MiR-424-5p suppresses tumor growth and progression by directly targeting CHEK1 and activating cell cycle pathway in Hepatocellular Carcinoma

Objectives The aim of this study is to elucidate the functional mechanism of the miRNA-424-5p/CHEK1 pathway in hepatocellular carcinoma (HCC), thereby offering novel insights for the development of targeted therapeutic strategies for HCC. Methods We employed a combination of bioinformatics analysis and data from the GEO to construct a regulatory network between miRNA and mRNA. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to assess the expression levels of miR-424-5p and CHEK1. Protein expression of CHEK1 was determined using Western blot analysis. The targeting relationship between miR-424-5p and CHEK1 was validated through a dual-luciferase reporter assay. Furthermore, the effects of miR-424-5p on HCC cell proliferation, migration, and invasion were evaluated using the Cell Counting Kit-8 assay, wound healing assay, and Transwell invasion assay, respectively. Apoptosis of HCC cells was measured by flow cytometry. Results Bioinformatics analysis revealed that miR-424-5p was significantly downregulated, while CHEK1 was upregulated respectively in GEO dataset. Furthermore, this inverse expression pattern was observed in both HCC tissues and cell lines. Specifically, downregulation of miR-424-5p was found to promote the proliferation, migration, and invasion of HCC cells, while also inhibiting their apoptosis. The dual-luciferase reporter assay confirmed a direct targeting relationship between miR-424-5p and CHEK1. Inhibition of miR-424-5p was shown to counteract the suppressive effects on HCC cell proliferation, migration, and invasion that result from CHEK1 silencing. Additionally, experimental verification indicated that the activation of the cell cycle pathway is implicated in the oncogenic function of miR-424-5p/CHEK1 in HCC. Conclusions The present study demonstrates that miR-424-5p exerts a suppressive effect on HCC cell proliferation, migration, and invasion by downregulating the expression of CHEK1. This finding may offer a theoretical foundation for improving the prognosis and developing novel therapeutic strategies for HCC patients.


Introduction
Liver cancer is the second most common malignant tumor and ranks as the fourth leading cause of cancer-related deaths worldwide [1,2].HCC is a type of liver cancer, accounting for 85%-90 % of all primary liver cancers [3].Patients with early-stage HCC have the option of effective treatments such as surgical resection, liver transplantation, and chemotherapy [4].However, the treatment for patients with advanced metastatic HCC remains unsatisfactory [5].Furthermore, the molecular mechanisms underlying HCC recurrence and metastasis are not yet fully understood.As a result, it is a critical issue for clinicians to delve deeper into the pathogenesis of liver cancer and to identify new therapeutic targets that could enhance the efficacy of HCC treatment.
MicroRNAs (miRNAs) are non-coding RNA molecules that are 17-25 nucleotides in length [6] and can negatively regulate gene expression by inducing mRNA degradation or inhibiting translation [7].A substantial body of research has demonstrated that miRNAs are implicated in the pathogenesis of numerous diseases and are involved in the initiation, progression, metastasis, and drug resistance of various types of cancer [8][9][10][11].
Checkpoint kinase 1 (CHEK1) is a serine/threonine protein kinase that plays a pivotal role in the cellular response to DNA damage.When the integrity of the genome is compromised, CHEK1 functions to prevent DNA replication, thereby maintaining genomic stability and preventing abnormal cell division.This action is crucial for inhibiting the formation of tumors.However, in some cancers, including liver cancer, tumor cells have been observed to upregulate CHEK1 [12,13].This upregulation allows the cells to evade the DNA damage response that would normally be triggered by treatments such as radiotherapy and chemotherapy.As a result, these tumor cells may exhibit increased resistance to drugs and enhanced invasiveness, which poses significant challenges in cancer therapy.
The miR-424-5p has also been implicated in the regulation of various cancers, including its role in liver cancer [14].While its involvement in cancer development has been noted, the specific mechanism by which miR-424-5p might influence the malignant behavior of liver cancer cells has not been fully elucidated.Specifically, it was previously unknown whether miR-424-5p could target CHEK1 to modulate its expression levels in liver cancer.
Our study sheds new light on this question.We have confirmed that miR-424-5p plays a significant role in the progression of liver cancer.Importantly, our findings indicate that miR-424-5p negatively regulates the expression of CHEK1.By doing so, it inhibits the invasive and proliferative capabilities of liver cancer cells.This discovery not only uncovers a potential regulatory link between miR-424-5p and CHEK1 but also suggests miR-424-5p as a promising molecular target for the development of new therapeutic strategies against liver cancer.

Specimen collection
HCC tissues and their adjacent non-cancerous liver tissues were collected from 30 patients who had undergone surgical resection at the Second Affiliated Hospital of Anhui Medical University.All HCC cases were pathologically confirmed.Prior to surgery, none of the patients had received any form of systemic anti-cancer therapy, ensuring that the tissues were free from treatment-induced alterations.Immediately following surgical excision, the tissues were snap-frozen in liquid nitrogen to preserve their molecular integrity and then stored at − 80 • C to maintain their stability until further analysis.

Cell culture
The human normal liver cell line L-02 and the HCC cell lines HepG2, SMCC7721, Huh-7, and HepG3B were procured from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.These cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) (GibcoBRL, Gaithersburg, MD, USA), supplemented with 10 % fetal bovine serum (FBS).The cultures were maintained in a humidified incubator with an atmosphere of 5 % CO2 at 37 • C. Upon reaching 80 %-90 % confluence, the cells were dissociated using 0.25 % trypsin (Shanghai Rugi Biotechnology) for subculturing.

Plasmid construction and cell transfection
The sequences for miR-424-5p and CHEK1 were obtained from the NCBI database.Constructs including the miR-424-5p mimic, miR-424-5p inhibitor, negative control mimic (mimic NC), negative control inhibitor (inhibitor NC), and three distinct siRNAs targeting CHEK1 (si-CHEK1-1#, si-CHEK1-2#, si-CHEK1-3#) as well as a non-targeting siRNA control (si-CHEK1-NC) were synthesized by Shanghai Genepharma Co., Ltd.(Shanghai, China).HepG2 cells were transfected with these constructs using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol and then cultured in a humidified incubator with 5 % CO2 at 37 • C. A transfection concentration of 50 nM was applied for all constructs.Cells were maintained in complete growth medium for at least 24 h prior to transfection and washed with phosphate-buffered saline (PBS, pH 7.4) to eliminate serum that might interfere with the transfection process.The efficiency of transfection was evaluated using RT-qPCR, and cells were collected between 24 and 48 h post-transfection for subsequent analysis.

RT-qPCR
Total RNA was extracted from HCC samples and cells using TRIzol reagent (catalog number 15596018, Life Technologies, Carlsbad, CA, USA).The miRNA 1st Strand cDNA Synthesis Kit (tolobio, China) and the ToloScript All-in-one RT EasyMix (tolobio, China) were used to reverse transcribe RNA into cDNA for the amplification of miR-424-5p and CHEK1 genes, following the manufacturers' instructions.The qPCR reaction mixture, prepared according to the 2 × Q3 SYBR qPCR Master Mix (tolobio, China) protocol, contained 0.4 μL of each primer, 10 μL of 2 × Q3 SYBR qPCR Master Mix, 1 μL of cDNA, and 8.2 μL of ddH2O.The cycling conditions were 95 • C for 1 min, followed by 40 cycles of 95 • C for 20 s and 60 • C for 60 s.The relative expression levels of the target miRNA and mRNA were determined using the 2 − ΔΔCt method, where ΔCt = Ct (target gene) -Ct (reference gene-β-actin for CHEK1 and U6 for miR-424-5p), and ΔΔCt = ΔCt (HCC group) -ΔCt (control group).The primer sequences are listed in Table 1.

Western blot
Cells were lysed using a radio immunoprecipitation assay lysis buffer (RIPA) from Beyotime (P0013B, Shanghai, China).The lysate was homogenized and centrifuged at 12,000 rpm for 15 min to obtain the supernatant, which contained the total protein.Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit.Protein samples were then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane.The membrane was blocked with 5 % skimmed milk at room temperature for 2 h to prevent non-specific binding.
The relative expression levels of the target proteins were quantified by analyzing the gray values of the protein bands using ImageJ software (National Institutes of Health, Bethesda, MD, USA).The gray value ratios to GAPDH (TA-08, Zsbio, China) were calculated to normalize the protein expression levels.

Cell Counting Kit 8 (CCK-8) assay
Cells in the logarithmic growth phase were seeded into 96-well plates at a density of 1 × 10 4 cells per well.The plates were then incubated at 37 • C in a humidified atmosphere containing 5 % CO2 for 12 h.Following treatment for 24 h, 10 μl of CCK-8 reagent (BA00208, Bioss, China) was added to each well.The plates were further incubated for 2 h under the same conditions.Cell viability was assessed by measuring the absorbance at 450 nm using a microplate reader (Infinite M200, Tecan).The absorbance values obtained were used to calculate the percentage of viable cells relative to the control group.

Transwell assays
Cells were adjusted to a concentration of 2 × 10 5 cells/mL in serum-free medium.An aliquot of 100 μl of this cell suspension was then carefully added to the upper chamber of a transwell insert (Catalog #08416047, Corning, USA).The lower chamber was filled with complete growth medium supplemented with 10 % fetal bovine serum (FBS) to serve as a chemoattractant.The assembled transwell system was incubated for 48 h at 37 • C in a humidified atmosphere containing 5 % CO2.
Following the incubation period, non-migratory cells on the upper surface of the membrane were gently removed using a damp cotton swab.The cells that had migrated to the lower surface of the membrane were fixed with 4 % paraformaldehyde for 30 min at room temperature.Subsequently, the fixed cells were stained with 0.5 % crystal violet solution for 15 min, followed by gentle elution with distilled water to facilitate visualization.The migratory cells on the underside of the upper chamber's membrane were examined using an optical microscope (Olympus, Japan), and representative images were captured.

Wound healing assay
A wound healing assay was conducted to assess the migratory capacity of HepG2 cells.Cells were seeded into 6-well culture plates at a density sufficient to achieve 90 % confluence.Upon reaching the desired confluence, a standardized wound was introduced by scraping the cell monolayer with a 10 μl pipette tip, ensuring uniform wound width across all wells.Following the creation of the wound, the wells were gently washed three times with sterile phosphate-buffered saline (PBS) to remove any detached cells.Fresh complete growth medium was then added to each well, and the cells were allowed to migrate into the wound area for 24 h in a 37 • C, 5 % CO2 incubator.
The migratory response of the cells was monitored by capturing images of the wound area at 0 h and after 24 h of culture using an inverted microscope.The distance migrated by the cells was measured from the images, and the data were used to calculate the rate of cell migration.

Flow cytometry for apoptosis analysis
HepG2 cells were harvested using trypsin without the addition of EDTA.The detached cells were collected and centrifuged at 300×g for 5 min at room temperature.Following centrifugation, the supernatant was discarded.Cells were then resuspended in 500 μl of binding buffer.Subsequently, 5 μl of Annexin V-FITC (Catalog #AP101, MULTISCIENCES) was added, followed by the addition of 5 μl of Propidium Iodide (PI).The cells were incubated for 15 min in the dark at room temperature to allow for the binding of the fluorescent probes.Apoptotic cells were quantified using a flow cytometer (CytoFLEX, Beckman Coulter) within 1 h post-staining.

Flow cytometry for cell cycle analysis
After a 48-h transfection period, cells were trypsinized and collected by centrifugation at 300×g for 5 min at 4 • C. The supernatant was removed, and the cell pellet was fixed in 70 % ethanol at 4 • C overnight.The fixed cells were then centrifuged again at 300×g for 5 min to pellet the cells.The ethanol was aspirated, and the cells were resuspended in 0.5 mL of a staining solution containing PI and RNase A. The cells were incubated at 37 • C for 15 min in the dark to allow for DNA staining.The cell cycle distribution was analyzed by measuring red fluorescence at 488 nm using the same flow cytometer (CytoFLEX, Beckman Coulter).To ensure the reproducibility of the results, the experiment was independently repeated three times.

Dual-luciferase reporter assay
The target plasmids, including miR-424-5p, NC-mimic, CHEK1-3′UTR-WT (wild-type), and CHEK1-3′UTR-Mut (mutant), were transfected into 293T cells.Luciferase activity was assessed 48 h post-transfection using a dual-luciferase reporter assay system.The specific protocol for detection is as follows: Preparation of Cell Lysate: Dilute the 5 × Passive Lysis Buffer (PLB) with distilled water to a final concentration of 1 × PLB.Add 100 μl of the diluted PLB to each well of a 96-well plate.Cells were lysed by gentle pipetting using a pipette gun, followed by incubation on a room temperature shaking bed for 15 min with slow shaking to ensure thorough lysis.Collection of Cell Lysate: After lysis, the cell lysate was transferred into a 1.5 ml centrifuge tube and centrifuged at 300×g for 10 min at 4 • C to remove cellular debris.The supernatant, containing the soluble proteins, was then transferred to a new tube.
Luciferase Assay: To the 96-well plate, 100 μl of Luciferase Assay Reagent II (LAR II) working solution was added.Subsequently, 20 μl of the prepared cell lysate was added to each well, and the mixture was gently pipetted and mixed 2-3 times to ensure homogeneity.
The firefly luciferase activity, serving as the internal control, was measured and recorded.
Renilla Luciferase Assay: Finally, 100 μl of Stop & Glo® Reagent was added to each well.The mixture was again pipetted and mixed 2-3 times, and the Renilla luciferase activity, representing the reporter gene luminescence, was measured and recorded.
This dual-luciferase assay was performed to determine the relative expression levels of the reporter gene under the influence of miR-424-5p and its potential interaction with the CHEK1-3′UTR.To ensure the reproducibility and reliability of the results, the experiment was independently repeated three times.

Tumor xenograft model in BALB/c nude mice
Thirty-six BALB/c nude mice, aged 4 weeks and weighing between 18 and 25 g, were procured from the Experimental Animal Center of the Department of Medicine at Nanchang University (Jiangxi, China).No specific gender was selected for this study.HepG2 cells (3.0 × 10 6 ) were stably transfected with either lentivirus-miR-424-5p (LV-miR-424-5p) or lenti-MOCK.These cells were then subcutaneously injected into the lower flank region of the mice.The anesthesia was administered using pentobarbital sodium at a dosage of 0.1-0.2ml per 10 g of body weight, following the guidelines provided by the China Laboratory Animal Information Network.
The lentivirus used for transfection was sourced from Shanghai GenePharma (Shanghai, China).Ten days post-inoculation, the tumor volume was measured and calculated using the formula: Volume = (Length × Width 2 )/2 (units in mm 3 ).At the end of the 6-week experimental period, the mice were humanely euthanized using carbon dioxide asphyxiation, and the tumors were surgically excised and weighed.The data obtained were used to plot a curve representing the average tumor volumes at various time points throughout the study.This xenograft model was utilized to assess the effect of miR-424-5p on tumor growth in vivo.

Statistical analysis
All statistical analyses were conducted using SPSS software version 26.0 (IBM Corporation, Armonk, NY, USA).Graphical representations of the data were generated using GraphPad Prism software version 9.0 (GraphPad Software, La Jolla, CA, USA).Data are presented as the mean ± standard deviation (SD).To determine the statistical significance of differences between two groups, a Students t-test was employed.A p-value of less than 0.05 was considered to indicate statistical significance.

Construction of the miRNA-mRNA regulatory network and screening of hub genes
The datasets GSE31568 and GSE61144 were analyzed using R software (version 4.1.2),resulting in the identification of 209 differentially expressed miRNAs (DEMs) and 173 differentially expressed genes (DEGs) (Fig. 1A-B).The miRNA-mRNA regulatory network was constructed based on the principle of negative regulation between miRNAs and their target mRNAs (Fig. 1C).Subsequently, the protein-protein interaction (PPI) network analysis of DEGs was conducted utilizing the STRING database, revealing that CHEK1 occupied a central hub position within the PPI network (Fig. 1D).Further investigation using the GEPIA database indicated that the overexpression of CHEK1 was associated with poor prognosis in HCC (Fig. 1G-H).Consequently, we resolved to further validate the miR-424-5p/CHEK1 regulatory axis both in vivo and in vitro, and to explore its impact on the cell cycle signaling pathway.

CHEK1 is highly expressed, while miR-424-5p is poorly expressed in HCC tissues and cell lines
The expression levels of miR-424-5p and CHEK1 in HCC tissues and adjacent normal tissues, collected from 30 HCC patients, were determined using RT-qPCR.The results showed that miR-424-5p was less expressed, whereas CHEK1 was more highly expressed in HCC tissues compared to adjacent normal tissues (Fig. 2A-B).Additionally, we detected the expression levels of miR-424-5p and CHEK1 in the normal cell line L-02 and various HCC cell lines using RT-qPCR.As shown in Fig. 2C-D, the expression levels of miR-424-5p in the HCC cell lines were significantly lower, with the following order: HepG2, HepG3B, HuH-7, SMMC-7721, and L-02.Conversely, CHEK1 expression levels were significantly higher in the HCC cell lines, with the following order: HepG2, HepG3B, HuH-7, SMMC-7721, and L-02.Therefore, the HepG2 HCC cell line was chosen for subsequent experiments.

MiR-424-5p can act as a gene expression regulator by binding to the 3′UTR of CHEK1
We identified a binding site between miR-424-5p and CHEK1 using FunRich software.Subsequently, a dual-luciferase reporter assay was conducted to assess the regulatory interaction between miR-424-5p and CHEK1.As depicted in Fig. 2E-F, the luciferase activity of the CHEK1 wild-type (WT) construct was significantly suppressed in the presence of the miR-424-5p mimic (p < 0.05), whereas the activity of the CHEK1 mutant (MT) construct did not change significantly (p > 0.05).Further studies involved RT-qPCR and Western blot analyses in HCC cell lines treated with miR-424-5p mimic or inhibitor, as well as in control cells.The results showed that CHEK1 expression was significantly reduced in the miR-424-5p mimic-treated cells, while it was markedly elevated in the miR-424-5p inhibitor-treated cells (Fig. 2H).Additionally, we observed that the miR-424-5p expression level was significantly higher in the si-CHEK1 group compared to the si-CHEK1-NC group, and its expression was significantly reduced upon co-transfection with miR-424-5p inhibitor and si-CHEK1 (Fig. 2J-K).These findings suggest that CHEK1 is a target gene of miR-424-5p.
The CCK-8 assay, wound healing assay, and transwell assay were employed to evaluate the impact of miR-424-5p and CHEK1 on HepG2 cell proliferation, migration, and invasion, respectively.Transfection with the miR-424-5p inhibitor significantly enhanced the proliferation, migration, and invasion of HepG2 cells compared to the matched controls (Fig. 3A-B,E-G).No significant difference was observed between the control group and the inhibitor-NC group.Conversely, transfection with si-CHEK1 resulted in suppressed proliferation, migration, and invasion compared to the matched controls(Fig.3A-B,E-G).No significant difference was noted between the control group and the si-NC group.Furthermore, the inhibitory effects of CHEK1 silencing on HepG2 cell proliferation, migration, and invasion were reversed by the miR-424-5p inhibitor(Fig.3A-B,E-G).

The effects of miR-424-5p and CHEK1 on the apoptosis and cell cycle in HepG2 cells
To elucidate the effects of miR-424-5p and CHEK1 on HepG2 cells, apoptosis and cell cycle analysis were conducted on cells transfected with various plasmids, including inhibitor-NC, miR-424-5p inhibitor, si-CHEK1-NC, si-CHEK1, and a combination of miR-424-5p inhibitor and si-CHEK1.Flow cytometry was utilized to assess these cellular processes.
The results indicated that, compared to the inhibitor-NC group, the miR-424-5p inhibitor group had a reduced proportion of cells in the G1 phase, an increased proportion in the S phase, and a decreased rate of apoptosis (Fig. 3C-D, H-I).In contrast, silencing CHEK1 led to an accumulation of cells in the G1 phase, a decrease in the S phase population, and an elevated rate of apoptosis (Fig. 3C-D, H-I).There was no significant difference between empty plasmid group and si-NC group (Fig. 3C-D, H-I).Additionally, the inhibition of miR-424-5p was found to alleviate cell cycle arrest and rescue the apoptosis induced by si-CHEK1(Fig.3C-D, H-I).Collectively, these findings suggest that miR-424-5p silencing restrains apoptosis in HepG2 cells, whereas CHEK1 silencing promotes it.

Activation of the cell cycle pathway is implicated in the oncogenic functions of the miR-424-5p/CHEK1 regulatory axis in HCC
KEGG enrichment analysis revealed that CHEK1 is significantly enriched in the cell cycle pathway in HCC (Fig. 1E-F).To further elucidate the molecular mechanisms underlying its functional role in HCC, Western blot analysis was conducted to examine proteins associated with the cell cycle pathway.As depicted in Fig. 4A-B, compared to the NC group, the expression levels of Cyclin B, CDK1, p-P53, and ATR were found to be differentially regulated in the miR-424-5p inhibitor and si-CHEK1 groups in vitro.These findings suggest that the miR-424-5p/CHEK1 regulatory axis contributes to HCC tumorigenesis, at least in part, by activating the cell cycle signaling pathway.

miR-424-5p negatively regulates CHEK1 and impacts the development of HCC via the cell cycle pathway
To further explore the effect of miR-424-5p on HCC proliferation, a nude mice xenograft tumor model was utilized.Nude BALB/c mice were xenografted with a HepG2 stable cell line overexpressing miR-424-5p.A significant reduction in miR-424-5p expression was observed in the inhibitor group (Fig. 5C), which was associated with an increase in tumor size and weight compared to the control HepG2 cells or NC group (Fig. 5A-B, E).Western blot analysis showed a significant decrease in the expression of proteins related to the cell cycle signaling pathway in the inhibitor group compared to the control group (Fig. 4C-F).Importantly, the application of the cell cycle pathway inhibitor topoisomerase II further enhanced this difference(Fig.4C-F).Collectively, these results suggest that miR-424-5p can suppress HCC proliferation in vivo, at least in part, by regulating the cell cycle pathway.

Discussion
At present, liver cancer is the fourth leading cause of cancer related deaths in the world, and the number of deaths is increasing year by year [17].About 90 % of liver cancer patients are HCC [18].Although the comprehensive treatment strategy of liver cancer has benefited some patients, however, the prognosis of liver cancer patients is still poor [19].Therefore, it is particularly important to find new therapeutic targets for liver cancer for patients with poor surgical treatment effect or anti-tumor drug resistance.This study explored the new molecular mechanism of liver cancer progression, to provide a theoretical basis for the development of new therapeutic targets for liver cancer.
MicroRNAs (miRNAs), as pivotal noncoding RNAs, have been demonstrated to influence the biological behaviors of numerous tumors, including proliferation, apoptosis, migration, and invasion [20,21].Among them, miR-424-5p has emerged as a tumor suppressor with significant implications in various human cancers.Its dysregulation in malignant tumors is strongly correlated with the initiation and progression of cancer.In thyroid cancer, overexpression of miR-424-5p is significantly linked to distant metastasis [22].In breast cancer, diminished levels of miR-424-5p are associated with advanced clinical staging, increased tumor volume, a higher metastatic lymph node count, the presence of distant metastases, and a lower histological grade [23].Recent findings suggest that miR-424-5p may promote endometrial cancer progression by modulating the CLDN4/PI3K/AKT signaling pathway [24].Additionally, in colorectal cancer, miR-424-5p has been shown to enhance cell proliferation and metastasis by directly targeting SCN4B [25].Previous studies have established that miR-424-5p can suppress tumor biological behaviors in liver cancer, while CHEK1 promotes the proliferation, migration, and invasion of liver cancer cells.However, the regulatory relationship between miR-424-5p and CHEK1 in HCC remains elusive.Utilizing bioinformatics analysis of the GEO database, we identified differential expression of miR-424-5p and CHEK1 in liver cancer tissues.Our subsequent experiments using RT-qPCR confirmed these findings in HCC tissues and multiple HCC cell lines.Functional assays, along with FunRich software predictions and dual-luciferase reporter experiments, demonstrated that miR-424-5p binds to the 3′UTR of CHEK1, negatively regulating its expression at the post-transcriptional level.This regulation was further validated by RT-qPCR and Western blot analysis.Through cell functional experiments, we explored the specific molecular mechanism of the miR-424-5p/CHEK1 regulatory axis, providing insights into the pathogenesis of HCC.
The results of the cell function experiment demonstrate that the proliferation, migration, and invasion of HepG2 cells were significantly enhanced, the apoptosis was reduced, the number of cells staying in G1 phase was reduced, and more cells stayed in S phase after transfection with miR-424-5p inhibitor compared with NC group and inhibitor NC group.On the contrary, silencing CHEK1 can significantly inhibit the proliferation, migration, and invasion of HepG2 cells, and significantly increase apoptosis.The rescue experiment further showed that inhibition of CHEK1 could partially reverse the effect of miR-424-5p-inhibitor on the biological behavior of HepG2 cells.
In addition, we also conducted nude mouse tumorigenesis experiments and found that miR-424-5p inhibitor can significantly promote tumor growth, and the expression level of CHEK1 is significantly increased, which indicate that miR-424-5p, as a tumor suppressor gene for HCC, plays a regulatory role by negatively regulating the expression of CHEK1.To further explore the molecular mechanism of the influence of miR-424-5p/CHEK1 regulatory axis on HCC, we also conducted functional enrichment analysis on DEGs and found that CHEK1 was mainly enriched in the cell cycle signaling pathway.Subsequently, we detect the alteration of cell cycle signaling pathway related proteins through Western blot analysis.As shown in the figure, with changes in the expression levels of miR-424-5P and CHEK1, the levels of cell cycle signaling pathway related proteins (such as Cyclin B, CDK1, p-P53, and ATR) also changed.Based on the results of cell cycle analysis, we speculate that the miR-424-5p/CHEK1 regulatory axis can further affect the proliferation, migration, invasion, and apoptosis of HCC cells through the cell cycle signaling pathway.
In summary, our study underscores the clinical and biological relevance of the miR-424-5p/CHEK1 regulatory axis in HCC, revealing its critical downstream influence on the cell cycle pathway.This axis exerts a pivotal control over tumorigenesis and progression through the modulation of cellular proliferation and cell cycle regulation, with significant repercussions on the cell cycle pathway.While prior studies have noted the irregular expression patterns of miR-424-5p and CHEK1 in HCC, our research presents the first comprehensive elucidation of miR-424-5p′s direct inhibitory effect on CHEK1 in HCC.Furthermore, our bioinformatics analysis unveils a potentially broader regulatory network, where miR-424-5p may also govern the expression of additional target genes, including RGP1, FAM189B, and KIF23.Intriguingly, CHEK1 expression in liver cancer cells appears to be under the dual regulation of miR-424-5p and miR-199-5p.These insights not only spotlight the intricate pathophysiology of HCC but also chart new avenues for future investigative pursuits.

Fig. 1 .
Fig. 1.The results of Bioinformatics analysis.A-B: Volcano plot of DEMs and DEGs in normal group and tumor group of HCC from GEO database; C: miRNA-mRNA regulatory network; D: PPI network analysis of DEGs; E-F: The result of GO and KEGG analysis;G: The expression level of CHEK1 in liver cancer and non-tumor tissues from TCGA database; H: Kaplan-Meier analysis of overall survival from TCGA-LIHC cohort based on CHEK1 expression.

Fig. 5 .
Fig. 5. Impacts of miR-424-5p suppression on HepG2 cell growth in vivo.HepG2 cells were co-transfected with miR-424-5p inhibitor or inhibitor NC and miR-424-5p inhibitor + Topoisomerase II inhibitor 9:C-D: The expression levels of MiR-424-5p and CHEK1; miR-424-5p-inhibitor-NC was the control group; A-B and E: Tumor size or weight in the mice of the different experimental groups was assessed on day 35 after injection; miR-424-5p-inhibitor-NC was the control group; *P < 0.05.

Table 1
Primer sequences for RT-qPCR.