Long Intergenic Noncoding RNA 00641 Promotes Growth and Invasion of Colorectal Cancer through Regulating miR-450b-5p/GOLPH3 Axis

Background Long noncoding RNAs (lncRNAs) have a vital function in tumor onset and progress. For instance, long intergenic noncoding RNA 00641 (LINC00641) has been linked to cancer modulation. Nonetheless, the precise biological roles of LINC00641 in colorectal cancer (CRC) remain elusive. Methods The expression levels of LINC00641 as well as the docking sites for LINC00641 and miR-450b-5p were analyzed using public data resources and web-based analytic tools. The putative downstream targets of miR-450b-5p were also predicted. Next, we evaluated the biological functions and the contents of LINC00641 in CRC both in vivo and in vitro. We next explored the influence of LINC00641 on the growth, migration, and infiltration of CRC cells via cell proliferation, migration, and invasion experiments. Besides, qRT-PCR, western blotting, flow cytometry, luciferase enzyme reporter assay, and in vivo tumorigenicity assays were conducted. Results Our results confirmed that LINC00641 was markedly upmodulated in CRC tissues and CRC cell lines, and the upmodulation was linked to poor survival. Notably, the proliferative and migratory abilities of HCT-116 and SW480 cells were significantly inhibited by the knockdown of LINC00641 both in vitro and in vivo, illustrating that LINC00641 exerted a tumor-promotion role in CRC. Mechanistically, LINC00641 could competitively bind miR-450b-5p, thereby expunging its inhibitory effect on GOLPH3 expression. Moreover, miR-450-5p and GOLPH3 were able to reverse LINC00641-mediated cellular processes. Conclusions Overall, the findings of this study suggest that LINC00641 promotes the proliferative and migratory abilities of CRC through sponging the miR-450b-5p/GOLPH3 axis.


Introduction
Among cancer-related deaths globally, colon cancer (CRC) is the third most common type which ranks second among cancer-related deaths [1]. It is worth noting that CRC development is a multistep process involving various oncogenes and tumor suppressor genes. Specifically, the process involves protein-coding genes, such as P53, APC, RAS, and GOLPH3, and other types of noncoding RNAs. e interaction between these genes regulates downstream protein expression and affects CRC's progression, metastasis, and chemoresistance. However, the distinct underlying mechanisms of the regulation have not yet been fully elucidated. erefore, this necessitates a greater knowledge of the mechanisms behind CRC formation and progression, with the overarching goal of generating more effective treatments and finding novel ways for early detection.
Long intergenic noncoding RNAs (lncRNAs) constitute a class of RNA transcripts with more than 200 nucleotides but cannot be converted into proteins [2]. Emerging evidence has revealed that lncRNAs harbor vital modulatory role among numerous biological processes, including cancer. In addition, recent studies have discovered that lncRNAs regulate numerous cellular processes in CRC, for instance cell cycle progress, apoptosis, invasion, and metastasis [3][4][5]. And they have been documented to participate in the modulation of cancer stem-like cells (CSC) and epithelial-mesenchymal transition (EMT) phenotypes in CRC [6,7]. Among the various lncRNAs, a novel lncRNA termed as long intergenic noncoding RNA 00641 (LINC00641) has attracted our attention. LINC00641 was first discovered by Liang et al. [8] as a disease-associated lncRNA. It is located at 14q11.2 in the chromosome and has been documented to harbor an oncogenic role and a tumorrepressive function in various human cancers. For example, LINC00641 is involved in regulating the onset and progress of non-small-cell lung cancer [9], renal cell carcinoma [10], and gastric adenocarcinoma [11]. Nonetheless, the underlying mechanisms of LINC00641 in CRC involve a complex process, which remains unclear to date.
LncRNAs modulate gene expression at multiple stages, such as the levels of transcription, pre-transcription, and post-transcription, to modulate numerous biological processes. Previous research evidence confirms that Golgi phosphoprotein 3 (GOLPH3) is the first Golgi resident oncoprotein and drives cancer through binding tightly at the trans-Golgi to PtdIns(4)P [12,13]. In our previous investigations, we established that the overexpression of GOLPH3 promotes the growth, migration, and chemoresistance of colon cancer cells by activating multiple intracellular signal pathways [14][15][16]. Besides, elevated GOLPH3 expression has been linked with a poor outcome in colon cancer, [17] and is a possible predictor of 5-FU chemosensitivity [18]. However, only a handful of investigations have explored the upstream regulation of GOLPH3 expression. In the recent times, it has been documented that noncoding RNAs, for instance miR-126, miR-3150b, and miR-3150b-3p may downmodulate GOLPH3 expression and decrease growth by interacting with the promoter in many malignancies [19][20][21]. However, there is little knowledge on the regulation of GOLPH3 expression by lncRNAs in CRC. e main purpose of this research work was to assess the expression and precise role of LINC00641. starBase postulated that LINC00641 may target miR-450b-5p. We established that the contents of miR-450b-5p were downmodulated in recurrent CRC tissues. It has been documented that miR-450b-5p dampens stemness and 5-FU chemoresistance development in CRC via targeting SOX2 [22]. Mechanically, LINC00641 might compete with miR-450b-5p, leading to the upregulation of its targets N-cadherin, SOX2, and Twist that are involved in EMT and CSC regulation. In addition, we explored the potential underlying mechanism to verify whether LINC00641 can upregulate GOLPH3 expression through sponging miR-450b-5p, ultimately exerting an oncogene function in colorectal cancer. e exploratory cohort comprising of e Cancer Genome Atlas (TCGA) cases were abstracted from the GDC data resource (https://portal.gdc.cancer.gov) and UCSC Xena data resource (https://tcga.xenahubs.net). Next, we standardized and compared the data of LINC00641 expression levels in CRC tissue; if the obtained value was greater within CRC tissue than healthy tissue, the expression would be regarded as "high expression." Otherwise, it was "low-expression." e overall survival (OS) and disease-free interval survival (DFIS) rates in CRC subjects were subjected to Kaplan-Meier analysis. e patients' information was abstracted from the public data resource. Notably, this research premise was exempted from ethical approval by the database administrators due to its retrospective nature. Datasets GSE81581 and GSE from the GEO database were used to screen downregulated miRNAs and upregulated mRNAs, respectively. Differentially expressed miRNAs or mRNAs between nonmalignant and CRC tissues were identified by R (version 4.0.5) package limma (version 3.44.3) based on a false discovery rate (FDR) cutoff <0.05 and a |log2fold change| cutoff >1.

qRT-PCR.
e isolation of total RNA from CRC cell lines or xenograft tumors was done with the TRIzol reagent (Invitrogen, Carlsbad, United States, CAT#15596026) as described by the manufacturer. ereafter, the generation of cDNA was done with the TaqMan ® MicroRNA Reverse

Transwell Migration and Invasion Assays.
Transwell plates were utilized to perform transwell migration and invasion assays (CAT#3422, Corning, Corning, United States) without Matrigel (CAT#356234, BD Biosciences, Sparks, Ubited sates) for migration assays or with Matrigel for invasion assays. Briefly, we suspended the cells with FBS-free DMEM at a density of 5 × 10 5 /mL, followed by the addition of 100 μL cell suspension to the upper compartment and 500 μL of DMEM enriched with 10% FBS to the lower compartment. After 24 h, the migrated and invasive cells were stained with 0.2% crystal and then photographs were captured with a light microscope MOTIC AE31 (MOTIC, Hongkong, China). Finally, cell numbers were counted with the ImageJ 1.52v (NIH, Bethesda, MD, USA).

Xenograft Tumor
Model. BALB/c-nude mice (6-8 weeks old) from Shanghai SLAC Laboratory Animal Co., Ltd were used in the xenograft tumor model. For orthotopic assays, we subcutaneously inoculated 5 × 10 6 cells into the lower back regions of the mice (n � 6 per group). ereafter, we euthanized the mice 32 days post-orthotopic inoculation, and the growth of subcutaneous tumors was quantified. Weighing of the tumor tissues was performed, followed by Journal of Oncology grinding with grinder Tissuelyser-24 (Jingxin, Shanghai, China). ereafter, qRT-PCR, western blotting analysis, and IHC assay were used to assay the homogenates.
2.14. Statistical Analysis. All statistical analyses were implemented in GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA), and all experimental data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA), followed by Tukey's post hoc test was utilized for multiple comparisons among three groups, whereas Student's t-test was done to compare the difference between two groups. e generation of survival curves was done with the Kaplan-Meier approach, and the significance was assessed via the log-rank test. P < 0.05 denoted statistical significance.

LINC00641 Was Upregulated in CRC.
To assess the difference in LINC00641 expression, we retrieved CRC and nonmalignant tissues from the GEO database. Data illustrated that LINC00641 were remarkably upmodulated in the 90 CRC tissues in contrast with the 6 nonmalignant tissues (Figure 1(a)). qRT-PCR was then adopted to assess the mRNA contents of LINC00641 in four CRC cell lines (SW480, HT-29, HCT-116, and SW620) and normal human colon mucosal epithelial cell line (NCM460). It was found that the contents of LINC00641 were remarkably increased in all CRC cell lines in contrast with NCM460 cells (Figure 1(b)). Subsequently, the TCGA-COAD database was used to assess the relationship of the LINC00641 contents with the survival time. Although LINC00641 expression level failed to predict the overall survival (OS) (Figure 1(c)), CRC subjects with higher level of LINC00641 tend to have disease-free interval (DFI; Figure 1(d)).
Altogether, these data indicate that LINC00641 was upmodulated in CRC and its expression was inversely linked to the patients' survival.

Knockdown of LINC00641 Inhibited CRC Cell Malignant
Activities In Vitro. To evaluate the biological function of LINC00641 in CRC, endogenous LINC00641 was silenced in SW480 and HCT-116 cells by a lentivirus-based antagomir expression platform. e qRT-PCR analysis illustrated that LINC00641 expression was reduced by shLINC00641-1 and shLINC00641-2 compared to the control group (Figure 2(a)). Furthermore, the MTTassay and flow cytometry revealed that the knockdown of LINC00641 repressed cell proliferation (Figure 2 ese results suggest that the knockdown of LINC00641 inhibits the growth, migration, and infiltration of CRC cells and promotes apoptosis of CRC cells in vitro.

miR-450b-5p Was a Direct Target of LINC00641.
Given that lncRNAs' biological roles were dependent on their cellular distribution, [23], we first explored the localization of LINC00641. On the basis of the subcellular fractionation assay, LINC00641 was primarily located in the cytoplasm (Figure 3(a)), which was further confirmed by in silico prediction data from an online database ( Figure S1) and fluorescent in situ hybridization (FISH; Figure S2). Because cytoplasmic lncRNAs commonly work as ceRNAs to modulate the levels of target miRNAs [24], we checked the potential miRNAs targeted by LINC00641. After overlapping the miRNAs downregulated in the GSE81581 dataset with miRNAs that were predicted as candidate targets of LINC00641 in LncBase and starBase databases, only miR-450b-5p was enriched (Figure 3(b)). To examine whether miR-450b-5p serves as the valid target of LINC00641, we generated luciferase enzyme reporter vectors containing the wild-type (WT) or mutant (MUT) of LINC00641 (Figure 3(c)) to examine whether miR-450b-5p was the valid target of LINC00641. Results revealed that the luciferase enzyme activities of LINC00641-WT, but not LINC00641-MUT, were significantly decreased in SW480 and HCT-116 cells upon overexpression of miR-450b-5p (Figure 3(d)). qRT-PCR data exhibited that the LINC00641 mRNA contents were remarkably decreased in SW480/miR-450b-5p cells and HCT-116/miR-450b-5p compared to SW480/ctrl and HCT-116/ctrl cells (Figure 3(e)). Meanwhile, qRT-PCR illustrated that the contents of miR-450b-5p were remarkably increased by the knockdown of LINC00641 in both SW480 and HCT-116 cells in contrast with the controls (Figure 3(f )). In addition, the contents of miR-450b-5p in CRC tissues were inversely linked to the contents of LINC00641 in the CRC tissues (Figure 3(g)). Collectively, these data revealed that cytoplasmic LINC00641 targeted miR-450b-5p and negatively regulated its level.

miR-450b-5p Dampened the Growth, Migration, and
Infiltration of CRC Cells In Vitro. Next, the biological function of miR-450b-5p in CRC was explored. Both the SW480 and HCT-116 cell lines demonstrated a significant reduction in cell proliferation when miR-450b-5p was expressed ectopically (Figures 4(a) and 4(b)). Increasing miR-450b-5p repressed cell G1/S phase transition (Figure 4(c)) and facilitated apoptosis (Figure 4(d)), which was determined by flow cytometry analysis. Moreover, SW480 and HCT-116 cell migration (Figure 4(e)) and invasion (Figure 4(f )) was significantly reduced in vitro when miR-450b-5p was overexpressed, which was demonstrated by transwell experiments. Overall, miR-450b-5p suppressed the malignant activities of CRC cells and promoted apoptosis.

Overexpression of GOLPH3 Reversed the Repressive Effects of LINC00641 Silencing in CRC Cells.
To determine whether GOLPH3 overexpression could reverse the function of LINC00641 in SW480 and HCT-116 cells, we measured GOLPH3 re-expression in SW480/shLINC00641 and HCT-116/shLINC00641 cell lines (Figure 6(a)). It was found that GOLPH3 expression was upmodulated in SW480 and HCT-116 cells co-transfected with GOLPH3 OE and shLINC00641 compared to cells co-transfected with vector and shLINC00641 (Figures 6(b) and 6(c)). e overexpression of GOLPH3 remarkably enhanced cell growth, cell cycle G1/S phase transition, migration, and infiltration and repressed cell apoptosis compared to the knockdown of LINC00641 (Figures 6(d)-6(h)). is suggests that the reexpression of GOLPH3 in SW480/shLINC00641 and HCT-116/shLINC00641 cells reverted the inhibitory impacts of LINC00641 silencing in CRC cells.

Knockdown of LINC00641 Dampened Tumor Growth In
Vivo. To explore whether the knockdown of LINC00641 could suppress the tumor growth of CRC cells in vivo, 5 × 10 6 SW480/shLINC00641 and control cells were inoculated into the orthotopic sites of nude mice, respectively. As the results shown in Figure 7(a), LINC00641 knockdown dramatically suppressed tumor growth. A dramatic decrease in tumor volume and weight was reported in group shLINC00641 (Figures 7(b) and 7(c)). e downmodulation of LINC00641 was verified in the tumor tissues (Figure 7(d)), which was accompanied with an elevated content of miR-450b-5p and a reduced level of GOLPH3 (Figures 7(e)-7(h)). Moreover, the protein contents of Bax, cleaved caspase 3, and cleaved caspase 9 were elevated in group shLINC00641, contrary to the Bcl-2 protein level, which indicated that LINC00641 knockdown promoted cell apoptosis (Figures 7(g) and 7(h)). LINC00641 also modulated the protein contents of EMT markers consisting of N-cadherin and E-cadherin (Figures 7(g) and 7(h)). Besides, the contents of stemness genes SOX2 and Twist1 were determined. e data illustrated that LINC00641 deficiency reduced the protein levels of SOX2 and Twist1 (Figures 7(g) and 7(h)). Immunohistochemical data illustrated that the contents of Ki-67 were remarkably decreased after LINC00641 silencing (Figures 7(i) and 7(j)). Altogether, these data suggest LINC00641 promotes the tumorigenesis of CRC in vivo.

Discussion
To date, the effect of advanced CRC treatment is still unsatisfactory, despite the rapid development of technologies and therapeutic strategies.
us, understanding the molecular mechanisms underlying CRC progress and metastasis is critical. Growing research evidence illustrates that lncRNAs play a remarkable role in regulating gene expression, cell signaling cascades, and chemoresistance in CRC [25][26][27]. Studies have reported that LINC00641 exerts the opposite effect in the progression of numerous types of cancer largely due to targeting different miRNAs. For example, LINC00641 was found to enhance the ability of cell growth and infiltration through sponging miR-340-5p in renal cell carcinoma [10], miR-429 in gastric cancer [11], and miR-378a in acute myeloid leukemia [28]. However, it has also been shown to suppress tumor growth and migration capacities via interacting with miR-365a-3p in prostate cancer [29], miR-4262 in glioma [30], miR-424 in cutaneous squamous cell cancer [31], miR-194-5p in breast cancer [32], miR-424-5p in NSCLC [9], and miR-197-3p in bladder cancer [33]. Herein, results indicated that LINC00641 contents were correlated with dismal prognosis in CRC, and LINC00641 silencing remarkably dampened growth and infiltration of colon cancer cells in vitro and in vivo. Additionally, it was revealed that LINC00641 promotes EMT and CSC stemness in vivo   (e), (f ) Ectopic expression of miR-450b-5p inhibited the migration and invasion ability of SW480 and HCT-116 cells, which was detected using transwell assay. Data are represented as mean ± SD of three biological replicates. Compared with mimic NC group, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001. Scale bar: 10 μm.  assay. erefore, LINC00641 shows pivotal effects in the promotion of tumor in CRC. Several investigations have documented that lncRNAs can sequester miRNAs through acting as ceRNAs or as RNA sponges [5,34]. MicroRNAs (miRNAs) constitute endogenous small 18-25 nucleotide noncoding RNA molecules that attach to the 3′UTR of particular target mRNAs, causing mRNA degradation and translation. Evidence exhibits that miRNAs harbor remarkable roles in the onset and progress of numerous diseases, including cancer. In this context, we utilized bioinformatics to uncover miRNAs that can cross talk with LINC00641. Subsequently, miR-450b-5p, which had the highest binding score, was chosen to perform further experiments. In various forms of human malignancies, such as rhabdomyosarcoma, CRC, hepatocellular cancer, nasopharyngeal carcinoma, and breast cancer, miR-450b-5p is downmodulated and functions as a tumor repressor [22,[35][36][37][38][39]. Herein, we established that miR-450b-5p dampens the growth, migration, and infiltration of CRC cells in vitro, which suggests that miR-450b-5p works as a tumor repressor in CRC. A previous study showed miR-450b-5p inhibits stemness and progress of chemoresistance to 5-FU via targeting SOX2 in CRC [22]. Herein, the luciferase reporter gene validated the targeting relationship of LINC00641 with miR-450b-5p. And, in vivo assay, LINC00641 also regulated the protein contents of EMT markers, for instance N-cadherin and E-cadherin. e data illustrated that LINC00641 deficiency reduced the protein levels of SOX2 and Twist1. is indicated that LINC00641 enhances EMTand CSC stemness through acting as a sponge of miR-450b-5p in CRC.
Furthermore, the 3′UTR of GOLPH3 has been discovered to contain highly conserved miR-450b-5p binding sites. Recent studies have confirmed that the GOLPH3 gene plays the role of an oncogene, and upregulation of GOLPH3 expression promoted colon cancer cell growth, invasion, and chemoresistance [16,[40][41][42]. Herein, the luciferase enzyme reporter assay verified that GOLPH3 is the downstream target of miR-450b-5p. Moreover, GOLPH3 was positively regulated by LINC00641, but was inversely linked to the expression of miR-450b-5p. Moreover, the rescue assays proved that the overexpression of GOLPH3 reversed LINC00641 silencing-mediated proliferation, apoptosis, migration, and infiltration in CRC cells. us, we concluded that LINC00641 acts as a ceRNA in CRC via sponging miR-450b-5p to upmodulate GOLPH3. In addition, the cross talk of LINC00641 with miR-450b-5p upregulated GOLPH3 expression, ultimately contributing to the progress of CRC.
is research has some limitations. For example, we did not directly detect the LINC00641 levels in human tissues because of lack of clinical samples. We are seeking for Ethics Committee approval to collect clinical samples for    LINC00641 detection using qRT-PCR. Additionally, we will also purchase tissue microarrays for LINC00641 detection using FISH after the COVID-19 pandemic, which is breaking out in Quanzhou City now. In addition, SW480 cells containing luciferase vector should be used to construct in situ xenograft and metastatic models to elucidate the effect of LINC00641 on CRC progression.

Conclusions
In summary, we established that LINC00641 was upmodulated in CRC and was associated with dismal prognosis. Results revealed that it played the function of an oncogene and upregulated GOLPH3 expression through sponging miR-450b-5p in colon cancer. Overall, our results suggested that LINC00641/miR-450b-5p/GOLPH3 axis might serve as a novel therapeutic target for CRC.
Data Availability e analyzed datasets generated during this work are accessible upon request from the corresponding author.
Ethical Approval e experimental protocol was established as per the ethical guidelines of the Helsinki Declaration. It was authorized by the Ethics Committee of the Second Affiliated Hospital of Fujian Medical University (approval no. 2021146, Quanzhou, Fujian, China).  Figure S1. Lnclocator (http://www.csbio.sjtu.edu.cn/bioinf/ lncLocator/) predicted the nucleocytoplasmic localization of lncRNA LINC00641, and the prediction data illustrated that it was in the cytoplasm. Figure S2. Images of Fluorescence in situ hybridization (FISH) illustrating the location of LINC00641. Table S1. Primer sequences for qRT-PCR.