Long Noncoding XLOC_006390 Regulates the Proliferation and Metastasis of Human Colorectal Cancer via miR-296/ONECUT2 Axis

Long noncoding RNA (LncRNA) XLOC_006390 has been shown to be dysregulated in cancer tissues and regulates cancer growth and development. Nonetheless, the molecular role of lncRNA-XLOC_006390 in colorectal cancer via modulation of miR-296/ONECUT2 axis is still unclear. Against this backdrop, the current study was designed to explore the role of lncRNA-XLOC_006390 in colorectal cancer proliferation and metastasis. The results revealed significant (P < 0.05) overexpression of lncRNA-XLOC_006390 in colorectal cancer tissues and cell lines, and the transcript levels increased with the advancement of the disease. Moreover, its high expression was shown to be associated with poor patient survival. Silencing of lncRNA-XLOC_006390 in colorectal cancer cells significantly (P < 0.05) suppressed their viability via onset of apoptosis and restricted cancer cell migration and invasion. In vivo tumor growth was significantly (P < 0.05) inhibited under lncRNA-XLOC_006390 repression. LncRNA-XLOC_006390 was shown to sponge the expression of miR-296-3p which in turn acted via post-transcriptional suppression of ONECUT 2 transcription factor to regulate the growth of colorectal cancer. Taken together, the results revealed the oncogenic role of lncRNA-XLOC_006390 in colorectal cancer via modulation of miR-296/ONECUT2 axis. The results also point towards its prognostic and therapeutic potential in the treatment of colorectal cancer.


Introduction
For their surprisingly immense expression in eukaryotes, the noncoding RNAs were earlier described as the unavoidable transcriptional noise emerging out of exceptionally low reliability of RNA polymerase [1]. However, the advancement in transcriptomics has led to ascription of functional roles to a great majority of noncoding RNAs [2]. e long noncoding RNAs (lncRNAs) constitute a relatively heterogenous class of single-stranded noncoding RNAs arbitrarily greater than 200 nucleotides in size [3]. e lncRNAs are regulatory in function and profoundly affect the expression of specific target genes through varying mechanisms in both the nucleus and the cytoplasm, depending upon their subcellular localization [4]. It is well understood that lncRNAs through their coordination with other molecules affect the eukaryotic physiology and development, and even little functional deviation of lncRNAs has been shown to result in many pathological conditions including cancer [5,6]. Many lncRNAs regulate one or several key aspects of human tumorigenesis like uncontrolled cell division, avoidance of apoptotic cell death, and metastatic behavior of cancer cells [7]. e cancer biologists have proposed to explore the essence of lncRNAs as biomarkers and feasible therapeutic targets to assist in combating crucial facets of cancer [8]. From the recent research investigations, several lncRNAs were found to be associated with pathogenesis of human colorectal cancer and proved to be involved in either promoting or restricting the growth and metastasis of colorectal cancer cells [9,10]. Colorectal cancer is highly prevalent in developed countries, annually affecting about 100 million people with mortality rate nearing to 33% [11]. e disease of colorectal cancer is seen with high recurrence and re-appears in half of the patients within five years of disease treatment and results in their death [11]. Scientists are looking at lncRNAs with tremendous possibilities to serve as potential diagnostic and therapeutic biological agents against colorectal cancer [12]. In the present study, the functional role of lncRNA-XLOC_006390 was illustrated in colorectal cancer. LncRNA-XLOC_006390 acts as the crucial regulator of tumorigenesis of human cancers like cervical cancer and pancreatic cancer [13,14]. However, its functionality is yet to be understood in colorectal cancer. e results of present study were supportive of oncogenic behavior of lncRNA-XLOC_006390 in promoting the growth and metastasis of colorectal cancer cells. LncRNA-XLOC_006390 was shown to be abundantly expressed in colorectal cancer and its silencing induced apoptosis in cancer cells and inhibited their proliferation. e downregulation of lncRNA-XLOC_006390 also restricted cell migration and invasion in vitro and restrained the in vivo tumor growth. LncRNA-XLOC_006390 was shown to be interacting with and sponging the expression of microRNA-296 (miR-296) in colorectal cancer. e latter was found to be targeting ONECUT 2, a homeobox transcription factor in colorectal cancer. ONECUT 2 acts as a key transcriptional regulator of human tumorigenesis [15]. e present study thus indicated that the enhancement in miR-296 sponging by lncRNA-XLOC_006390, due to overexpression in colorectal cancer, alleviates posttranscriptional targeting of ONECUT 2, and the latter promotes the cancer growth and proliferation.

Collection of Tissue Specimens.
e 79 colorectal cancer tissues and adjacent normal tissues were obtained during the period of 2013-2016 from the patients prior to application of radio/chemotherapies at the Second Affiliated Hospital of Harbin Medical University Harbin, China, after informed consent of the patients. e tissues were frozen using liquid N 2 and stored at ultra-low temperatures for future use. e characteristics of the patients are presented in Table 1. e pathologists of the hospital assisted in the pathological staging of the cancerous specimens. All standard ethical guidelines were followed, and the study was approved by the research ethics committee of the Second Affiliated Hospital of Harbin Medical University Harbin, China, under approval number HMU-3381IV-2020.

Transfection.
e overexpression vector pcDNA3.1 ( ermo Fisher Scientific) was used for generating the construct ONECUT 2, while empty pcDNA3.1 vector served as expression control. All other expression constructs including si-XLOC_006390, miR-296 mimics, and si-ONE-CUT 2 together with the respective negative controls were ordered from Gemma Pharmaceutical Technology Co., Ltd. (Shanghai, China).

Quantitative Real Time-PCR (qRT-PCR).
TRIzol reagent ( ermo Fisher Scientific) was used for extracting total RNA from tissues and cells. For expression analysis of lncRNA-XLOC_006390 and ONECUT 2, cDNA was synthesized with the help of first strand cDNA synthesis kit (Applied Biosystems Inc., Foster City, CA). e qRT-PCR was performed on QuantStudio 3.0 RT-PCR system ( ermo Fisher Scientific) using SYBR Green PCR mix ( ermo Fisher Scientific). e U6 and GAPDH were used as references for the determination of expression. e relative gene expression was calculated by 2 −ΔΔCt method. e sequences of the primers used in the study are presented in Table 2.

Western
Blotting. e transfected cancer cells were treated with RIPAlysis buffer ( ermo Fisher Scientific) to isolate total proteins which were quantified by Lowry method. Equal amounts of protein lysates were separated on 10% SDS-PAGE. e PAGE gels were blotted to PVDF membranes, and 5% skimmed milk powder was used to block the membranes. e membranes were subsequently incubated with primary antibodies e membranes were washed three times with TBST and incubated for 2 h with appropriate HRP-conjugated secondary antibody anti-rabbit IgGHRP (7074s, Cell Signaling Technology, Danvers, USA) (Dilution; 1 : 2000). Finally, protein bands were detected through chemiluminescence. Human β-actin protein acted as the protein expression control.

Scratch Healing Migration Assay.
e migration of transfected colorectal cancer cells was measured with the help of scratch-heal method.
e transfected cells were cultured in 6-well plates for 72 h, following which the cell surface was scratched perpendicularly with 10 μl pipette-tip. After 24 h of incubation at 37°C, the width of the scratch was observed and compared with width at 0 h to estimate the cell migration.

Transwell Invasion Assay.
e cancer cell invasion was deduced using transwell chambers (Corning, New York, USA). 250 μl of cell suspension in serum-free RPMI-1640 medium carrying 2.5 × 10 5 cancer cells was added to the upper chamber. e lower chamber was added with 750 μlRPMI-1640 medium supplemented with serum. e transwell chamber plate was incubated at 37°C for 24 h, and then the cells invading the lower chamber were ethanol fixed and subsequently stained with 0.1% crystal violet. Light microscope was used for visualizing the cells, and five random microscopic fields were used for cell counting.

Mice Xenograft Study.
e xenograft transplantation of colorectal cancer cells was carried out in 5-week old BALB/c nude mice procured from Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing China. Well aerated rooms were used for maintaining the experimental animals in Animal House.
e Animal care and experimentation committee approved the in vivo study. e animals were randomly divided into two groups. e CaCo 2 cancer (5 × 10 6 ) cells at exponential growth stage were injected subcutaneous into the dorsal flank of nude mice. e respective mice groups were administered with intratumor injections carrying si-XLOC_006390 or negative control, si-NC. e tumor volume was examined after every 3 days and the mice were finally sacrificed at the end of 3 weeks. e xenograft tumors were then excised; their size and average weight were measured.

Immunohistochemistry (IHC) Assay.
e xenograft tumors were excised from each animal, sliced, and fixed in 10% v/v formal saline. e tumors were subsequently embedded in paraffin; sections of 4 μm were prepared. ese Table 2: List of primers used in the study.

Primer
Direction Sequence sections were subjected to IHC staining with anti-Ki67 and anticleaved caspase-3 (Abcam) and mounted on slides previously coated with Mayer's albumin. Cover slip was mounted using DPX and observed using OLYMPUS BX51 microscope, and the images were captured with OLYMPUS DP 72 camera attached to the microscope.

Bio-Informatics Analysis and Dual Luciferase
Assay. e predictions of the corresponding molecular targets of lncRNA-XLOC_006390 and miR-296 were made through online bioinformatics. LncBase Experimental v.2 prediction tool was used for predicting the miRNA specifically interacting with lncRNA-XLOC_006390. e mRNA specifically targeted by miR-296 was predicted through TargetScan Human 7.2 (https://www.targetscan.org/vert_72/) online tool. e interaction of lncRNA-XLO_006390 and miR-296 was studied in vitro using dual luciferase reporter assay system (Promega) as per manufacturer's guidelines.

Statistical Analysis.
e experiments were performed in triplicate and expressed as mean ± SD. SPSS 22.0 (IBM, SPSS, Chicago, IL, USA) software was used for performing the statistical analyses. Student's t-test was used for analyzing the difference between two data sets. P < 0.05 was considered as statically significant difference. e Kaplan-Meier method was employed to appraise the survival rate and the analysis was carried out using the log-rank test.

LncRNA-XLOC_006390 Is Overexpressed in Colorectal
Cancer. To elucidate the expression of lncRNA-XLOC_006390 in colorectal cancer, qRT-PCR was used. e results showed that the cancer tissues possessed significantly (P < 0.05) higher expression of lncRNA-XLOC_006390 in comparison to the normal adjacent tissues (Figure 1(a)). Interestingly, the expression of lncRNA-XLOC_006390 was found to increase with the advancement of colorectal cancer (Figure 1(b)). Based on the expression of lncRNA-XLOC_006390, the patients from whom tissue collection was made were categorized into high expression or low expression group. e survival period was found to be markedly higher for the patients with lower expression of lncRNA-XLOC_006390, while the high-expression patients survived for lesser periods. e relationship between the patient survival and expression of lncRNA-XLOC_006390 was analyzed with the help of Kaplan-Meier test (Figure 1(c)). qRT-PCR was also performed to analyze the expression of lncRNA-XLOC_006390 in colorectal cancer cell lines (CaCo 2 , HCT-15, and SW-40) and normal epithelial colorectal cell line (CRL-1790). All three colorectal cancer cell lines were found to express significantly (P < 0.05) higher levels of lncRNA-XLOC_006390 in comparison to the normal colorectal cells (Figure 1(d)). Together, the results suggest that colorectal cancer is associated with lncRNA-XLOC_006390 upregulation, and the expression of lncRNA-XLOC_006390 negatively correlated with the survival of colorectal cancer patients.

Silencing of lncRNA-XLOC_006390 Induced Apoptosis in
Colorectal Cancer Cells. To find out whether lncRNA-XLOC_006390 regulates the growth and proliferation of colorectal cancer cells, its expression was silenced in HCT-15 and CaCo 2 cancer cells, and the same was confirmed by qRT-PCR expression study (Figure 2(a)). e MTT assay showed that silencing of lncRNA-XLOC_006390 suppresses the proliferation of both HCT-15 and CaCo 2 (Figure 2(b)).
e AO/EB staining revealed that lncRNA-XLOC_006390 silencing triggered apoptosis of HCT-15 and CaCo 2 cancer cells (Figure 2(c)). Similar inference was made by the flow cytometric study where the percentage of apoptotic cells was deduced to be significantly (P < 0.05) higher under lncRNA-XLOC_006390 silencing in HCT-15 and CaCo 2 cancer cells when compared with the negative control cells (Figure 2(d)).
e levels of pro-apoptotic proteins like Bax and cleaved caspase-3 were also shown to be enhanced under lnc-XLOC_006390 repression (Figure 2(e)).
e results thus support that lncRNA-XLOC_006390 silencing suppresses the proliferation of colorectal cancer via induction of apoptosis.

LncRNA-XLOC_006390 Knockdown Inhibited the Migration and Invasion of Colorectal Cancer Cells.
e effect of the transcriptional knockdown of lncRNA-XLOC_006390 in colorectal cancer cells was further investigated on the migration and invasion of cancer cells. It was found that silencing of lncRNA-XLOC_006390 caused significant (P < 0.05) inhibition of HCT-15 and CaCo 2 cancer cell migration (Figure 3(a)). e invasion of cancer cells was also reduced significantly (P < 0.05) under lncRNA-XLOC_006390 silencing (Figure 3(b)). e western blotting of the metastasis marker proteins revealed that downregulation of lncRNA-XLOC_006390 decreased the expression of N-cadherin, metalloproteinase 2 (MMP-2), and metalloproteinase 9 (MMP-9), while the expression of E-cadherin protein increased (Figure 3(c)). Collectively, lncRNA-XLOC_006390 positively regulates the colorectal cancer metastasis suggesting its therapeutic value.

LncRNA-XLOC_006390 Downregulation Limited in Vivo Growth of Xenograft Mice Tumors.
e xenograft mice model was used for assessing in vivo role of lncRNA-XLOC_006390 in regulating the tumorigenesis of colorectal cancer. e mice xenografted tumors administered to repress the expression of lncRNA-XLOC_006390 exhibited significantly (P < 0.05) lower tumor size in comparison to the negative control mice (Figure 4(a)). e repression was also shown to negatively affect the average tumor volume (Figure 4(b)). After the animal sacrifice, the tumors were excised, and it was found that the average weight was significantly (P < 0.05) lower for the mice tumor xenografts downregulating lncRNA-XLOC_006390 as that of the negative control animals (Figure 4(c)). e mice tumors 4 Journal of Oncology were seen to express higher levels of apoptosis marker protein, cleaved caspase-3, while the proliferation marker protein, ki67, showed exceptionally low expression under lncRNA-XLOC_006390 downregulation (Figure 4(d)). Taken together, the results are supportive of growth regulatory role of lncRNA-XLOC_006390 in colorectal cancer, further indicating it as a potential therapeutic target in colorectal cancer.

LncRNA-XLOC_006390 Sponges miR-296-3p Expression in Colorectal
Cancer. e in silico analysis predicted that lncRNA-XLOC_006390 interacts in a sequence specific manner with miR-296-3p ( Figure 5(a)). To validate the prediction, luciferase reporter assay was performed. e significant (P < 0.05) reduction in the luciferase activity of colorectal cancer cells co-transfected with miR-296 mimics and native reporter construct of lncRNA-XLOC_006390, in comparison to that when mutated (MUT) sequence site of lncRNA-XLOC_006390 was used, thereby confirming the interaction betweenlncRNA-XLOC_006390 and miR-296-3p in colorectal cancer ( Figure 5(b)). e miR-296-3p expression was found to be significantly (P < 0.05) lower in colorectal cancer tissues relative normal tissues ( Figure 5(c)). e expression was also significantly (P < 0.05) downregulated in the colorectal cancer cell lines in comparison to normal cell line, CRL-790 ( Figure 5(d)). Further, the CaCo 2 cancer cells exhibited significantly (P < 0.05) higher transcript levels of miR-296-3p under lncRNA-XLOC_006390 downregulation ( Figure 5(e)). e miR-296-3p was overexpressed in CaCo 2 cancer cells as confirmed by its relative expression ( Figure 5(f )). e cancer cells overexpressing miR-296-3p proliferated at minimal rate in comparison to the corresponding control cells ( Figure 5(g)).
Collectively, the results suggest that lncRNA-XLOC_006390 sponges miR-296-3p expression in colorectal cancer and its overexpression declines miR-296-3p expression to improve the proliferative ability of cancer cells.

LncRNA-XLOC_006390 Regulates ONECUT 2 Expression through miR-296-3p Sponging.
To identify the downstream target of miR-296-3p, online bioinformatics was used. e homeobox transcription factor ONECUT 2 was identified to be the regulatory target of miR-296-3p in colorectal cancer. e miR-296-3p was shown to bind 3′UTR of ONECUT 2 in sequence dependent mode (Figure 6(a)). As expected, the expression of ONECUT 2 gene was significantly (P < 0.05) low in cancer tissues and cell lines (Figures6(b) and 6(c)). e levels of ONECUT 2 protein level was found to decrease under miR-296-3p overexpression (Figure 6(d)). Similar reduction in the expression of ONECUT 2 protein was observed under lncRNA-XLOC_006390 silencing (Figure 6(e)). Transcriptional repression of ONECUT 2 in CaCo 2 colorectal cancer cells declined the cell proliferation significantly (P < 0.05) (Figure 6(f )). Conversely, the   Journal of Oncology overexpression of ONECUT 2 in colorectal cancer cells rescued them from the proliferation loss both under miR-296-3p overexpression or lncRNA-XLOC_006390 silencing (Figures 6(g) and 6(h)). Taken together, the results suggest that lncRNA-XLOC_006390 executes its regulatory role in colorectal cancer through miR-296/ONECUT 2 regulatory axis. Both lncRNA-XLOC_006390 and miR-296-3p might be utilized as potential therapeutic targets against colorectal cancer.

Discussion
Colorectal cancer is ranked as the third most diagnosed human cancer and considered as the fourth most death causing cancer at the global level [16]. e lack of effective prognostic measures and disease recurrence are the dominant factors responsible for higher mortality of colorectal cancer. In depth understanding of various regulatory mechanisms controlling the growth and progression of colorectal cancer is thus indispensable to formulate better screening and treatment procedures against this lethal malignancy. e lncRNA-XLOC_006390 was found to be overexpressed in colorectal cancer in the present study. e overexpression of lncRNA-XLOC_006390 has been shown to be associated with several human cancers, and lncRNA-XLOC_00630 was proven to have oncogenic role in cancer tumorigenesis [14,17]. Further, it was previously shown that overexpression of lncRNA-XLOC_006390 marks the pathological advancement of human cancer and is linked with the disease metastasis [18]. Supporting the same, the results of our present study showed that the expression of lncRNA-XLOC_006390 increases with the progression of colorectal cancer and promotes the cancer metastasis which highlights the potency of lncRNA-XLOC_006390 to serve in the disease prognosis and therapeutic formulations. LncRNAs are involved in miRNA sponging, and, through this ability, they effectively alter the expression levels of some key miRNA molecules which gets manifested in malignancies like cancer [19]. e sponging of miRNAs by lncRNA-XLOC_006390 has been shown to promote tumorigenicity of cervical cancer [13]. Here, it was revealed that miR-296-3p expression negatively correlated with lncRNA-XLOC_006390 expression, and sponging role of the latter was made clear through in vitro experimentation. e miR-296-3p has tumor-suppressive role in colorectal cancer and inhibits the cancer cell metastasis [20]. e lower expression of miR-296-3p was expounded to be involved in chemotherapeutic resistance and marks poor clinical outcomes of colorectal cancer [21]. e miR-296-3p was shown to interact and target ONECUT 2 transcription factor for its translational silencing. ONE-CUT 2 is a homeodomain type of transcription factor whose expressional elevation promotes the growth and metastasis of colorectal cancer cells [22].
e results of the current study pointed out that colorectal cancer is linked with high ONECUT 2 expressions resulting from miR-296-3p downregulation which in turn emerges from the sponging action of lncRNA-XLOC_006390. In sum, the present work elucidated that overexpression of lncRNA-XLOC_006390 in colorectal cancer leads to repression in the expression of tumor-suppressor miR-296-3p which in turn enhances transcript and protein levels of ONECUT 2 transcription factor, and the latter enables high proliferation and metastasis of colorectal cancer cells.

Conclusion
Collectively, the current study evaluated the regulatory control exercised by lncRNA-XLOC_006390 on growth and propagation of colorectal cancer. e results showed that lncRNA-XLOC_006390 silencing induces apoptosis of colorectal cancer cells via modulation of miR-296/ONECUT 2 axis. ese results point towards the therapeutic implication of lncRNA-XLOC_006390 in the treatment of colorectal cancer. Data Availability e data used to support the findings of the stuyd can be obtained from the corresponding author upon request.
Ethical Approval e study was approved by the ethics committee of Cancer Institute and Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing China, and the informed consent was obtained from all of the patients. All patients enrolled in this study have approved the publication of this study.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
TM, TQ, XZ, and XY designed the study and applied for Research Ethics Board approval. TM, TQ, GY, RH, MW, ZY, YZ, and HW performed the experiments, analyzed the data, and prepared the manuscript. Dr. XZ and XW provided some theoretical and experimental guidance for designing and performing the experiments.