LncRNA SLC16A1‐AS1 contributes to the progression of hepatocellular carcinoma cells by modulating miR‐411/MITD1 axis

Abstract Background Hepatocellular carcinoma (HCC) is a common malignancy with high morbidity. The current study aimed to explore the molecular mechanism of lncRNA SLC16A1‐AS1 in the tumorigenesis of HCC. Material and methods The expression of SLC16A1‐AS1 and miR‐411 was examined in clinical HCC tissues. HCC cell lines Hep3B and Huh‐7 were employed and transfected with si‐SLC16A1‐AS1. The correlation between SLC16A1‐AS1 and miR‐411 was verified by luciferase reporter assay. Cell viability was detected by CCK‐8 assay. Cell migration and invasion capacity were examined by transwell assay. The protein level of MITD1 was analyzed by western blotting. Results The expression of SLC16A1‐AS1 markedly increased in HCC tissues and cell lines. Subsequent studies identified SLC16A1‐AS1 as a downstream target of miR‐411. In addition, SLC16A1‐AS1 knockdown and miR‐411 overexpression significantly stagnated the progression of HCC cells. SLC16A1‐AS1 knockdown also downregulated MITD1 levels. Conclusion Our findings showed that SLC16A1‐AS1 was overexpressed in HCC cells and tissues. SLC16A1‐AS1 promoted the malignant characteristics of HCC cells and acted as an oncogene. Its regulatory effect may be associated with miR‐411/MITD1 axis. Therefore, SLC16A1‐AS1 has the potential to be used as a biomarker or therapeutic target for the treatment of HCC.

HCC. Therefore, finding new targets for therapeutic and diagnostic methods will help in the early diagnosis and intervention of HCC.
Studies have shown that less than 1.5% of the human genome has a protein-coding function, and some of the rest contain functionally conserved non-coding series. 5 A large part of the genome is transcribed into non-coding RNAs (ncRNAs), with no or low protein-coding functions. NcRNA can be divided into several families according to its molecular size. 6 LncRNA is defined as a transcript with a length of more than 200 nucleotides. 7 Recently, a lot of attention has been focused on lncRNA. With the development of genome-wide transcriptomics analysis, lncRNA has been confirmed to participate in a variety of biological activities, such as cell cycle regulation, apoptosis, differentiation, and other life activities. 8,9 The latest research shows that lncRNA can affect HCC metastasisrelated molecules at the transcription level and post-transcription level, and provide potential diagnostic indicators and therapeutic targets for HCC patients. 10 The current study aims to better understand the molecular mechanism of SLC16A1-AS1 in the occurrence and development of HCC. The expression level of SLC16A1-AS1 in clinical specimens and cells was determined. Function assay uncovered the role of SLC16A1-AS1 in the malignant phenotypes of HCC cells. Further study investigated the molecular interaction between SLC16A1-AS1 and miR-411/MITD1 axis. This article analyzes in detail the role of SLC16A1-AS1 in the progression of HCC, which will help to understand the pathogenesis of HCC.

| Clinical specimens
The present study was conducted with the permission of the Ethics Committee of Wannan Medical College and carried out according to the guidelines of ethical management. All participants provided written informed consent. A total of 30 HCC specimens and paired normal tissues were obtained from Yijishan Hospital of Wannan Medical College from 2019 to 2021. The including criteria were as follows: (1) all patients pathologically diagnosed with primary HCC, and diagnosis was based on the World Health Organization criteria; (2) no surgical treatment, radiotherapy, chemotherapy, and biotherapy were performed before operation; (3) complete clinical records and follow-up information were available in all cases. Patients were excluded if any of the following conditions were met: (1) presence of cancers other than HCC; (2) autoimmune hepatitis or toxic hepatitis; (3) refusal to participate.

| Cell culture and reagents
The human HCC cell lines (Hep3B and Huh-7) and human normal hepatic cell line (LO2) were purchased from ATCC (American Type Culture Collection). The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (GIBCO).

| Quantitative real-time PCR
The total RNA was isolated from cells and tissues using Trizol reagent (Life Technologies). First-strand cDNA was carried out using reverse transcription reagents (ABI). Quantitative real-time PCR (qRT-PCR) was performed using SYBRH Select Master Mix for CFX (Invitrogen) and using the CFX Connet TM real-time PCR system (Bio-Rad). GAPDH and U6 were chosen as the internal standard. The primer sequences used are shown in Table 1.

| Cell viability assay
Cell viability was measured using Cell Counting Kit-8 assay (Beyotime). After being inoculated into 96-well plates at a density of 2 × 10 3 cells/well, cells were stained with 20 μl of CCK8 reagent 48 h after transfection. Cell viability was determined at the 450 nm absorbance.
Hep3B and Huh-7 cells were used for the dual-luciferase reporter assay. Luciferase activity was measured and compared 48 h later.
Luciferase assay was performed 48 h after transfection using a Dual-Luciferase Reporter Assay System (Promega).

| Western blot
Cells were washed twice with PBS, and total protein was extracted using RIPA buffer (Beyotime Biotechnology anti-GAPDH antibody, 1:5000; Santa Cruz). ECL (Millipore) After incubation with secondary antibodies, an enhanced chemiluminescence reagent was applied for detection.

| SLC16A1-AS1 is upregulated in HCC cells and tissues
To determine the expression of SLC16A1-AS1 in HCC tissues, we explored the expression of SLC16A1-AS1 in the TCGA data portal of starbasever3.0. As shown in Figure 1A, compared to normal tissues, SLC16A1-AS1 increased significantly in HCC tissues. Accordingly, the expression of SLC16A1-AS1 in HCC tissues and cells was significantly higher than that in normal cells ( Figure 1C,D). Kaplan-Meier analysis was performed, and the data revealed that compared to patients with low SLC16A1-AS1, patients with high SLC16A1-AS1 had a worse overall survival time ( Figure 1B). In addition, the subcellular distribution experiment was used to study the expression area of SLC16A1-AS1 in HCC cells. The results showed that SLC16A1-AS1 was mainly expressed in the nucleus rather than the cytoplasm ( Figure 1E,F). In summary, our findings suggest that SLC16A1-AS1 is overexpressed in HCC.

| SLC16A1-AS1 regulates proliferation and invasion of HCC in vitro
Considering the increased expression of SLC16A1-AS1 in HCC, we

| SLC16A1-AS1 acts as a ceRNA in HCC cells
It has been confirmed that lncRNA regulates the expression of miRNA through the sponge effect. Therefore, we used the TargetScan database to identify candidate miRNAs that interact with SLC16A1-AS1.
The potential binding site of miR-411 is shown in Figure 3A. QRT-PCR analysis suggested that miR-411 is lower expressed in HCC tissues compared to paired normal tissues ( Figure 3B). Then, a Pearson's correlation analysis was performed. Our data suggested that miR-411 expression level was negatively associated with the expression of SLC16A1-AS1 in 30 HCC patients ( Figure 3C). The expression level of miR-411 in HCC cells was also detected and found that its expression level was downregulated as expected ( Figure 3D). To further verify our hypothesis, we performed a luciferase reporter analysis to explore the correlation between miR-411 and SLC16A1-AS1.
The results showed that miR-411 significantly inhibited the luciferase activity of the cells transfected with SLC16A1-AS1-Wt, while the luciferase activity of the SLC16A1-AS1-Mut group was not affected ( Figure 3E,F). As mentioned above, the above data indicate that SLC16A1-AS1 acts as a ceRNA to regulate the expression of miR-411 in HCC cells.

| MiR-411 regulates malignant phenotypes of HCC cell
The TCGA data portal of starbasever3.0. As shown in Figure 4A,B, compared to normal tissues, miR-411 decreased and MITD1 increased significantly in HCC tissues. To study the biological functions of miR-411 in vitro. After transfection with miR-411 mimic, the expression of miR-411 in HCC cells was detected. The data showed that miR-411 was significantly increased in HCC cells ( Figure 4C,D).
Cell viability assays showed that miR-411 overexpression remarkedly stagnated cell proliferation ( Figure 4E,F). Then the cell invasion experiment showed that in the two HCC cell lines, the number of invaded cells was obviously reduced after the miR-411 mimic was transfected compared to the miR-411 mimic NC ( Figure 4G). In summary, these findings suggest that miR-411 plays a tumor suppressor role in the proliferation and invasion of HCC cells.

| MiR-411 directly targets MITD1
With the help of open access databases (TargetScan, MiRanda and Starbase3.0), we determined that MITD1 is a potential downstream target of miR-411. Therefore, a dual-luciferase reporter plasmid containing WT or MITD1 3′UTR mutants was synthesized ( Figure 5A).
The plasmid containing MITD1 3′UTR was co-transfected with miR-411 mimic or NC mimic into Hep3B and Huh-7 cells. Determination of luciferase activity. The data from the dual-luciferase reporter gene analysis showed that the 3′UTR mutation of MITD1 abolished the inhibitory ability of miR-411 compared to the wild type ( Figure 5B,C).

| DISCUSS ION
Numerous evidences showed that lncRNAs regulate a variety of biological processes and play an important role in tumorigenesis and MITD1 encodes a protein that regulates the activity of ESCRT-III and is necessary for normal cytokinesis. MITD1 has been shown to be involved in the shedding phase of cytokinesis. 16 Disorders of shedding may lead to tumorigenesis and genetic instability, especially when combined with mitotic stress caused by oncogenes. 17,18 In our study, we identified MITD1 as a downstream target of miR-411. SLC16A1-AS1 regulated MITD1 expression by interacting with miR-411. These data suggested that SLC16A1-AS1 exerts its function through the miR-411/MITD1 axis.

| CON CLUS ION
In conclusion, our study shows that SLC16A1-AS1 is upregulated in HCC tissues and cells. SLC16A1-AS1, as an oncogene in HCC, exerts a carcinogenic effect by regulating the miR-411/MITD1 axis. Our research helps to understand the role of SLC16A1-AS1 in HCC, suggesting that SLC16A1-AS1 may be used as a target for liver cancer treatment.

CO N FLI C T O F I NTE R E S T
All authors declare no conflicts of interest.

CO N S E NT TO PA RTI CI PATE
Prior written consent was well informed and signed by all participants.

CO N S E NT FO R PU B LI C ATI O N
All patients signed consent for publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.