LINC01614 Promotes Colorectal Cancer Cell Growth and Migration by Regulating miR-217-5p/HMGA1 Axis

Colorectal cancer (CRC) substantially contributes to cancer-related deaths worldwide. Recently, a long non-coding RNA (lncRNA), LINC01614, has emerged as a vital gene regulator in cancer progression. Yet, how LINC01614 affects CRC progression remains enigmatic. Here, we defined LINC01614 expression in CRC, investigated the performance of CRC cells lacking LINC01614, and elucidated the underpinning mechanism. We observed that LINC01614 was upregulated in both CRC tissues and cell lines. LINC01614 knockdown repressed the proliferation and metastasis capacity of CRC cell lines. Consistently, an in vivo LINC01614 deficiency model exhibited slow tumor growth rate. Moreover, luciferase reporter assay, RNA pull-down, and immunoprecipitation confirmed that LINC01614 targeted miR-217-5p. LINC01614 knockdown reduced the expression of HMGA1 and N-cadherin, while increasing that of E-cadherin, resulting in decreased viability, proliferation, migration, and invasion capacity of CRC cells. Our results demonstrate that LINC01614 regulates CRC progression by modulating the miR-217-5p/HMGA1 axis, thus holding great potential as a prognostic biomarker for CRC diagnosis and treatment.


Introduction
Colorectal cancer (CRC) accounts for 10% of new cancer cases globally, with one to two million newly diagnosed patients each year [1]. And it ranks third in incidence and second in cancer mortality with 700,000 deaths annually [2]. Approximately, 25% of CRC cases are familial [3]. Aging is another major risk factor of developing CRC, and the probability of developing CRC increases significantly in people aged 50 years older [4]. Recently, noncoding RNAs (ncRNAs) have been found to be essential for the pathogenesis of cancers [5]. Long non-coding RNAs (lncRNAs) are a type of ncRNA and have emerged as a critical gene regulator in cancer pathogenesis and progression [6,7].
LncRNAs are important gene regulators in CRC. LncRNA DLEU1 contributes to CRC progression by activating KPNA3 [8]. Overexpression of lncRNA DANCR is associated with tumor progression and poor prognosis [9]. Similarly, lncRNA MALAT1 is upregulated in CRC, leading to poor prognosis in patients [10]. A circulating lncRNA, SNHG11, has been reported as a novel biomarker for the diagnosis of early CRC [11]. Moreover, lncRNAs have been proposed as a linker between RNA regulation and CRC progression [12].
Long intergenic non-coding RNA 01614 (LINC01614) is located on chromosome 2q35, encoding a lncRNA of 2904 nucleotides (nt) [13]. LINC01614 dysregulation is associated with cancer development. For instance, LINC01614 is upregulated in breast cancer, which is associated with poor prognosis [14]. Similarly, upregulation of LINC01614 has been proposed as a prognostic indicator of survival in nonsmall cell lung cancer (NSCLC) [15], and LINC01614 knockdown inhibits lung adenocarcinoma progression [13]. Moreover, LINC01614 is upregulated in glioma [16]. However, the role of LINC01614 in CRC has not yet been explored. Elucidation of the function and regulation mechanism of LINC01614 can help design new therapeutic strategies for CRC treatment and provide a new biomarker for CRC diagnosis. Here, using a combination of cell biology and molecular techniques, we dissected the role of LINC01614 in the pathogenesis and progression of CRC.

Materials and Methods
2.1. Cell Culture and Transfection. NCM460 cells were cultured in RPMI-1640 (Thermo Fisher Scientific, USA), SW620 cells in DMEM (Thermo Fisher Scientific, USA), HCT116 and HT-29 cells in McCoy's 5a Modified Medium (Thermo Fisher Scientific, USA), LOVO cells in ATCCformulated F-12K medium (ATCC, USA), and SW480 cells in ATCC-formulated Leibovitz's L-15 medium (ATCC, USA) under standard laboratory conditions in an incubator at 37°C with 5% CO 2 . All the media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco-Thermo Fisher Scientific, USA). Cells were grown to 70-90% confluence at transfection. Lipofectamine reagent 2000 (Invitrogen, USA) and transfection constructs were diluted separately in the Opti-MEM ® Medium (Thermofisher, USA) and mixed at 1 : 1 followed by incubation for 5 minutes at room temperature. The resulting lipid complex was incubated with the cells at 37°C for 48 hours.

Collection of Clinical Samples.
Thirty-six paired CRC and normal tissue samples were collected from patients by surgery with written informed consent at Yantai Yuhuangding Hospital from May 2017 to October 2020. CRC samples were confirmed by histopathological examinations. None of the patients were treated with chemotherapy or radiotherapy before the surgery. Paired adjacent non-tumor tissues were dissected from a healthy area 5 cm away from the tumor edge and were confirmed to be negative for tumor cells. The samples were frozen at −80°C immediately after collection. All experimental procedures were approved by the ethical committee for human experimentation at Yantai Yuhuangding Hospital and were performed in accordance with the declaration of Helsinki.
2.3. Cell Viability Assay. The cell counting kit 8 assay (CCK-8) was used to determine the cell viability. Briefly, approximately 1000 cells/well were seeded into 96 well cell culture plates and cultured overnight. After treatments, cells were incubated for 24, 48, and 72 hours. Subsequently, 10 μL of CCK-8 (Dojindo, Japan) was added to each well, followed by incubation for 1 hour at 37°C. Finally, the absorbance at 450 nm wavelength was measured using the Elx800 absorbance reader (BioTek Instruments).

Colony Formation
Assay. The proliferation of CRC cells was examined by the colony formation assay. Approximately 500 cells/well with or without transfection were seeded into 24-well plates and cultured for 10 days under standard laboratory conditions. Further, the cells were fixed with 100% methanol (Sigma, USA), stained with 0.1% crystal violet solution (Solarbio, China), and manually counted under a laboratory microscope (Olympus, Japan).

Flow Cytometry Analysis.
Annexin V-FITC/PI Apoptosis Detection Reagent (Invitrogen, USA) was used to calculate cell apoptosis ability following the supplier's protocol. The apoptotic cells were calculated using a Beckman Coulter FACS flow cytometer (Beckman Coulter, USA) coupled with the FlowJo software system (Ashland, USA).
2.6. Western Blot. Cells were lyzed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Servicebio, China) containing a protease inhibitor cocktail (Sigma, USA). Following quantification by the bicinchoninic acid (BCA) method, the proteins were applied to a 4-20% polyacrylamide gel (GenScript, USA). Following electrophoresis, the separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, USA), which was then blocked in 5% skim milk at room temperature. The primary antibody (1 : 500) was incubated with the membrane overnight at 4°C. Following three washes in 1 × PBS buffer (pH 7.4), the membrane was treated with the secondary antibody (1 : 2000) for 2 hours at room temperature. The blots were developed using Enhanced Chemiluminescence Kit (FDbio Science, China), and the intensities of protein bands were measured using ImageJ software.

Quantitative Real-Time Polymerase Chain Reaction.
Total RNAs were isolated using TRIzol reagent (Invitrogen, USA) and were quantified by the Nano-drop spectrophotometer. The RNA was reverse-transcribed into cDNA by the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with 40 cycles of alternate temperatures of denaturation, annealing, and extension using the SYBR Green PCR Master Mix (Thermo Fisher Scientific, USA). The relative gene expression levels were calculated by the 2 −ΔΔCt method with GAPDH as the reference gene for LINC01614 and HMGA1 and U6 as the reference gene for miR-217-5p. All the experiments were performed in triplicate.
2.8. Subcellular Fractionation. First, cytoplasmic and nuclear RNAs were isolated by the PARIS Kit (Life Technologies, MA, USA). Then, qRT-PCR was performed to quantify LINC01614 in cytoplasm and nucleus using GAPDH and U6 as respective references.
2.9. Luciferase Reporter Gene Assay. Approximately 5 × 10 4 cells were seeded into a 96-well plate followed by incubation for 24 hours. After transfection/co-transfection for 48 hours with Lipofectamine 2000 (Invitrogen, USA), luciferase activity was measured using a Luciferase assay kit (Promega, USA) according to the provided protocol. In short, 100 μL of luciferase assay solution was added to an equal volume of cell suspension followed by incubation for 20 minutes at room temperature. Luciferase activity was determined using the Synergy H4 Hybrid Reader (BioTek, Winooski, USA) [17].

Analytical Cellular Pathology
Sangon Biotech (Shanghai, China). The cells were lysed, and one-half of the lysate was used for input control, while the other half was incubated with magnetic Dynabeads M-280 Streptavidin beads (Invitrogen, USA) overnight at 4°C. Finally, miR-217-5p enrichment was determined by qRT-PCR assay.
RNA immunoprecipitation (RIP) was performed using the Magna RNA Immunoprecipitation (RIP) Kit (Millipore, Bedford, MA) according to the manufacturer's guidelines. Briefly, cell lysates were fully mixed with Sepharose beads coupled with the AgO 2 -specific antibody (Cell Signaling Technology, USA) at 4°C for 4 hours. IgG antibody was used as the control. Finally, RNA was extracted from the beads followed by qRT-PCR to determine the relative enrichment [19].
2.11. Transwell Assay. The migration and invasion potential of differently treated CRC cells were examined by the transwell assay as per previously described procedures [20]. For invasion assay, the cells were suspended in a serum-free cell culture medium in an upper chamber with a porous membrane containing Matrigel solution (BD, USA). The lower chamber was immersed in a solution containing a complete cell growth medium. Following incubation for 24 hours at 37°C, the cells at the upper side of the porous membrane were gently removed, while the cells retained at the lower side were collected. The collected cells were fixed with 4% formaldehyde solution, stained with 0.1% crystal violet solution, and counted under a light microscope (Olympus, Japan). Migration analysis was performed using the same protocol without the usage of Matrigel.  2.14. Statistical Analysis. Statistical analysis was conducted by GraphPad Prism software version 6. Kaplan-Meier analysis was used to compare the survival difference between patient groups. Student's t test was used for comparisons between two groups, while one-way ANOVA was used for the statistical analysis of multiple groups. P values less than 0.05 were considered significant. The data were presented as the mean ± SD. Each experiment was repeated at least three times.

LINC01614 Is Upregulated in CRC Tissues and Cell
Lines. First, we analyzed LINC01614 expression in 41 pairs of cancer and normal tissues in the TCGA database and found that LINC01614 was upregulated in cancer tissues (Figures 1(a) and 1(b)). To confirm this, we performed qRT-PCR to quantify LINC01614 abundance in 36 clinical samples of CRC. LINC01614 expression was significantly higher (P < 0:01) in the CRC than in the control tissues (Figure 1(c)). Then we investigated the correlation of LINC01614 expression levels with the survival of the patients with CRC. We divided the 36 patients into a high expression group (n = 18) and a local expression group (n = 18) according to the median expression level of LINC01614. Kaplan-Meier survival analysis indicated that CRC patients with high expression of LINC01614 had low survival rates and poor prognosis (P = 0:0434) ( Figure 1(d)). The high expression group had significantly bigger tumor size, greater Tumor Node Metastasis (TNM) stages, and higher incidences of lymph node and distant metastasis than the low expression group (P < 0:01), and age and gender were not significantly different between the two groups (Table 1). Moreover, we determined LINC01614 expression in the CRC cell lines (SW620, HCT116, LOVO, HT-29, and SW480) and the normal colon epithelial cell NCM460 using qRT-PCR. We found that LINC01614 expression was significantly (P < 0:01) upregulated in CRC cell lines, a result that was in line with LINC01614 expression in cancer tissues (Figure 1(e)).

LINC01614 Knockdown Inhibits CRC Cell Proliferation and Invasion.
To explore the function of LINC01614, we first defined the subcellular localization of LINC01614 in the SW620 and HT-29 cells that exhibit the highest expression levels of LINC01614. Using qRT-PCR, we detected that LINC01614 was mostly enriched in the cytoplasm (Figure 2(a)). Further, we designed two shRNAs targeting LINC01614 (sh-LINC01614#1 and #2) to knock down LINC01614 expression in the SW620 and HT-29 cell lines (Figure 1(e)). QRT-PCR revealed that the knockdown efficiency of both shRNAs was more than 50% (Figure 2(b)). LINC01614 knockdown decreased CRC cell viability, which was manifested by decreased absorption values at 450 nm after 0, 24, 48, and 72 hours of treatment (Figure 2(c)). In addition, the colony formation assay revealed that LINC01614 knockdown decreased the proliferation of CRC cells (Figure 2(d)). Moreover, using the flow cytometry analysis, we found that LINC01614 knockdown enhanced the apoptosis of SW620 and HT-29 cells (Figure S1(a)). Furthermore, LINC01614 silencing significantly decreased the migration (Figure 2(e)) and invasion of SW620 and HT-29 cells (P < 0:01) (Figure 2(f)). In order to further explore the role of LINC01614 in CRC progression, cell cycle was investigated after LINC01614 knockdown in vitro. The cell cycle analysis showed that more CRC cells were distributed in G1 phase and less in S phase after silencing LINC01614, which suggested that CRC cells were arrested at G1 phase by sh-LINC01614#1/2 (Figure 2(g)).  Next, we sought to identify the target of LINC01614. Using LncBase v.2, we predicted that LINC01614 contained a potential binding site for miR-217-5p (Figure 3(a)). We first used qRT-PCR to detect the expression of miR-217-5p in both SW620 and HT-29 cells transfected with miR-217-5p mimics ( Figure S1(d)). The results have shown that in cells transfected with miR-217-5p mimics, the level of miR-217-5p increased significantly than the control. We then performed the luciferase reporter gene assay in SW620 and HT-29 cells and found that miR-217-5p mimics decreased luciferase activity (Figure 3(b)); however, the mutant of the putative binding site did not (Figure 3(b)). The RNA pulldown assay indicated that LINC01614 probe enriched greater proportion of miR-217-5p in SW620 and HT-29 cells (P < 0:01) (Figure 3(c)). Moreover, anti-Ago2 enriched greater amounts of miR-217-5p than did Anti-IgG (P < 0:01) (Figure 3(d)). Furthermore, LINC01614 knockdown significantly increased miR-217-5p expression in SW620 and HT-29 cells (P < 0:01) (Figure 3(e)). Interestingly, CRC tissues exhibited significantly lower miR-217-5p levels (Figure 3(f)). In addition, LINC01614 expression levels were inversely correlated with miR-127-5p expression levels (R 2 = 0:4995, P < 0:001) (Figure 3(g)).

LINC01614 Knockdown Inhibits CRC Tumor Growth In
Vivo. To examine how LINC01614 affects tumor growth in vivo, we established a tumor xenograft model in nude mice. Because the knockdown efficiency of sh-LINC01614#1 and sh-LINC01614#2 and the cell phenotype they caused were similar, we just chose the cells transfected with sh-LINC01614#1 for in vivo experiments. HT-29 cells were stably transfected with sh-NC and sh-LINC01614#1 and were injected subcutaneously into nude mice. Tumor volume detection revealed a decrease in subcutaneous tumor volume in the sh-LINC01614#1 group compared to the sh-NC group ( Figure S1(b)). Similarly, we found that LINC01614 knockdown significantly reduced CRC tumor weight ( Figure S1(c)). Collectively, the above data suggest that LINC01614 knockdown inhibits CRC tumor growth in vivo.

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Our results indicated that HMGA1 was the target of miR-217-5p. Low levels of HMGA1 were correlated with weak cell proliferation and invasion, while high levels of HMGA1 were correlated with poor patient survival. HMGA1 was first discovered in aggressive cervical cancer cells [28] and is involved in the pathogenesis of various malignant cancers [29]. Increasing studies have shown that HMGA1 expression is elevated in malignant cancers including hepatocellular carcinoma [30], breast cancer [31,32], gastric cancer [33], and uterine cancer [34], as well as glioblastoma [35], and osteosarcoma [36]. HMGA1 was found to be upregulated in CRC [37,38], which corroborates our finding that HMGA1 is dysregulated in CRC. The decrease in HMGA1 expression caused by miR-217 overexpression was associated with decreased Ncadherin expression and increased E-cadherin expression in CRC cells. E/N-cadherin has been suggested as a toggle to regulate tumor progression [39]. The decreased expression of Ecadherin has been reported to be associated with poor prognosis in CRC [40] and the invasiveness of colon cancer [41]. Hence, we can conclude that E-cadherin, whose expression is regulated by miRNAs/HMGA1 axis, plays an inhibitory role in CRC progression. In conclusion, our results suggest that LINC01614 promotes CRC progression through the HMGA1/miR-217-5p axis, potentiating it as a prognostic biomarker for CRC.

Data Availability
The data are available from the corresponding author upon reasonable request.

Ethical Approval
All the procedures were approved by the ethical committee for human experimentation at Yantai Yuhuangding Hospital and were in accordance with the guidelines of the declaration of Helsinki.