Long non-coding RNA TP73-AS1 sponges miR-194 to promote colorectal cancer cell proliferation,migration and invasion via up-regulating TGF α

Abstract

OBJECTIVE:

Colorectal cancer (CRC) is the 3 ${}^{\text{rd}}$ most common cancer worldwide. Recently, long non-coding RNAs (lncRNAs) were found to be critical modulators in the CRC progression. The aim of this study is to investigate the potential roles of lncRNA P73 antisense RNA 1T (TP73-AS1) in CRC development and progression.

METHODS:

Quantitative real-time PCR (qRT-PCR) was performed to determine relevant gene expression levels; western blot was performed to determine protein expression levels; CCK-8, colony formation, wound healing and Transwell invasion assays were used to determined CRC cell proliferation, migration and invasion; in vivo tumor growth was assessed in xenograft mice model.

RESULTS:

TP73-AS1 was up-regulated in both CRC tissues and CRC cell lines. Overexpression of TP73-AS1 was associated with metastasis and advanced clinical stages in CRC patients. Overexpression of TP73-AS1 promoted CRC cell growth, proliferation, migration and invasion in vitro; and knockdown of TP73-AS1 significantly inhibited CRC cell growth, proliferation, migration and invasion in vitro as well as tumor growth in vivo. Bioinformatics analysis and luciferase reporter assay indicated that TP73-AS1 could bind directly with miR-194, and TP73-AS1 negatively regulated the expression of miR-194 in CRC cells. Further study indicated that miR-194 negatively regulated the downstream target of transforming growth factor alpha (TGF $\alpha$ ) via targeting its 3’ untranslated region, and TP73-AS1 positively regulated the expression of TGF $\alpha$ in CRC cells. Moreover, overexpression of miR-194 suppressed CRC cell proliferation and invasion, and attenuated the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion. Silence of TGF $\alpha$ inhibited CRC cell proliferation and invasion, and also reversed the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion.

CONCLUSIONS:

this study demonstrated that TP73-AS1 regulated CRC progression by acting as a competitive endogenous RNA to sponge miR-194 to modulate the expression of TGF $\alpha$ .

1. Introduction

Colorectal cancer (CRC) is the third most common cancer and is one of the leading cause of cancer death worldwide [1]. Up to date, although many achievements have been made in the diagnosis and therapy of CRC, a great majority of the CRC patients are diagnosed at an advanced stage that has missed the optimal time for a radical operation, and CRC still causes significant morbidity and mortality [2, 3, 4]. A growing evidence revealed that CRC carcinogenesis is a complicated biological process involving many genomic mutations and complex biological signaling networking [5, 6, 7]. In this regard, to further understand the molecular mechanisms underlying the development and progression of CRC is critical for us to find novel approach for the management of CRC.

Recently, studies have found that protein-coding genes constitute only small portions of the genome encoding proteins, and most of the human genome is transcribed into noncoding RNAs such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) [8]. The lncRNAs are a class of newly discovered non-coding RNAs, and the deregulation of lncRNAs have been implicated in the pathogenesis of various human diseases, particularly in cancer [9, 10]. In the aspect of CRC, lncRNAs have been suggested for their important roles in the CRC diagnosis, therapy, and prognosis. For instance, the lncRNA HOTAIR was found to regulate polycomb-dependent chromatin modification and was associated with poor prognosis in CRC [11]. Takahashi et al., showed that amplification of PVT-1 inhibited CRC cell apoptosis and contributed to poor prognosis in CRC [12]. The lncRNA MALAT1 was shown to promote tumor growth and metastasis in colorectal cancer through binding to SFPQ and releasing oncogene PTBP2 from SFPQ/PTBP2 complex [13]. Recently, lncRNAs were found to function as a competitive endogenous RNA (ceRNA) to sponge miRNAs, which in turn regulated the CRC progression. Huang et al., showed that the novel long intergenic noncoding RNA UCC promoted colorectal cancer progression by sponging miR-143 [14]. The lncRNA FBXA19-AS functioned as an oncogene by sponging miR-203 in CRC [15]. The lncRNA P73 antisense RNA 1T (TP73-AS1) is a newly discovered lncRNA, and TP73-AS1 is the antisense of the coding gene TP73, which is located on human chromosomal band lp36.32 [16]. As far as we know, TP73-AS1 was found to play oncogenic role in various types of cancers including myeloma, esophageal squamous cell carcinoma, glioma, liver cancer, and breast cancer [16, 17, 18, 19, 20, 21]. However, no study has investigated the role of TP73-AS1 in CRC.

Given the oncogenic role of TP73-AS1 in CRC, in the present study, we found that TP73-AS1 was up-regulated in the CRC tissues and the overexpression of TP73-AS1 was associated with metastasis and advanced clinical stage. Further in vitro and in vivo studies also showed that TP73-AS1 had enhanced effects on the CRC cell proliferation, migration and invasion in vitro, and knock-down of TP73-AS1 suppressed tumor growth in vivo. In addition, the mechanistic studies also revealed that TP73-AS1/miR-194/transforming growth factor alpha (TGF $\alpha$ ) axis may contribute the development and progression of CRC.

2. Materials and methods

2.1 Clinical samples

The CRC tissues and adjacent normal colon tissues were obtained from 84 CRC patients who underwent surgical resection in The First Affiliated Hospital of Xi’an Medical University from January 2013 to December 2015. None of the patients received any treatment before surgery, and all the clinical samples were confirmed by the pathologists. The clinical and pathological features of the CRC patients were obtained from patients’ history record. This study was approved by the Ethics Committee of The First Affiliated Hospital of Xi’an Medical University and all patients provided informed consent before participating in this study.

2.2 Cell lines

The human embryonic kidney (HEK) 293T cells, the normal colon epithelial cell line, NCM460 and CRC cell lines including HT29, SW480 and HCT116 were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA) and cells were kept in a humidified incubator with 5% CO ${}_2$ at 37 ${}^{\circ }$ C.

2.3 Vector construction, miRNAs, siRNAs and cell transfection

The pcDNA3.1 vector and TP73-AS1 overexpression vector (pcDNA3.1-TP73-AS1) were commercially purchased from Genepharma (Shanghai, China). The miRNAs including mimics control, miR-194 mimics, inhibitors control and miR-194 inhibitors, and the siRNAs including control siRNA and TGF $\alpha$ siRNA were purchased from Ribobio (Guangzhou, China). Cell transfection and co-transfection were performed by using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and 24 h after transfection, cells were processed for further experimentation.

2.4 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis

Total RNA from cells or tissues were extracted by using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA) was used to detect miR-194 expression level according to the manufacturer’s instruction. The reverse transcription for TP73-AS1 and TGF $\alpha$ was performed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed by using SYBR Green Real time PCR MasterMix (Takara, Dalian, China). GAPDH was used as the internal control for TP73-AS1 and TGF $\alpha$ , and U6 was sued as the internal control for miR-194. The relative expression of relevant genes was calculated by the comparative Ct method.

2.5 CCK-8 assay

Cell proliferation was detected by the CCK-8 assay (Beyotime, Beijing, China). Briefly, cells were seeded on a 96-well plate and cells were then incubated with CCK-8 solution for 2 h at room temperature according to the manufacturer’s instructions. The cell proliferative index was measured by detecting the absorbance at the wavelength of 450 nm.

2.6 Colony formation assay

Cell growth was detected by the colony formation assay. Briefly, cells were trypsinized and re-suspended in DMEM with 10% FBS, and the cells were then seeded in the six-well plates and cultured in a humidified incubator containing 5% CO ${}_2$ at 37 ${}^{\circ }$ C for 14 d. Cell colonies were fixed with methanol for 30 min and stained with 0.1% crystal violet for 20 min. The number of colonies were counted under a microscope.

2.7 Wound healing assay

The cell migration was detected by the wound healing assay. Briefly, cells were trypsinized and seeded in the 6-well plates, and 12 h later, an artificial wound was created by using a sterile 200 $\mu$ l pipette tip. The wound width was measured at 0 h and 24 h, and the fraction of cell coverage across the line were calculated for the migration rate.

2.8 Cell invasion assay

The cell invasion was determined by the Transwell cell invasion assay. Briefly, cells were trypsinized and cells in serum-free medium were seeded on the upper chamber with 8 $\mu$ m pore-size insert coated with Matrigel (BD Biosciences, San Jose, CA, USA). The DMEM medium with 10% FBS was added into the lower chamber. After culturing for 24 h, cells that invaded into the lower chamber were fixed with methanol and stained with 0.1% crystal violet. The number of invaded cells was counted under a microscope.

2.9 Lentivirus packaging

The retrovirus system (Clontech Laboratories, Mountain View, CA, USA) was used to establish cell lines with stable down-regulation of TP73-AS1. The sh-TP73-AS1 was subcloned into the pLNCX2 plasmids, and GP293 cells were transfected with pLNCX2-sh-TP73-AS1 or pLNCX2 and the package plasmids according to the manufacturer’s instructions. Virus particles were harvested 48 h after transfection. HCT116 cells were infected with virus particles and maintained in DMEM containing G418. After 2 weeks of selection, cells with stable down-regulation of TP73-AS1 or control cells were pooled.

Figure 1.

TP73-AS1 is significantly up-regulated in CRC tissues and cell lines. (A) Relative expression of TP73-AS1 in CRC tissues ( $n=$ 84) and adjacent normal tissues ( $n=$ 84) (*** $P<$ 0.001). (B) Relative expression of TP73-AS1 in CRC tissues from patients with metastasis ( $n=$ 46) and from patients without metastasis ( $n=$ 38) (** $P<$ 0.01). (C) Relative expression of TP73-AS1 in CRC tissues from patients with different clinical stages (Stage I: $n=$ 22; Stage II: $n=$ 17; Stage III: $n=$ 22; Stage IV: $n=$ 23; * $P<$ 0.05). (D) Relative expression of TP73-AS1 in normal colon cell line and CRC cell lines ( $n=$ 3; *** $P<$ 0.001). Error bars represent the mean $\pm$ SD.

2.10 In vivo tumor growth assay

The 5-week-old BABL/c nude mice were obtained from the the Animal Center of the Chinese Academy (Shanghai, China). Briefly, 1 $\times$ 10 ${}^6$ cells with stable knockdown of TP73-AS1 (sh-TP73-AS1) or negative control cells (sh-NC) were inoculated into the right flanks of each mouse. Tumor diameter (mm) was measured every 7 d, and the tumor volumes were calculated using the formula V $=$ (shortest diameter) ${}^2$ $\times$ (longest diameter) $\times$ 0.5. After 42 d, the mice were killed and the tumors were exercised. All animal procedures were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Xi’an Medical University.

2.11 Luciferase reporter assay

The fragment from TP73-AS1 containing the predicted miR-194-binding site and the fragment from 3’UTR of TGF $\alpha$ containing the predicted miR-194-binding site were amplified using PCR and subcloned into a pmirGLOluciferase Target Expression Vector (Promega, Madison, WI, USA) to form the wild-type pmirGLO-TP73-AS1 vector and the wild-type pmirGLO-TGF $\alpha$ 3’UTR vector. The mutant miR-194-binding sequence was constructed that was named as mutant pmirGLO-TP73-AS1 vector and mutant pmirGLO-TGF $\alpha$ 3’UTR vector. The HEK293T cells were co-transfected with wild-type pmirGLO-TP73-AS1 vector, mutant pmirGLO-TP73-AS1 vector, wild-type pmirGLO-TGF $\alpha$ 3’UTR vector or mutant pmirGLO-TGF $\alpha$ 3’UTR vector and miR-194 mimics, miR-194 inhibitors or the respective negative controls using Lipofectamine 2000 reagent (Invitrogen), and the relative luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Promega) after 48 h.

2.12 Western blot assay

The protein levels were detected by western blot assay. Total cellular proteins were extracted and separated by using SDS-PAGE, and western blotting was performed in accordance with standard procedures. The primary antibodies including anti-TGF $\alpha$ (Abcam, Cambridge, MA, USA), anti- $\beta$ -actin (Abcam), and $\beta$ -actin was used as an internal control.

Figure 2.

Effects of TP73-AS1 on CRC colony formation, cell proliferation, migration and invasion. (A) Transfection of pcDNA3.1-TP73-AS1 up-regulated TP73-AS1 expression as measured by qRT-PCR assay in HT29 cells ( $n=$ 3; *** $P<$ 0.001). (B) Transfection of TP73-AS1 siRNA down-regulated TP73-AS1 expression as measured by qRT-PCR in HCT116 cells ( $n=$ 3; *** $P<$ 0.001). (C) Overexpression of TP73-AS1 promoted colony formation as indicated by colony formation assay in HT29 cells ( $n=$ 3; ** $P<$ 0.01). (D) Knockdown of TP73-AS1 inhibited colony formation as indicated by colony formation assay in HCT116 cells ( $n=$ 3; ** $P<$ 0.01). (E) Overexpression of TP73-AS1 promoted cell proliferation as indicated by CCK-8 assay in HT29 cells ( $n=$ 3; * $P<$ 0.05). (F) Knockdown of TP73-AS1 inhibited cell proliferation as indicated by CCK-8 assay in HCT116 cells ( $n=$ 3; * $P<$ 0.05). (G) Overexpression of TP73-AS1 promoted cell migration as indicated by wound healing assay in HT29 cells ( $n=$ 3; * $P<$ 0.05). (H) Knockdown of TP73-AS1 inhibited cell migration as indicated by wound healing assay in HCT116 cells ( $n=$ 3; * $P<$ 0.05). (I) Overexpression of TP73-AS1 promoted cell invasion as indicated by cell invasion assay in HT29 cells ( $n=$ 3; ** $P<$ 0.01). (J) Knockdown of TP73-AS1 inhibited cell invasion as indicated by cell invasion assay in HCT116 cells ( $n=$ 3; * $P<$ 0.05). Error bars represent the mean $\pm$ SD.

Figure 3.

Knockdown of TP73-AS1 inhibits tumor growth in vivo. (A) Knockdown of TP73-AS1 significantly inhibited tumor growth of HCT116 cells in nude mice model. The tumor volume formed by HCT116 sh-TP73-AS1 cells was significantly lower than that of HCT116 sh-NC cells ( $n=$ 6; ** $P<$ 0.01). (B) Knockdown of TP73-AS1 significantly decreased TP73-AS1 expression in tumor tissues taken from nude mice ( $n=$ 6; ** $P<$ 0.01). Error bars represent the mean $\pm$ SD.

2.13 Statistical analysis

The statistical analysis and graph plotting were performed by using the GraphPad Prism software version 6.0. Significant differences between the in vivo and in vitro data were determined by using Student’s t tests or one-way ANOVA. The correlation between TP73-AS1 expression and miR-194 expression in CRC tissues were analyzed by Pearson correlation test. The data are presented as mean $\pm$ standard deviation (SD), and the threshold for statistical significance was set at $P<$ 0.05.

3. Results

3.1 The expression of TP73-AS1 in CRC tissues and cell lines

The expression of TP73-AS1 in CRC tissues and cell lines was detected by qRT-PCR assay. We found that TP73-AS1 was significantly up-regulated in 84 CRC tissues compared with 84 paired adjacent normal tissues (Fig. 1A). To further examine the clinical significance of TP73-AS1 in CRC, the expression of TP73-AS1 was compared among different groups based on clinical features. The expression level of TP73-AS1 in CRC tissues from patients with distant metastasis was significantly higher than that from patients without distant metastasis (Fig. 1B). In addition, the expression level of TP73-AS1 was significantly increased with more advanced clinical stage (Fig. 1C). In addition to the expression of TP73-AS1 in the tissues, TP73-AS1 was up-regulated in CRC cell lines including HT29, SW480 and HCT116 compared with normal colon cell line, NCM460 (Fig. 1D).

3.2 Effects of TP73-AS1 on CRC colony formation, cell proliferation, migration and invasion

Based on the expression features of TP73-AS1 in clinical samples and CRC cells lines, we hypothesized that TP73-AS1 might play an oncogenic role in CRC. Firstly, we performed the cell transfection experiments to transiently overexpress the TP73-AS1 by transfecting HT29 cells with TP73-AS1-overexpressing vector (pcDNA3.1-TP73-AS1) (Fig. 2A), and to transiently knockdown TP73-AS1 by transfection HCT116 cells with TP73-AS1 siRNA (Fig. 2B). Then, we conducted colony formation, CCK-8, wound healing and cell invasion assays, and the results showed that ectopic TP73-AS1 overexpression significantly promoted colony formation (Fig. 2C), cell proliferation (Fig. 2E), cell migration (Fig. 2G) and cell invasion (Fig. 2I) in HT29 cells compared with control group (pcDNA3.1 group). On the other hand, knockdown of TP73-AS1 by TP73-AS1 siRNA transfection significantly suppressed colony formation (Fig. 2D), cell proliferation (Fig. 2F), cell migration (Fig. 2H) and cell invasion (Fig. 2J) in HCT116 cells. Collectively, these results demonstrated that TP73-AS1 is a positive regulator of cell proliferation, migration and invasion in CRC cells.

3.3 Knockdown of TP73-AS1 inhibits tumorigenesis in vivo

To further examine if TP73-AS1 could regulate tumorigenesis in vivo, cells (HCT116/sh-NC and HCT116/sh-TP73-AS1) were subcutaneously injected into the flank of the nude mice, and the tumor volume was monitored every 7 d for 42 d post inoculation. At the end of the experiment, the mice were killed and the tumors were taken out. The results showed that the tumor volume formed by HCT116/sh-TP73-AS1 cells were markedly less than that formed by HCT116/sh-NC cells (Fig. 3A). The qRT-PCR results confirmed that the expression of TP73-AS1 in tumor tissues from sh-TP73-AS1 group was significantly higher than that from sh-NC group (Fig. 3B).

Figure 4.

Effects of TP73-AS1 on miR-194 expression. (A) Schematic representation of the predicted target site for miR-194 in TP73-AS1.(B) Luciferase reporter assay in HEK293T cells co-transfected with reporter plasmid and the indicated miRNAs. Overexpression of miR-194 significantly decreased the luciferase activity in the wild-type reporter plasmid (wild-type pmirGLO-TP73-AS1); knockdown of miR-194 significantly increased the luciferase activity in the wild-type reporter plasmid ( $n=$ 3; * $P<$ 0.05, ** $P<$ 0.01). (C) Overexpression/knockdown of miR-194 had no effect on the luciferase activity in the mutant reporter plasmid (mutant pmirGLO-TP73-AS1). (D) Overexpression of TP73-AS1 decreased miR-194 expression in HT29 cells ( $n=$ 3; ** $P<$ 0.01). (E) Knockdown of TP73-AS1 increased miR-194 expression in HCT116 cells ( $n=$ 3; ** $P<$ 0.01). (F) Relative expression of miR-194 in CRC tissues ( $n=$ 84) and adjacent normal tissues ( $n=$ 84) (*** $P<$ 0.001). (G) Correlation between TP73-AS1 expression and miR-194 expression in CRC tissues ( $r=$ $-$ 0.2544, $P<$ 0.05). Error bars represent the mean $\pm$ SD.

3.4 Interactions between TP73-AS1 and miR-194

Studies have demonstrated that lncRNAs may involve in the ceRNA regulatory network. By using the online software program (DIANA tools; www.microrna.gr/LncBase), we found that TP73-AS1 formed complementary base pairing with miR-194 (Fig. 4A). To confirm the direct binding relationship between TP73-AS1 and miR-194, we further performed the luciferase reporter assay. We found that miR-194 mimics transfection in HEK293T cells markedly suppressed the luciferase activities of wild-type pmirGLO-TP73-AS1 compared to control group (mimics control), and miR-194 inhibitors transfection in HEK293T cells significantly increased the luciferase activities of wild-type pmirGLO-TP73-AS1 compared to control group (inhibitors control) (Fig. 4B). However, miR-194 mimics or miR-194 inhibitors transfection had no effect on the luciferase activity of mutant pmirGLO-TP73-AS1 in HEK293T cells (Fig. 4C). Overexpression of TP73-AS1 significantly suppressed the expression of miR-194 in HT29 cells (Fig. 4D), and knockdown of TP73-AS1 markedly increased the expression of miR-194 in HCT116 cells (Fig. 4E). The expression of miR-194 in CRC tissues and adjacent tissues were also determined, and the expression of miR-194 was significantly down-regulated in CRC tissues compared with adjacent normal tissues (Fig. 4F), and the correlation analysis showed that the expression of miR-194 was inversely correlated with TP73-AS1 expression in CRC tissues (Fig. 4G).

Figure 5.

Effects of miR-194 on TGF $\alpha$ expression. (A) Schematic representation of the predicted target site for miR-194 in 3’UTR of TGF $\alpha$ . (B) Luciferase reporter assay in HEK293T cells co-transfected with reporter plasmid (wild-type pmirGLO-TGF $\alpha$ 3’UTR) and the indicated miRNAs. Overexpression of miR-194 significantly decreased the luciferase activity in the wild-type reporter plasmid; knockdown of miR-194 significantly increased the luciferase activity in the wild-type reporter plasmid ( $n=$ 3; * $P<$ 0.05). (C) Overexpression/knockdown of miR-194 had no effect on the luciferase activity in the mutant reporter plasmid (mutant pmirGLO-TGF $\alpha$ 3’UTR). (D) and (E) Overexpression of miR-194 decreased mRNA and protein expression of TGF $\alpha$ in HCT116 cells ( $n=$ 3; * $P<$ 0.05). (F) and (G) Knockdown of miR-194 increased mRNA and protein expression of TGF $\alpha$ in HT29 cells ( $n=$ 3; * $P<$ 0.05). (H) and (I) Overexpression of TP73-AS1 increased mRNA and protein expression of TGF $\alpha$ in HT29 cells ( $n=$ 3; ** $P<$ 0.01). (J) and (K) Knockdown of TP73-AS1 decreased mRNA and protein expression of TGF $\alpha$ in HCT116 cells ( $n=$ 3; ** $P<$ 0.01). Error bars represent the mean $\pm$ SD.

Figure 6.

MiR-194 and TGF $\alpha$ mediated the biological effects of TP73-AS1 in HT29 cells. (A) CCK-8 assay following co-transfection with different plasmids (pcDNA3.1 or pcDNA3.1-TP73-AS1) and miRNAs (mimics control or miR-194 mimics) in HT29 cells ( $n=$ 3; * $P<$ 0.05).(B) Cell invasion assay following co-transfection with different plasmids (pcDNA3.1 or pcDNA3.1-TP73-AS1) and miRNAs (mimics control or miR-194 mimics) in HT29 cells ( $n=$ 3; * $P<$ 0.05). (C) CCK-8 assay following co-transfection with different plasmids (pcDNA3.1 or pcDNA3.1-TP73-AS1) and siRNAs (control siRNA or TGF $\alpha$ siRNA) in HT29 cells ( $n=$ 3; * $P<$ 0.05). (D) Cell invasion assay following co-transfection with different plasmids (pcDNA3.1 or pcDNA3.1-TP73-AS1) and siRNAs (control siRNA or TGF $\alpha$ siRNA) in HT29 cells ( $n=$ 3; * $P<$ 0.05). Error bars represent the mean $\pm$ SD.

3.5 TGF $\alpha$ is a direct target of miR-194

Studies have showed that miR-194 repressed gene expression by targeting the 3’UTR, and the TargetScan software (www.targetscan.org) analysis showed that miR-194 formed complementary base pairing with the 3’UTR of TGF $\alpha$ (Fig. 5A). To confirm the direct binding relationship between miR-194 and TGF $\alpha$ , we further performed the luciferase reporter assay. We found that miR-194 mimics transfection in HEK293T cells markedly suppressed the luciferase activities of wild-type pmirGLO-TGF $\alpha$ 3’UTR compared to control group (mimics control), and miR-194 inhibitors transfection in HEK293T cells significantly increased the luciferase activities of wild-type pmirGLO-TGF $\alpha$ 3’UTR compared to control group (inhibitors control) (Fig. 5B). However, miR-194 mimics or miR-194 inhibitors transfection had no effect on the luciferase activity of mutant pmirGLO-TGF $\alpha$ 3’UTR in HEK293T cells (Fig. 5C). Overexpression of miR-194 significantly suppressed the mRNA and protein expression of TGF $\alpha$ in HCT116 cells (Fig. 5D and E), and knockdown of miR-194 markedly increased the mRNA and protein expression of TGF $\alpha$ in HT29 cells (Fig. 5F and G). Consistently, overexpression of TP73-AS1 significantly increased the mRNA and protein expression of TGF $\alpha$ in HT29 cells (Fig. 5H and I), and knockdown of TP73-AS1 markedly decreased the mRNA and protein expression of TGF $\alpha$ in HCT116 cells (Fig. 5J and K).

3.6 MiR-194 and TGF $\alpha$ mediated the biological effects of TP73-AS1 in HT29 cells

As shown in Fig. 6A and B, co-transfection with TP73-AS1 and mimics control significantly promoted cell proliferation and invasion compared with co-transfection with pcDNA3.1 and mimics control in HT29 cells; while co-transfection with pcDNA3.1 and miR-194 mimics significantly suppressed cell proliferation and invasion compared with co-transfection with pcDNA3.1 and mimics control in HT29 cells (Fig. 4A and B). Co-transfection with TP73-AS1 and miR-194 mimics significantly reversed the effects of TP73-AS1 overexpression on cell proliferation and cell invasion in HT29 cells (Fig. 6A and B). Consistently, co-transfection with pcDNA3.1 and TGF $\alpha$ siRNA significantly suppressed cell proliferation and invasion compared with co-transfection with pcDNA3.1 and control siRNA in HT29 cells (Fig. 6C and D), and knockdown of TGF $\alpha$ significantly reversed the effects of TP73-AS1 overexpression on cell proliferation and cell invasion in HT29 cells (Fig. 6C and D).

4. Discussion

The lncRNAs are a new class of non-coding RNAs, which play important roles in the pathogenesis of human cancers. Recent studies have suggested the oncogenic role for TP73-AS1 in several types of human cancers [16, 17, 18, 19, 20, 21]. In this study, we for the first time found that TP73-AS1 levels were up-regulated in CRC tissues compared with adjacent normal colon tissues, and overexpression of TP73-AS1 in CRC tissues was positively correlated with metastasis and advanced clinical stage. Functional and mechanistic studies revealed that TP73-AS1 promoted cell growth, cell proliferation, cell migration and invasion by acting as a ceRNA of miR-194 in CRC.

TP73-AS1 was first reported to be down-regulated in oliogdendroglial tumors and its knockdown by siRNA induced chemoresistance in glioma cells [22]. Zang et al., showed that TP73-AS1 was up-regulated in esophageal cancer tissues and were strongly correlated with tumor location and TNM stage in clinical samples, and TP73-AS1 knockdown also suppressed esophageal cancer cell proliferation in vitro and in vivo [19]. In brain glioma, TP73-AS1 was up-regulated in glioma tissues and cell lines and was associated with poorer prognosis in patients with glioma. In addition, TP73-AS1 knockdown also suppressed glioma cell proliferation and invasion via sponging miR-142 and modulating HMGB1/RAGE pathway [20]. Similarly, TP73-AS1 was also up-regulated in hepatocellular carcinoma (HCC) tissues and cell liens, and high expression of TP73-AS1 was correlated with worse clinicopathological features, poorer prognosis and shorter survival [17], and further mechanistic study revealed that TP73-AS1 modulated HCC cell proliferation via miR-220a-depedent HMGB1/RAGE regulation [17]. More importantly, TP73-AS1 also was found to regulate breast cancer progression via interacting with miR-200a [18, 21]. In the present study, we consistently found that TP73-AS1 was up-regulated in CRC tissues and TP73-AS1 overexpression was positively correlated with metastasis and advanced clinical stage. In vitro studies showed that TP73-AS1 promoted CRC cell growth, cell proliferation, cell migration and invasion, and in vivo study also demonstrated that knockdown of TP73-AS1 suppressed tumor growth in the nude mice. These results suggest that TP73-AS1 may play an oncogenic role in CRC.

To further understand the mechanisms of TP73-AS1-mediated CRC progression, we performed the bioinformatics analysis and the results showed that miR-194 was among of these predicted miRNAs, which may have potential interaction with TP73-AS1. In the present study, the physical interaction between TP73-AS1 and miR-194 was confirmed by luciferase reporter assay, and overexpression of TP73-AS1 down-regulated the expression of miR-194, while knockdown of TP73-AS1 increased the expression of miR-194 in CRC cells. In the aspect of clinical samples, miR-194 was found to be down-regulated in CRC tissues; and the expression of TP73-AS1 was inversely correlated with miR-194 expression in CRC tissues. Overexpression of miR-194 suppressed CRC cell proliferation and invasion, and also reversed the effects of TP73-AS1 overexpression in CRC cells. Indeed, miR-194 was found to be frequently down-regulated in CRC [23], and the down-regulation of miR-194 contributed to colorectal carcinogenesis via targeting AKT2 pathway [23]. In addition, studies also revealed that miR-194 suppressed tumor growth by regulating the MAP4K4/c-Jun/MDM2 signaling pathway [24]. Collectively, these data suggest miR-194 as a tumor suppressor in CRC, and TP73-AS1 exerted its oncogenic function possibly via acting as a ceRNA to sponge miR-194 in CRC cells. However, we need to be cautious that TP73-AS1 may also target other miRNAs, which may require further investigation.

As miRNAs regulated the targeted genes by targeting the 3’UTR, we performed bioinformatics analysis to predict the downstream targets if miR-194, and TGF $\alpha$ was found to be one of the downstream targets. Luciferase reporter assay confirmed that miR-194 physically targeted the 3’UTR of TGF $\alpha$ , and overexpression of miR-194 suppressed the mRNA and protein expression of TGF $\alpha$ in CRC cells; while knockdown of miR-194 increased the mRNA and protein expression of TGF $\alpha$ in CRC cells. Silence of TGF $\alpha$ suppressed the CRC cell proliferation and invasion, and also attenuated the effects of TP73-AS1 overexpression in CRC cells. Previous studies have demonstrated that TGF $\alpha$ and its receptor (epidermal growth factor receptor) played critical roles in the development and progression of cancers [25, 26, 27]. Overexpression of TGF $\alpha$ was also well-documented in the CRC, and it played oncogenic role in the CRC progression [28, 29]. In the present study, our results suggested that TP73-AS1 promoted CRC progression may be mediated via up-regulation of TGF $\alpha$ . As the downstream targets of miR-194 was not limited to TGF $\alpha$ , other targets of miR-194 may be explored to further understand underlying molecular mechanisms of TP73-AS1-mediated CRC progression.

In summary, we demonstrated the first time that TP73-AS1 was up-regulated in CRC tissues, and TP73-AS1 up-regulated miR-194-targeted gene TGF $\alpha$ by acting as a ceRNA to sponge miR-194, and then promoted CRC cell proliferation, migration and invasion. Targeting TP73-AS1 may be a promising approach for developing novel therapy for the treatment of CRC.