Coptisine inhibits the proliferation of hepatocellular carcinoma by promoting ubiquitination of IGF2BP1

doi:10.21203/rs.3.rs-1821713/v1

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Research Article

Coptisine inhibits the proliferation of hepatocellular carcinoma by promoting ubiquitination of IGF2BP1

https://doi.org/10.21203/rs.3.rs-1821713/v1

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Background

Hepatocellular carcinoma (HCC) is a leading cause of death in humans. Clinical investigations have reported that insulin-like growth factor 2 (IGF2) mRNA binding protein 1 (IGF2BP1) is overexpressed in HCC, promoting oncogene activation, and is associated with cancer progression and poor prognosis. Previous research from our laboratory found that coptisine (COP) could inhibit the proliferation of HCC cells, but the exact mechanism was unknown.

Methods

The activity of COP against HCC was evaluated by the experiments with HCC cells and HuH7 xenografted mice. Based on transcriptome analysis, cellular thermal shift assay (CETSA), and mass spectrometry analysis, the key direct target of COP in inhibiting the proliferation of HCC were discovered. In vitro and in vivo assays were performed to study the biological function of IGF2BP1 in HCC. Screening of E3 ligases and deubiquitin enzyme interacting with IGF2BP1 by IP-mass spectrometry and website prediction. The molecular mechanism of COP causes protein ubiquitination and degradation of IGF2BP1 was demonstrated by CO-IP, immunofluorescence and protein stability assays.

Result

The results disclosed that COP could inhibit the growth of liver cancer cells and tumors and IGF2BP1 was identified as the direct target of COP. IGF2BP1 overexpression and silencing in vivo and in vitro confirmed that IGF2BP1 could significantly influence the growth of liver cancer cells and tumors. Mechanistic studies revealed that COP could either increase the interaction between E3 ligase Pre-mRNA Processing Factor 19 (PRPF19) and IGF2BP1 or decrease the deubiquitin function of the deubiquitin enzyme Ubiquitin Specific Peptidase 10 (USP10), resulting in increased ubiquitin degradation of IGF2BP1.

Conclusions

These findings suggest that COP has an anti-HCC activity by reducing IGF2BP1 expression. It opens up new avenues for research into modern pharmacology and the molecular mechanisms of traditional Chinese medicine against tumors.

Hepatocellular carcinoma

COP

IGF2BP1

USP10

PRPF19

According to the International Agency for Research on Cancer, primary liver cancer is the sixth most common cancer and the third leading cause of cancer death worldwide in 2020, trailing only lung cancer and colorectal cancer^[1]. Numerous risk factors for HCC include chronic liver injury caused by chronic hepatitis B or C virus infection, alcohol consumption, aflatoxin exposure, or metabolic diseases (obesity, diabetes, etc.)^[2]. Sorafenib, a tyrosine kinase inhibitor (TKI), has been the most common first-line targeted therapy approved by the US Food and Drug Administration (FDA) for patients with advanced liver cancer since 2007. Within six months, oral sorafenib can cause drug resistance^[3]. Therefore, developing safe and effective drugs for HCC treatment is critical. Many clinical studies have revealed that natural monomers of traditional Chinese medicine have distinct advantages in cancer treatment. For example, paclitaxel can treat ovarian cancer, breast cancer, non-small cell lung cancer, head and neck cancer, esophageal cancer, and seminoma^{[4, 5]}. Berberine has been demonstrated to lower the risk of recurrence of colorectal adenomas and polypoid lesions in patients following polypectomy^[6]. This indicates that screening natural compounds from traditional Chinese medicine for HCC treatment has great potential. Coptidis Rhizoma is bitter, and it clears heat and dampness while also eliminating heat and purging fire. Coptisine (COP) is the second most abundant isoquinoline alkaloid found in Coptidis Rhizoma, and it shares the same mother nucleus as berberine^[7]. COP has anti-inflammatory, hypolipidemic, hypoglycemic, and other medicinal properties^{[8, 9]}. In addition, COP has antitumor properties. According to some studies, COP not only promotes DNA damage by increasing the level of pH2AX protein (DNA damage marker) in non-small cell lung cancer cell line A594 cells, but it also induces apoptosis by up-regulating ROS, caspase-3/-9, bax/bcl-2, and PARP cleavage^[10]. This study used the experimental method of detecting the direct effect of small molecular drugs on the target to clarify the molecular mechanism of COP anti-HCC and discovered that the direct target of COP was insulin-like growth factor 2 (IGF2) mRNA-binding protein 1 (IGF2BP1). Research has shown that IGF2BP1 is overexpressed in HCC, promotes the activation of oncogenes, and is associated with cancer progression and poor prognosis^{[11, 12]}. For instance, SOX12 can promote the growth, apoptosis, invasion, and metastasis of HCC cells by up-regulating IGF2BP1 expression^[13]. Therefore, targeting IGF2BP1 may become a potential strategy for effectively controlling the process of HCC.

In this study, we found that COP can inhibit the growth of HCC cells in vitro and in vivo. Further research revealed that IGF2BP1 is a direct target of COP against HCC. Subsequently, we excavated the deubiquitinase and E3 ligase involved in the action of COP on IGF2BP1. It was found that COP could increase the ubiquitination degradation of IGF2BP1 and reduce IGF2BP1 expression by promoting the action of IGF2BP1 and E3 ligase PRPF19 and reducing the action of IGF2BP1 and deubiquitinase USP10. The study opens up new avenues for research into modern pharmacology and the molecular mechanisms of antitumor treatment in traditional Chinese medicine ^[14].

Antibodies and reagents

The primary antibodies used in this research work included, anti-IGF2BP1 (14796S, Cell Signaling), anti-PRPF19 (4937, Cell Signaling), anti-USP10 (T8203, Sigma), ab82411, anti-GAPDH Abcam), and anti-Ub (4393S, Cell Signaling). The reagents utilized in the experiments consisted of purified coptisine (> 98%, Chongqing Bositai, 6020-18-4), Z-Leu-Leu-Leu-CHO (MG132, MCE, HY-13259), cycloheximide (CHX, Glpbio, GC34329). Sorafenib was obtained from Chongqing Beibei District Hospital of Traditional Chinese Medicine (Chongqing, China).

Cell culture and transfection

HuH7, 7404, HepG2 and MHCC97-H cell lines were supplied by the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were grown in DMEM culture medium, enriched with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin, under 5% CO2 and 37 ℃ settings. Transfection of SiRNA was done at a final level of 50 nM and plasmids was performed at a final concentration of 1 µg with the Lipofectamine 8000 Platform (Beyotime Biotechnology, Cat.: C0533) as documented by the vendor. All siRNAs utilized in this research work were supplied by Sangon (Shanghai, China), and the sequences of the siRNAs are provided in Supplementary Table 1 pCDH-CMV-IGF2BP1(human)-HA-EF1a-CopGFP-T2A-Puro (P29894), pLVX-USP10-FLAG-IRES-Neo (P16085), pLVX-EnCMV-mCherry-PRPF19(human)

-FLAG-PGK-Puro (P33192), pRK5-6×His-Ubiquitin-K48 (P37539), pCMV-VSV-G (P0269), pLP1 (P0264) and pLP2 (P0265) were purchased from Wuhan Miaoling Biotechnology (Wuhan, China).

Cell viability assay

Cell viability of HuH7, 7404 and HepG2 cells was explored via MTT (Beyotime Biotechnology, Cat.:ST316). Cells adhered to 96-well plates were treated with 0, 35, 70, 140, and 280 µM of COP for 24, 48, and 72 hours, respectively. After the cells had been cultured for 4 hours with MTT, they were subsequently added with 150 µL of DMSO An EXL808 microplate analyzer was used to measure each well's absorbance at 490 nm.

Colony formation, flow cytometry and EdU staining Assays

The colony formation, flow cytometry and EdU staining assays were performed as documented previously^[15].

Animal study design for xenografted tumor model

Four-weeks old, male BALB/c nude mice weighing 16-18g were provide by Weitonglihua (Beijing, China). The Southwest University Animal Care and Use Review Committee authorized all animal experiments, which were done as per guidelines of the NIH. Naked mice were kept in a pathogen-free environment. We randomly assigned 40 nude mice with 1×10⁷ HuH7 cells inoculated in the right foreleg into five groups: tumor control (TC), coptisine low-dose (COP-L), coptisine medium-dose (COP-M), and coptisine high-dose (COP-H) and Sorafenib (a clinical liver cancer positive drug) group. In order to make it comparable to the oral dosage for humans, the COP-L, COP-M, and COP-H groups were given COP orally once daily at 30 mg/kg, 60 mg/kg, and 120 mg/kg, respectively. The Sorafenib group was treated with 120 mg/kg. Similarly, 40 male BALB/c nude mice were randomly divided into five groups:control group (lV-NC), coptisine treatment in control group (LV-NC + COP), overexpression IGF2BP1 group (LV-ov-IGF2BP1), coptisine treatment in overexpression IGF2BP1 group (LV-ov-IGF2BP1 + COP), silence IGF2BP1 group (LV-ShIGF2BP1). The oral dosage of coptisine was 120 mg/kg. Every three days, the mice body weight, as well as volume of tumors were assessed. We sacrificed the nude mice after 26 days, then dissected the tumors. Finally, the tumor was imaged followed by weighing, and all relevant data documented.

TCGA database analysis

Liver hepatocellular carcinoma (LIHC) gene expression information along with the matching clinical data were abstracted from the TCGA data resource (https://portal.gdc.cancer.gov/). We utilized the R ‘limma’ package (3.46.0) and a p < 0.05 and a log2FOLDChange > 2 to assess the genes that were differentially expressed in colon cancer.

mRNA sequencing

Sequencing of mRNA was done by Novogene (Beijing, China). The Illumina protocols were adopted in sequencing. The quality of the sequencing reads was assessed via FastQC. We only mapped the reads with a quality score > 28 to human genome, as per the genome annotation. In the analysis of DEGs, the uniquely localized reads were utilized. Differential expression of the target genes can reflect the changes in biological casacades and biological roles, as revealed by KEGG and GO analyses. ClusterProfiler (3.18.1), DOSE (3.16.0), as well as enrichplot (1.10.2) were utilized to visualize the KEGG and GO enrichment analyses.

Western blot and real-time PCR

Western blotting and RT-qPCR were done as documented previously^[15]. Primer sequences are provided in Supplementary Table 2

Cellular thermal shift assay (CETSA)

To assess the prospective target accounting for COP against HCC, we carried out CETSA. Concisely, lysis of HuH7 cells was done via repeated thawing thrice in liquid nitrogen. After that, we aliquoted the cell lysates into two; the control and the other was inoculated for 30 minutes with COP (0–50 µM) at room temperature (RT). Afterwards, the lysates were incubated for 5 min at 57°C, then cooled on ice. Fractionation of proteins was done on the SDS-PAGE gels, followed with staining using Coomassie Blue. After inoculation with COP, we isolated the remarkably different bands, subjected them to digestion with trypsin and detection using LC-MS/MS. Besides, validation of direct target protein was done via inoculating 90 µL of the cell lysates for five minutes with COP (12.5 µM) at 54–72°C, then cooling on ice until completion of the range of heat treatments. Then the protein bands were detected with western-blotting.

Active ingredients -target protein docking

Active ingredients-target protein docking was conducted as described previously reported^[15].

Drug affinity responsive target stability (DARTs) assay

We collected the HuH7, 7404 and HepG2 cells, followed 30 minutes inoculation with RIPA lysis buffer enriched with a cocktail of protease inhibitors cocktail, and then spinning for 10 min at 15,000 g. We diluted the cell lysates with 10 X TNC buffer (500 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 100 mM CaCl2). the aliquots (90 µL) of the cell lysates with COP (0-500µM, 10µL) treatment 30 min, then incubated with 2 µL pronaseE (1:2000) (Solarbio, Cat.: P8360) for 30 min at 40°C, followed by introducing the sample loading dye, then boiling for 10 min at 98°C. Moreover, the aliquots (90 µL) of the cell lysates with COP (125µM, 10µL) treatment 30 min, then inoculated with 2 µL pronaseE (1:1000 ~ 1:6000) for 30 min at 40°C. Finally, an equal measure of every sample was fractionated onto SDS-PAGE gels.

Cycloheximide chase experiment

Transfection of the HuH7, 7404, and HepG2 cells with specified siRNAs or plasimids was done for 24 hours, then inoculated in DMEM culture media enriched with 100 µg/ml CHX. At the designated points of time, we collected the cell, lysed them with RIPA buffer, and then the cell lysates were submitted to western blotting to assess protein expression. ImageJ was utilized to determine the relative expression level.

Co-immunoprecipitation (Co‐IP)

We harvested the cells, followed by lysing on ice in cell lysis buffer for IP enriched with a cocktail of protease inhibitors for 30 min, and spinning for 10 mins at 15,000 g at 4°C. The cell lysate supernatants were inoculated overnight with 2 µg of anti-IGF2BP1 or and normal rabbit IgG, anti‐USP10 and anti‐PRPF19 antibodies at 4°C under rotation. Afterwards, rotation of the bead suspensions was done for two hours using 40 µL Dynabeads® Protein A/G (MCE, Cat.: HY-K0202) at 4°C. Thereafter, we rinsed the beads four times with PBS + 0.5% Tween-20 (pH 7.4) followed by final elution via direct boiling in 1× SDS loading buffer.

Immunofluorescence

HuH7 cells were inoculated in fluorescent chamber slides, then fixed (in 4% PFA) after 24 h. After that, we rinsed the slides for five mins with PBS thrice. Permeabilization of the cells was done for 30 mins with the blocking buffer (0.1% Triton-X100 in PBS) at RT, then overnight inoculated with anti-IGF2BP1 antibody and anti-USP10 antibody or anti-PRPF19 antibody at 4°C. Thereafter, we rinsed the slides thrice PBS, followed by inoculation for two hours with Alexa 488-linked and 549-linked secondary antibodies at RT in the dark. Subsequently, PBS was washed thrice, the slides were sealed with antifade mounting medium with DAPI (Beyotime Biotechnology, Cat.: P0131) and observed with a confocal microscope.

Lentivirus production and infection

Generation of lentivirus was done in HEK 293FT cells. Lentiviral packaging vectors pCMV-VSV-G, pLP1 and pLP2 and the transfer plasmid were mixed 1:1:1:1 and transfected with the Lipofectamine 8000. We collected the lentivirus supernatant, followed by filtering for 48 hours with a 0.45 µM filter. HuH7 cells was inoculated in DMEM (Hyclone) enriched with 10% FBS and inoculated with lentivirus. Following elapse of 48 h, we introduced puromycin (2 µg/mL) for positive selection of infected cells.

Statistical analysis

Data are provided as the means ± standard deviation (SD). ANOVA, repeated measures, s well as student’s t-tests were implemented in the GraphPad Prism 8.0 software, with P < 0.05 signifying statistical significance.

COP repressed the growth of HCC cells and tumor in vitro and in vivo.

To elucidate if COP can impacts the growth of HCC cells, we assessed the impact of COP on the viability of HuH7, 7404, HepG2 and LO2 cells after treatment in different time periods by MTT. It was found that COP gradually reduced the viability of HuH7, 7404 and HepG2 cells in a concentration-dependent, as well as time-dependent approach (Fig. 1A-D). Of the tested cells, COP is the most sensitive to HuH7 and less toxic to LO2 of normal human hepatocytes. Based on the IC50 of HuH7 as determined by MTT, we administered 0, 25, 50 and 100 µM for 24 hours in the downstream assays. Colony-formation and EdU assay validated that COP repressed the growth of HuH7, 7404 and HepG2 cells in a dose-dependent approach (Fig. 1E and F, Supplementary Figure. 1A). After 24 hours of COP treatment, the cell cycle, as well as apoptosis of HCC cells were analyzed via flow cytometry, and showed that COP inhibited the cell cycle progress and triggered apoptosis of HCC cells dose-dependently (Supplementary Figure. 1B and C). In order to further assess the antitumor properties of COP in vivo, we generated a xenograft tumor model using HuH7 cells. In contrast with the TC group, the COP-M, COP-H and Sorafenib groups illustrated remarkable repression of tumor growth (Fig. 1G-J). The body weight as well as the ratio of organ weight to body weight did not show significant differences among the five groups, illustrating that COP is less toxic in vivo (Supplementary Figure. 4A-E). Altogether, our data suggest that COP may inhibit the growth of HCC cells and tumors in vitro and in vivo.

IGF2BP1 is a potential target of COP against HCC

In this experiment, the HuH7 cell of the control group (NC) without coptisine treatment and the 100 µM coptisine-treated group (COP) were subjected to transcriptome sequencing, with three replicates in each group. The criteria for differential gene screening were Fold Change ≥ 0 and p-value (adj) < 0.05. The volcano plot visually shows the differential gene distribution with each comparison combination (Fig. 2A). The cluster analysis is revealed in the supplementary Fig. 2A. In total, 2052 genes were obtained by crossing differential genes of transcriptome data and TCGA data (Fig. 2B). GO enrichment and KEGG analysis revealed that these 2052 genes were primarily involved in cell proliferation, cell cycle, migration, and invasion, consistent with our previous cell phenotype (Fig. 2C and D). To investigate further the potential target of COP in HCC, we used CETSA in conjunction with proteomic technology to identify the target protein that COP directly binds to HCC cells. CETSA is a newly published technology for target verification and discovery that can detect the binding of targets and small molecules in cells, tissues, and animals. The band marked by the arrow in Fig. 2E is the target one. Following mass spectrometry identification, 205 proteins were obtained, which intersected with the differential genes of liver cancer in the TCGA database, and a total of 35 essential target genes were obtained. Through the screening of protein molecular weight and literature research, we focused on IGF2BP1(Fig. 2F). The molecular docking results between COP and IGF2BP found that COP can interact with lysine at positions 538 and 487 and glutamic acid at positions 489 and 520 in the KH3/4 domain of IGF2BP1 (Fig. 2G). IGF2BP1 is an RNA-binding protein that binds to mRNA and stabilizes it to ensure its normal translation into protein. To confirm whether IGF2BP1 is a key target of COP in HCC, we predicted the downstream targets of IGF2BP1 and compared them with transcriptome data and TCGA data, as shown in Fig. 2H. There are 7774 overlapping genes between the predicted target genes of IGF2BP1 and the transcriptome differential genes, which means that the key target of COP is most likely IGF2BP1. GO enrichment and KEGG analysis were performed on 1190 intersection genes from three data sets. These genes are mainly associated with cell proliferation, cell cycle, and cell migration, consistent with the transcriptome data (Supplementary Fig. 2B and C). We performed transcriptomic verification of the 10 target genes of IGF2BP1. The accuracy rate was 90%, indicating the reliability of the transcriptome data (Supplementary Fig. 2D). IGF2BP1 expression in liver cancer was analyzed through the website (http://ualcan.path.uab.edu) (Fig. 2I). The analysis showed that IGF2BP1 is lowly expressed in normal tissues, highly expressed in liver cancer tissues, and increases gradually with the tumor stage (Supplementary Fig. 2E). The survival and prognosis of IGF2BP1 in liver cancer were further analyzed through the website (https://kmplot.com/analysis/). High expression of IGF2BP1 is significantly correlated with the poor prognosis of liver cancer (Fig. 2J). In conclusion, we hypothesize that IGF2BP1 is the direct target of COP and plays a key role in COP against HCC.

IGF2BP1 is confirmed as the direct target of COP by CETSA and DARTs

We used CETSA (including temperature gradient and drug concentration gradient) and DARTs (including enzyme concentration gradient and drug concentration gradient) to confirm that IGF2BP1 is the direct target of COP. The drug concentration gradient results of CETSA in different cell protein samples showed that at the same temperature of 57°C, IGF2BP1 protein gradually increases with the increasing COP concentration, indicating that COP can bind to IGF2BP1(Fig. 3A). The temperature gradient experimental results of CETSA showed that the decline rate of IGF2BP1 after COP treatment was slower than that without COP treatment, indicating that IGF2BP1 can combine with COP and stabilize IGF2BP1 conformation and reduce IGF2BP1 degradation with increasing temperature (Fig. 3B). DARTs assessed protein stability by adding pronaseE to determine the direct binding ability of small molecules to proteins. The drug concentration gradient test results of DARTs showed that after treatment with 1:2000 pronaseE, IGF2BP1 protein in different cell protein samples gradually increased with increasing COP concentration, as shown in Fig. 3C. The pronaseE concentration gradient test results of DARTs showed that the decline rate of IGF2BP1 after COP treatment was slower than that without COP treatment (Fig. 3D). This indicates that COP can bind to IGF2BP1 and inhibits the degradation of IGF2BP1 by pronaseE. Finally, we used WB to detect IGF2BP1 expression after COP administration in HuH7 and 7404 live cells. After 24 h of COP administration, IGF2BP1 was dramatically reduced with a dose-dependent trend (Fig. 3E and F). Concurrently, COP can reduce the protein expression of IGF2BP1 in animal tumors (Fig. 3G and H). We concluded that COP could directly bind to IGF2BP1 by CETSA and DARTs and confirmed that COP treatment could significantly reduce IGF2BP1 expression.

Effects of IGF2BP1 on the growth of HCC cells and tumors in vitro.

To verify the function of IGF2BP1 gene in HCC cells, we detected the protein expression of IGF2BP1 in different HCC cells. The results displayed that IGF2BP1 expression was the highest in HuH7 cells and the lowest in HepG2 (Fig. 4A). HuH7 and 7404 were selected for IGF2BP1 silencing. The silencing efficiency of IGF2BP1 siRNA was detected by a qPCR experiment. We set up 100 µM COP treatment as controls. Three IGF2BP1 siRNAs effectively reduced the expression levels of IGF2BP1 gene mRNA in HuH7 and 7404 cells (Fig. 4B and C). Clone formation assay and EdU staining assay showed that three IGF2BP1 siRNAs inhibited the proliferation of HuH7 and 7404 cells (Fig. 4D, supplementary Fig. 3A). In the following experiments, we constructed shRNA based on the siRNA fragment sequence of si-IGF2BP1-1. shRNA of IGF2BP1 significantly increased the number of cells in G0/G1 phase and apoptotic cells in HuH7 and 7404 cells (Supplementary Fig. 3C and D). To further verify the function of IGF2BP1 gene in HCC cells, we overexpressed IGF2BP1 gene in HepG2 and 7404 cells. The overexpression efficiency of IGF2BP1 was assessed by WB, as shown in Fig. 4E. Clone formation assay and EdU staining assay showed that overexpression of IGF2BP1 rescued the reduction in cell proliferation caused by COP in 7404 and HepG2 (Fig. 4F, Supplementary Fig. 3B). IGF2BP1 overexpression significantly reduces COP-induced S phase arrest in 7404 and G2/M phase arrest in HepG2 and significantly improves the number of apoptotic cells induced by COP treatment in 7404 and HepG2 cells (Supplementary Fig. 3E and F). Notably, there are many overlaps between the downstream target genes in IGF2BP1, and the differential genes changed after COP treatment. We selected predictive target genes (E2F7, BCRA2, FEPMT1, NES, PTP4A3, CK2), which changed significantly after COP treatment and are related to cancer, for verification. After overexpression of IGF2BP1 in HuH7 and 7404 cells, the predicted target genes E2F7, BCRA2, FEPMT1, and NES in the ov-IGF2BP1 group were significantly up-regulated compared to the Vector group. However, the ov-IGF2BP1 group of PTP4A3 and CK2 only showed significant up-regulation in single cells (Fig. 4G-M). Our findings suggest that IGF2BP1 may affect the growth of HCC cells in vitro.

Effects of IGF2BP1 on the growth of HCC cells and tumors in vivo.

To study the antitumor effect of IGF2BP in nude mice, we constructed a stable expression cell line based on the HuH7 cell by lentiviral packaging. WB experiments confirmed that HuH7 cells infected with LV-ovIGF2BP1 or LV-ShIGF2BP1 could stably overexpress or silence IGF2BP1 (Fig. 5A). We subcutaneously injected lentivirus-infected HuH7 cell lines LV-NC, LV-ovIGF2BP1, and LV-ShIGF2BP1 into the right front leg of 4-week-old nude mice, eight nude mice in each group. At the end of the experiment, there was no significant difference in the body weight changes of nude mice among each group (Fig. 5B). Compared to LV-NC group, the tumor weight of LV-NC + COP group and LV-ShIGF2BP1 group was significantly decreased, and the tumor weight of LV-ovIGF2BP1 group increased significantly (Fig. 5C and D). Simultaneously, the experimental results revealed that the organ ratios of LV-NC + COP, LV-ovIGF2BP1, LV-ovIGF2BP1 + COP, and LV-ShIGF2BP1 groups did not differ significantly from LV-NC group (supplementary Fig. 4F-G). All the above results in vivo and in vitro show the critical role of IGF2BP1 in HCC. IGFBP1 expression in tumor tissue was then detected by immunofluorescence staining to confirm the successful overexpression of IGF2BP1, and COP intervention could reduce IGF2BP1exprssion (Fig. 5E). We also detected the expression of target genes (E2F7, BCRA2, FEPMT1, NES, PTP4A3, and CK2) in tumor tissues, and the results were consistent with those of cells (Fig. 5F). Although there was no significant difference in the decrease of target genes E2F7, BCRA2, and NES after COP treatment, there remained a downward trend. These findings show that IGF2BP1 plays a vital role in liver cancer and that E2F7, BCRA2, FEPMT1, and NES may be downstream target genes of IGF2BP1 and play an essential role in COP's anti-HCC activity.

COP causes protein ubiquitination and degradation of IGF2BP1

To investigate how COP reduced the expression of IGF2BP protein, we first measured changes in IGF2BP1 mRNA levels in HuH7, 7404, and HepG2 after different concentrations of COP were added. After 24 h of COP treatment, the mRNA level of IGF2BP1 in HuH7, 7404, and HepG2 cells were increased at 100 µM (Fig. 6A-C). It indicates that COP cannot reduce the mRNA level of IGF2BP1 but can increase the mRNA level of IGF2BP1 at high concentrations, which shows an adverse influence on protein expression. As a result, we hypothesize that the decrease in IGF2BP1 protein levels is due to the direct effect of COP. The stability of IGF2BP1 protein was then determined following COP intervention. After 24 h of intervention with 100 µM COP, cycloheximide (CHX: 50 µg/µL) was added, and the total cell protein was collected at 0, 2, 4, and 8 h. The results showed that the protein stability of IGF2BP1 in the HuH7, 7404, and HepG2 cells decreased significantly after COP intervention (Fig. 6D-F). We further investigated the potential reasons for the decline of IGF2BP1 protein stability caused by COP and found the changes in IGF2BP1 ubiquitin level after COP intervention. HuH7 cells with simultaneous overexpression of IGF2BP1 and ubiquitin (Ub) were treated with COP for 24 h. Protein was collected after immunoprecipitation with MG132 (a proteasome inhibitor) to pull-down IGF2BP1. The results showed that after the COP intervention, the ubiquitin level of IGF2BP1 increased significantly (Fig. 6G). We also added MG132 to HuH7 and 7404 cells to identify if IGF2BP1 degradation is related to proteasomal degradation. Compared with COP group, the protein level of IGF2BP1 in COP + MG132 group was significantly up-regulated (Fig. 6H and I). This indicated that MG132 could restore the protein level of IGF2BP1 decreased by COP. Simultaneously, MG132 can slow down the decrease of IGF2BP1 protein stability caused by COP (Fig. 6J). In conclusion, we showed that the reduction in IGF2BP1 protein stability induced by COP is primarily due to increased ubiquitination and degradation.

Effects of overexpression of USP10 and silencing of PRPF19 on the protein level and stability of IGF2BP1

To identify the key enzymes responsible for IGF2BP1 ubiquitination and degradation after COP treatment, we performed an IGF2BP1 pull-down experiment on 7404 cells and analyzed the pull-down protein using mass spectrometry (Fig. 7A). The website (http://ubibrowser.bio-it.cn/) predicted the E3 ligases and deubiquitinases that can interact with IGF2BP1. Four E3 ligases and two deubiquitinases were obtained by intersecting the predicted related enzymes with the protein mass spectrometry data (Fig. 7B and C). According to the scores of predicted sites and the results of protein mass spectrometry; we speculate that E3 ligase PRPF19 and deubiquitinase USP10 play a vital role in the ubiquitination and degradation of IGF2BP1 by COP. We used co-immunoprecipitation (CO-IP) experiments to investigate whether IGF2BP1 interacts with PRPF19 and USP10. Endogenous IGF2BP1 can bind to endogenous USP10 and PRPF19 in 7404 cells, as shown in Fig. 7D. An immunofluorescence assay was performed to observe the cellular localization of IGF2BP1 and USP10 or PRPF19. The overlapping signal (yellow) from IGF2BP1(green) and USP10 or PRPF19 (red) confirms the co-localization between IGF2BP1 and USP10 or PRPF19 (Fig. 7E). The changes in IGF2BP1 protein level after overexpression of USP10 or silencing PRPF19 were observed. This showed that overexpression of USP10 or silencing PRPF19 in HuH7 and 7404 cells could reduce COP-induced ubiquitination and IGF2BP1 degradation (Fig. 7F and G). Simultaneously, overexpression of USP10 or silencing PRPF19 could restore the half-life of IGF2BP1 protein caused by COP and increase the stability of IGF2BP1 protein (Fig. 7H and I).Taken together, USP10 and PRPF19 can regulate the protein level of IGF2BP1 in HCC cells and restore the protein stability of IGF2BP1 decreased by COP, indicating that USP10 and PRPF19 play a key role in the direct effect of COP on IGF2BP1.

Coptidis Rhizoma is the dry rhizome of Coptis Chinensis Franch, which is commonly used and famous in traditional Chinese medicine with more than 2000 years of history. COP has been widely studied in the treatment of cancer. For example, COP can inhibit HCT116 cell survival, adhesion, and migration, as well as MMP3 and MMP9 expression, and down-regulate the expression of PI3K and Akt, as well as changes in downstream markers of epithelial-mesenchymal transformation (EMT), such as E-cadherin, N-cadherin, vimentin, and snail^[16]. In this study, we systematically evaluated the inhibitory effect of COP on HCC cells in vitro and in vivo through cell experiments and a tumor-bearing nude mouse model.

CETSA was originally developed to assist in the research of anticancer drug targets. For the first time, it was used to study the interaction of cells and drug targets using a label-free method^[17]. Combined with CETSA and multiple quantitative mass spectrometry (MS-CETSA), the changes in protein thermal stability of the whole proteome under drug treatment can be monitored simultaneously. Therefore, proteins interacting with drugs can be identified in MS-CETSA without a prior understanding of relevant pathways or mechanisms^{[18, 19]}. DARTs is a label-free method for direct recognition of target proteins developed in recent years. Compared with non-ligand-bound proteins, ligand-bound proteins exhibit different stability in the case of protein hydrolysis. Simultaneously, for different protease exposure, the change in protein stability can be detected by changing the proteolysis mode^[20]. DARTs do not require chemical modification of natural products for target recognition, allowing natural products and their extracts to determine their direct binding protein targets in this way^[21]. In this study, CETSA and DARTs experiments showed that COP could directly interact with IGF2BP1, and COP could down-regulate the expression of IGF2BP1 protein in HCC cells.

There are three IGF2BPs (IGF2BP1, IGF2BP2, and IGF2BP3) involved in regulating RNA transport and translation in mammals. IGF2BP1 is a carcinoembryonic protein, which is highly expressed between a fertilized egg and the embryonic stage and almost disappears in normal adult organisms^[22]. IGF2BP1 has been reported to be highly expressed in gallbladder carcinoma (GC), hepatocellular carcinoma (HCC), and fibrous hepatocellular carcinoma (FL-HCC). IGF2BP1 overexpression can promote the proliferation, migration, and invasion of HCC cells^[23], associated with poor survival and prognosis^[11]. Studies have reported that IGF2BP1 can bind to long-chain noncoding RNA HULC and promote its degradation through interaction with CNOT1^[24]. Therefore, IGF2BP1 is considered one of the potential targets for cancer treatment, and IGF2BP1 inhibitors will become a potential strategy for cancer treatment. Our findings indicate that IGF2BP1 can affect HCC cell proliferation, cycle, and apoptosis, though the effect of IGF2BP1 silencing on HCC cells is not as good as that after COP treatment. This could be due to the the limited silencing of IGF2BP1, or it could be that COP has more than one key target.

Molecular docking showed that COP mainly binds to KH3/4 domain of IGF2BP1. The dual-domain of KH3/4 is crucial for IGF2BP1 to bind to targeted mRNA in N6 methyl adenosine (m6A)-dependent manner, where KH domain can recognize the sequence of common GG (m6A) mRNA^[25]. Several in vivo and in vitro studies have reported that IGF2BP1 plays an important role in the growth of tumor tissues and the adhesion, apoptosis, migration, and invasion of tumor cells by regulating mRNAs related to cancer, including PTEN, ACTB, MAPK4, MKI67, c-MYC, and CD44^{[22, 26]}. There are 7774 cross genes in the predicted target of IGF2BP1 and transcriptome as an RNA binding protein. We selected six prediction target genes with large changes for verification and found that the levels of E2F7, BCRA2, NES, and FEPMT genes were changed in the IGF2BP1 overexpression. E2F7 plays a key role in DNA damage-dependent transcriptional regulation of cell cycle genes. It is reported that E2F7 is recruited to the location of DNA fracture and is considered to inhibit the damaged DNA repair process directly^[27]. FEPMT1 is primarily expressed in epithelial cells and can activate integrin with Talin, increase integrin affinity to extracellular ligands, and regulate integrin-mediated adhesion and signal transduction by stimulating the conformational changes of these receptors^[28]. BRCA1 and BRCA2 maintain genomic stability, especially the homologous recombination pathway of double-stranded DNA repair^[29]. We preliminarily speculate that IGF2BP1 can control the process of hepatocellular carcinoma by acting with E2F7, BCRA2, NES, and FEPMT, which requires more experimental verification. More research is required to determine how COP directly works on IGF2BP1 to reduce its expression. Studies have found that the ubiquitin-proteasome system (UPS) is the targeted degradation mode of most cellular proteins (70 ~ 80%)^[30]. Ubiquitin binding is carried out through an enzymatic cascade, which helps regulate the half-life of substrate proteins, ranging from seconds to days. This process is strictly regulated by three groups of enzymes, including E1, E2, and E3 ligases^{[31, 32]}. E3 ligase has the most species and the most robust specificity. Ubiquitination can be reversed by another group of enzymes known as deubiquitinase (DUBs), mediated by conjugation reaction^[33]. DUBs cleave Ub from the C-terminal of the target protein and maintain the dynamic balance of Ub in cells. Deubiquitination is necessary to provide sufficient free Ub molecules in cells^{[34, 35]}. In this study, the protein stability of IGF2BP1 decreased significantly, and the ubiquitination of IGF2BP1 increased after COP treatment. Simultaneously, MG132 inhibitor can significantly increase the protein level of IGF2BP1 reduced by COP. All these indicate that COP reduces the protein level of IGF2BP1 through proteasome degradation.

Through website prediction and protein mass spectrometry identification, E3 ligase PRPF19 and deubiquitination enzyme USP10 are most likely to increase IGF2BP1 ubiquitination. PRPF19 is a 55 kDa RNA binding protein involved in mRNA pre-processing and DNA damage repair^[36]. In 2001, Hatakeyama et al. demonstrated that N-terminal U-box domain of PRPF19 has E3 ligase activity in vitro^{[37, 38]}. The central coiled domain of PRPF19 promotes the formation of the homologous tetramer, and the dimer U-box domain and substrate recognition WD40 domain are attached at both ends^[39]. This quaternary complex brings the distance between the substrate protein and U-box domain close to ensure effective ubiquitination^{[40, 41]}. DUBs are a large class of enzymes divided into six distinct subfamilies based on their sequence and functional similarities. USP is the most prominent family consisting of more than 50 members of human origin^[35]. USP10 is evolutionarily conserved, and human USP10 shares 99% homology with rat and mouse amino acid sequences. USP10 is a cysteine protease that relies on the hydrolysis of ester, thioester, amide, or peptide bonds, and USP10 mediates the formation of thiols from carboxy-terminal glycine residues of Ub to remove thiols from target proteins^[42]. In this study, overexpression of USP10 or silencing PRF19 could save the protein level and protein stability of IGF2BP1 reduced by COP. Concurrently, we also observed that PRPF19 and USP10 decreased after COP treatment. We speculated that COP could promote the increase of IGF2BP1 ubiquitin due to the increase in the interaction between PRPF19 and IGF2BP1 or the decrease of the de ubiquitin function of USP10, resulting in IGF2BP1 degradation.

In conclusion, we found that COP can inhibit the growth of HCC cells both in vitro and in vivo. Further research has revealed that IGF2BP1 is a direct target of COP against HCC.COP has an anti-HCC effect by either promoting the action of IG2BP1 and the E3 ligase PRPF19 or decreasing the impact of IGF2BP1 and the deubiquitin enzyme USP10 to increase IGF2BP1 ubiquitin degradation (Fig. 8). It opens up new avenues for research into modern pharmacology and the molecular mechanisms of traditional Chinese medicine against tumors.

CETSA, Cellular thermal shift assay, DMEM, Dulbecco's modified Eagle's medium, DMSO, Di-methyl sulfoxide, DARTs, Drug affinity responsive target stability, FBS, Fetal bovine serum, HCC, Hepatocellular carcinoma, IGF2BP1, insulin-like growth factor 2 (IGF2) mRNA binding protein 1, LIHC, Liver hepatocellular carcinoma, PRPF19, Pre-mRNA Processing Factor 19, PBS, Phosphate-buffered saline, SPF, Specific pathogen-free, USP10, Ubiquitin Specific Peptidase 10 TCGA, The cancer genome atlas, WB, Western blot, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,

Ethics approval and consent to participate

The animal studies were approved by the Laboratory Animal Care and Use Committee of Southwest University, Chongqing, China (SCXK-YU2020-0006). All animal studies were carried out in accordance with the NIH guidelines in 2011 for the care and use of laboratory animals.

Consent for publication

all authors reviewed the manuscript and approved the publication of the manuscript in the present form.

Availability of data and material

All data were generated in-house, and no paper mill was used. All authors agree to be

accountable for all aspects of work ensuring integrity and accuracy. All data generated

or analysed during this study are included in this published article and its supplementary information files. The datasets used during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no conflict of interest.

Funding

This work was financially supported by the National Key Research and Development Program of China for Traditional Chinese Medicine Modernization (2017YFC1702600, 2017YFC1702605), the County-University cooperation Innovation Funds of Southwest University (SZ201901), the Industrial Technology System Program for Traditional Chinese Herbs of Chongqing Municipal Agricultural Commission (2017 [5]), the Special Key Project of Technology Innovation and Application Development in Chongqing (CSTC2021jscx-gksb-N0033，cstc2020jscx-tpyzxX0022), the Science Foundation of School of Life Sciences SWU (20212005425201).

Authors' contributions

XLY and XGL conceived the idea and supervised the research, JHF, LMZ, LH, ZCM, XDL, KWL, JL, ZWG, JWL, MYX, QJW and XC performed the experiments, JHF, MMX, HMW and XTC participated in the design of the study and performed the statistical analysis. JHF wrote the manuscripts. All authors read and approved the final manuscript

Acknowledgments

Not applicable

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GraphicalAbstract.png
A schematic diagram about the central role of coptisine inhibits the proliferation of hepatocellular carcinoma by promoting ubiquitination of IGF2BP1.
SupplementaryTable1.docx
SupplementaryTable2.docx
Supplementary1.tif
Supplementary Figure 1. COP inhibits HCC cell proliferation and induced cell apoptosis and cycle arrest in vitro. (A) Effect of COP on DNA replication of HCC cells detected by EdU staining. (B)The effect of COP on cell apoptosis of HCC cells was detected by flow cytometry. (C)The effect of COP on cell cycle of HCC cells was detected by flow cytometry. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 compared with 0 μM control group. All data are presented as means ± SD (n = 3).
Supplementary2.tif
Supplementary Figure 2. Bioinformatics analysis. (A) Clustering heat map of differentially expressed genes in cells (COP/NC).(B) GO enrichment analysis plot of overlaps between between TCGA dataset and transcriptome data and downstream target genes of IGF2BP1. (C) KEGG enrichment analysis of overlaps between between TCGA dataset and transcriptome data and downstream target genes of IGF2BP1. (D) Validation of cellular transcriptome sequencing results. (E) Expression levels of IGF2BP1 in HCC tissues of different tumor grades.
Supplementary3.tif
Supplementary Figure 3. Effects of IGF2BP1 on the HCC cell proliferation and induced cell apoptosis and cycle arrest in vitro. (A) Effects of IGF2BP1 siRNA on the proliferation of HuH7 and 7404 cells by EdU staining. (B) Effects of overexpression of IGF2BP1 on the proliferation of 7404 and HepG2 cells by EdU staining. (C) Effects of Sh-IGF2BP1 on the cell cycle of HuH7 and 7404 cells. (D) Effects of overexpression of IGF2BP1 on the cell cycle of 7404 and HepG2 cells. (E) Effects of Sh-IGF2BP1 on apoptosis in HuH7 and 7404 cells (F) Effect of overexpression on apoptosis of IGF2BP1 on 7404 and HepG2 cells * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 compared with Si-control control group or Vector control group, #P<0.05, ##P<0.01, ###P<0.001 and ####P<0.0001 compared with Vector+COP control group.
Supplementary4.tif
Supplementary Figure 4. Organ index of animals in vivo experiments. (A) The effect of COP on the organ index of HCC in vivo experimental. (B) The effect of IGF2BP1 on the organ index of HCC in vivo experimental. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 compared with control group (TC) and LV-NC group.

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Coptisine inhibits the proliferation of hepatocellular carcinoma by promoting ubiquitination of IGF2BP1

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Abstract

Background

Methods

Result

Conclusions

Figures

Background

Materials And Methods

Antibodies and reagents

Cell culture and transfection

Cell viability assay

Colony formation, flow cytometry and EdU staining Assays

Animal study design for xenografted tumor model

TCGA database analysis

mRNA sequencing

Western blot and real-time PCR

Cellular thermal shift assay (CETSA)

Active ingredients -target protein docking

Drug affinity responsive target stability (DARTs) assay

Cycloheximide chase experiment

Co-immunoprecipitation (Co‐IP)

Immunofluorescence

Lentivirus production and infection

Statistical analysis

Results

IGF2BP1 is a potential target of COP against HCC

IGF2BP1 is confirmed as the direct target of COP by CETSA and DARTs

COP causes protein ubiquitination and degradation of IGF2BP1

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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