Overexpressed lncRNA LINC00893 Suppresses Progression of Colon Cancer by Binding with miR-146b-3p to Upregulate PRSS8

Background Long noncoding RNAs (lncRNAs) play a significant role in the progression and metastasis of various cancers. LINC00893 has been reported to exert antitumor effect on various cancers such as gastric cancer and thyroid cancer. Bioinformatics analysis also predicted that LINC00893 was downregulated in colon cancer. However, the clinical significance and regulating mechanism of LINC00893 in colon cancer remain unknown. Methods Expression of LINC00893, miR-146b-3p, and PRSS8 was detected in colon cancer tissues and adjacent nontumor tissues by RT-qPCR, and clinical significance was analyzed by receiver operating characteristic curve. The regulatory mechanism of LINC00893, miR-146b-3p, and PRSS8 was investigated by dual luciferase reporter and RNA pull-down assays. Proliferation, migration, invasion, and apoptosis were measured in HCT116 and SW620 cells by MTT, EdU staining, wound healing, Transwell, TUNEL, and flow-cytometry assays. Moreover, the effect of LINC00893 on colon cancer progression was further evaluated in tumor-bearing mice. Results LINC00893 and PRSS8 were significantly downregulated, while miR-146b-3p was upregulated in colon cancer tissues compared to control group. LINC00893, miR-146b-3p, and PRSS8 had significant diagnostic value with area under curve of 0.9383, 0.7300, and 0.9644, respectively. Overexpressed LINC00893 or silenced miR-146b-3p suppressed the proliferation, migration, and invasion while promoting apoptosis in colon cancer cells (HCT116, SW620). Moreover, miR-146b-3p overexpression reversed the inhibitory effect of LINC00893, while PRSS8 knockdown rescued the suppressive effect of miR-146b-3p inhibitor on malignant cell behaviors in colon cancer. Furthermore, the tumor growth in mice was significantly reduced by LINC00893 overexpression. Conclusion LINC00893 overexpression suppressed the progression of colon cancer by binding with miR-146b-3p to upregulate PRSS8. LINC00893 and its downstream molecules miR-146b-3p and PRSS8 may serve as novel biomarkers and therapeutic targets of colon cancer, providing new treatment options and research approaches towards colon cancer.


Introduction
Colon cancer is a malignant tumor and one of the leading causes of cancer morbidity and deaths worldwide, posing a severe danger to human health [1]. The prevalence of colon cancer varies among countries. It is more common in developed nations than in nonindustrialized nations, and in males than females [2]. Furthermore, the incidence of colon cancer continues to increase among persons under the age of 50, and the risk rises with age. The advancements in cancer detection and therapy, however, have not brought substantial increment in survival time of colon cancer patients [3,4]. It is imperative to explore useful biomarkers to improve the colon cancer therapy [2].   Journal of Oncology A small percentage (1.2%) of the mammalian genomes encode proteins, whereas the rest of the genome is translated to a variety of noncoding RNAs (ncRNAs) [5][6][7][8]. Small ncRNAs (20-200 nucleotides) and long ncRNAs (lncRNAs, >200 nucleotides) are the two primary types of ncRNAs [9]. lncRNAs are deemed as regulating molecules and linked to a wide variety of biological functions and disorders [10][11][12][13][14][15]. lncRNAs have been indicated to influence the expression of genes through chromatin modification, transcriptional regulation, and microRNA (miRNA) sponging. A study reported that lncRNA DNAJC3-AS1 might promote colon cancer progression by mediating the miR-214-3p/ Livin axis [16]. LINC00893 overexpression suppresses the proliferation, migration, and invasion of gastric cancer cells and regulates epithelial-mesenchymal transition by binding with RBFOX2 [17]. LINC00893 inhibits proliferation and migration of papillary thyroid cancer cells by inactivation of the AKT pathway via stabilizing PTEN [18]. miR-146b-3p has previously been reported to be implicated in some malignancies and exerts oncogenic effects in the growth of tumors such as colorectal cancer and thyroid cancer [19,20]. Moreover, miR-146b is involved in cancer metastases [21,22]. Whereas the expression of PRSS8 is decreased in the esophagus and colorectal malignancies and is inversely related to prognosis in both tumors [23,24]. lncRNAs play a significant role in the progression and metastasis of various types of cancer via mediating miRNAs and their targeted genes. However, the clinical significance and regulating mechanism of lncRNA LINC00893 in the development of colon cancer remain to be unknown. In this study, we aimed to explore the function and regulatory mechanism of LINC00893 in colon cancer, which may provide insight on the colon cancer therapy.

Materials and Methods
2.1. Patients. Thirty pairs of colon cancer and normal adjacent tissue samples were retrospectively obtained from the patients in Affiliated Huaian No. 1 People's Hospital of Nanjing Medical University. The subjects were enrolled from July 2017 to July 2020. According to the Declaration of Helsinki, the study was carried out under the approval of the ethics committee of the Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University. All patients had signed the informed consents before the conduction of the study. The clinical significance of LINC00893, miR-146b-3p, and PRSS8 was measured in colon cancer patients by ROC curves. (i) Expression correlation of LINC00893, miR-146b-3p, and PRSS8 in colon cancer tissues (n = 30) was analyzed. * P < 0:05, * * * P < 0:001. Extraction Reagent, ELK Biotechnology, Wuhan). The concentrations of RNA were determined using a spectrophotometer (ND1000, NanoDrop Technologies, USA). The total RNA was then reverse-transcribed into cDNA using the reverse transcript kit (EQ003, ELK Biotechnology, Wuhan). The following were the thermal cycle conditions involved in the PCR reaction: predenaturation was carried out for 10 minutes at 95°C; denaturation was carried out for 15 seconds at 95°C; annealing was carried out for 15 seconds at 60°C; and elongation was carried out for 20 seconds at 72°C, for a total of 40 cycles. When the temperature reached 4°C, the processes were halted. For each specimen, these three steps were followed, and quantitative analysis of the data was performed based on the 2 -△△CT method [25].

MTT Assay.
MTT assay was carried out to evaluate the viability of colon cancer cells following the manufacturer's protocols (Beyotime Institute of Biotechnology). HCT116 and SW620 cell lines were incubated in 96-well plates at the density of 5 × 10 3 cells/well and then treated with LINC00893 OE, empty vector, miR-146b-3p mimics, miR-146b-3p inhibitors, si-PRSS8, and si-NC plasmid for 24 h. Next, 10 μL of MTT reagent (Beyotime, Shanghai, China) was added into each well and further incubated for 4 h at 37°C. The optical density (OD) value was evaluated by a microplate reader (Promega, WI, USA) at 450 nm. Results from three independent experiments were normalized to the control group and expressed as the mean ± SD.
2.5. EdU Assay. The 10 μmol/L EdU was added into a growth medium and cultured in the incubator for 24 h after cell transfection. In the following step, 1 mL of 0.5% triaxone solution was added, followed by 0.5 mL reaction solution and 0.5 mL Hoechst 33342 solution for 30 minutes of incubation, after which the photographing and counting of the stained cells were carried out using an inverted fluorescent microscope.
2.6. Wound Healing Assay. After the cell transfection, the colon cancer cells were treated with trypsin, plated into the 6-well plates, and incubated till reaching 80% confluence of the medium. Then, the sterile pipette (200 μL) tip was used to scratch each well followed by washing with PBS solution several times to abolish cell debris. In the following 48 h, cells were incubated in medium (serum-free), and the migrated cells to the surface of the wound were considered as fabricating an in vitro healing process. The images of the wound healing were obtained by an inverted microscope (Olympus Corporation, magnification: ×100), and the     500 μL of medium containing 10% FBS was put into the lower chamber. Then, cells on the upper chamber were wiped away. Afterward, the culture medium was discarded, and cells were stained with crystal violet for 5 minutes. Subsequently, after washing with PBS and drying, images were taken by a microscope (Olympus, Japan).

Flow-Cytometry Analysis.
A Cell Cycle Kit (Beyotime, China) was used to conduct cell cycle analyses. Immediately following transfection, the cells were extracted and fixed in 70% ethanol for 2 h at 4°C. A centrifuge was used to treat these cells for 5 minutes at 1000 g. These cells were then treated with 25 μL of propidium iodide liquid and 10 μL of RNase A for 30 minutes at 37°C. The cell cycle was measured using the NovoCyte Flow Cytometer (ACEA Biosciences, China).
2.9. TUNEL Apoptotic Assay. Apoptosis of cells after indicated transfection was examined under various circumstances using a TUNEL apoptosis kit (Roche, Basel, Switzerland). In brief, cells were fixed in 4% paraformaldehyde. Afterward, the cells were cleaned with PBS and treated with a fluorescein-labeled TUNEL reaction mixture in a moistened chamber for 1 h at room temperature. A fluorescent microscope was used to count the number of apoptotic cells (×200). Subsequently, apoptotic cells were counted under an optical microscopy.

Dual-Luciferase
Assay. The binding sequences between LINC00893 and miR-146b-3p, miR-146b-3p, and PRSS8 were obtained from bioinformatics tools, LncBase V2, and TargetScan. Then, the targeted fragment was amplified from cDNA by PCR and inserted into a pmirGLO vector. Afterward, HCT116 and SW620 cells were seeded into a 96-well plate at the density of 1 × 10 5 cells/well and cotransfected with pmirGLO vectors and miR-146b-3p mimics using 3000 Lipofectamine (Invitrogen, Carlsbad, USA). Fortyeight hours posttransfection, relative firefly luciferase activities were standardized to luciferase activities of Renilla.
2.11. Pull-Down Assay. miR-146b-3p was labeled with biotin and was transfected into HCT116 and SW620 cells with bio-NC for 48 h. At the end of the process, the cells were lysed, and the complex was incubated with streptavidin magnetic beads for 4 h followed by rinsing three times with wash buffer. Following extraction with Trizol, RT-qPCR assay was used to assess the attached RNA in the complex.         2.14. Bioinformatics and Statistical Analysis. Bioinformatics tool GEPIA [26] was used to create a box plot of LINC00893 expression in colon cancer. Graphpad prism and SPSS softwares Version 20.0 were used in the study for statistical analysis, and all the data were presented as the mean ± standard deviation. T-test, one-way, and two-way ANOVA analyses were used to compare differences in two or more groups. LINC00893, miR-146b-3p, and PRSS8 clinical significances were explored using the receiver operating curve (ROC) based area under the curve (AUC) in the peripheral blood samples. P < 0:05 was considered to have statistical significance.

LINC00893 Expression and Its
The key indicators for tumor metastasis are migration and invasion of cells. The present study used wound healing and Transwell assays to detect the migrative and invasive capacities of colon cancer cells. Wound healing assay showed that the LINC00893 OE significantly inhibited the migration of HCT116 and SW620 cells compared to empty  vector and control groups (Figure 2(c), P < 0:05). Subsequently, Transwell assay was performed to determine the invasive ability of HCT116 and SW620, which showed that LINC00893 OE significantly suppressed the invasion of HCT116 and SW620 cells compared to control groups (Figure 2(d), P < 0:05). Meanwhile, an EdU assay showed similar suppression of proliferation in colon cancer cells by LINC00893 OE (Figure 2(e), P < 0:05). The TUNEL assay demonstrated that the apoptosis rate of HCT116 and SW620 cells was significantly increased in LINC00893 OE group compared to vector and control groups (Figure 2(f), P < 0:05). The cell-cycle arrests were demonstrated by flow-cytometry analysis, which showed that transfection of LINC00893 OE significantly increased the number of cells in G1 phase (Figures 2(g) and 2(h), P < 0:05). Taken together, LINC00893 may act as a tumor suppressor in colon cancer.
Transwell assay was used to determine the effect of LINC00893 OE + miR − 146b − 3p mimics on invasion of HCT116 and SW620 cells. The result demonstrated that miR-146b-3p overexpression reversed the inhibitory effect of LINC00893 upregulation on the invasion of HCT116 and SW620 cells (Figure 2(e), P < 0:05). The MTT assay also showed that the cell viability of colon cancer cells transfected with LINC00893 OE + miR − 146b − 3p mimics was increased compared to the LINC00893 OE groups (Figure 3 3.4. miR-146b-3p Targeted PRSS8 and Their Roles in Colon Cancer Cells. The predictive targeted gene of miR-146b-3p was analyzed by Targetscan [28], and the binding site between miR-146b-3p and PRSS8 was shown in Figure 4(a). To investigate the regulatory mechanism of miR-146b-3p on PRSS8, a series of assays were performed.

Effects of LINC00893 in Colon Cancer
In Vivo. To determine the effects of LINC00893 in colon cancer in vivo, the BALB/c colon cancer cell-based xenograft model was established using HCT116 and SW620 cells. Tumor weight and volume under the influence of LINC00893 OE were evaluated. Mice injected with LINC00893 OE showed significantly smaller tumor sizes in vivo compared with the vector and control group in Figure 5(a). The tumor weight and volume of mice were significantly reduced in LINC00893 OE group compared to other groups ( Figures 5(b) and 5(c), P < 0:05).

Discussion
The disparity in the expression of genes across tumor tissues and normal tissues is important for reflecting cancer biology [29]. Previous investigations have shown that many lncRNAs are aberrantly expressed in tumors compared with that in normal tissues [29][30][31]. Basic characteristics of tumorigenesis include the ability to maintain proliferation, to activate invasion and metastasis, to induce angiogenesis, and to resist apoptosis [32]. In certain cases, lncRNAs may interfere with or inhibit cell growth [33] or engage in the complex interaction of malignant cells [34]. In addition, lncRNAs have been shown to exert impact on cell 16 Journal of Oncology angiogenesis [34]. lncRNAs have the potential to be used as potential biomarkers for the diagnosis, prognosis, and therapy of a variety of diseases. lncRNAs are known to be involved in several regulatory processes [35]. In colon cancer, many lncRNAs were irregularly expressed [36]. The lncRNA RP11 is discovered to be diminished in colorectal cancer tissues and is substantially connected to tumor metastases [4]. Furthermore, upregulation of MALAT1 enhances HIF-1 and endothelial cell protein levels, which influence colon cancer progression [37]. Our study had determined and confirmed the dysregulated expression of lncRNA LINC00893 in colon cancer tumors and adjacent tissues samples. Meanwhile, lncRNA regulates miRNA, which governs protein expression and cell function [38]. Researchers have found numerous human miRNAs, which modulate gene expression levels [39]. The link between miRNAs and colon cancer has gained attention. miRNAs would be potential colon cancer therapy targets [40]. The miR-146b-5p, miR-21, and miR-221 have been linked to lung cancer development and metastasis [30]. miR-146b-3p is a prognostic marker for various tumor types and has been found in several studies [41,42]. Besides, PRSS8 is a tumor suppressor that has been shown to have essential roles in the prevention of colorectal cancer development and metastasis [43]. Present study has revealed the upregulated expression of miR-146b-3p in colon cancer and its downregulation on PRSS8. LINC00893 is demonstrated to be downregulated in tumor tissues and cell lines of colon cancer. On the other hand, LINC00893 took part in colon cancer tumorigenesis by binding with miR-146b-3p to upregulate PRSS8. Moreover, our study found that lncRNA LINC00893, miR-146b-3p, and PRSS8 were valuable in distinguishing colon cancer tumors and adjacent tissue samples. Furthermore, the tumor weight and volume of bxenograft earing mice were significantly reduced after LINC00893 overexpression. Thus, LINC00893, miR-146b-3p, and PRSS8 genes might take part in colon cancer pathogenesis, providing novel diagnostic biomarkers of colon cancer.
Previous research showed that lncRNA LINC00662 overexpression targeting miR-340-5p/CLDN8/IL22 axis promoted proliferation, migration, and invasion and suppressed apoptosis of colon cancer cells via activating the ERK signaling pathway in vitro and in vivo [44]. Similarly, lncRNA HAGLR targeting the miR-185-5p/CDK4/CDK8 axis promoted proliferation, migration, and invasion of colon cancer in vitro and in vivo [45]. Our study has evaluated the biological functions and molecular regulations of LINC00893 in colon cancer cells. Overexpression of LINC00893 or miR-146b-3p inhibition suppressed cell growth, migration, and invasion of HCT116 and SW620 colon cancer cells and promoted the apoptotic activity of cells. Whereas miR-146b-3p overexpression reversed the antitumor effect of LINC00893 upregulation on colon cancer. PRSS8 knockdown rescued the inhibitory effect of miR-146b-3p knockdown on colon cancer progression.
Nonetheless, the current study has some limitations. First, the determination of LINC00893 was screened by an online database, which may present biased microarray results or samples. Second, detection of LINC00893, miR-146b-3p, and PRSS8 was conducted in a small sample sizebased cohort, and therefore, future studies are needed for validation in a larger size-based cohort. Third, the present study did not reveal more clinical characteristics and risk factors of colon cancer patients. Fourth, the function and molecular mechanisms of LINC00893 were evaluated using only two cell lines without further validation of other up/ downregulated genes involved in signaling pathways. Thus, further studies are needed to determine and assess LINC00893, miR-146b-3p, and PRSS8 in detail from various aspects.

Conclusion
LINC00893 overexpression can suppress the progression of colon cancer by binding with miR-146b-3p to upregulate PRSS8. LINC00893, miR-146b-3p, and PRSS8 may serve as novel biomarkers and therapeutic targets of colon cancer, providing new treatment options and research approaches towards colon cancer.

Data Availability
Data archiving will be made available on reasonable request, all of the authors are responsible to the data.