INTRODUCTION:

Hepatocellular carcinoma (HCC) is one of the primary liver cancers with high mortality worldwide¹. The prevalence of HCC is increasing each year. HCC is a multifactorial disease caused by a combination of multiple risk factors such as liver cirrhosis², hepatitis B virus (HBV) and hepatitis C virus (HCV) infection³, Type 2 diabetes mellitus (T2DM)⁴, alcohol abuse and smoking⁵.

Other less common risk factors include genetic factors including autoimmune hepatitis, Wilson’s disease, hereditary hemochromatosis and alpha-1-antitrypsin deficiency⁶. At present, treatment for HCC is mainly based on curative treatments such as surgical resection, liver transplantation, cryoablation, trans-arterial embolization (TAE) and trans-arterial chemoembolization (TACE)⁷. Meanwhile, palliative treatments such as systemic therapy, molecular-targeted therapy and immunotherapy^8,9,10. Despite great advancements in HCC treatment, the prognoses of HCC patients are still dismal and effective therapeutic options for HCC are also limited¹¹. Therefore, it is urgently needed to explore therapeutic strategies for HCC. The mechanism of HCC growth and progression is still largely undiscovered. Thus, understanding the HCC mechanisms would provide potential therapeutic strategies for HCC.

In recent years, research has focused on non-coding RNA (RNA), such as long non-coding RNA (lncRNA) and microRNA (miRNA), as important regulators of human cancers, including HCC. LncRNAs are untranslated transcripts typically 1000–10,000 nucleotides in length, which are referred to as miRNA host genes (MIRHG). The lncRNAs served as the precursors and regulators of miRNAs. miRNA inhibits gene expression via binding to the 3’untranslated region (3’UTR) of their target mRNAs. However, less is known regarding the biological function of the lncRNA-miRNA and mRNA axis. In cancer and disease, altered lncRNA expression is often caused by deregulation of miRNA expression. For example, the lncRNA MALAT1 is highly expressed in HCC, and its high expression is significantly promoting cell proliferation and metastasis.

In this study, we focused on the lncRNA, microRNA-195-497 cluster host gene (MIR497HG) (Gene ID: 100506755; Ensembl: ENSG00000267532). Altered lncRNA MIR497HG expression has been documented in various cancers such as glioma cancer, bladder cancer and colorectal cancer^12,13,14. Nevertheless, MIR497HG expression in HCC is still elusive. MicroRNAs (miRNAs) are a class of endogenous small ncRNAs with approximately 20-22 nucleotides in length that regulate gene expression by binding to the 3’untranslated region (3’UTR) of the target mRNA¹⁵. MicroRNA-3182 (miR-3182) is located at chromosome 16 and belongs to families of miRNA, which are highly conserved sets of single-stranded ncRNA. miR-3182 has played an important role in the growth and development of cancer. Expression of miR-3182 shows high tissue and cell type specificity, which has been found to be downregulated in osteosarcoma¹⁶, retinoblastoma¹⁷, melanoma¹⁸, and lung cancer¹⁹. In contrast, miR-3182 has been found to be upregulated in nasopharyngeal carcinoma²⁰. Thus, miR-3182 acts as a tumour suppressor or oncogene in different types of cancers. Nevertheless, miR-3182 expressions in HCC have not been investigated yet. The exact roles of miR-3182 have yet to be elucidated, as there are limited publications on its role in cancer, especially HCC.

Currently, there is an emergence of effective treatment modalities for HCC. Thus, understanding the lncRNA-miRNA-mRNA of HCC allows the discovery of non-invasive and specific biomarkers of HCC. Apart from that, the regulatory role of MIR497HG and its miRNA in HCC remains unclear. To our knowledge, for the first time we reported the regulatory role of MIR497HG and miR-3182 in HCC. This study applied the assay to confirm and validate the lncRNA/miRNA/mRNA axis pathways in HCC through bioinformatic analysis and in vitro experimental approaches. Taken together, this study helps to better understand the role of miR-3182 in HCC.

MATERIALS AND METHODS:

Maintenance of Cell Culture:

Human hepatocellular carcinoma (HepG2) and normal liver (WRL-68) cell lines were purchased from the iCell Bioscience, Shanghai, China. All cells were routinely maintained in a sterile-filtered mixture of Minimum Essential Medium (MEM) (Sigma, USA) supplemented with 10% fetal bovine serum (FBS) (Tico, Europe), 1% L-glutamine (Sigma, USA) and 1% of penicillin/streptomycin antibiotic (Nacalai Tesque, Japan). Cells were maintained and kept grown in a humidified incubator with 5% CO₂ atmosphere at 37^oC. The cell growth, condition and morphology were observed under a light-inverted microscope (Olympus, Japan). All other chemicals were of analytical grade unless otherwise stated.

Sequence Retrieval and Computational Analysis:

The conservative sequence and annotation of the miR-3182 (miRbase: MIMAT0015062) (mature miR-3182 sequence: 5’-GCUUCUGUAGUGUAGUC-3’) was obtained from the miRbase database (https://www.mirbase.org/) and the full-length of the MIR497HG sequence was obtained from the National Centre for Biotechnological Information (NCBI) RefSeq database (https://www.ncbi.nlm.nih.gov/) (Gene ID: 100506755) (Ensemble: ENSG00000267532). TargetScan 8.0 (https://www.targetscan.org/vert_80/) was used for the identification and prediction of the miR-3182 binding sites in the 3’UTR of MIR497HG.^21,22

Cells Transient Transfection:

The miR-3182 mimic (sequence: 5’-GCUUCUGUAGUGUAGUC-3’) and miR-3182 inhibitor (a single-stranded antisense RNAs targeting miR-3182) were synthesized by Qiagen, USA. Briefly, 2 x 10⁵ cells per well were seeded in 96-well cultured in an antibiotic-free medium and transfected with miR-3182 mimic and miR-3182 inhibitor (10 nM) using the FugeneHD transfection reagent (Promega, USA) according to the manufacturer’s protocol. Cells were harvested at 24 and 72 h post-transfection for use in subsequent experiments.

Cell Proliferation Assay:

The cell proliferation was performed using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, USA)²³. HepG2 cells were seeded in the 96-well plates and transfected with miR-3182 mimic and miR-3182 inhibitor. Then, cells were incubated for 24 and 48 h at 37˚C with 5% CO₂ atmosphere. Subsequently, a 20μl of cell proliferation assay reagent was added to cells. Cells were incubated for 2 hours. The optical density was measured at 490nm using a microplate reader, Tecan Infinite M200 Pro (DKSH, Austria).

Total RNA Extraction, cDNA Synthesis and RT-qPCR:

Total RNA in HCC and normal liver cell lines were extracted using the miRNeasy^™ Mini Extraction kit (Qiagen, USA) according to the manufacturer’s protocol. The concentration was determined by using the QIAxpert (Qiagen, USA). The RNA was separated by 1% of 1x TAE agarose gel electrophoresis and fragments were visualized using AlphaImager™ Gel Imaging System (Alpha Innotech). Based on the quality and concentration of RNA, the RNA was reverse-transcribed with the miRCURY LNA RT kit (Qiagen, Germany) and Quantitect Reverse Transcription kit (Qiagen, USA) according to the manufacturer’s protocol. The resultant cDNA was used for the RT-qPCR. The expression level of miR-3182 was quantified using the miRCURY LNA SYBR® Green PCR kit (Qiagen, USA). Meanwhile, expression of MIR497HG, Bcl-2 and CCND2 were quantified using the Quantinova SYBR Green PCR kit (Qiagen, USA) respectively according to the manufacturer’s protocol. The RT-qPCR was performed using the Rotor-Gene Q (Qiagen, USA). The following PCR cycling program for miRNA was employed as follows; initial activation step at 95^oC for 2 min, followed by 40 cycles of denaturation at 95^oC for 10 s, and annealing at 56^oC for 60 s. Meanwhile, PCR cycling program for mRNA was employed as follows: initial activation step at 95^oC for 2 min, followed by 40 cycles of denaturation at 95^oC for 5 s, and annealing at 60^oC for 10 s. Melting curve analysis of the PCR products was performed to assess the specificity of the PCR products. Primers used in this study were purchased from the Qiagen, USA. As controls, human glyceraldehyde-3-phosphate (GAPDH) and U6snRNA (U6RNA) were used for the mRNA and miRNA normalization respectively. Relative expressions were calculated by the 2 delta-delta ct (2^-∆∆Ct) method²⁴. The list of specific primers used were summarized in Table 1.

Statistical Analysis

The data are expressed as means ± standard deviation using the GraphPad^® Prism Version 9.0 analysis software (GraphPad Software Inc, USA). Data analysis was carried out using a student’s t-test (two-tailed) and one way ANOVA with Tukey’s post hoct test. P<0.05 was considered as a statistically significant difference.

RESULT:

miR-3182 Targeting the 3’UTR of MIR497HG, Bcl-2 and CCND2:

Using the TargetScan, the miR-3182 binding site in the 3’UTR of the MIR497HG region was predicted and matched with at least one binding site. Our results suggested that miR-3182 possessed a potential binding ability to the 3’UTR of MIR497HG, Bcl-2 and CCND2. The target prediction shown in (figure 1).

Figure 1: The miR-3182 binding site A) the miR-3182 binding sites in the Bcl-2 B) the miR-3182 binding sites in the CCND2 as predicted by the TargetScan C) MIR497HG/miR-3182/Bcl-2 axis and D) MIR497HG/miR-3182/CCND2 axis. The red highlighted nucleotides are the complementary miR-3182 seed sequence.

miR-3182 and MIR497HG are Downregulated in HCC Cell Line:

To evaluate and validate the observation from computational analysis, in vitro cell validation was carried out in this study. We first examined the expression of miR-3182 and lncRNA MIR497HG in HepG2 and WRL-68 cell lines. The HepG2 exhibited significant decreases of miR-3182 and MIR497HG compared to the WRL-68 cell line (P<0.05) as depicted in (figure 2). These results indicate that deregulation of miR-3182 and MIR497HG in the HCC cell line suggesting a potential role for both miR-3182 and MIR497HG in HCC.

Figure 2: The expression level A) miR-3182 and B) MIR497HG in WRL-68 and HepG2 cell lines. Data are represented as the mean ± standard deviation (n=3). *P<0.05 compared to the WRL-68 cell line.

Bcl-2 and CCND2 are Upregulated in HCC Cell Line:

The expression of anti-apoptotic genes, Bcl-2 and CCND2 in HCC and normal cell lines were further assessed. Notably, significant increases in Bcl-2 and CCND2 expressions were observed in the HCC cell line compared with the normal liver cell line (P<0.05) as shown in (figure 3).

Figure 3: The expression level A) Bcl-2 and B) CCND2 in WRL-68 and HepG2 cell lines. Data are represented as the mean ± standard deviation (n=3). *P<0.05 compared to the WRL-68 cell line.

miR-3182 Regulates HCC Cell Proliferation:

We continued to explore the effect of overexpression of miR-318 on the proliferation of HepG2 cells at two time points. There was significant decrease of cell proliferation in miR-3182 mimic groups at 24 h post- transfection compared to the control (P<0.05). There was no significant change in groups treated with miR-3182 inhibitor at 24 h post-transfection compared to control (P>0.05). Meanwhile, there was no significant change in the proliferation of the proliferation of cells in miR-3182 mimic and miR-3182 inhibitor at 72 h post-transfection compared to control (P>0.05). The results reveal that overexpressing miR-3182 reduces the cell proliferation in HCC cells. The results depicted as in (figure 4).

Figure 4: Overexpression of miR-3182 inhibits cell proliferation. Data are represented as the mean ± standard deviation (n=3). **P<0.01 compared to control.

miR-3182 Mimic and Inhibitor Regulate Expression of Endogenous miR-3182 in HCC Cell Line:

The transfection efficiency of miR-3182 mimics and miR-3182 inhibitors was determined by examining the expression of endogenous miR-3182 using RT-qPCR (figure 5). At 24 h post-transfection, miR-3182 expression was found to be significantly higher in the miR-3182 mimic-treated group compared to the untreated control (P<0.001). Meanwhile, miR-3182 expression was significantly lower in the miR-3182 inhibitor-treated group compared to the untreated control (P<0.05). On the contrary, there was no significant difference in miR-3182 expression in the miR-3182 mimic and miR-3182 inhibitor group at 48 h post-transfection compared to the control (P>0.05). The result suggests that transfection of miR-3182 mimics and miR-3182 inhibitors could influence and regulate the expression of endogenous miR-3182 in HepG2 cells in a time-dependent manner.

Figure 5: The expression level of endogenous miR-3182 at 24 and 48 h post transfection. Data are represented as the mean ± standard deviation (n=3). **P<0.01, ***P<0.001, ****P<0.0001 compared to the control.

miR-3182 Mimic and Inhibitor Regulate Expression of Bcl-2 in HCC Cell Line:

As described earlier, through computational analysis, miR-3182 directly targets the Bcl-2 and CCND2. There was a significant decreased in Bcl-2 level in miR-3182 mimic group at 24 h post-transfection in HepG2 cells compared to control (P<0.01). Meanwhile, there was no significant change in the Bcl-2 in miR-3182 mimic groups at 48 and 72 h post-transfection compared to control (P>0.05). Meanwhile, we observed that there were significant increases in Bcl-2 level in cells treated with 50 nM miR-3182 inhibitors at 24, 48 and 72 h post-transfection compared with controls (P<0.0001), (P<0.0001) and (P<0.01). Data suggests that overexpression and inhibition of miR-3182 could influences Bcl-2 in HCC cell line (figure 6).

Figure 6: The expression of Bcl-2 following Transfection of 50 nM of miR-3182 mimic and miR-3182 inhibitor. Data are represented as the mean ± standard deviation (n=3). **P<0.01, *P<0.05 compared to control.

miR-3182 Mimic and Inhibitor Regulate Expression of CCND2 in HCC Cell Line:

There was significant decreased in CCND2 level in HepG2 cells treated with 50 nM miR-3182 mimic at 24 h post-transfection compared to control (P<0.05). However, there was no significant change in CCND2 level in HepG2 cells treated with miR-3182 mimic at 48 and 72 h compared to control (P>0.05). Conversely, there were significant increase in CCND2 levels in miR-3182 inhibitor-treated cells at 24, 48 and 72 h post-transfection compared to control groups (P<0.01), (P<0.0001) and (P<0.01) respectively. Data suggests that inhibition and overexpression of miR-3182 could influences the CCND2 in HCC cells through time-dependent manner (figure 7).

Figure 7: The expression of Bcl-2 following Transfection of 50 nM of miR-3182 mimic and miR-3182 inhibitor. Data are represented as the mean ± standard deviation (n=3). ****P<0.0001, **P<0.01, *P<0.05 compared to the control.

DISCUSSION:

Mounting numbers of lncRNAs and miRNAs have been reported to be associated with HCC growth and progression. However, the potential effect of miRNAs on HCC still needs further exploration. Our previous report demonstrated that miR-3182 potentially targeting the 3’UTR of MIR497HG by computational analysis²⁵. In this regard, the present study experimentally revealed the mechanism by which miR-3182 influences the 3’UTR of MIR497HG and its target genes. We identified MIR497HG as direct target of miR-3182. Additionally, we also found that miR-3182 as a direct target of Bcl-2 and CCND2. These data suggests that miR-3182 may be an important regulator in the expression of MIR497HG, Bcl-2 and CCND2. Thus, these findings could help in understanding the lncRNA/miRNA/mRNA regulatory network.

Numerous studies have reported that lncRNAs such as TGLC15²⁶, LINC00467²⁷ and DUXAB8²⁸ play an important role in HCC growth and progression. However, the exact underlying molecular mechanism of HCC remains largely unexplored. For example, the lncRNA MIR497HG expression was found to be downregulated in glioma¹², bladder cancer¹³ and colorectal cancer¹⁴. In this study, we found that MIR497HG expression was observed to be downregulated in the HCC cell line, consistent with other studies²⁹.

miR-3182, a member of the miRNA family has been found as a tumour suppressor miRNA in various cancers including melanoma¹⁸, lung cancer¹⁹ and nasopharyngeal carcinoma²⁰. However, the function of miR-3182 in HCC has not yet been investigated and is still elusive. Thus, our study fills the gap in miRNA research, particularly for miR-3182. Here, computational analysis and in vitro study confirmed that miR-3182 was notably downregulated in the HCC cell line. This subsequently suggests that miR-3182 might be a key miRNA in HCC. Our study shows that miR-3182 is one of the tumour suppressors (tsmiRNA) in HCC, which is consistent with the findings on the inhibitory role of miR-3182 in other cancers^18,19. Here, we report that miR-3182 potentially attenuates HCC proliferation by modulating the expression of MIR497HG as well as target genes; Bcl-2 and CCND2 in the HCC.

In this study, Bcl-2 and CCND2 were identified as novel downstream targets of miR-3182. We reported here that Bcl-2 and CCND2 were targeted by miR-3182 via computational analysis and in vitro experiments. Bcl-2 is an anti-apoptosis factor that is important in HCC growth and apoptosis inhibition³⁰. Previous evidence revealed that Bcl-2 has detectable mRNA levels in HCC^31,32. Additionally, we found that Bcl-2 was highly expressed in HCC cell line, consistent with other studies. Furthermore, this finding is similar to the finding by³³ demonstrating CCND2 expression is higher in HCC compared to normal liver cells.

MIR497HG was observed to be downregulated in the HCC cell line and inversely correlated with the expression of Bcl-2 and CCND2. miRNAs have been identified as tumour suppressor and oncogene based on their modulating effect on the expression of their target genes. For instance, overexpression of oncogenic miRNAs (oncomiRs) blocks tumour suppressor genes and leads to tumour growth. Meanwhile, the downregulation of tsmiRs increases the translation of oncogenes. In this study, miR-3182 was found to act as a tsmiR that modulates the expression of MIR497HG and target genes; Bcl-2 and CCND2. Single miRNA in cancer cells is not caused by its strong effect on single target genes but reflects the enrichment of its effects on multiple target genes involved in a specific signaling pathway³⁴. Importantly, we found that miR-3182 could target genes aberrantly expressed Bcl-2 in HCC cells when restored. Therefore, overexpression miR-3182 can be considered as promising agents to suppresses apoptosis-related genes³⁵.

For instance, it was observed that miR-3182 can significantly alter the expression pattern of MIR497HG and its downstream target genes, Bcl2 and CCND2 in a dosage and time dependent manner. Here, 10 and 50 nM of miR-318 mimic and miR-3182 inhibitor was used, which respond to the manufacturer’s suggestion and literature search as common ranges of miRNA for gain-of-function and loss-of-function studies. For instance, different targets can be suppressed by different concentration and incubation times, which result in selective downregulation of target genes³⁶.

Nevertheless, our study has several limitations. First, Bcl-2 and CCND2 were shown to regulate the HCC by activating the apoptosis and cell cycle progression signalling pathways respectively. Therefore, the downstream signalling pathways of the MIR497HG/miR-3182/ Bcl-2 and MIR497HG/miR-3182/ CCND2 axes should be further explored for future study. In addition, we demonstrated the function of the MIR497HG/miR-3182/Bcl-2 and MIR497HG/miR-3182/CCND2 axes in vitro only. Its function should be deeply investigated using animal models in the future. For instance, the present study confirmed that the regulation of miR-3182 in HCC could serve as a therapeutic target for HCC. Our results proved that miR-3182 mimics could serve as therapeutic agents to exert their anticancer effects on HCC.

CONCLUSION:

We demonstrate the functional role of miR-3182 in HCC. Overall, miR-3182 could be explored as a non-invasive biomarker and potential therapeutic target for HCC. However, further studies are needed to confirm it. For example, in the future, the effects observed in this study will be confirmed on a protein level using Western blot. Importantly, our data may facilitate a deeper understanding of the mechanisms associated with HCC and target miRNAs as potential cancer therapies.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this study.

ACKNOWLEDGMENTS:

The study was supported by Faculty of Pharmacy, Universiti Teknologi MARA, UiTM Puncak Alam Campus and The Ministry of Higher Education (MOHE) Malaysia, under the Fundamental Research Grant Scheme, (FRGS/1/2019/SKK10/UITM/02/4).

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Received on 22.03.2024 Revised on 18.03.2025

Accepted on 04.10.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):61-68.

DOI: 10.52711/0974-360X.2026.00010

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Primers	Sequences	Sources
MIR497HG		GeneGlobe ID: SBH0663009 (Qiagen, USA)
Bcl-2	Forward: 5’-GATGACTGAGTACCTGAACCG-3’ Reverse: 5’-AGCCAGGAGAAATCAAACAGAG-3’	Integrated DNA Technologies (Hs.PT.56a2905156)
CCND2	Forward: 5’-GACATCCAACCCTACATGCG-3’ Reverse: 5’-CCAAGAAACGGTCCAGGTAA-5’	Integrated DNA Technologies (Hs.PT.58.28257)
GAPDH	Forward: 5’-ACATCGCTCAGACACCATG-3’ Reverse: 5’-TGTAGTTGAGGTCAATGAAGGG-3’	Integrated DNA Technologies (Hs.PT.39a.22214836)
miR-3182	miRBase Accession: MIMAT0015062 5’- GCUUCUGUAGUGUAGUC-3’	GeneGlobe ID: YP02115918 (Qiagen, USA)