Long non-coding RNA BCYRN1 exerts an oncogenic role in colorectal cancer through regulating miR-204-3p/KRAS axis

Background: It has been well documented that long non-coding RNAs (lncRNAs) regulate numerous characteristics of cancer, including proliferation, migration, metastasis, apoptosis, and even metabolism. LncRNA BCYRN1 (BCYRN1) is a newly identi�ed brain cytoplasmic lncRNA with 200 nucleotides that was discovered to be highly expressed in tumour tissues, including those of hepatocellular carcinoma, gastric cancer and lung cancer. However, the roles of BCYRN1 in colorectal cancer (CRC) remain obscure. This study was designed to reveal the role of BCYRN1 in the occurrence and progression of CRC. Methods: RT-PCR was used to detect the expression level of BCYRN1 in tumour tissues and CRC cell lines. BCYRN1 was knocked down in CRC cells, and cell proliferation changes were evaluated by cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), and Ki-67 and proliferating cell nuclear antigen (PCNA) expression assays. Cell migration and invasion changes were evaluated by wound healing, Transwell and invasion-related protein expression assays. Flow cytometry analysis was used to assess whether BCYRN1 regulates the apoptosis of CRC cells. The dual luciferase reporter gene detects the competitive binding of BCYRN1 to miR-204-3p. In vivo experiments were performed to evaluate the effect of BCYRN1 on tumour development. TargetScan analysis and dual luciferase reporter gene assays were applied to detect the target gene of miR-204-3p. Rescue experiments veri�ed that BCYRN1 affects CRC by regulating the effect of miR-204-3p on KRAS. Results: We found that compared with normal tissues and human intestinal epithelial cells (HIECs), CRC tumour tissues and cell lines had signi�cantly increased BCYRN1 levels. We further determined that knockdown of BCYRN1 inhibited the proliferation, migration, and invasion and promoted the apoptosis of CRC cells. In addition, bioinformatics analysis and dual luciferase reporter assay showed that BCYRN1


Background
Colorectal cancer (CRC) has a high incidence and mortality worldwide; speci cally, it ranks third in cancer morbidity and second in cancer mortality globally [1,2].CRC has become one of the most common and dangerous malignancies in China [3].The mainstay treatment for CRC is surgery, chemotherapy, radiotherapy and other comprehensive therapies, but the 5-year survival rate has not changed signi cantly in recent decades and is still poor [4,5].Therefore, delivering high-e ciency CRC diagnosis and treatment methods should be the aim of all physicians in internal medicine research.The present study aims to provide an up-to-date molecular mechanism of CRC occurrence.
Recently, epigenetic changes have been reported to play important roles in the occurrence and progression of malignancies [6,7].LncRNAs are a class of transcripts with lengths greater than 200 nucleotides and have limited or non-protein coding potential [8][9][10].Various studies have demonstrated that lncRNAs participate in the multilevel regulation of gene expression by targeting microRNAs (miRNAs), mRNAs, and even proteins [11,12].Furthermore, growing evidence has proven that lncRNAs regulate multiple characteristics of cancer, such as proliferation, migration, apoptosis, and even metastasis [13,14].BCYRN1 is a brain cytoplasmic lncRNA that is newly identi ed and activated by c-MYC [15].It has been reported that BCYRN1 is highly expressed in various tumour tissues [16], such as hepatocellular carcinoma [17], gastric cancer [18], and lung cancer [15] but is expressed at lower levels in healthy control (HC) tissues.In addition, BCYRN1 plays an important role in inhibiting smooth muscle differentiation and vascular development in the cardiovascular system [19,20].However, little is known about the role of BCYRN1 in CRC, which aroused our great interest.
MiRNAs are a group of small, non-coding RNAs with nucleotides of approximately 22 and are identi ed as negative regulators [21,22].miRNAs can act as targets of lncRNAs and participate in the regulation of gene expression.Speci cally, miRNA is involved in gene expression by binding to the complementary sequence location of the 3'-untranslated region (3'-UTR) of target mRNA to disturb the structural stability and translation of mRNAs [23,24].Increasing evidence has demonstrated that miRNAs play crucial roles in cancer, cardiovascular disease and other diseases [22,25] by regulating relative cell proliferation, migration, invasion and apoptosis [23].On the other hand, lncRNAs competitively bind to miRNAs as ceRNAs, resulting in the inability of the miRNAs to combine with their corresponding mRNAs, thus regulating gene expression and function [11,26,27].
The present study aimed to determine the function and underlying mechanism of BCYRN1 in CRC progression.Our research determined that BCYRN1 plays an oncogenic role in CRC by regulating the miR-204-3p/KRAS axis, revealing a novel mechanism for the occurrence and development of CRC and providing a novel therapeutic and prognostic target for CRC.

Experimental human tissues and animals
The research tissues of CRC patients and healthy controls were obtained from The A liated Cancer Hospital of Nanjing Medical University, and all patients signed written consent forms.Tissue debris was immediately frozen in liquid nitrogen after surgery and stored at −80°C.All animal experiments were designed according to the standards of the Guide for the Care and Use of Laboratory Animals (NIH, 8th edition, 2011).The Ethics Committee of The A liated Cancer Hospital of Nanjing Medical University approved the present research.

RNA extraction and qRT-PCR
Total RNA was extracted from human tissues, mouse tissues and cells with TRIzol reagent (Life Technologies, Gaithersburg, MD, USA).Reverse transcription experiments were performed with PrimeScript® RT reagent Kits (Takara, Otsu, Shiga, Japan) and a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).Next, quantitative reactions were performed with SYBR Green RT-PCR (Takara Biotechnology Co., Ltd., Tokyo, Japan) using the StepOnePlus™ system.GAPDH and U6 snRNA were used as the internal controls.The primer sequences used for qPCR are shown in supplementary Table S1.

Evaluation of cell proliferation
Cell proliferation was detected by cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) incorporation test, and nucleoprotein Ki-67 and proliferating cell nuclear antigen (PCNA) expression analysis.First, for the CCK-8 experiment, cells were seeded into a 96-well plate with 10% FBS DMEM for 24 h.Then, the medium was changed with serum-free DMEM for CCK-8 kit (Beyotime Biotechnology, Shanghai, China) detection.A microplate reader (Model ELX800, BioTek, Vermont, USA) was used to determine the absorbance at 450 nm, which re ects cell viability and proliferation.Second, cells were seeded into a 24-well plate for EdU assessment.After 48 h of transfection, DNA synthesis was examined with an EdU incorporation assay (Guangzhou RiboBio, Guangzhou, China).The proportion of EdUpositive cells was analysed by uorescence microscopy (DP70, Olympus Optical, Tokyo, Japan).Finally, cell proliferation was evaluated by Ki-67 and PCNA expression.Ki-67 is a nucleoprotein that is a marker of tumour proliferation [28].PCNA acts on chromatin and participates in all aspects of the DNA replication chain [25].The expression levels of Ki-67 and PCNA can be used to evaluate the status of cell proliferation.

Evaluation of cell migration and invasion
Cell migration and invasion were assessed with the wound healing assay, Transwell assay and invasionrelated protein expression.For the wound healing assay, cells were seeded into a 6-well plate.A sterile 1-mL pipette tip was used to scratch the bottom of the plate to form a gap.The images of cell migration were captured at 0 h and 48 h after transfection, respectively, with an inverted microscope (Axio Vert.A1, Zeiss, Oberkochen, Germany).The scratch distance at 48 h was subtracted from the scratch distance at 0 h to calculate the percentage closure of the wound [29].The average distance of migration re ects the cell migration capacity.For the Transwell assay, cells were plated into the no FBS medium in the upper chamber of a 12-well Transwell with an 8-μm pore size (Merck kGaA, Darmstadt, Germany).FBS medium (10%) was added to the lower chamber.After 24 h, the cells that migrated to the submembrane surface were stained with crystal violet.Stained cells were counted in 6 randomly selected regions.

Flow cytometric analysis
Flow cytometric analysis was designed to evaluate whether BCYRN1 regulated the apoptosis of CRC cells.Consistently, cells were seeded into a 6-well plate.The cells were collected and stained with annexin V-FITC (Beyotime Biotechnology, Shanghai, China) and propidium iodide (PI) reagent after 48 h transfection according to the manufacturer's instructions.Then, cell apoptosis was determined by ow cytometry (BD FACSCalibur cytometer, Becton Dickinson, San Jose, CA, USA).

Dual luciferase reporter assay
The partial sequences of BCYRN1 and the 3'-untranslated region (3'-UTR) of KRAS containing the putative binding sites of miR-204-3p were synthesized and inserted into a luciferase reporter vector plasmid (GenePharma, Shanghai, China).According to the instructions, re y luciferase reporter plasmids and equal amounts of miR-204-3p mimics or NC mimics were transfected into cells.Then, the relative luciferase activities were detected by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) on a Luminometer 20/20n (Turmer Biosystems, Sunnyvale, CA, USA) after transfection for 48 h.Renilla luciferase activity was employed as an internal control for cellular density and transfection e ciency.

In vivo experiments
The nude mice were injected subcutaneously with CRC cells (approximately 10 6 cells) into the right anks.Then, sh-NC or sh-BCYRN1 was directly injected into the mice to knockdown BCYRN1.Tumour size was measured every 5 days for a total of 6 measurements.At the end of the experiments, mice were humanely sacri ced with overdose anaesthesia for the collection of tumours.Each tumour was weighed to evaluate the effects of BCYRN1 on the development of tumours.

Immunohistochemistry
Tumours were obtained, xed in 4% paraformaldehyde, embedded in para n, and then cut into 4 mm sections.After depara nization and blocking, the sections were incubated with primary anti-Ki-67 (1:100; Abcam; Cambridge, MA, USA) overnight at 4°C.The immuno uorescence images were captured with an Olympus BX51 microscope (Olympus, Tokyo, Japan) after incubating horseradish peroxidase-conjugated goat anti-rabbit antibody.

HE staining and TUNEL staining
Haematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nickend labelling (TUNEL) staining were performed to detect cancer cell apoptosis in mouse tumour tissues.Brie y, mouse tumour tissues were pre xed, and the para n-embedded sections were stained with haematoxylin-eosin and TUNEL (containing 2 mL TdT enzyme, 48 mL Fluorescent labelling solution and 50 mL TUNEL detection solution) according to the manufacturer's instructions.The images were obtained with an Olympus BX51 microscope (Olympus, Tokyo, Japan) coupled with an Olympus DP70 digital camera.

Western blot analysis
Cyclooxygenase-2 (Cox-2) is an enzyme complex that plays signi cant roles in metastasis and invasion of malignancies [30].The matrix metalloproteinase (MMP) family, especially the gelatinases MMP-2 and MMP-9, are recognized as markers of tumour migration and invasion [31].The expression of Cox-2, MMP-2 and MMP-9 can re ect the status of cell migration and invasion.The protein expression levels of Ki-67, PCNA, Cox-2, MMP-2, MMP-9, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9 and KRAS were detected by Western blot analysis.Total protein was extracted, and equal amounts of protein (30 mg) were separated by SDS-PAGE and then transferred to PVDF membranes.Protein bands were visualized with the Enhanced Chemiluminescence Detection Kit (Thermo Fisher Scienti c, Rockford, IL, USA).All primary antibodies were obtained from Abcam (Cambridge, MA, USA).The dilution of PCNA antibody was 1:5,000, and the dilution ratio of other primary antibodies was 1:1,000.

Statistical analysis
Experiments were conducted in a randomized, double-blinded situation.Data are expressed as the mean±SD.Student's unpaired t-test was used to compare the differences between two groups.One-way or two-way ANOVA followed by post hoc Bonferroni test was employed for multiple comparisons.Statistical signi cance was considered as *P < 0.05, **P < 0.01, and ***P < 0.001.

Results
BCYRN1 is highly expressed in the tissues and cell lines of CRC In our preliminary study, we noted that BCYRN1 was highly expressed in CRC tissues (Figure 1A).Then, we examined the expression levels of BCYRN1 in HIECs and four types of human CRC cell lines.Consistently, BCYRN1 expression was signi cantly increased in CRC cell lines (Figure 1B).These data aroused our interest, as they suggested that BCYRN1 levels may be closely related to the development of CRC.

Knockdown of BCYRN1 inhibits the proliferation and promotes the apoptosis of CRC cells
To further illuminate the roles of BCYRN1 in CRC, a series of experiments were designed in LoVo and SW480 cells.First, we used sh-BCYRN1 to downregulate endogenous BCYRN1, and the knockdown e ciency is shown in Figure 2A.CCK-8 and EdU assays showed that downregulation of BCYRN1 evidently decreased the proliferation of LoVo and SW480 cells (Figure 2 B & C).Furthermore, knockdown of BCYRN1 obviously inhibited the expression of proliferation-related proteins, such as PCNA and Ki-67 (Figure 2D).Subsequently, we detected cell apoptosis by ow cytometry analysis.The results showed that knockdown of BCYRN1 signi cantly increased apoptosis of LoVo and SW480 cells (Figure 2E).In addition, several pro-apoptosis proteins, including Bax, cleaved caspase-3 and cleaved caspase-9 [32,33], were highly expressed in LoVo and SW480 cells when they were treated with sh-BCYRN1.In contrast, the expression of Bcl-2, an inhibitor of cell apoptosis [33], was abolished in the sh-BCYRN1 group (Figure 2F).These results indicated that BCYRN1 knockdown reduced proliferation while promoting the apoptosis of CRC cells.

Downregulation of BCYRN1 decreases the migration and invasion of CRC cells
Next, we investigated the effects of BCYRN1 expression on CRC cell migration and invasion.Both wound healing and Transwell assays revealed that the migration and invasion of LoVo and SW480 cells were effectively suppressed by sh-BCYRN1 transfection (Figure 3 A & B).Consistently, migration-and invasionrelated proteins, such as Cox-2, MMP-2 and MMP-9, were inhibited in LoVo and SW480 cells treated with sh-BCYRN1 (Figure 3C).These results elucidated that BCYRN1 levels were closely related to the migration and invasion properties of CRC cells.

miR-204-3p is a target gene of BCYRN1
A recent report showed that lncRNAs function as ceRNAs to competitively bind to miRNAs [11].To illuminate the potential regulatory mechanism through which BCYRN1 affects CRC progression, we performed bioinformatic analysis with LncBase Predicted V.2 and found that BCYRN1 possesses a putative binding site for miR-204-3p (Figure 4A).We further retrieved potential targets of BCYRN1 in different databases (Table S2).To determine the interaction of BCYRN1 and miR-204-3p, we performed dual luciferase reporter analysis and found a signi cant reduction in the luciferase activity of BCYRN1 WT when LoVo or SW480 cells were transfected with miR-204-3p mimics.However, there was no notable difference in the luciferase activity of BCYRN1 Mut between the miR-204-3p mimic group and the negative control (NC) group (Figure 4B).Accordingly, downregulation of BCYRN1 distinctly increased the expression of miR-204-3p in LoVo and SW480 cells (Figure 4C).In addition, qRT-PCR results revealed that To evaluate the therapeutic potential of BCYRN1 in CRC patients, we performed BCYRN1 knockdown by using sh-BCYRN1 in CRC model mice.Encouragingly, knockdown of BCYRN1 signi cantly decreased the tumour volume and weight of CRC model mice (Figure 8 A-C).HE staining, immunohistochemical staining and TUNEL staining showed that downregulation of BCYRN1 inhibited Ki-67 expression and promoted cancer cell apoptosis (Figure 8D).More importantly, sh-BCYRN1 treatment obviously promoted the expression of miR-204-3p and decreased the expression of KRAS (Figure 8E).These results revealed that knockdown of BCYRN1 inhibited the development of CRC.

Discussion
Cancer mortality remains an enormous public health challenge on a global scale [35].Although new advances have been made in the genomic changes in most different cancers [36], the molecular mechanisms underlying the development of CRC are poorly understood.Moreover, compared with patients with other malignancies, a large number of CRC patients have distant metastasis at the early stage of diagnosis, which leads to a low 5-year survival rate of approximately 10%-20% [37].In addition, therapeutic methods are very limited in advanced CRC patients and mainly involve combinations of toxic drugs, such as 5-uorouracil, irinotecan and/or oxaliplatin, for chemotherapy.Therefore, the molecular mechanism involved in the occurrence and development of CRC needs to be explored urgently.Our current study revealed an important new mechanism of the occurrence of CRC and provided a novel idea for the effective treatment of CRC, brie y, to control CRC progression by blocking BCYRN1.
LncRNAs have been shown to play vital roles in the regulation of cancer progression [13,14], and the main functions of lncRNAs are recruiting chromatin modi cation complexes for transcriptional regulation [38] and interacting with miRNAs, mRNAs, and/or proteins for posttranscriptional regulation [11,12].It has been proven that one of most common and crucial roles of lncRNA is their functions as a "miRNA sponges" [27,39].This means that lncRNA can be used as a bait to bind with a speci c miRNA, resulting in the inability of the miRNA to combine with its corresponding mRNA.BCYRN1 is a newly identi ed lncRNA with similar characteristics [15].Previous studies have revealed that miRNAs, such as miR-125-5p, miR-149 and miR-490-3p, act as targets of BCYRN1 in hepatocellular carcinoma, human glioma and lung cancer, respectively [40,41,17].Recently, the relationship between BCYRN1 and CRC has been gradually observed.Gu L et al rst found that BCYRN1 levels were closely related to the occurrence, development and prognosis of CRC, and microarray bioinformatics analysis con rmed that the possible target of BCYRN1 was the NRP3 gene [42].Moreover, Yu JH and Chen Y proved that BCYRN1 levels were signi cantly increased in tumour tissues and cell lines (SW620) of CRC by a series of well-designed in vitro experiments [43].However, it remains obscure whether BCYRN1 can control miRNAs to regulate the progression of CRC.Our present study indicated that BCYRN1 is involved in the occurrence and development of CRC through speci c binding to a new target miRNA: miR-204-3p.We found that BCYRN1 was highly expressed, while the expression levels of miR-204-3p were signi cantly reduced in the tissues and cell lines of CRC compared to those of controls.In addition, knockdown of BCYRN1 distinctly increased miR-204-3p expression.These ndings provide new insights into the mechanism of CRC progression and the treatment of CRC.
As mentioned above, miRNAs are involved in the regulation of cancer [22,23].Interestingly, miRNAs can act as tumour suppressor genes [44] or oncogenes [45] to regulate the biological characteristics of cancer.Cui ZH et al reported that miR-204-3p acts on its potential target gene bronectin 1 (FN1) and inhibits its expression, thereby decreasing the growth of hepatocellular carcinoma tumour endothelial cells [46].Recently, several studies have reported the roles of miR-204-3p in ovarian cancer [47], lung adenocarcinoma [48] and bladder cancer [49], but the functions of miR-204-3p in CRC remain obscure.Our current research showed that miR-204-3p is a potential target gene of BCYRN1 and is negatively regulated by BCYRN1.We further investigated whether miR-204-3p overexpression inhibited CRC cell proliferation, migration and invasion while exacerbating CRC cell apoptosis.In addition, bioinformatics analysis and dual luciferase reporter assay proved that KRAS was a potential target gene of miR-204-3p.
KRAS is an important oncogene and a key tumour maintenance gene in many carcinomas [34].Then, we showed that KRAS was highly expressed in CRC tissues and cell lines, and overexpression of miR-204-3p eliminated this effect.These results indicated that overexpression of miR-204-3p and downregulation of KRAS can be considered new approaches to the treatment of CRC.
In summary, these ndings demonstrated that BCYRN1 participates in the occurrence and development of CRC.In terms of functional regulation, BCYRN1 promotes CRC progression at least in part by regulating the miR-204-3p/KRAS axis.Our study broadens the current understanding of BCYRN1 function in CRC and provides a novel therapeutic target for CRC.

Conclusion
1. BCYRN1 is highly expressed in CRC tissues and cell lines compared to normal controls.

Downregulation of BCYRN1 decreases the migration and invasion of CRC cells. Knockdown of
BCYRN1 promotes the apoptosis of CRC cells.

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