Role of lncRNA LINC01194 in hepatocellular carcinoma via the miR-655-3p/SMAD family member 5 axis.

Long non-coding RNAs (lncRNAs) are involved in developing hepatocellular carcinoma (HCC). The present study explored the role of lncRNA LINC01194, which is upregulated in HCC tissues and might be a vital regulator in HCC progression. Levels of LINC01194, microRNA (miR)-655-3p, and SMAD family member 5 (SMAD5) were assessed using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The bioactivity of Huh-7 cells was assessed using cell counting kit-8 and transwell assays and flow cytometry. Western blotting was conducted to measure the expression of invasion- and apoptosis-related proteins. The relationships between lncRNA LINC01194 and miR-655-3p, and miR-655-3p and SMAD5 were predicted using StarBase and TargetScan, and further verified using a dual-luciferase reporter assay. LINC01194 was overexpressed in HCC cells and in clinical samples. ILINC01194 silencing suppressed proliferation and migration; however, it promoted apoptosis in HCC cell lines. We also confirmed that miR-655-3p could bind to LINC01194, and miR-655-3p was downregulated in HCC. The upregulation of miR-655-3p suppressed HCC cell invasion and migration, and enhanced the number of apoptotic cells. SMAD5, which was overexpressed in HCC cell lines, was directly targeted by miR-655-3p. Therefore, LINC01194 promoted HCC development by decreasing miR-655-3p expression and may serve as a promising therapeutic target for HCC patients.

Introduction

Hepatocellular carcinoma (HCC), a malignant tumor with high recurrence and metastasis rates, is the second leading cause of cancer-related mortality worldwide [1,2]. Several elements may lead to liver cancer, including hepatitis B and C viruses, alcoholism, and aflatoxin. Nonalcoholic fatty liver disease is also a risk factor for hepatoma [3]. The prognosis of patients with HCC is poor, and the 5-year survival rate in China is approximately 40%. Currently, HCC diagnosis is generally achieved using blood indicators, imaging, and histopathology. However, HCC screening is not sufficiently sensitive [4-6]. Notable advancements have been made in HCC treatment; however, the 5-year recurrence rate of postoperative patients with HCC remains high [7]. Therefore, elucidating the pathogenesis of HCC is urgently required.

Long non-coding RNAs (lncRNAs) are critical epigenetic regulatory mechanisms, including DNA methylation and genomic imprinting. lncRNAs are RNA molecules > 200 nucleotides in length with a low translation ability, and exhibit crucial biological functions in tumors, including epithelial mesenchymal transformation, invasion and metastasis cascade, proliferation, and drug resistance [8-11]. For instance, the lncRNA insulin-like growth factor 2 antisense 1 (Igf2as) was upregulated in HCC and dominated liver cancer via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway [12]. lncRNA HOXD-AS2 could promote glioblastoma cell proliferation, migration and invasion by regulating the miR-3681-5p/mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) signaling pathway [13]. lncRNA PCED1B-AS1 promotes gastric cancer cells proliferation, migration, invasion and Epithelial-to-Mesenchymal Transition (EMT) [14]. LINC01194 is a new lncRNA that promotes cell growth and invasion in non-small cell lung carcinoma (NSCLC) by targeting the microRNA (miRNA/miR)-486-5p/cyclin-dependent kinase 4 pathway [15]. Although previous studies have demonstrated the functions of LINC01194 in lung cancer, the underlying mechanism of its regulation in HCC remains unknown.

miRNAs are evolutionarily conserved non-coding small RNAs with a length of 18–20 base pairs (bp) [16]. Evidence has shown that miRNAs are associated with the progression of several diseases by targeting post-transcriptional translation [17,18]. A previous study reported that miR-655-3p acts as a sponge for taurine upregulated 1 to participate in the regulation of bioactivity in acute myeloid leukemia [19]. Xin et al. [20] revealed that miR-655-3p was regulated via sponging by the lncRNA NFIA antisense RNA 2 to regulate glioma. However, to the best of our knowledge, the biological functions of miR-655-3p in HCC have not yet been investigated.

Therefore, further exploration of the pathogenesis of HCC could help develop novel treatment strategies, which is of great significance. We hypothesized that lncRNA LINC01194 might play a role in HCC via the regulation of miR-655-3p. Thus, our study was designed to determine whether lncRNA LINC01194 regulates HCC progression via the miR-655-3p/SMAD family member 5 (SMAD5) axis. The present study provides novel biomarkers for the treatment and HCC prognosis.

Materials and methods

Clinical tissue collection

Samples of 40 HCC tissues and paired adjacent normal tissues (located > 5 cm away from the cancer edge) were obtained from surgical patients at the Affiliated Hospital of Jianghan University (Wuhan, China). Inclusion criteria included: i) No patients had received preoperative antitumor therapy, such as radiotherapy or chemotherapy, and ii) the final diagnosis was confirmed by routine pathological examination. Exclusion criteria included: i) Patients receiving preoperative radiotherapy or chemotherapy. The clinicopathological features for HCC patients were presented in Table 1. This study was approved by the Ethics Committee of the Affiliated Hospital of Jianghan University. Written informed consent was obtained from all patients. The obtained samples were stored at −80°C until subsequent use.

Table 1.

The clinicopathological features for HCC patients

			lncRNA LINC01194
Characteristics	n = 40		Low expression (n = 20)	High expression (n = 20)	P
Age (y)	< 50	17	7	10	>0.05
	≥50	23	13	10
Sex	Male	32	15	17	>0.05
	Female	8	5	3
HBV	Absent	13	8	5	>0.05
	Present	27	12	15
Serum AFP level (ng/mL)	< 20	12	7	5	>0.05
	≥20	28	13	15
Tumor size (cm)	< 5	16	12	4	<0.05
	≥5	24	9	15
No. of tumor nodules	1	33	17	16	>0.05
	≥2	7	3	4
Cirrhosis	Absent	16	9	7	>0.05
	Present	24	11	13
Venous infiltration	Absent	24	13	11	>0.05
	Present	16	7	9
Edmondson-Steiner grading	I + II	30	17	13	<0.05
	III + IV	10	3	7
TNM tumor stage	I + II	30	18	12	<0.05
	III + IV	10	2	8

Cell culture

Huh-7 and HCCLM3 human HCC cell lines, and THLE-2 and 293 T human normal hepatocytes for the dual-luciferase reporter assay were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, CA, USA) containing 1% penicillin, streptomycin, and 10% fetal bovine serum (FBS; Gibco, CA, USA) at 37°C in the presence of 5% CO2.

Cell transfection

To regulate miR-655-3p expression, miR-655-3p inhibitor and negative control oligonucleotide inhibitor (miR-655-3p inhibitor, 5ʹ-UGAGGAGAGGUUAUCCGUGUU-3ʹ; control inhibitor, 5ʹ-CAGUACUUUUGUGUAGUACAA-3ʹ), miR-655-3p mimic (5ʹ-AUAAUACAUGGUUAACCUCUUU-3ʹ), and a control mimic (5ʹ-AAUUCGUAGCUUGCAUGCAAGC-3ʹ) were obtained from Invitrogen (Thermo Fisher Scientific, Inc.). To knock down LINC01194, small interfering RNA (siRNA/si) against LINC01194 (si-LINC01194, 5ʹ-GCAAGTTTGCTACATGGGTAA-3ʹ) was used, with nonspecific RNA serving as the negative control (control siRNA, 5ʹ-CACGTAGAGGCATGATGCCGT-3ʹ; Invitrogen, USA). All vectors and reagents were transfected into cells grown to 70%–80% confluence using Lipofectamine® 3000 (Invitrogen). After incubation for another 48 h, the transfected cells were harvested for subsequent use.

RT-qPCR analysis

Total RNA was obtained from the different cells using TRIzol® reagent (Life Technologies, USA) according to the manufacturer’s protocol. Then, the total RNA was reverse transcribed to cDNA using a PrimeScript RT Reagent Kit (TaKaRa, China). miRNA expression was detected using a Hairpin-itTM MicroRNAs Quantitation kit (Shanghai GenePharma Co., Ltd.) and SYBR Green reagents (Vazyme Biotech Co., Ltd.) were used for RT-qPCR analysis. Relative expression levels were calculated using the 2−ΔΔCq method [21]. The following primers were used for RT-qPCR: U6, 5ʹ-CGCTTCGGCAGCACATATACTA-3ʹ forward and 5ʹ-CGCTTCACGAATTTGCGTGTCA-3ʹ reverse; β-actin, 5ʹ-CCTCGCCTTTGCCGATCC-3ʹ forward and 5ʹ-GGATCTTCATGAGGTAGTCAGTC-3ʹ reverse; LINC01194, 5ʹ-AGGCCTGACACGTTTACTAA-3ʹ forward and 5ʹ-AGGTCGTAGTGTTAGTGCAT-3ʹ reverse; cyclin D1, 5ʹ-TACTACCGCCTCACACGCTTC‐3ʹ forward and 5ʹ‐TTCGATCTGCTCCTGGCAG‐3ʹ reverse; MMP-9, 5ʹ- GAGTGGCAGGGGGAAGATGC-3ʹ forward and 5ʹ- CCTCAGGGCACTGCAGGATG-3ʹ reverse; and SMAD5, 5ʹ-CCAGCAGTAAAGCGATTGTTGG-3ʹ forward and 5ʹ-GGGGTAAGCCTTTTCTGTGAG-3ʹ reverse.

Cell counting kit-8 (CCK-8) assay [22]

Cell proliferation was assessed using the CCK-8 assay (Beijing Solarbio Science & Technology Co., Ltd.). Transfected cells were treated and cultured in 96-well plates at 5 × 103 cells/well, followed by incubation with CCK-8 solution (10 µL) in accordance with the manufacturer’s instructions. The optical density was measured at 450 nm using an ultraviolet spectrophotometer (Agilent Technologies, USA).

Transwell assay

A transwell assay was performed as previously described [20]. In brief, for the invasion assay, cells were seeded in a 50 µL Matrigel®-coated upper transwell chamber with a pore (BD Biosciences; Thermo Fisher Scientific, Inc.), and cell migration was evaluated in the transwell chamber without matrigel coating. Medium without 10% FBS was added to the upper chamber, and medium with 10% FBS was attached to the lower chamber. Following a 24-h incubation, the cells that migrated and invaded the membrane surface were fixed with 4% paraformaldehyde, followed by staining with crystal violet staining solution. In total, five randomly selected fields were summed using a microscope (Olympus Corporation).

Cell apoptosis assay [23]

After treatment, 1 × 106 transfected HCC cells were measured using an annexin V-fluorescein isothiocyannate (FITC)/propidium iodide (PI) apoptosis detection kit (Beyotime) for 30 min at 37°C without light, following the manufacturer’s protocol. Then, apoptotic cells were quantified using a flow cytometer (BD Biosciences, USA) and analyzed using FlowJo analysis software.

Western blotting [24]

Total protein extraction from the HCC cells was performed using Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease inhibitor and measured using a Bicinchoninic acid (BCA) protein assay kit (Invitrogen). Protein samples were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Millipore Sigma). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies at 4°C overnight. After washing in Tris-buffered saline with Tween solution (TBST, 50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, and 0.1% Tween-20), the membranes were treated with secondary antibodies for 1 h. Protein bands were imaged using ECL reagent (Millipore Sigma). The following primary antibodies were used: Anti-Bcl-2 (Abcam), anti-Bax (Abcam), anti-MMP9 (Cell Signaling Technology, Inc.), anti-cyclin D1 (ProteinTech Group, Inc.), anti-SMAD5 (Abcam), and anti-GAPDH (Abcam). β-actin was used as the reference gene.

Dual-luciferase reporter assay [25]

The complementary sequences between LINC01194 and miR-655-3p were forecasted using StarBase, and SMAD5 fragments containing miR-655-3p binding sites were predicted using TargetScan V 7.0. Wild-type or mutant LINC01194 and SMAD5 fragments containing miR-655-3p binding sites were synthesized and placed into the pGL3-basic plasmid (Sangon) to generate LINC01194-wild-type (WT) or LINC01194-mutated (Mut), and SMAD5-WT or SMAD5-Mut. 293 T cells were co-transfected with these reporter vectors and miR-655-3p or control mimics. After 48 h of transfection, the dual-luciferase reporter system (Promega, USA) was used to detect luciferase activity.

Statistical analysis

Statistical analyses were conducted using SPSS 20.0. All results are expressed as mean ± standard deviation from three independent experiments. Mean differences among groups were analyzed by one-way analysis of variance or Student’s t-test. *P < 0.05 and **P < 0.01 indicated a significant difference.

Results

LINC01194 was highly expressed in HCC tissues

To determine the expression of LINC01194 in HCC, RT-qPCR was conducted to determine the LINC01194 levels in HCC tissues and cell lines. The results showed that LINC01194 levels were higher in HCC samples than in adjacent normal tissues (Figure 1a; p < 0.01). In addition, LINC01194 expression was increased in Huh-7 and HCCLM3 HCC cell lines compared to THLE-2 cells (Figure 1b; p < 0.01).

Figure 1.

lncRNA LINC01194 is highly expressed in HCC. (a) RT-qPCR analysis of lncRNA LINC01194 in HCC tumor tissues, with non-tumor tissues serving as the negative control. (b) RT-qPCR analysis of lncRNA LINC01194 in Huh-7 and HCCLM3 HCC cells, with THLE2 cells serving as the negative control. *P < 0.01 vs. normal adjacent tissue; ##P < 0.01 vs. THLE2 cells.

miR-655-3p was bound to LINC01194

To investigate the relationship between LINC01194 and miR-655-3p, the bioinformatics prediction tool StarBase was used, and miR-655-3p harbored the potential LINC01194 binding sites (Figure 2a). To further determine the relationship between miR-655-3p and LINC01194, a dual-luciferase reporter assay was performed and demonstrated that the miR-655-3p mimic remarkably enhanced miR-655-3p expression in the 293 T cells (Figure 2b; p < 0.01). Figure 2c shows that the luciferase activity of LINC01194-WT was suppressed in miR-655-3p mimic transfected cells. However, when the putative binding sites were mutated, the miR-655-3p mimic exerted modest effects. These findings confirmed that miR-655-3p directly targeted LINC01194.

Figure 2.

lncRNA LINC01194 functions as an miR-655-3p sponge in HCC cells. (a) StarBase software predicted the lncRNA LINC01194 on miR-655-3p 3ʹUTR. (b) miR-655-3p expression in the control mimic or miR-655-3p mimic transfected 293 T cells. (c) Direct binding relationship between lncRNA LINC01194 and miR-655-3p were confirmed using a dual-luciferase gene reporter gene assay. **P < 0.01 vs. control mimic.

miR-655-3p was downregulated in HCC

To further determine the expression of miR-655-3p in HCC, RT-qPCR was conducted to determine the miR-655-3p levels in HCC tissues and cell lines. The RT-qPCR results revealed that miR-655-3p was lower in HCC tissues than in corresponding para-carcinoma tissues (Figure 3a; p < 0.01). The same results were observed in HCC cells compared to THLE-2 cells (Figure 3b; p < 0.01).

Figure 3.

miR-655-3p was downregulated in HCC tissues and cells. miR-655-3p expression in (a) pairs of tumor and matched non-tumor tissues from HCC patients, and (b) Huh-7 and HCCLM3 HCC cells, with THLE2 cells serving as the negative control were determined using RT-qPCR. **P < 0.01 vs. normal adjacent tissue; ##P < 0.01 vs. THLE2 cells.

LINC01194 negatively regulated miR-655-3p in Huh-7 cells

We then explored whether LINC01194 could negatively regulated miR-655-3p in Huh-7 cells. RT-qPCR analysis showed that LINC01194 expression in Huh-7 cells was reduced following si-LINC01194 transfection, compared to the control siRNA group (Figure 4a; p < 0.01). The downregulation of miR-655-3p inhibited miR-655-3p expression in Huh-7 cells compared to the control inhibitor group (Figure 4b; p < 0.01). Moreover, miR-655-3p expression was increased in Huh-7 cells transfected with si-LINC01194, whereas co-transfection with miR-655-3p inhibitor decreased the expression (Figure 4c; p < 0.01). These results indicate that LINC01194 negatively regulated miR-655-3p levels in Huh-7 cells.

Figure 4.

LINC01194 negatively regulates miR-655-3p in Huh-7 cells. (a) The efficiency of LINC01194 knockdown was verified using RT-qPCR. Detection of miR-655-3p levels following transfection with miR-655-3p inhibitor (b), and LINC01194 siRNA and miR-655-3p co-transfection group (c) using RT-qPCR. **P < 0.01 vs. control siRNA; ##P < 0.01 vs. inhibitor control; &&P < 0.01 vs. LINC0119-siRNA + control inhibitor. miR, microRNA.

miR-655-3p knockdown reversed the effects of si-LINC01194 on the malignant behavior of Huh-7 cells

To explore the influence of LINC01194 on the advancement of Huh-7 cells, CCK-8, transwell, and cell apoptosis assays and Western blotting were conducted. At first, the relative cell viability of Huh-7 cells was notably lower in the si-LINC01194-transfected group than that in the siRNA-transfected group; however, miR-655-3p knockdown rescued the inhibitory action of si-LINC01194 transfection (Figure 5a). Western blotting and RT-qPCR results indicated that cyclin D1 and MMP9 were significantly downregulated following si-LINC01194 transfection in Huh-7 cells (all p < 0.01), whereas this effect was reversed by the miR-655-3p inhibitor (Figure 5b-d). The transwell assay suggested that invading and migrating cells were decreased when Huh-7 cells were transfected with si-LINC01194 (all p < 0.01), whereas co-transfection with miR-655-3p inhibitor and si-LINC01194 reversed this inhibition (Figure 5e-h).

Figure 5.

lncRNA LINC01194 knockdown inhibits HCC cell migration and invasion via miR-655-3p. (a) CCK-8 assay was performed to check Huh-7 cell growth. (b-d) Determination of cyclin D1 and MMP9 levels using Western blotting and RT-qPCR analysis, respectively. (e-h) Migrated and invaded Huh-7 cells were analyzed using a transwell assay. **P < 0.01 vs. control siRNA; ##P < 0.01 vs. LINC0119-siRNA + control inhibitor.

Flow cytometry revealed that the increased Huh-7 cell apoptosis caused by transfection with si-LINC01194 was recovered following co-treatment with the miR-655-3p inhibitor (Figure 6a and b). Bax was upregulated and Bcl-2 was downregulated in si-LINC01194 transfected cells (Figure 6c). The Bax/Bcl-2 ratio was increased in the si-LINC01194 group (Figure 6d; p < 0.01), an effect that was reversed following co-transfection with the miR-655-3p inhibitor. Therefore, the LINC01194 knockdown blocked the progression and induced more apoptotic cells.

Figure 6.

lncRNA LINC01194 knockdown promotes HCC cell apoptosis by regulating miR-655-3p. (a and b) Flow cytometry analysis of apoptotic Huh-7 cells. (c) Western blotting analysis of apoptosis-related proteins Bax and Bcl-2 levels. (d) Semi-quantitative analysis of Bax, Bcl-2, and Bcl-2/Bax expression. **P < 0.01 vs. control siRNA; ##P < 0.01 vs. LINC0119-siRNA + control inhibitor.

miR-655-3p was involved in HCC progression through negatively regulating SMAD5

The molecular mechanism of miR-655-3p in HCC was determined using a bioinformatics assay. Using TargetScan, SMAD5 harbored the potential miR-655-3p binding sites (Figure 7a). A dual-luciferase reporter assay further revealed that the miR-655-3p mimic impaired the luciferase activity of SMAD5-WT, whereas SMAD5-Mut did not respond to the miR-655-3p mimic, further confirming that miR-655-3p was directly bound to SMAD5 (Figure 7b). miR-655-3p expression was upregulated by miR-655-3p mimic transfection compared to the control mimic transfection (Figure 7c; p < 0.01). Moreover, the miR-655-3p inhibitor downregulated its expression in HCC cells compared to that in the inhibitor group (Figure 4b; p < 0.01). It was also shown that the miR-655-3p mimic markedly decreased the protein and mRNA expression levels of SMAD5 in Huh-7 cells (Figure 7d and e; p < 0.01). The opposite results were observed for miR-655-3p knockdown, which increased SMAD5 levels (Figure 7f and g; p < 0.01). Our observations demonstrate that SMAD5 directly targeted miR-655-3p and that its expression was negatively regulated by miR-655-3p in Huh-7 cells.

Figure 7.

miR-655-3p negatively targets SMAD5 in Huh-7 cells. (a) TargetScan software predicted the miR-655-3p on SMAD5 3ʹUTR. (b) A dual-luciferase reporter gene assay was conducted to verify the binding sites between miR-655-3p and SMAD5. (c) miR-655-3p mimic efficiency was verified using RT-qPCR assay. (d and e) SMAD5 expression was evaluated using RT-qPCR and Western blotting in miR-655-3p overexpression Huh-7 cells. (f and g) RT-qPCR and Western blotting analysis of SMAD5 level in miR-655-3p inhibitor transfected Huh-7 cells. **P < 0.01 vs. mimic control; ##P < 0.01 vs. control inhibitor.

Discussion

In the present study, the mechanism underlying the promotion of HCC progression by lncRNA LINC01194 was elucidated. The schematic diagram of summary of this study was presented in Supplementary Figure S1. This study demonstrated that lncRNA LINC01194 accelerated HCC progression through the miR-655-3p/SMAD5 axis, showing that lncRNA LINC01194 served as a promising biomarker for patients with HCC.

LncRNAs are a new type of non-coding RNA, consisting of more than 200 nucleotides that regulate various molecular functions [26]. LncRNAs are associated with developing various diseases by regulating target gene expression via sponging miRNAs [27]. Previous studies have suggested that lncRNAs are abnormally expressed, regulate many miRNAs and genes, and modulate various biological processes [28-30]. Thus, one or more of these lncRNAs will become latent therapeutic candidates for HCC treatment.

The development and progression of HCC are associated with various factors. lncRNAs are significant biomarkers and molecular targets for HCC. Pan et al. [31] revealed that lncRNA could be used as a biomarker of poor prognosis in HCC, guiding clinical research. Chao et al. [32] found that lncRNA D16366 was a potential target for HCC treatment. In our study, we illustrated the functions of LINC01194 in HCC and provided a potential biomarker and therapeutic target for HCC treatment. Wang et al. [33] reported that lncRNA LINC01194 was a diagnostic biomarker for colorectal carcinoma. Liu et al. [34] reported that LINC01194 was associated with laryngeal squamous cell carcinoma. Song et al. [35] demonstrated that LINC01194 was involved in prostate cancer. However, only a few studies have investigated the role of LINC01194 in HCC. In our study, we found that the LINC01194 inhibitor alleviated HCC. The present findings broaden our current understanding of the vital functions of LINC01194 in regulating HCC, which may assist in the study of the pathological mechanism of HCC.

Several studies have shown that lncRNAs regulate the evolution of various types of cancer through miRNA patterns. Luo et al. [36] reported that lncRNA CACS11 contributed to the development of bladder cancer by sponging miR-150. He et al. [37] demonstrated that lncRNA promoted prostate cancer metastasis through an miRNA-to-mRNA network. Furthermore, Wang et al. [38] revealed that miR-655-3p promoted NSCLC through the PTTG1 regulator of sister chromatid separation, securin. Zha et al. [39] found that miR-655-3p inhibited ovarian cancer by targeting the Ras-related protein Rab-1A. The present investigation revealed that miR-655-3p was downregulated and negatively related to LINC01194 expression in HCC tissues and cells. Functionally, miR-655-3p overexpression inhibits HCC cell progression.

SMAD5 is closely related to the occurrence and progression of various cancers. Chen et al. [40] reported that SMAD5 was highly expressed in bladder cancer. We found that SMAD5 was highly expressed in HCC, suggesting that SMAD5 might play a positive role in the progression of HCC. In addition, SMAD5, which serves as a target gene, is targeted by miRNAs to regulate tumor progression. For instance, miR-145 accelerates esophageal cancer progression by regulating SMAD5 [41]. Our data indicated that miR-655-3p negatively targeted SMAD5, and lncRNA LINC01194 promotes HCC metastasis and inhibits apoptosis via the miR-655-3p/SMAD5 axis.

However, there are some limitations of the current study. This study is only a preliminary in vitro study of the role of LINC01194 in HCC. In order to make the role of LINC01194 in HCC more convincing, more in-depth research is needed. For example, it is necessary to explore the role of LINC01194 in HCC animal models. Besides, the prognosis value of LINC01194 in HCC should be further analyzed. We will perform these issues in the future.

Conclusion

LINC01194 is upregulated in HCC cell lines and can bind to miR-655-3p. These findings suggest that LINC01194 regulates the proliferation and migration of HCC cells via the miR-655-3p/SMAD5 axis, providing a promising and novel biomarker for diagnosing and treating HCC.

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