of colon cancer cells. These effects were related to the activation of the NF-kB pathways that mediated by the miR-214-3p/LIVIN axis. These results enhanced our understanding of lncRNA functions, a better understanding of the pathogenesis of colon cancer, and promote the development of diagnosis and treatment of colon cancer.

Highlights
• DNAJC3-AS1 affects the proliferation and metastatic ability of colon cancer cells.