In Hepatocellular Carcinoma, miRNA-296-3p Targets MSL2 and Suppresses Cell Proliferation and Invasion

Hepatocellular carcinoma (HCC) is the third-highest cause of cancer-related death in the world. miRNAs have a role in cell division, differentiation, and death biological processes. They are typically dysregulated in cancers, affecting tumor progression. miRNA-296-3p appears to play a crucial role in cancer control, according to new research. However, its expression and roles in HCC are unknown. This study used qRT-PCR and western blotting to detect the miRNA-296-3p and male-specific lethal 2 (MSL2) expression. In addition, cell proliferation, migration, invasion, and apoptosis were studied using CCK-8, flow cytometric analysis, colony formation assay, wound healing test, and transwell assays. The results show that miRNA-296-3p is underexpressed in HCC cell lines, particularly in Huh-7 and HepG2 cells. miRNA-296-3p overexpression lowers the ability of HCC cells to proliferate, migrate, and invade while increasing cell death. Luciferase reporter experiments revealed that the MSL2 is a direct target of miRNA-296-3p. Furthermore, overexpression of miRNA-296-3p reduced MSL2 mRNA and protein levels considerably, according to our findings. Furthermore, the rescue experiments showed that the MSL2 overexpression partially blocked the inhibition effects of miRNA-296-3p mimic on the proliferation and migration of HCC cells. The above results show that miRNA-296-3p may have a repressive effect in HCC by targeting MSL2 and could be used as a therapeutic target for HCC treatment.


Introduction
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-associated death worldwide [1][2][3]. Despite the significant advancements achieved in HCC treatment, the current survival rate of HCC patients remains poor [4,5]. Consequently, to improve HCC treatment and the quality of patients' lives, it is critical to investigate the molecular mechanisms behind HCC progression and look for new HCC treatment targets.
According to the previous research, chronic hepatitis B virus (HBV) infection is a significant risk factor for HCC [20][21][22]. Gao et al. [23] reported that MSL2 is extensively expressed in clinical HBV-related HCC tissues, implying that it could be a key mediator in the HBV-host interaction. Given the inhibition effects of miRNA-296-3p on tumors and the interaction of MSL2 and HBV, we assumed that miRNA-296-3p and MSL2 were involved in the procession of HCC. However, the interaction between miRNA-296-3p and MSL2 was not yet well documented.
Our study first identified the miRNA-296-3p expression characteristics in HCC. It confirmed the miRNA-296-3p beneficial effects on the proliferation, apoptosis, migration, and invasion of HCC cells. Second, we discovered that miRNA-296-3p had MSL2 as a target gene. Finally, we demonstrated that overexpression of MSL2 rescued the inhibition effect of miRNA-296-3p overexpression on HCC progression. In summary, our study revealed that miRNA-296-3p inhibited cell proliferation and invasion by targeting MSL2 in HCC cells and provided novel therapeutic targets against HCC.

Cell Culture and Transfection.
e Chinese Academy of Sciences (CAS; Shanghai, China) provided the average human liver cells HL-7702. e American Type Culture Collection provided the human HCC cell lines Huh-7, MHCC97-H, HepG2, and SMMC-7721 (ATCC; Manassas, VA, USA). All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin 100 IU/mL and streptomycin) (Gibco, Grand Island, NY, USA). ese cells were incubated at 37°C in a humidified environment containing 5% CO 2 .

Western Blotting Assay.
Total proteins were extracted from cells in RIPA buffer with protease inhibitor and then quantified using a BCA protein assay kit (Pierce, Santa Cruz, CA, USA). SDS-PAGE was used to separate equivalent amounts of protein and then deposited onto PVDF membranes (Millipore, Billerica, USA). After blocking with 5% nonfat milk, the primary antibodies were incubated with the PVDF membrane overnight at 4°C, followed by 1 h at 25°C with the appropriate secondary antibody. Enhanced chemiluminescence was used to see protein bands (Millipore, Billerica, MA, USA). Proteintech provided the primary antibodies against Cyclin D1, CDK2, P21, and β-actin (Chicago, IL, USA). Abcam provided antibodies against PCNA, Cox-2, MMP-2, and MMP-9 (Cambridge, MA, USA). Novus Biologicals provided the antibody against MSL2 (Littleton, CO, USA). β-Actin was used as the internal reference. Protein bands were visualized using enhanced chemiluminescence (MilliporeSigma) and quantified using ImageJ software (version 4.3; National Institutes of Health).

Cell Counting Kit-8 (CCK-8) Assay.
e CCK-8 kit was used to assess the ability of cells to multiply (Beyotime, Shanghai, China). Cells (3000 cells/well) were seeded in 96well plates and transfected with miRNA-296-3p mimic or NC mimic for 24, 48, and 72 hours, after which they were incubated in the dark for 2 hours with CCK-8 solution (10 μL). An ultraviolet spectrophotometer was used to measure optical density (OD) values at 450 nm ( ermo Fisher Scientific, USA).

Flow Cytometric Analysis of Apoptosis Rate and Cell Cycle.
Flow cytometric analysis was used to evaluate cell apoptosis and the cell cycle. e transfected cells were simply rinsed twice with PBS before being collected with trypsin. e cells were then resuspended and centrifuged at 4°C for 5 minutes at 1000 rpm, and the supernatant was discarded. After that, the cells were fixed in 75% ethanol and stored at 20°C overnight. Finally, the cells were rinsed with PBS and incubated with RNase before being stained with propidium iodide (PI) reagent (Sigma-Aldrich, USA) for 15 minutes at room temperature in the dark. According to the manufacturer's recommendations, flow cytometry (Beckman Coulter, CA, USA) was used to detect the cell cycle or apoptosis.

Colony Formation Assay.
e cells that had received different treatments were incubated in a six-well plate (500 cells/well) for the colony formation assay. e cells were rinsed twice in PBS, fixed with 10% formaldehyde, and stained for 10 minutes with 0.1% crystal violet. en, the colonies (>50 cells) were counted and photographed (Olympus, ×40, Tokyo, Japan).

Wound Healing Assay.
e ability of cells to migrate was assessed using a wound healing experiment. First, Huh-7 or HepG2 cells were sown on six-well plates, and a scratch was made using a 100 L pipette tip when the cells reached about 80% confluency. After that, the floating cells were rinsed multiple times with PBS, and a new medium was introduced. Next, we imaged the exact position of the wounds at two time points (0 and 48 h) with a microscope (Olympus, Tokyo, Japan). Finally, the migrated distance was calculated using a standard caliper.

Transwell Assay.
e transwell chambers with 8 μm pores (Merck KGaA, Darmstadt, Germany) were inserted into a 24-well plate to access the cells' migration and invasion. Cells were seeded onto the upper chamber with serum-free DMEM, while DMEM (10% FBS) was added to the lower chamber. For determining miRNA-296-3p overexpression effects on HCC cell migration or invasion, Huh-7 and HepG2 cells were transfected with miRNA-296-3p or NC mimic for 24 h. Nonmigrated cells on the upper surface of the membrane were removed with cotton swabs after a 24hour incubation at 37°C, and migrated or invaded cells were fixed with 4% paraformaldehyde for 10 minutes before being stained with 0.1% crystal violet for 30 minutes. Finally, cells were counted in five random fields using an Olympus microscope (Tokyo, Japan).

Statistical Analysis.
Every experiment was carried out at least three times. e mean and standard deviation were used to present all of the data (SD). e significance between the two groups was determined using the Student's t-test; statistical analysis was performed using one-way ANOVA. e comparison between the two groups was done with a post hoc test for data analysis among multiple groups. * P < 0.05, * * P < 0.01 were considered statistically significant differences.

miRNA-296-3p Is a Low-Expression Gene in HCC at
Inhibits Cell Proliferation When Overexpressed. Firstly, to analyze the role of miRNA-296-3p in HCC, we detected normal human liver cells (HL-7702) and 4 HCC cell lines (Huh-7, MHCC97-H, HepG2, and SMMC-7721) and the expression level of miRNA-296-3p with qRT-PCR. e results revealed that HCC cell lines had much lower levels of miRNA-296-3p expression than HL-7702 cells (Figure 1(a)). As a result, Huh-7 and HepG2 cells were used in the following tests. Next, we transfected miRNA-296-3p mimic into Huh-7 and HepG2 cells to investigate the effects of miRNA-296-3p on HCC progression. e expression level of miRNA-296-3p in the miRNA-296-3p mimic was considerably upregulated, according to the qRT-PCR study after transfection (Figure 1(b)). Furthermore, the CCK-8 test and colony formation assay revealed that overexpression of miRNA-296-3p decreased proliferation in Huh-7 and HepG2 cells (Figures 1(c) and 1(d)), implying that miRNA-296-3p inhibits HCC cell proliferation.

Overexpression of miRNA-296-3p Promotes HCC Cell
Cycle. miRNA-296-3p mimics dramatically accelerated apoptosis of Huh-7 and HepG2 cells, as demonstrated in Figure 2(a) compared to the NC-mimic group. Furthermore, flow cytometry analysis revealed that overexpression of miRNA-296-3p resulted in a considerable increase in G0/G1 phase cell numbers and a decrease in S or G2/M phase cell numbers (Figure 2(b)). Compared to the NC-mimic group, miRNA-296-3p mimics dramatically accelerated apoptosis of Huh-7 and HepG2 cells, as seen in Figure 2(a). According to flow cytometry studies, overexpression of miRNA-296-3p also resulted in a significant increase in G0/G1 phase cell numbers and a decrease in S or G2/M phase cell counts (Figure 2(b)).

miRNA-296-3p Negatively Regulates the Migration and Invasion of HCC Cells.
e effects of miRNA-296-3p on the migration and invasion capacities of HCC cells were investigated using wound healing assays, transwell assays, and western blot analysis. Transfection with miRNA-296-3p mimic reduced the wound closure rate of Huh-7 and HepG2 cells, as demonstrated in Figure 3(a). Furthermore, the transwell experiment revealed that the number of migrated/ invaded cells was reduced compared to the control group, whereas miRNA-296-3p was overexpressed in these cells (Figure 3(b)). Finally, a similar conclusion was reached after detecting the expression of migrated-associated proteins (Cox-2, MMP-9, and MMP-2) using western blot analysis (Figure 3(c)). All of these findings revealed that miRNA-296-3p played a critical role in inhibiting HCC cell motility and invasion.

MSL2 Is a Target
Gene of miRNA-296-3p. MSL2 has been proposed as a target for the therapy of HBV-related HCC in previous research [22]. To explore whether miRNA-296-3p inhibited HCC progression by targeting MSL2, we predicted the potential binding sites between miRNA-296-3p and MSL2 with the help of the TargetScan database. e 3′-UTR region of MSL2 mRNA had a binding site for miRNA-296-3p, which was discovered. A dual-luciferase reporter experiment in HEK-293T cells was used to further investigate the relationship between miRNA-296-3p and MSL2. When cells were transfected with miRNA-296-3p mimic, the luciferase activity of MSL2 3′-UTR was considerably reduced, as seen in Figure 4(b). However, when the putative binding sites were mutated, the miRNA-296-3p mimic exhibited modest effects. ese findings determined that MSL2 was a target of miRNA-296-3p. Furthermore, we discovered that overexpression of miRNA-296-3p significantly altered MSL2 mRNA and protein levels in Huh-7 and HepG2 cells (Figures 4(c) and 4(d)). In Huh-7 and HepG2 cells, the   Journal of Oncology amount of MSL2 was found to be inversely linked with miRNA-296-3p.

Discussion
Currently, it remains a significant challenge to search for novel therapeutic strategies in HCC. Several improperly expressed miRNAs in HCC tumors and adjacent nontumorous tissues have been identified by miRNA profiling of HCC tumor tissues and adjacent nontumorous tissues [25][26][27][28]. us, exploring the relevant mechanisms between miRNA and HCC has become a novel strategy to find HCC therapy. miRNA-296-3p was found to be underexpressed in HCC cell lines in this investigation. miRNA-296-3p overexpression inhibited cell proliferation, migration, and invasion in a practical sense. All these data demonstrate that miRNA-296-3p possesses antitumor effects in HCC. Previous studies have shown that miR-296-5p reduces tumor growth and spread in HCC [19]. Increasing evidence has confirmed that miRNA-296-3p exerts beneficial effects on regulating the biological behavior of human cancer cells. miRNA-296-3p expression is reduced in malignant choroidal melanoma [29]. miRNA-296-3p inhibits tumor cell proliferation, invasion, and metastasis in lung adenocarcinoma (LADC) [18]. Moreover, miRNA-296-3p participates in suppressing clear cell renal cell carcinoma (ccRCC) metastasis [30]. Concerning the progression of HCC, the functions and molecular mechanisms of miRNA-296-3p are still poorly understood. miRNA-296-3p overexpression in HCC cell lines inhibited cell proliferation and migration/invasion but enhanced cell death, according to the findings. Furthermore, we discovered that miRNA-296-3p overexpression reduced MSL2 mRNA and protein levels in HCC cell lines [21,22].
Male-specific lethal 2 (MSL2) exerts crucial roles in several molecular processes, such as targeting histone H2B and the tumor suppressor p53 [31]. MSL2 can enhance the growth of HepG2 cells in vitro and in vivo [23]. We used bioinformatics and a luciferase assay further to understand Values were mean ± SE, * P < 0.05 or * * P < 0.01 vs. NC mimic, # P < 0.05 vs. miRNA-296-3p mimic, n � 3 per group.
the mechanism of MSL2 on HCC cells. MSL2 was discovered to be a direct target of miRNA-296-3p, according to the findings. e level of MSL2 was shown to be adversely linked with miRNA-296-3p in our research. Overexpression of MSL2 decreased the effects of miRNA-296-3p overexpression on HCC cell proliferation, apoptosis, and migration, according to rescue experiments. It is well understood that p53's cytoplasmic location is essential for p53-mediated apoptosis and autophagy [31]. As a result, MSL2 functions as a critical ubiquitin ligase, mediating p53 ubiquitination and promoting p53 cytoplasmic localization. However, more research was needed to see if MSL acted as a regulator of p53-mediated apoptosis in HCC. Taken together, we found that miRNA-296-3p inhibited the proliferation, migration, and invasion in HCC by targeting MSL2. us, MSL2 was validated as a novel target of miRNA-296-3p. Furthermore, our findings revealed the therapeutic goals of the novel HCC. ey revealed that its clinical efficacy was worthy of further study.

Data Availability
All data generated or analyzed during this study are included in this published article.
Ethical Approval e experimental protocol was established according to the ethical guidelines of the Helsinki Declaration. It was approved by the Ethics Committee of the Qingpu Branch of Zhongshan Hospital.

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

Authors' Contributions
MA and XL conceived and designed the study. MA performed the literature search. XL performed the data extraction. MA drafted the manuscript. All authors read and approved the final manuscript. XL and MA contributed equally to this work.