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. 2023 Nov 6;6(12):1817–1828. doi: 10.1021/acsptsci.3c00114

MicroRNA-204-5p Inhibits Hepatocellular Carcinoma by Targeting the Regulator of G Protein Signaling 20

Yanqing Su †,‡, Yao Lu ‡,§, Honglin An ‡, Jinhong Liu ‡,∥,⊥, Feimin Ye ‡, Jiayu Shen ‡, Zhuona Ni ‡, Bin Huang ‡,∥,⊥,*, Jiumao Lin ‡,∥,⊥,*
PMCID: PMC10714421  PMID: 38093845

Abstract

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Although the oncogenic roles of regulator of G protein signaling 20 (RGS20) and its upstream microRNAs (miRNAs) have been reported, their involvement in hepatocellular carcinoma (HCC) remains unexplored. We utilized the starBase, miRDB, TargetScan, and mirDIP databases, along with a dual-luciferase reporter assay and cDNA chip analysis to identify miRNAs targeting RGS20. miR-204-5p was selected for further experiments to confirm its direct targeting and downregulation of the RGS20 expression. To study the miR-204-5p/RGS20 axis in HCC, RGS20 and miR-204-5p were increased in PLC/PRF/5/Hep3B cells, and the viability, hyperplasia, apoptosis, cell cycle, and invasion/migration of the cells were assessed. RGS20 exhibited optimism, while miR-204-5p exhibited pessimism in tumors. miR-204-5p directly targeted RGS20 and downregulated its expression, whereas high RGS20 expression indicated a poor prognosis. Transfection of miR-204-5p inhibited the hyperplasia, migration, and invasion of HCC cells, but promoted apoptosis and influenced the levels of cyclin-dependent kinase 2 (CDK2), cyclin E1, B-cell lymphoma-2 (Bcl-2), Bax, and cleaved caspase-3/8. These effects were reversed by overexpression of RGS20. We recognized miR-204-5p as an upstream regulator targeting RGS20, thereby inhibiting HCC progression by downregulating RGS20 expression. RGS20 may prove to be a potential target for HCC treatment, and miR-204-5p might seem like to be a potential miRNA in gene therapy.

Keywords: hepatocellular carcinoma, miR-204-5p, RGS20, proliferation and apoptosis, migration and invasion

The proportion of hepatocellular carcinoma (HCC) diagnoses among patients with primary liver cancer ranges from 70–90% and is associated with poor prognoses. The average survival time is reported to be 6–20 months, with a mortality rate approaching 100%.1 The incidence rate is in a higher numerical value in East Asia than in Western countries. However, the latter has exhibited an increasing trend over the past several years.2,3 The vast majority of patients with HCC are typically diagnosed at an intermediate or later period of disease progression. Numerous studies have reported a maximum 5-year survival of only 18% for symptomatic people.1,4 HCC ranks as the third leading factor in the astronomically alarming high cancer death rate.5 The G protein-coupled receptor family comprises cell surface receptors that are involved in cell proliferation, survival, and motility. As such, this receptor family is closely associated with tumor growth, neovascularization, and metastasis in carcinoma development.6 G protein signal transduction regulators are Gα subunit GTPase activity sensitizers that amplify GTPase activity and are important factors in the G protein-coupled receptor protein signal transduction pathway.7 Regulator of G protein signaling 20 (RGS20) is a G protein signal regulator protein and a GTPase activator that can increase GTPase activity 100-fold; it participates in the G protein-coupled receptor protein signal transduction pathway and regulates cell functions.8 It has been shown that RGS affects the process of cell development by adjusting downstream cell signaling pathways.9 Although RGS20 is acknowledged as a proto-oncogene in certain cancers, its precise role in HCC has not been fully elucidated.10−12

MicroRNAs (miRNAs) are endogenous noncoding RNAs, usually between 20 and 25 nucleotides in length, that are ubiquitously found in eukaryotic cells.13 More than 70% of human gene expression is regulated by miRNAs; there are multiple and complex regulatory relationships between target genes and miRNAs.

miRNAs participate in a succession of cellular biological processes with significant impact.14 miRNAs can regulate gene expression post-transcriptionally, which influences the expression of both onco- and tumor-suppressor genes.15 Abnormal expression of miRNAs can lead to the differential expression of regulated target genes or an imbalance in signaling pathways, resulting in a variety of diseases.16 Research has revealed an incredible effect of miR-204-5p in various cancers, which constrains the multiplication of HCC cells by controlling the target genes of E2F1, SIX1, and SIRT1.17−19 However, the regulatory relationship between miRNAs and RGS20 in HCC has not been reported.11,12,20 Therefore, it is important to clarify whether miRNAs can directly affect the expression of RGS20 and how miRNAs and RGS20 are engaged in regulating the biological processes of HCC cells. This has important implications for the clinical treatment of HCC and future research on miRNA gene therapy. This study validates that miR-204-5p specifically targets the 3′-untranslated region (UTR) of RGS20 and downregulates the expression of RGS20 expression. The implications of miR-204-5p and RGS20 expression on the changes of HCC cell state reveal the importance of the miR-204-5p/RGS20 axis in the process of HCC and provide a new direction for miRNA gene therapy.

Results

RGS20 is Highly Expressed in HCC Tissue

The starBase database was applied to compare the mRNA levels of RGS20 to explore the relationship between RGS20 and HCC. RGS20 was dramatically higher for tumors than for the corresponding control tissues (P < 0.0001) (Figure 1A). Patients with different Child–Pugh grades exhibited no differences in RGS20 mRNA levels (Figure 1B), likely due to the limited number of samples. However, patients with macrovascular invasion exhibited higher RGS20 mRNA levels than those without vascular invasion (Figure 1C). Moreover, higher levels of RGS20 mRNA occasionally correlated with a poorer histological grade (Figure 1D) and tumor-node-metastasis (TNM) stage. RGS20 mRNA levels were higher in G3 and stage II and III tumors than in the corresponding control tissues (Figure 1D,E). Additionally, higher RGS20 expression levels often indicated a poorer prognosis, with a hazard ratio of 1.77 (Figure 1F). These results suggest that RGS20 is a proto-oncogene associated with HCC.

Figure 1.

Figure 1

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RGS20 expression is frequently upregulated in patients with liver carcinoma. (A) RGS20 was found to have huge gaps in individuals who were healthy (n = 50) and patients with liver carcinoma (n = 369). (B) RGS20 expression in HCC tissues was independent of the Child–Pugh grade. (C) Vascular invasion upregulated RGS20 mRNA expression in tumors. (D) RGS20 were substantially higher in G3 tumors than in the corresponding controls. (E) TNM stage II and III tumors had higher RGS20 mRNA levels than the corresponding control. (F) The impact of RGS20 expression on overall survival rate. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.

miR-204-5p is Lowly Expressed in HCC

Six miRNAs targeting RGS20 were screened using four databases (miRDB, TargetScan, starBase, and mirDIP) to predict (Figure 2A) their anticancer effects in HCC by regulating the expression of RGS20. A weak negative correlation has come to light between RGS20 and miR-204-5p (Figure 2B). Meanwhile, miR-204-5p was dramatically lower for tumors than for normal cells (Figure 2C). Further analysis indicates that miR-204-5p in HCC were closely associated with alpha-fetoprotein (AFP) levels, vascular invasion, and histological grade (Figure 2D,F,G), but not with Child–Pugh grade or TNM stage (Figure 2E,H). Furthermore, higher miR-204-p expression indicated longer overall survival (Figure 2I). On the basis of the results, we conclude that miR-204-5p exerts an anticancer effect in HCC by targeting RGS20 expression.

Figure 2.

Figure 2

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miR-204-5p is frequently downregulated in patients with HCC. (A) Venn diagram showing miRNA targeting RGS20 expression. (B) The mRNA expression between RGS20 and miR-204-5p manifested a slight negative correlation. (C) miR-204-5p were found with huge gaps in normols (NT, n = 50) and tumors (n = 370). (D) miR-204-5p levels is lower for AFP-positive HCC tissues than for AFP-negative HCC tissues. (E) miR-204-5p expression was independent of Child–Pugh grade. (F) miR-204-5p expression was less in macro vascular invasion compared with that in no or micro vascular invasion. (G) The expression of miR-204-5p was higher in G1 than in G2, G3, and G4. (H) miR-204-5p was entirely unrelated to TNM stage. (I) Higher presentation of miR-204–5p predicted better livability. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.

Targeting RGS20 Expression by miR-204-5p

The targetedness to RGS20 expression by miR-204-5p was confirmed by constructing the wild-type RGS20 3′-UTR (3′-UTR WT) reporter vector and introducing mutations at two potential binding sites (7mer-m8 and 8mer) to create the mutant RGS20 3′-UTR (3′-UTR MUT) vector (Figure 3A). The luciferase activity of 3′-UTR WT was sharply declined after cotransfection with miR-204-5p mimic compared to that of the NC mimic in PLC/PRF/5 and Hep3B cells, but miR-204-5p exhibited entirely unrelated to the reporter activity of 3′-UTR MUT (Figure 3B). In HCC cells transfected with miR-204-5p, RGS20 was enormously debased (Figure 3C–E). According to the cDNA microarray data, there was a slight negative relationship between the mRNA expression of RGS20 and miR-204-5p (Figure 3F). Patients with HCC tended to express higher RGS20 mRNA levels than healthy individuals. Additionally, mRNA levels of RGS20 were dramatically higher for liver cancer samples than those for the adjoining hepatic tissues (Figure 3G). miR-204-5p expression exhibited an opposite tendency, however (Figure 3H). The results suggest that miR-204-5p acts directly on the 3′-UTR of RGS20, leading to downregulation of RGS20 expression.

Figure 3.

Figure 3

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miR-204-5p targets RGS20 and downregulates its expression. (A) Figure displaying the combination of miR-204-5p with RGS20 3′-untranslated region (UTR). (B) dual-luciferase reporter assay. (C) Detection of miR-204-5p using RT-qPCR. (D) Transfection of miR-204-5p downregulated RGS20 mRNA. (E) Transfection of miR-204-5p downregulated RGS20 protein expression. (F) miR-204–5p and RGS20 showed a weak negative correlation according to the cDNA microarray data set analysis. (G) RGS20 is sharply higher in tumors than in healthy individuals/adjacent liver tissue (ANT). (H) miR-204-5p was enormously less expressed in tumors than in healthy individuals/adjacent liver tissues. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.

miR-204-5p Inhibits Cell Proliferation by Targeting RGS20

The findings also confirmed that miR-204-5p targets RGS20 and exerts a suppressive role in HCC. The miR-204-5p mimic was transfected into PLC/PRF/5/Hep3B to resolve the influence of miR-204-5p on cellular function, followed by overexpression of RGS20 in rescue experiments (Figure 4A–C). MTT assay results verified that high levels of miR-204-5p tremendously constrain proliferation (Figure 4D). EdU assay results verified that miR-204-5p constrains cell replication (Figure 4E). The proportion of EdU+ PLC/PRF/5 cells decreased from 54.1 ± 1.2 to 43.8 ± 4.8% (P = 0.0223), and the proportion of EdU+ Hep3B cells decreased from 42.6 ± 2.1 to 34.2 ± 3.1% (P = 0.0186) after transfection of the miR-204-5p mimic. Meanwhile, cotransfection of miR-204-5p and the RGS20 expression vector increased the proportion of EdU+ PLC/PRF/5 and Hep3B cells from 43.8 ± 4.8 to 64.63 ± 2.6% (P = 0.0027, Figure 4F left) and from 34.2 ± 3.1 to 49.57 ± 2.4% (P = 0.0025, Figure 4F right), respectively. Cell cycle assays validated that miR-204-5p caused cells to arrest in the G0/G1 phase (Figure 4G). When miR-204-5p was transfected into PLC/PRF/5 cells, it increased the ratio of cells in the G0/G1 phase from 48.8 ± 1.9 to 65.35 ± 1.3% (P = 0.0001, Figure 4H). After transfection of miR-204-5p into Hep3B cells, the ratio of G0/G1 phase cells increased from 50.5 ± 1.2 to 56.1 ± 0.7% (P = 0.0024, Figure 4H). Additionally, increased miR-204-5p levels downregulated the key cell cycle proteins CDK2 and cyclin E1 (Figure 4I,J). However, RGS20 overexpression reversed these effects (Figure 4D–J). The findings show that miR-204-5p induces G0/G1 phase arrest by downregulating RGS20, thereby constraining cell proliferation.

Figure 4.

Figure 4

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miR-204-5p restricts cell proliferation by targeting RGS20. (A) Detection of miR-204-5p using RT-qPCR. (B) RGS20 mRNA was detected by using RT-qPCR. (C) RGS20 protein was detected using Western blot. (D) miR-204-5p inhibited cell viability. (E) Representative images of the EdU experiment. (F) miR-204-5p inhibited cell replication. (G) Univariate histograms of the flow cytometry detection. (H) miR-204-5p transfection causes G0/G1 phase arrest. (I) Representative Western blot image of cell cycle proteins. (J) miR-204-5p inhibits CDK2 and cyclin E1. *P < 0.05, **P < 0.01, *** and ****P < 0.001, vs mimic NC + vector; ns, not significant; #P < 0.05, ##P < 0.01, ### and ####P < 0.001, vs miR-204-5p mimic + vector.

miR-204-5p Promotes Apoptosis by Targeting RGS20

The action of miR-204-5p and RGS20 expression levels on apoptosis was analyzed by flow cytometry. Increased levels of miR-204-5p promoted apoptosis, whereas the proportion of apoptotic cells was reduced after overexpression of RGS20 (Figure 5A). For PLC/PRF/5 cells, the proportions of apoptotic cells in the mimic NC + vector and miR-204-5p mimic + vector groups were 8.6 ± 0.3 and 16.9 ± 3.7%, respectively; the difference in these proportions was significant (P = 0.0195). After RGS20 overexpression, the latter group proportion significantly decreased to 3.8 ± 0.5% (P = 0.0041). For Hep3B cells, the apoptotic cells in the mimic NC + vector, miR-204-5p mimic + vector, and miR-204-5p mimic + RGS20 groups were 8.1 ± 0.7, 12.9 ± 0.4, and 4.9 ± 0.3%, respectively. In addition, statistically significant differences were found between the mimic NC + vector and miR-204-5p mimic + vector groups (P = 0.0016) as well as between the miR-204-5p mimic + vector and miR-204-5p mimic + RGS20 groups (P = 0.0006). The levels of Bax, cleaved caspase-3 and cleaved caspase-8 proteins were dramatically higher and Bcl-2 was prominently lower in miR-204-5p mimic + vector group compared with mimic NC + vector group, whereas this phenomenon was subverted in miR-204-5p mimic + RGS20 group (Figure 5B). Summarizing these results essentially provides the viewpoint that miR-204-5p promotes apoptosis by targeting RGS20 straightly.

Figure 5.

Figure 5

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(A) miR-204-5p promotes apoptosis by targeting RGS20 expression. (A) Representative images of apoptosis detected using flow cytometry. Transfection of miR-204-5p mimic increased the proportion of apoptotic cells. (B) Representative Western blot images of apoptosis-related proteins. Transfection of miR-204-5p mimic increased Bax, cleaved caspase-3, and cleaved caspase-8, but decreased Bcl-2. *P < 0.05, **P < 0.01, ***P < 0.001, vs. mimic NC + vector; ##P < 0.01, ### and ####P < 0.001, vs miR-204-5p mimic + vector.

miR-204-5p Inhibits Cell Migration/Invasion by Targeting RGS20

The role of miR-204-5p/RGS20 in the pathogenesis and development of HCC was further investigated using a Transwell migration assay to observe and elaborate the influence on cells. miR-204-5p mimic inhibited cell migration (Figure 6A), and the number of PLC/PRF/5 cells that moved from the upper chamber to the lower chamber decreased from 232 ± 10 to 121 ± 11 (P = 0.0002). The number of PLC/PRF/5 cells increased to 274 ± 11 (P < 0.0001) after the restoration of RGS20 expression. The number of migrated Hep3B cells decreased from 189 ± 12 to 86 ± 6 (P = 0.0002) with miR-204-5p mimic and then increased to 267 ± 9 after RGS20 expression was restored (P < 0.0001). The invasion assay also showed that the upregulation of miR-204-5p prevented cells from being invaded (Figure 6B). For PLC/PRF/5 cells, the number of invaded cells in the mimic NC + vector, miR-204-5p mimic + vector, and miR-204-5p mimic + RGS20 groups was 111 ± 10, 47 ± 5, and 176 ± 9, respectively. Statistically confirmed, there is a significant difference between mimic NC + vector and miR-204-5p mimic + vector groups (P = 0.0007), as well as between the miR-204-5p mimic + vector and miR-204-5p mimic + RGS20 groups (P < 0.0001). In Hep3B cells, there has a sharp reduction in the number of invasive cells in the miR-204-5p mimic + vector group, for which the value was 31 ± 7, comparing with the control (73 ± 5; P = 0.0013) and the miR-204-5p mimic + RGS20 group (94 ± 8; P = 0.0005). Corresponding to these results, the miR-204-5p mimic + vector group for both the PLC/PRF/5 and Hep3B cells exhibited significantly decreased expression of the pro-cellular metastasis-associated proteins, N-cadherin and vimentin, and extremely raised expression of the inhibitory transfer protein E-cadherin. These effects were reversed in the miR-204-5p mimic + RGS20 group (Figure 6C). Our results taken together confirm that miR-204-5p directly targets RGS20 expression to inhibit HCC cell migration and invasion.

Figure 6.

Figure 6

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miR-204-5p prevents cells from being migrated and invaded by targeting RGS20 expression. (A) Representative images of crystal violet-stained cells migrating from the upper to the lower compartment. Scale: 50 μm. Transfection of miR-204-5p mimic reduced the number of migrating cells. (B) Representative images of crystal violet-stained cells invading from the upper into the lower chamber. Scale: 50 μm. Transfection of miR-204-5p mimic reduced the number of invading cells. (C) Western blot technology for verifying proteins related to cell migration and invasion. *P < 0.05, **P < 0.01, ***P < 0.001 vs mimic NC + vector; ##P < 0.01, ### and ####P < 0.001 vs miR-204-5p mimic + vector group.

Discussion

HCC is among the five most prevalent cancers, and primary liver cancer ranges from 70–90%, making it the third major factor contributing to the extremely high mortality rate from cancer. In China, the incidence rate and mortality rate of liver cancer in malignant tumors rank fourth and second respectively, with a 5-year survival rate of merely 18%.21 Presently, various therapies are available for the treatment of HCC. Resection is considered the first-line therapy for HCC, whereas other treatment modalities include interventional procedures, local ablative techniques, precision radiotherapy, molecular-targeted and biological therapies, immunotherapy, and symptomatic support.22,23 Immune checkpoint inhibitors (ICIs) represent a new type of immunotherapy. Some studies have shown that early mortality following first-line ICI treatment is a clinically significant occurrence observed in various patients with solid tumors, and that the use of ICI significantly increases the chance of achieving a complete response.24 However, this outcome can be mitigated by supplementing ICI with additional treatments.25 Reportedly, a combination of immunotherapy with VEGF blockade and radiation therapy may benefit patients with HCC.26 However, owing to their limitations, predictive biomarkers for identifying HCC immunotherapy responses have not achieved satisfactory clinical results.27,28 The emerging role of miRNA in the evolution of HCC, making miRNA more eye-catching. miRNAs have attracted increasing attention owing to their emerging roles in the cellular evolution and biological processes of HCC. That is, the expression levels of some miRNAs are upregulated in malignant tissues, playing roles similar to oncogenes.16 For example, miR-21 promoted the production and progression of HCC cells by initiating the Akt/ERK pathway and inducing epithelial–mesenchymal transition (EMT).29 Additionally, miR-135b promotes HCC through the APC axis.30 Addition of inhibitors or antinucleotide drugs targeting these highly expressed miRNAs to treatment regimens can effectively inhibit the evolution and development of HCC. Conversely, the expression levels of some miRNAs are downregulated in HCC, playing a role similar to that of tumor suppressor genes.31 The introduction of these miRNAs by viruses or liposomes can effectively inhibit HCC and other cancers. miR-520e has been verified to suppress the growth of HCC and colorectal cancer cells,32,33 and overexpression of miR-122 significantly reduced HCC development.34

Analysis of starBase databases indicated that miR-204-5p was more pessimistically expressed in tumors than in normals. miR-204-5p restrained HCC cell proliferation by altering E2F1, SIX1, and SIRT1 activities.17−19 In this study, cell function experiments in vitro confirmed that miR-204-5p passably also targets RGS20 and downregulates its activity, thereby inhibiting HCC cell proliferation, migration, and invasion and promoting HCC cell apoptosis, which exerts a tumor suppressor effect. This achievement indicates the theory that miR-204-5p is a prospective candidate for gene therapy targeting RGS20.

Cyclin-dependent kinases (CDKs) and their associated cyclins regulate the cell cycle,35 and cyclin E1 is the key regulatory cell cycle protein of CDK2. The cyclin E1/CDK2 complex has an important role in the G1/S transition, centrosome replication, and other processes. In addition, cyclin E1 and CDK2 have been identified as key factors in HCC development.36 RGS20 promoted the upregulation of N-cadherin and downregulation of E-cadherin, favoring EMT37 and providing a potential new target for HCC therapy. However, no mechanistic research has been conducted on the role of RGS20 in HCC. We tested the content of genes referring to the cell cycle (cyclin E1 and CDK2), apoptosis (cleaved caspase-3/8 and Bcl-2, Bax), and EMT (vimentin, E-cadherin, and N-cadherin). Transfection of miR-204-5p mimic gave an upbeat protein value of Bax and E-cadherin and reacted with a downbeat protein value of Bcl-2, CDK2, cyclin E1, N-cadherin, and vimentin. However, these were counter-attacked by the overexpression of RGS20. Investigation results elucidate that miR-204-5p alters apoptosis-, cell cycle-, and EMT-related gene levels by targeting RGS20, and thereby adjusts apoptosis, the cell cycle, migration, and invasion.

Our experiments elucidate that miR-204-5p alters the multiplication of PLC/PRF/5 and Hep3B cells by targeting RGS20. Notwithstanding, the effect of miR-204-5p in inflammation and animal models was not analyzed. Thus, the exact effect of miR-204-5p in the treatment of HCC was not fully established, which is very constrained in this pilot project.37 In our next direction, we will use lentiviral technology to construct stable HCC cell lines and confirm the impact of the miR-204-5p/RGS20 axis on HCC gene therapy in animal models.

Although our experiments demonstrated that miR-204-5p targets the 3′-UTR of RGS20 and downregulates RGS20 expression. There was only a weak negative correlation between miR-204-5p and RGS20 in human HCC tissues as detected using tissue cDNA arrays. This suggests that the regulation of RGS20 expression is not solely dependent on miR-204-5p, and that other factors may influence its expression. For example, the known studies miR-204-5p has been proven to inhibit esophageal squamous cell carcinoma by targeting IL-1138 and HMGA2.39 Previous findings have indicated that other miRNAs, like miR-36537 and miR-96,40 may also play a role in regulating RGS20 expression.

Noncoding RNA (regulatory ncRNAs) involved in the regulation of the same type of gene are known as miRNAs. There are two main types of ncRNAs: long noncoding RNAs (lncRNAs) and short noncoding RNAs (piRNAs, miRNAs, and siRNAs).41 lncRNAs and miRNAs interact as competitive endogenous RNAs (ceRNAs), which allows the former to regulate the expression and biological functions of miRNAs.42 Lu et al., discovered that the lncRNA HOTAIR targeted miR-204-5p in cholangiocarcinoma cells, thereby inhibiting apoptosis and autophagy and promoting cell proliferation.43 HOTAIR is considered to be proto-oncogenic in HCC, suggesting that it may exert this pro-oncogenic role in HCC via the miR-204-5p/RGS20 axis. Exploring the worth of the HOTAIR/miR-204-5p/RGS20 axis in the onset and progression of HCC will be the focus of our ongoing research. Notably, RGS20 acts as a regulator of G-protein-coupled receptors, and thus there should be at least one regulated G-protein-coupled receptor under the miR-204-5p/RGS20 signaling axis; which is worth further exploration through bioinformatics predictions and cellular and animal experiments.

Conclusions

In summary, miR-204-5p is pessimistic in HCC, and elevated levels of miR-204-5p regulate the performance levels of cell cycle-, apoptosis-, and EMT-related genes, which inhibit proliferation, migration, and invasion of PLC/PRF/5/Hep3B. RGS20 is optimistic in HCC tissues, with its expression fluctuating with miR-204-5p, and its optimistic expression is interconnected with an unfavorable prognosis. RGS20 expression can significantly reverse the tumor suppressor gene miR-204-5p on the growth of tumor cells and the performance levels of cell cycle-, apoptosis-, and EMT-related genes and then promote the cell cycle to mitosis and inhibit cell apoptosis. We have prioritized providing evidence that miR-204-5p may keep HCC progression under control by targeting RGS20 and downregulating its expression. This study provides a new reference for HCC gene therapy, for which RGS20 as a presumable target and miR-204-5p as a promising candidate miRNA.

Materials and Methods

Bioinformatics Analysis

The starBase, miRDB, TargetScan, and mirDIP databases were applied to calculate the presumable target genes of miRNAs that interact with RGS20.

cDNA Chip Analysis of the Differential Expression of RGS20 and miR-204-5p

An HCC cDNA chip (HLivH060PG02) samples were purchased from Shanghai Outdo Biotechnology Co., Ltd. RT-qPCR analyses were executed to assess RGS20 and miR-204-5p using a Roche 480II real-time PCR system. The mRNA levels were evaluated utilizing threshold cycle values calculated as 2–ΔΔCT, where ΔCT = [CT (target gene) – CT (β-actin)]. The primers are shown in Table 1.

Table 1. Primers for cDNA Chip.

miR-204-5p-F 5′-GGCTTCCCTTTGTCATCCTAA-3′
miR-204-5p-R 5′-AGTGCAGGGTCCGAGGTATT-3′
RGS20-F 5′-CAGACTCGCCCGCCGCCC-3′
RGS20-R 5′-TCTGCTCTGAGTTCGTGGGAAC-3′
U6-F 5′-GCTTCGGCAGCACATATACTAAAAT-3′
U6-R 5′-CGCTTCACGAATTTGCGTGTCAT-3′
β-actin-F 5′-GAAGAGCTACGAGCTGCCTGA-3′
β-actin-R 5′-CAGACAGCACTGTGTTGGCG-3′
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Cell Culture

PLC/PRF/5 and Hep3B purchased from Shanghai Fuheng Biotechnology Co., Ltd., were uniformly inoculated in cell plates and cultured in complete DMEM.

Plasmid Construction

The wild-type RGS20 3′-UTR was amplified from the genomic DNA of PLC/PRF/5/Hep3B cells using the primers reflected in Table 1 and cloned downstream of the firefly luciferase gene in the pmirGLO plasmid (Promega) (3′-UTR WT). There are two miR-204-5p binding sites in RGS20 3′-UTR, which were mutated utilizing the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) (3′-UTR MUT). Total RNA was harvested from the PLC/PRF/5/Hep3B cells using an RNA separator (R401-01; Vazyme). RGS20 cDNA was amplified using the Titanium One-Step RT-PCR Kit (639504, Takara) and cloned into the lentiviral vector, pCDH-EF1α-MCS-T2A-Puro, to construct the RGS20 overexpression plasmid (pCDH-RGS20). The primer sequences are shown in Table 2.

Table 2. Primers for Plasmid Constructiona.

pmirGLO-RGS20 3′ UTR WT (F) 5′-CTCGCTAGCCTCGAGGATTTTTCAAATATATTTATTATTAAT-3′
pmirGLO-RGS20 3′ UTR WT (R) 5′-CATGCCTGCAGGTCGACGAGATAAAATTCTTGTG-3′
pmirGLO-RGS20 3′ UTR MUT1 (F) 5′-GATACTTTTGAAAAAAAAAAATAATCCCATATGGC-3′
pmirGLO-RGS20 3′ UTR MUT1 (R) 5′-CGCTATCTTTCTACAACAGCCATATGGGATTAT-3′
pmirGLO-RGS20 3′ UTR MUT2 (F) 5′-GCAGGTACATCCACCAGAGCATTCCCAACCAC-3′
pmirGLO-RGS20 3′ UTR MUT2 (R) 5′-CTCATGCAAAATAAAAGTGGTTGGGAATGCTCTGG-3′
RGS20 (F) 5′-CTAGAGCTAGCGAATTCATGCCCCAGCTTTCCC-3′
RGS20 (R) 5′-TCAGCGGCCGCGGATCCCTATGCTTCAATAG-3′
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a

The specific gene sequences are shown in italics.

Cell Transfection

miR-204-5p (sense strand: 5′-UUCCCUUUGUCAUCCUAUGCCU-3′, antisense strand: 5′-AGGCAUAGGUGACAAAGGGAA-3′) and NC (sense strand: 5′-UUGUACUACACAAAAGU

ACUG-3′, antisense strand: 5′-CAGUACUUUUGUGUAGUACAA-3′) mimics were purchased from Guangzhou Ruibo. PLC/PRF/5/Hep3B cells were uniformly inoculated in 6-well plates. EXfect Transfection Reagent (T101-01, Vazyme) was used for transfections, with 200 pmol of either miR-204-5p mimic or mimic NC per well. These constituted the miR-204-5p mimic and mimic NC groups, separately. Simultaneously, a cotransfection was performed with 4 μg of the overexpression vector or pCDH-RGS20, accordingly. Thus, there were three groups: mimic NC + vector, miR-204-5p mimic + vector, and miR-204–5p mimic + RGS20.

Dual-Luciferase Reporter Assay

Uniformly inoculate the transfected cells in 24-well plates and cotransfect at 80% confluence with miR-204-5p or mimic NC and pmirGLO-RGS20 3′-UTR WT or pmirGLO-RGS20 3′-UTR MUT. The Dual-Glo Luciferase Assay System (E1910, Promega) was utilized to specify firefly luciferase activity.

RT-qPCR

Total RNA was achieved from the cells spending an RNA isolation kit, and cDNA was synthesized by utilizing the cDNA Synthesis Kit (R211-01/02, Vazyme). Quantitative PCR was performed by employing the Bio-Rad CFX96 thermocycler. Amplification was performed under thermal cycling conditions. The primers employed to amplify the RGS20, miR-204-5p, and 18s rRNA and U6 internal reference genes were synthesized by Xiamen Botrin Biosynthesis as shown in Table 3.

Table 3. Primer Sequences for RT-qPCR.

miR-204-5p-RT 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGCAT-3′
miR-204-5p-F 5′-CGCGTTCCCTTTGTCATCCT-3′
miR-204-5p-R 5′-AGTGCAGGGTCCGAGGTATT-3′
U6-RT 5′-CGCTTCACGAATTTGCGTGTCAT-3′
U6-F 5′-GCTTCGGCAGCACATATACTAAAAT-3′
U6-R 5′-CGCTTCACGAATTTGCGTGTCAT-3′
RGS20-F 5′-CTTCCCACGAACTCAGAGCAGA-3′
RGS20-R 5′-TCCTTCCTGCTGGAGTGACCAT-3′
18S RNA-F 5′-CGACGACCCATTCGAACGTCT-3′
18S RNA-R 5′-CTCTCCGGAATCGAA CCCTGA-3′
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Western Blotting

RIPA lysis buffer (89900, Thermo Fisher Scientific) was used to obtain total protein from the cells. Proteins (40 μg) were separated via electrophoresis on a 12% SDS-PAGE gel (120 V, 1.5 h). The proteins were covered in a polyvinylidene fluoride (PVDF) membrane (3010040001; Roche) and incubated in 5% BSA (05470, Sigma) for 1 h. Primary anti-RGS20 (ab191500, Abcam) or anti-GAPDH (ab128915, Abcam) antibodies were incubated with the membranes. The membranes were washed five times with Tris-buffered saline (TBS) for 3 min each. The membranes then were immersed in secondary antibody (ab97051, Abcam) at 37 °C for 1 h, washed with TBS, and soaked in a color-developing solution for membrane color development.

Cell Viability Assay

Uniformly inoculate the transfected cells in a 96-well plate at 3000 cells in 200 μL of complete medium per well. At the specified time points, 20 μL of MTT reagent (40206ES76, Yeasen) was put into the cells, and incubation was continued for 4 h at 37 °C. Then, the liquid was cast off and dimethyl sulfoxide (DMSO) was placed into the cells. The cells were mixed to completely crack the crystals. The absorbance was measured at 490 nm in six replicate wells for each group of samples.

Cell Proliferation Assay

After 24–48 h of uniform inoculation of transfected cells into 96-well plates, 4% formaldehyde was used to fix cells, treated with 2 mg/mL glycine at 28 °C, and permeabilized with 0.5% Triton X-100. The experiment was conducted according to the instructions provided with the EdU kit (C10310-3; RiboBio). Finally, images were obtained under a fluorescence microscope (Motic).

Cell Cycle Test

Transfected cells were harvested by trypsin digestion and fixed in 70% ethanol at 4 °C overnight. The fixed cells were treated with 0.2% Triton X-100 and then stained with 1 mg/mL of RNase containing propidium iodide (PI) for 30 min. Enable NovoCell flow cytometry for cell cycle elucidation (ACEA, San Diego, CA, USA).

Cell Apoptosis Test

Cells were digested with 0.25% trypsin without ethylene diamine tetraacetic acid (EDTA) after they were transfected for 24–48 h, and wash them once with PBS. An Annexin V-FITC/PI apoptosis kit (A211, Vazyme) was spent on staining, and flow cytometry was invested in detecting cell apoptosis.

Migration/Invasion Assay

The migration/invasion of cells were counted using Transwell plates (3422, Corning) with or without Matrix (356234, Biosciences) following transfection for 24–48 h. Cells (5 × 104) were resuspended in serum-free DMEM and cultivated to the upper chamber of Transwell plate, and filled the lower chamber with complete DMEM. After incubating at 37 °C for 16–24 h, the migrating/invading cells were fastened and colored with 0.5% crystal violet. Then count the migrating/invading cells in three independent microscope fields.

Statistical Analyses

The statistical analyses of the data were completed utilizing SPSS software (IBM SPSS, Armonk, NY, USA). Use unpaired t tests to compare two sets of samples that fit the normal distribution, and Mann–Whitney or Wilcoxon tests to compare data that does not fit the normal distribution. The final result is represented by the mean ± standard deviation, with P < 0.05 as the setting for statistical significance.

Glossary

Abbreviations

RGS20

regulator of G protein signaling 20

miRNAs

microRNAs

HCC

hepatocellular carcinoma

CDK2

cyclin-dependent kinase 2

Bcl-2

B-cell lymphoma-2

UTR

untranslated region

TNM

tumor-node-metastasis

AFP

alpha-fetoprotein

ICIs

immune checkpoint inhibitors

EMT

epithelial–mesenchymal transition

Author Contributions

# Y.S., Y.L. and H.A. contributed equally to this work.

Author Contributions

Conceptualization: B.H. and J.L.; methodology: H.A. and J.L.; software and validation: Y.L., Y.S. and H.A.; data curation: F.Y., J.S., and Z.N.; writing-original draft preparation: Y.L.; writing-review & editing: Y.S.; visualization: B.H.; supervision: J.L.; funding acquisition: J.L.

This work was supported by the National Nature Science Foundation of China (grant no. 82204925) and the Natural Science Foundation of Fujian Province, China (2020J01748 and 2022J01368).

The data sets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

The authors declare no competing financial interest.

Notes

Ethics statement—Analysis of the differential expression of RGS20 and miR-204-5p using the cDNA chip was approved by the Ethics Committee of Shanghai Outdo Biotech Company (No. YBM-05-02).

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