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METTL16/IGF2BP2 axis enhances malignant progression and DDP resistance through up-regulating COL4A1 by mediating the m6A methylation modification of LAMA4 in hepatocellular carcinoma

Cell Division volume 20, Article number: 9 (2025) Cite this article

Abstract

Background

Hepatocellular carcinoma (HCC) is the third most common malignant tumor after gastric cancer and esophageal cancer, which is a serious threat to human health. Methyltransferase-like protein 16 (METTL16) regulates the occurrence and development of various cancers, but its molecular mechanism in HCC has not been fully investigated.

Methods

A series of databases were used to predict gene expression, methylation sites, correlation analysis, and protein interaction analysis. Gene expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC). What’s more, drug-resistant cell lines were established for drug resistance analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2’-deoxyuridine (EdU) staining. Flow cytometry, transwell and wound healing assays were used for apoptosis, invasion and migration, respectively. In addition, the regulatory mechanism of METTL16 in HCC was investigated by methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP) and co-immunoprecipitation (Co-IP). Finally, constructing subcutaneous transplanted tumor in nude mice confirmed the effect of METTL16 in vivo.

Results

METTL16 was up-regulated in HCC drug-resistant tissues and cells. Knockdown of METTL16 inhibited Cisplatin (DDP) resistance, proliferation, invasion and migration of HCC cells, but promoted apoptosis. Besides, laminin subunit alpha 4 (LAMA4), which was overexpressed in HCC drug-resistant tissues and cells, was selected as the target of METTL16. Mechanistically, METTL16 and m6A reader insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) co-regulated the m6A modification and mRNA stability of LAMA4, and LAMA4 weakened the effects of METTL16 knockdown on HCC drug-resistance. Meanwhile, LAMA4 bound to collagen type IV alpha 1 chain (COL4A1) and facilitated DDP resistance and HCC progression via COL4A1. Similarly, in vivo, METTL16 induced tumor growth, as well as LAMA4 and COL4A1 expression, and increased DDP resistance.

Conclusion

METTL16 and IGF2BP2 jointly mediated the m6A methylation modification of LAMA4, thereby promoting DDP resistance and malignant progression of HCC through regulation of COL4A1.

Introduction

Hepatocellular carcinoma (HCC) is one of the most frequent and highly lethal malignancy in the world [1], with high recurrence and metastasis rates and low survival rates [2]. During the growth of tumors, different tumor cells from the same source can show strong heterogeneity, that is, the growth rate, invasion ability and sensitivity to drug are different [3, 4]. The same is true for liver cancer, which is highly heterogeneous. Therefore, it is pressing to definite the biological mechanism of HCC evolution.

For the past few years, there had been increasing evidence that epigenetic modifications were essential for the development of liver cancer [5,6,7]. N6-methyladenosine (m6A) was the most common and important epigenetic modification in mammals [8], which was widely involved in regulating the nuclear output, splicing, translation and degradation of RNA [9, 10]. m6A played an vital regulatory role in the progression of many diseases by binding to its methyltransferase, demethylase and methyl-binding protein [11,12,13,14].

Methyltransferase-like protein 16 (METTL16) is a class I SAM methyltransferase that, like METTL3, contains Rossmann fold and uses SAM as a methyl donor [15, 16]. METTL16, as a conserved protein [17], exists in the nucleus and cytoplasm [18]. METTL16 had been recognized as a potential gene related to cancer development, and its expression varied across different cancers. For example, METTL16 was highly expressed in gastric cancer and promoted the proliferation of tumor cells [19]. In addition, study reported that METTL16 was also up-regulated in colorectal cancer [20] and esophageal cancer [21], and the survival rate of patients was low. However, low expression of METTL16 had also been reported in cancers such as pancreatic ductal adenocarcinoma [22] and endometrial cancer [23]. By detecting the mRNA of METTL16 in 66 HCC tissues and 21 paraneoplastic tissues, Wang et al.. found that METTL16 was significantly down-regulated in HCC and was associated with poor prognosis [24]. Nevertheless, the specific mechanism of METTL16 in HCC has not been clarified.

In a word, the objective of this research was to clarify the mechanism of METTL16 in HCC disease progression and drug resistance.

Results

METTL16 was elevated in HCC drug-resistant tissues and cells

Firstly, the CPTAC database predicted high expression of METTL16 in liver cancer samples (Fig. 1A), while the same result was obtained in the ENCORI database (Fig. 1B). Meanwhile, the high expression of METTL16 was positively correlated with advanced tumor, lymph node metastasis, and poor overall survival (Table 1; Supplementary Fig. 1). Compared with DDP sensitive tissues, both RNA and protein levels of METTL16 were up-regulated in HCC resistant tissues (Fig. 1C and D). The DDP resistance of HCC drug-resistant cells was enhanced compared with HCC cell lines (Fig. 1E), among which METTL16 was the highest in HCC drug-resistant cells (Hep3B/DDP and Huh- 7/DDP), followed by non-drug-resistant HCC cells (Hep3B and Huh- 7), and finally normal hepatocyte cells (THLE- 2) (Fig. 1F). Therefore, METTL16 was overexpressed in HCC drug-resistant tissues and cells, and was related to DDP resistance.

Fig. 1
figure 1

METTL16 was up-regulated in HCC tissues and cells.A Protein expression of METTL16 in HCC normal samples (Normal, n = 165) and tumor samples (Primary tumor, n = 165) from CPTAC database. B METTL16 expression with 374 cancer and 50 normal samples in HCC samples from ENCORI database. C mRNA expression of METTL16 in sensitive tissues (n = 29) and resistant tissues (n = 33). D Protein expression of METTL16 in sensitive tissues (n = 3) and resistant tissues (n = 3). E IC50 values of Hep3B, Hep3B/DDP, Huh- 7, and Huh- 7/DDP cells. F Protein expression of METTL16 in THLE- 2, Hep3B, Hep3B/DDP, Huh- 7, and Huh- 7/DDP cells. *P < 0.05

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Table 1 Relationship between METTL16 expression and clinicopathologic features of HCC patients
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Knockdown of METTL16 inhibited proliferation, metastasis, and DDP resistance in HCC drug-resistant cells

In order to further research the role of METTL16 in HCC drug-resistant cells, this study synthesized METTL16 si-RNA, and detected the interference efficiency. It was observed that after transfection with si-METTL16, the expression of METTL16 in HCC drug-resistant cells was significantly reduced (Fig. 2A), indicating that the si-METTL16 was effective. Then the loss of function test was performed. After METTL16 knockdown, the IC50 values of Hep3B/DDP and Huh- 7/DDP cells were significantly decreased (Fig. 2B), while the expression of drug-resistant related proteins multidrug resistance protein 1 (MDR1) and multidrug resistance associated protein 1 (MRP1) were decreased (Fig. 2C-D), indicating that down-regulation of METTL16 could reduce the DDP resistance of cells. The proliferation capacity of the cells was subsequently examined, and the MTT results demonstrated that the cell viability of the si-METTL16 group was lower than that of the si-NC group (Fig. 2E), which was also illustrated by the EdU assay (Fig. 2F). However, silencing METTL16 induced apoptosis of drug-resistant cells (Fig. 2G). In addition, invasion ability and migration ability were both decreased after METTL16 expression was reduced (Fig. 2H-J). In conclusion, METTL16 promoted the biological progression and DDP resistance of HCC drug-resistant cells.

Fig. 2
figure 2

METTL16 facilitated progress and resistance of HCC resistant cells.A The interference efficiency of si-METTL16 in Hep3B/DDP, and Huh- 7/DDP cells were measured by western blot. B IC50 values of Hep3B/DDP and Huh- 7/DDP cells after interference of METTL16. C, D The expression of MDR1 and MRP1 in Hep3B/DDP and Huh- 7/DDP cells with si-METTL16 was analyzed by western blot. E MTT assay of cell viability in Hep3B/DDP and Huh- 7/DDP cells after interference of METTL16. F EdU staining of Hep3B/DDP and Huh- 7/DDP cells after interference of METTL16: EdU (red) staining, nucleus (blue) staining. G Flow cytometry assay of apoptosis in Hep3B/DDP and Huh- 7/DDP cells after interference of METTL16. H, I Transwell assay was used for cell invasion in Hep3B/DDP and Huh- 7/DDP cells after interference of METTL16. J Cell migration of cells was detected by wound healing in Hep3B/DDP and Huh- 7/DDP cells after interference of METTL16. *P < 0.05

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Silencing METTL16 weakened LAMA4 expression

Gene expression of LAMA4 was significantly declined with lacking of METTL16 in HCC cells as analyzed by GSE224008 database (Fig. 3A). RNA and protein assays confirmed this result (Fig. 3B-C). What’s more, TCGA and CPTAC databases predicted that LAMA4 was highly expressed in HCC (Fig. 3D-E). The ENCORI database and GEPIA database obtained the same results in HCC tissue samples (Fig. 3F-G). Furthermore, the expression of LAMA4 in HCC drug-resistant tissues was detected, and it was found that the expression of LAMA4 was increased in resistant group (Fig. 3H), and was positively correlated with the expression of METTL16 (Fig. 3I). Meanwhile, at the cellular level, the expression of LAMA4 showed the same trend as METTL16, that is, LAMA4 expression was higher in Hep3B/DDP and Huh- 7/DDP cells than that in Hep3B and Huh- 7 cells and THLE- 2 cells (Fig. 3J). Summarizing the above experiments, LAMA4 was regulated by METTL16 and was highly expressed in HCC drug-resistant tissues and cells.

Fig. 3
figure 3

METTL16 increased LAMA4 expression.A Heat map of gene expression profile in Huh- 7 after METTL16 knockdown from GSE224008. B qRT-PCR assay for LAMA4 with si-METTL16 in Hep3B/DDP and Huh- 7/DDP cells. C Western blot assay for LAMA4 after transfection of si-METTL16 in Hep3B/DDP and Huh- 7/DDP cells. D LAMA4 expression with 371 HCC tumor tissues and 50 normal tissues from TCGA database. E Protein expression of LAMA4 in HCC normal samples (Normal, n = 165) and tumor samples (Primary tumor, n = 165) from CPTAC database. F LAMA4 expression with 374 cancer and 50 normal samples in HCC from ENCORI database. G LAMA4 mRNA expression in tumor tissues (n = 369) and normal tissues (n = 160) of HCC samples by GEPIA database. H mRNA expression of LAMA4 in sensitive tissues (n = 29) and resistant tissues (n = 33). I Correlation assay of METTL16 and LAMA4. J Protein expression of LAMA4 in THLE- 2, Hep3B, Hep3B/DDP, Huh- 7 and Huh- 7/DDP cells. *P < 0.05

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METTL16 mediated m6A methylation of LAMA4

The presence of binding methylation modification sites in LAMA4 was predicted by the SRAMP website (Fig. 4A), and the ENCORI database displayed a positive correlation between METTL16 and LAMA4 (Fig. 4B). RIP analysis revealed that METTL16 was combined with LAMA4 (Fig. 4C). At the same time, the m6A methylation level of LAMA4 was lowered after METTL16 was knocked down (Fig. 4D-E). The cells were treated with Act D and si-METTL16 was found to reduce mRNA stability of LAMA4 (Fig. 4F-G). Hence, METTL16 was involved in m6A modification of LAMA4 and regulated the stability of LAMA4 mRNA.

Fig. 4
figure 4

METTL16 mediated m6A methylation of LAMA4.A The SRAMP website predicted the presence of m6A methylation modification sites in LAMA4. B The expression correlation between METTL16 and LAMA4 was analyzed in 374 HCC samples from ENCORI database. C RIP assay of LAMA4 and METTL16 in Hep3B/DDP and Huh- 7/DDP cells. D, E The m6A methylation level of LAMA4 in Hep3B/DDP and Huh- 7/DDP cells after METTL16 knockdown was determined by MeRIP. F, G After silencing METTL16, mRNA expression levels of LAMA4 after treatment with Act D at 0 h, 3 h, 6 h and 9 h in Hep3B/DDP and Huh- 7/DDP cells. *P < 0.05

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m6A methylation of LAMA4 was mediated by IGF2BP2, a methylated reading protein

IGF2BP2 is an m6A reader gene involved in RNA stability and translation [25]. Therefore, in this study, ENCORI database was used to predict that IGF2BP2 might interact with LAMA4 (Fig. 5A), and their expressions were positively correlated in HCC tissue samples (Fig. 5B). Furthermore, RIP results confirmed the binding between IGF2BP2 and LAMA4 at the cellular level, and after the absence of METTL16, the binding between the two was weakened, implied that IGF2BP2 mediated the m6A modification process of LAMA4 with the participation of METTL16 (Fig. 5C-D). In the meantime, the interference efficiency of the si-RNA about IGF2BP2 was detected (Fig. 5E), and LAMA4 expression was reduced by si-IGF2BP2 (Fig. 5F). Furthermore, the direct binding of IGF2BP2 to LAMA4 mRNA was confirmed by RNA pull down (Supplementary Fig. 2). Above, the m6A modification and expression of LAMA4 were controlled by IGF2BP2.

Fig. 5
figure 5

m6A methylation of LAMA4 was regulated by IGF2BP2.A ENCORI database predicted the possible interaction between IGF2BP2 and LAMA4. B Correlation analysis of LAMA4 and IGF2BP2 in 374 samples from ENCORI database. C, D RIP assay of IGF2BP2 enrichment level after interference with METTL16 in Hep3B/DDP and Huh- 7/DDP cells. E Protein expression level of IGF2BP2 after transfection with si-IGF2BP2 in Hep3B/DDP and Huh- 7/DDP cells. F mRNA expression levels of LAMA4 after transfection with si-IGF2BP2 in Hep3B/DDP and Huh- 7/DDP cells. *P < 0.05

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METTL16 affected the biological function of HCC drug-resistant cells through LAMA4

For better explaining the role between METTL16 and LAMA4, the overexpression vector of LAMA4 was transfected into HCC drug-resistant cells and its efficiency was measured (Fig. 6A). si-METTL16 reduced the IC50 values in HCC drug-resistant cells, while LAMA4 overexpression abolished this effect (Fig. 6B). The suppression of si-METTL16 on MDR1 and MRP1 was also abrogated by LAMA4 (Fig. 6C-D). These results indicated that LAMA4 could alleviate the decline in DDP resistance caused by down-regulation of METTL16. Then the functional research experiments were carried out. Consistent results were obtained by MTT assay and EdU staining, namely, the inhibitory effect of METTL16 decline on cell proliferation was reversed under LAMA4 (Fig. 6E-F), while the promoting effect of METTL16 knockdown on apoptosis was blocked by LAMA4 (Fig. 6G-H). Besides, transfection of LAMA4 overexpressed vectors reversed the repression of METTL16 si-RNA on invasion and migration in Hep3B/DDP and Huh- 7/DDP cells (Fig. 6I-J). So METTL16 affected the expression of LAMA4 and thus regulated the biological function of HCC drug-resistant cells.

Fig. 6
figure 6

METTL16 affected the biological function of HCC drug-resistant cells through LAMA4.A Western blot assay of LAMA overexpression efficiency. B IC50 values of Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-METTL16, si-METTL16 + vector, si-METTL16 + LAMA4. C, D The expression of MDR1 and MRP1 in Hep3B/DDP and Huh- 7/DDP cells with si-NC, si-METTL16, si-METTL16 + vector, si-METTL16 + LAMA4 was analyzed by western blot. E MTT assay of cell viability in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-METTL16, si-METTL16 + vector, si-METTL16 + LAMA4. F EdU assay of cell proliferation in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-METTL16, si-METTL16 + vector, si-METTL16 + LAMA4. G, H Flow cytometry assay of apoptosis in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-METTL16, si-METTL16 + vector, si-METTL16 + LAMA4. I Transwell assay was used for cell invasion in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-METTL16, si-METTL16 + vector, si-METTL16 + LAMA4. J Cell migration of cells was detected by wound healing in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-METTL16, si-METTL16 + vector, si-METTL16 + LAMA4. *P < 0.05

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LAMA4 deletion impeded COL4A1 expression

LAMA4-associated proteins were screened using Genemania, STRING and Coexpedia databases (Fig. 7A), among which only COL4A1 had been studied in HCC, and COL4A1 was up-regulated in HCC and promoted malignant progression [26]. Hence, the interaction between LAMA4 and COL4A1 was studied. The TCGA and CPTAC databases predicted the high expression of COL4A1 in HCC tissue samples (Fig. 7B-C), while the same result was also forecasted by ENCORI database and GEPIA database (Fig. 7D-E), and LAMA4 was positively correlated with COL4A1 expression (Fig. 7F). Moreover, the effect on COL4A1 protein expression was measured by transfection of si-LAMA4 into cells (Fig. 7G). Compared with si-NC group, the expression of COL4A1 was abated in si-LAMA4 group (Fig. 7H). Co-IP further verified the interaction between LAMA4 and COL4A1 (Fig. 7I). Meanwhile, the COL4A1 and IGF2BP2 were up-regulated in HCC drug-resistant tissues, and the expression of COL4A1 was positively correlated with METTL16 and IGF2BP2 (Supplementary Fig. 3). Besides, by treating the HCC drug-resistant cells with cycloheximide (CHX), we found that LAMA4 promoted the stability of COL4A1 (Supplementary Fig. 4). As a result, COL4A1 could be used as a down-stream target for LAMA4 to participate in regulation.

Fig. 7
figure 7

LAMA4 induced COL4A1 expression.A Venn diagram of LAMA4 interacting proteins was analyzed by Genemania, STRING and Coexpedia databases. B COL4A1 expression with 371 HCC tumor tissues and 50 normal tissues from TCGA database. C Protein expression of COL4A1 in HCC normal samples (Normal, n = 165) and tumor samples (Primary tumor, n = 165) from CPTAC database. D COL4A1 expression with 374 cancer and 50 normal samples in HCC from ENCORI database. E COL4A1 mRNA expression in tumor tissues (n = 369) and normal tissues (n = 160) of HCC samples from GEPIA database. F Correlation analysis of LAMA4 and COL4A1 in 374 samples from ENCORI database. G Western blot analysis of LAMA interference efficiency in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-LAMA4. H The protein expression level of COL4A1 after interference with LAMA4 in Hep3B/DDP and Huh- 7/DDP cells. I Co-IP assay of LAMA4 and COL4A1 in Hep3B/DDP and Huh- 7/DDP cells. *P < 0.05

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LAMA4 induced DDP resistance and HCC drug-resistant cell progression via COL4A1

We examined the overexpression efficiency after overexpression of COL4A1 to further study the role of LAMA4 and COL4A1 in HCC drug-resistant cells (Fig. 8A). Overexpression of COL4A1 reversed the DDP resistance inhibition of LAMA4 interference in cells (Fig. 8B-D). Meanwhile, the inhibitory effect of si-LAMA4 on cell proliferation activity was retarded by COL4A1 (Fig. 8E-F), and the promotional effect on apoptosis was also mitigated (Fig. 8G-H). In addition, the retardation of invasion and migration by lacking of LAMA4 was reversed after transfection of COL4A1 overexpressing vectors (Fig. 8I-J). Taking all the above results into consideration, LAMA4 affected the biological function of HCC drug-resistant cells through COL4A1.

Fig. 8
figure 8

LAMA4 promoted the biological function via COL4A1.A Western blot analysis of COL4A1 overexpression efficiency in Hep3B/DDP and Huh- 7/DDP cells. B IC50 values of Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-LAMA4, si-LAMA4 + vector, si-LAMA4 + COL4A1. C, D The expression of MDR1 and MRP1 in Hep3B/DDP and Huh- 7/DDP cells with si-NC, si-LAMA4, si-LAMA4 + vector, si-LAMA4 + COL4A1 was analyzed by western blot. E MTT assay of cell viability in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-LAMA4, si-LAMA4 + vector, si-LAMA4 + COL4A1. F EdU assay of cell proliferation in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-LAMA4, si-LAMA4 + vector, si-LAMA4 + COL4A1. G, H Flow cytometry assay of apoptosis in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-LAMA4, si-LAMA4 + vector, si-LAMA4 + COL4A1. I Transwell assay was used for cell invasion in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-LAMA4, si-LAMA4 + vector, si-LAMA4 + COL4A1. J Cell migration of cells was detected by wound healing in Hep3B/DDP and Huh- 7/DDP cells after transfection with si-NC, si-LAMA4, si-LAMA4 + vector, si-LAMA4 + COL4A1. *P < 0.05

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Knocking down METTL16 in vivo suppressed tumor growth and DDP resistance

To verify the function of METTL16 in vivo, subcutaneous tumor models in nude mice were established using lentiviral vectors (Fig. 9A). In vivo, sh-MATTL16 reduced the volume and weight of tumors, this reduction was most significant in sh-METTL16 + DDP group, followed by sh-METTL16 + PBS group, then sh-NC + DDP group, and finally sh-NC + PBS group (Fig. 9B-C). However, we believed that the reason the sh-METTL16 + DDP group showed the lowest tumor volume and weight was that DDP itself inhibited tumor progression, which might be related to other proliferation-related and metastasis-related indicators such as Ki67 and MMP9 (Supplementary Fig. 5). Knockdown of MALLT16 down-regulated the expression of LAMA4 and COL4A1 (Fig. 9D-F), and immunohistochemistry showed the same results (Fig. 9G). Meanwhile, the inhibitory effects of METTL16 knockdown on tumor growth were weakened by LAMA4 up-regulation (Supplementary Fig. 6). These results indicated that METTL16 acted in vivo through the same mechanism as in vitro.

Fig. 9
figure 9

METTL16 Knockdown in vivo suppressed tumor growth A Western blot analysis of overexpression efficiency of sh-METTL16 in Huh- 7/DDP cells. B Tumor volume statistics at 8 Day, 12 Days, 16 Days, 20 Days, 24 Days, and 28 Days after treatment with sh-NC + PBS, sh-NC + DDP, sh-METTL16 + PBS, sh-METTL16 + DDP. C Tumor weight analysis after treatment with sh-NC + PBS, sh-NC + DDP, sh-METTL16 + PBS, sh-METTL16 + DDP. D Western blot analysis of METTL16 in tumor tissues after treatment with sh-NC + PBS, sh-NC + DDP, sh-METTL16 + PBS, sh-METTL16 + DDP. E Western blot analysis of LAMA4 in tumor tissues after treatment with sh-NC + PBS, sh-NC + DDP, sh-METTL16 + PBS, sh-METTL16 + DDP. F Western blot analysis of COL4A1 in tumor tissues after treatment with sh-NC + PBS, sh-NC + DDP, sh-METTL16 + PBS, sh-METTL16 + DDP. G The expressions of METTL16, LAMA4, and COL4A1 were detected by IHC after treatment with sh-NC + PBS, sh-NC + DDP, sh-METTL16 + PBS, sh-METTL16 + DDP. *P < 0.05

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Discussion

Although great progress has been made in improving cancer survival, there are still risks of recurrence, metastasis and drug resistance during treatment [27]. HCC accounts for the majority of primary liver cancers and is one of the most usual causes of cancer death [1]. Therefore, it is greatly significant to reduce the recurrence rate and heterogeneity of liver cancer and provide effective treatment strategies.

Previous study had shown that loss of METTL16 was associated with poor overall survival of HCC patients and with activation of multiple metabolic pathways in HCC [24]. Based on spatial transcriptome data [28], Xue et al.. reported that METTL16 was rose in HCC tumors, and by constructing liver-specific knockout mice for METTL16, it was observed that the deletion of METTL16 inhibited the formation and growth of HCC tumors [29]. In addition, METTL16 promoted the formation of HCC tumors [30]. These studies suggested that METTL16 might be a target for the treatment and prognosis of HCC. Through the analysis of HCC data from multiple databases, the present study observed the same results as previous studies, namely, METTL16 in HCC tumor tissues was higher than that in non-tumor tissues. Similarly, METTL16 was elevated in HCC drug-resistant tissues and cells. Researchers found that METTL16 was overexpressed in colorectal cancer [31], lung cancer [32], gastric cancer [19], breast cancer [33] and cholangiocarcinoma [34], and accelerated tumorigenesis. METTL16 also restrained drug resistance in bladder cancer [35]. It was found that interference with METTL16 enhanced the sensitivity of drug-resistant cell lines, impeded proliferation, invasion and migration, and induced apoptosis.

Literature study had shown that LAMA4 was significantly overexpressed in HCC and was associated with tumor metastasis and invasion [36]. Through bioinformatics analysis, LAMA4 could be used as a potential target of miRNA in liver cancer [37], and was associated with notch mediated HCC [38]. Moreover, LAMA4 was also associated with DDP resistance in gastric cancer [39]. Besides, LAMA4 also expedited drug resistance of cancers [40, 41]. Therewith, we mined the GSE224008 database to obtain the gene expression profile in HCC cells after METTL16 knockdown, and analyzed the top 10 differentially expressed genes, and observed that LAMA4 was the most significantly reduced with lacking of METTL16, which illustrated that the interaction might exist between METTL16 and LAMA4. Furthermore, LAMA4 was up-regulated in HCC drug-resistant tissues and cells from databases analysis and experimental results, which was consistent with previously reported results [36,37,38], and the expression of LAMA4 was positively correlated with METTL16. This further illustrated a possible interaction between METTL16 and LAMA4. However, whether METTL16 mediated the m6A methylation modification of LAMA4 has not been reported. Therefore, we predicted the existence of methylation modification sites of LAMA4 through the website, and verified the relationship between METTL16 and LAMA4 by using MeRIP and RIP, that is, knocking down METTL16 weakened the methylation of LAMA4 and reduced the mRNA stability of LAMA4.

As an m6A reader, IGF2BP2 was involved in cancer development by regulating transcription of miRNAs, lncRNAs, and other m6A related genes [42,43,44], and was associated with cancer prognosis. Both METTL3 and METTL14 were able to regulate mRNA m6A modification through an IGF2BP2-dependent mechanism [45,46,47,48,49]. So we hypothesized that METTL16 could also mediate LAMA4 methylation in this way. The correlation between IGF2BP2 and LAMA4 was predicted by the database, and the combination of the two was also shown by the RIP results. Meanwhile, knocking down IGF2BP2 inhibited LAMA4 expression. These results suggested that IGF2BP2 mediated m6A methylation of LAMA4. In addition, overexpression of LAMA4 reversed the inhibitory effect of METTL16 knockdown on HCC drug-resistant cells. This explained that METTL16 and IGF2BP2 co-mediated the m6A methylation of LAMA4, thereby contributing to HCC drug-resistant cells.

We further explored the molecular mechanism by which METTL16/IGF2BP2 was involved in HCC development by influencing LAMA4. Genemania, STRING and Coexpedia databases indicated that LAMA4 could interact with COL4A1. Bioinformatics analysis showed that COL4A1 was significantly correlated with the occurrence and prognosis of liver cancer [50], and its expression was up-regulated in HCC, at the same time, LAMA4 accelerated the growth and metastasis of HCC cells [26, 51]. As a tumor suppressor in pancreatic cancer, SIRT7 inhibited epithelial mesenchymal transformation (EMT) of pancreatic cancer cells by transcriptionally inhibiting the expression of COL4A1 [52]. In gastric cancer cells, COL4A1 blocked Hedgehog signaling to inhibit the aggressive phenotypes [53]. COL4A1 accelerated OSCC cell proliferation, migration, and EMT progression by binding to NID1 [54]. Additionally, Zhang et al.. reported that COL4A1 was negatively regulated by the XPD-miR- 29a- 3p axis and promoted HCC progression in vitro [51]. COL4A1 also facilitated HCC growth and metastasis by activating FAK-Src signaling Therefore, we speculated that LAMA4 could further affect the regulation of drug resistance of HCC by COL4A1. Consistent with previous results [26, 50, 51], database analysis revealed that COL4A1 was overexpressed in HCC tissues, and was positively relevant with LAMA4 expression, and Co-IP confirmed the interaction between the two, while silencing LAMA4 inhibited COL4A1 expression. In addition, recovery experiments were carried out, and overexpression of COL4A1 rescued the inhibition of HCC drug-resistant cells caused by LAMA4 knockdown, which manifested that LAMA4 functioned in HCC resistant cells through COL4A1. The mice experiment further explored the mechanism of METTL16 in vivo on tumor growth, and the results were consistent with the in vitro experiments, that is, deficiency of METTL16 hindered tumor growth and impeded the expression of LAMA4 and COL4A1.

To sum up, METTL16/IGF2BP2 mediated m6A methylation modification of LAMA4, which in turn affected COL4A1, thereby facilitating DDP resistance, cell proliferation, migration and invasion and blocking apoptosis in vitro, and stimulating tumor growth in vivo (Fig. 10). Therefore, the research concluded that METTL16 might be a therapeutic target for liver cancer and provided a certain reference for clinical diagnosis.

Fig. 10
figure 10

METTL16/IGF2BP2 influences the expression of COL4A1 by mediating m6A methylation modification of LAMA4, thereby regulating the proliferation and metastasis of HCC and DDP resistance

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Materials and methods

Data sources and bioinformatics analysis

Gene expression in HCC tissues were screened in Clinical Proteomic Tumor Analysis Consortium (CPTAC), Encyclopedia of RNA Interactomes (ENCORI), The Cancer Genome Atlas Program (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) databases. Download gene expression profile of GSE224008 from the Gene Expression Omnibus (GEO) public database. SRAMP website (http://www.cuilab.cn/sramp/) was used for Methylation sites prediction. Intergenic correlations were predicted by ENCORI database. In addition, Genemania (http://genemania.org/), STRING (https://cn.string-db.org/) and Coexpedia (https://www.coexpedia.org/) protein interaction databases were used to analyze interacting proteins.

Human samples

Cisplatin (DDP) sensitive HCC tissue samples (Sensitive, n = 29) and DDP resistant HCC tissue samples (Resistant, n = 33) were collected from the Second Hospital of Hebei Medical University. The process was authorized by the Ethics Committee of the Second Hospital of Hebei Medical University, and was taken with the informed consent of both the patients and their family.

Quantitative real-time polymerase chain reaction (qRT-PCR)

TRIzol solution (Invitrogen, Carlsbad, CA, USA) was used for extracting total RNA, and then the total RNA was reverted to cDNA by PrimeScript™ RT reagent Kit (Takara, Osaka, Japan), finally, quantitatively analyzed using Systemic Risk and Resilience (SYBR, Takara). The primers used in this experiment were shown in Table 2.

Table 2 Primers used for gene expression analysis
Full size table

Western blot

After protein extraction, the target bands were separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane and sealed at 37℃ for 2 h. Then the protein bands were incubated at 4℃ overnight for primary antibodies and were incubated at 37℃ of 2 h for secondary antibodies, and finally imaging was performed. The primary antibodies were used in this assay as followed: anti-METTL16 (1:1000, ab252420, Abcam, Cambridge, UK), anti-MDR1 (1:1000, ab170904, Abcam), anti-MRP1 (1:1000, ab233383, Abcam), anti-LAMA4 (1:200, sc- 130540, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-IGF2BP2 (1:2000, ab124930, Abcam), anti-COL4A1 (1:200, sc- 59814, Santa Cruz Biotechnology), anti-GAPDH (1:5000, ab8245, Abcam). The secondary antibodies were used in this assay as followed: anti-rabbit (1:5000, ab6721; Abcam) and anti-mouse (1:5000, ab6728; Abcam).

Cell culture

Human immortalized epithelial cell THLE- 2, human HCC cell lines Hep3B and Huh- 7 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. All cells were cultured in DMEM medium (Hyclone, Logan, Utah, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), in which penicillin (Hyclone) and streptomycin (Hyclone) were added. The cells were cultured in a constant temperature incubator (Thermo Fisher Scientific, Waltham, MA, USA), which at 37℃ and 5% CO2. The next day, the cell adhesion was observed and the fluid was changed. HCC cells with good adherent growth were digested by 0.25% trypsin (Gibco), and logarithmic growth cells were selected for the experiment.

Establishment of drug-resistant cell lines

DDP resistant HCC cell lines were established by gradual increase of drug concentration and continuous induction. Hep3B and Huh- 7 cells at logarithmic growth stage were replaced with fresh culture medium, and DDP (Yeasen Biotechnology, Shanghai, China) was added to make their acting concentration 100 ng/mL. The culture was continued, and the drug concentration of 100 ng/mL was maintained until the viable cells could grow and pass at this concentration stably. After appropriately increasing the DDP concentration every week, cells continued to culture, timely changed the fluid, and maintained the drug concentration until the survival cells could grow and pass at this concentration stably. The increase of DDP concentration in each stage was controlled within 200–500 ng/mL. Five months later, stable drug-resistant cell line Hep3B/DDP and Huh- 7/DDP were obtained. All the experiments were carried out with cells in logarithmic growth phase after 2 weeks of DDP discontinuation.

Cell drug sensitivity test

Hep3B, Hep3B/DDP, Huh- 7 and Huh- 7/DDP cells were inoculated into 96-well culture plates (Corning, Lowell, NY, USA), with 5 × 104 cells/mL. After 24 h culture in a constant temperature incubator, the dosing group was changed to complete culture medium containing different concentrations of drugs for further culture. After the treatment of the drug group for 24 h, 3-(4,5-Dimethyl- 2-Thiazolyl)− 2,5-Diphenyl Tetrazolium Bromide (MTT) was added, and the culture was continued for 4 h. The culture solution was carefully absorbed and abandoned, dimethyl sulfoxide (DMSO) was added, and the color was fully dissolved and oscillated for 10 min. The absorbance value (A490) was determined by a full-wavelength microplate reader (Thermo Fisher Scientific). The calculation formula was below: Growth inhibition rate (%) = (1-drug group A490/control group A490) × 100%. According to the growth inhibition rate, the drug half-inhibitory concentration (IC50) was calculated.

Interference and overexpression vectors construction and transfection

Small interfering (si)-METTL16, si-IGF2BP2, si-LAMA4, short hairpin (sh)-METTL16, LAMA4 overexpression vector (LAMA4), COL4A1 overexpression vector (COL4A1) and corresponding negative control (NC) vectors (si-NC, sh-NC, and vector) were synthesized by GENERAL BIOL (GENERAL BIOL, Anhui, China). All transfection procedures were performed using Lipofectamine™ reagent (Invitrogen). Briefly, According to the instructions, the LipofectamineTM reagent and the siRNAs or plasmids were mixed separately with the serum-free medium, and then the two mixtures were mixed. After standing at room temperature for 15 min, the configured transfection mixture was added to the cells of the corresponding treatment groups, and gently shaken evenly. After culture in the incubator, the cells were detected based on different experimental needs.

Cell viability assay

The cells were inoculated into 96-well plates (Corning) with 1 × 104 cells per well, and MTT solution was added to the plates (Corning). The cells were incubated for 4 h, then stopped the culture, carefully absorbed and discarded the culture supernatant, added DMSO to each well, and shook for 10 min to completely melt the crystals. The light absorption values were detected on the microplate reader (Thermo Fisher Scientific), and the results were recorded.

5-ethynyl-2′-deoxyuridine (EdU)

EdU staining was performed according to the BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China). Cells were cultured in a 6-well plate (Corning). 2× EdU working solution was prepared and preheated at 37℃, and the same volume with cells was added to the 6-well plate (Corning), so that the EdU concentration became 1×, and the cells were incubated for 2 h. Removed the culture solution, added fixing solution, and fixed at 37℃ for 15 min. Removed the fixing solution and incubated of permeable solution at 37℃ for 10–15 min. 1×Hoechst was nucleated and cells was incubated at 37℃ for 10 min away from light. Fluorescence detection could then be performed.

Apoptosis detection

According to the Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen Biotechnology), the cells were added with buffers successively, and then apoptosis was detected by flow cytometry (Thermo Fisher Scientific).

Transwell assay

Transwell cells were implanted in the upper chamber with serum-free medium and the upper chamber was pre-coated with matrigel (Corning), and the lower champer was added with medium containing 20% FBS (Gibco). After 48 h, the invasive cells were fixed with 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet for 20 min. Pictures were taken on the microscope (Thermo Fisher Scientific) and counted the number of cells penetrating to the submembrane.

Wound healing assay

The density of 2 × 105 cells per well were inoculated into 6-well plates (Corning) with and cultured in an incubator (Thermo Fisher Scientific). After the cells were evenly overgrown, the plates were removed, and the cells were crossed out with a sterile gun tip of the plates. The media in the holes were discarded, and continued to be cultured in the incubator (Thermo Fisher Scientific). Pictures were taken at fixed point under microscope (Thermo Fisher Scientific) at 0 h and 24 h after scratch.

Methylated RNA Immunoprecipitation (MeRIP)

According to the riboMeRIP™ m6A Transcriptome Profiling Kit (RiboBio, Guangzhou, China), DNase I digesters purified mRNA, which was then cleaved with RNA splitting reagents and incubated at 94℃ After crushing, standard ethanol was used to precipitate and collect. Anti-m6A (1:50, ab208577, Abcam) was pre-incubated at room temperature in IP buffer with magnetic beads for 1 h. Then, the fragment mRNA was added to the antibody magnetic bead mixture and incubated at 4℃ for 4 h. After digestion with protease K, RNA was extracted and qPCR analysis was performed.

RNA Immunoprecipitation (RIP)

According to the instruction of Magna RIP Kit (Sigma, St. Louis, MI, USA), anti-METTL16 (1:30, ab252420, Abcam), anti-m6A (1:50, ab208577, Abcam) and anti-rabbit IgG (1:50, 5415, Cell Signaling Technology, Danvers, MA, USA) were incubated with magnetic beads and then added to cell lysates. The RNA-protein IP complex was then washed and incubated with a protease K digestion buffer. Finally, RNA was extracted and qRT-PCR was performed.

mRNA stability detection

Cells were treated with si-METTL16, and then treated with actinomycin D (Act D, Sigma) for 0 h, 3 h, 6 h, and 9 h. RNA was extracted and mRNA levels were detected by qRT-PCR.

RNA pull down.

The RNA pull down was conducted according to the RNA-Protein Pull Down Kit (Sangon, Shanghai, China). Briefly, the cell pellet was collected and added with IP Lysis buffer and Protease inhibitor. After cracking in ice for 30 min, the samples were crushed by ultrasonic for 5 min. Following centrifugation at 4℃ 12,000 rpm for 10 min, the supernatant was collected for the follow-up experiments. The Nucleic-Acid Compatible Streptavidin Magnetic Beads were washed with Wash Buffer I and added with 1×RNA binding buffer to re-suspend the magnetic beads. Then, the Biotin-labeled probes were added to magnetic beads and incubated at room temperature for 2 h to bind the magnetic beads to RNA. Followed by the combination of RNA-magnetic bead complex and protein, the samples were washed with Wash Buffer I and added with Elution Buffer to boil for 10 min. Finally, the samples were added with 6×Loading Buffer and mixed for western blot assay.

Co-immunoprecipitation (Co-IP)

Protein samples were collected, the decoy protein was labeled with Flag, the protein was precipitated by anti-Flag (1:20000, 80010 - 1-RR, Proteintech, Wuhan, China), the protein complex was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then western blot was detected.

Mice model construction

The mice experiments of this research were reviewed and approved by the Animal Ethics Committee of the Second Hospital of Hebei Medical University. BALB/c-nude mice (male, 4 weeks old) were obtained from Vital River (Vital River, Beijing, China). 8 × 105 Huh- 7 cells transfected with lentivirus vectors containing sh-NC or sh-METTL16 were suspended in 1 mL PBS and inoculated subcutaneously into the axilla of nude mice. The next day, the two groups of mice were intraperitoneally injected with PBS or DDP (20 mg/kg), respectively. Finally, the mice were divided into four groups: sh-NC + PBS group, sh-NC + DDP group, sh-METTL16 + PBS group, and sh-METTL16 + DDP group, and 5 mice per group. After tumor formation in nude mice, the changes of tumor volumes were counted at 8 Days, 12 Days, 16 Days, 20 Days, 24 Days, and 28 Days, and the mice were killed and the tumor tissues were collected on day 28 for weighing and follow-up experiments.

Immunohistochemistry (IHC)

After xylene dewaxing, ethanol gradient hydration and citric acid repair, tissue sections were incubated with hydrogen peroxide for 10 min, closed at 37℃ for 30 min, then the tissue sections were incubated with primary antibodies at 4℃ overnight, and followed by secondary antibodies (Goat anti-Rabbit IgG H&L, ab6721, Abcam; Goat Anti-Mouse IgG H&L, ab6789, Abcam) at 37℃ for 20 min, color was developed by 3,3’-diaminobenzidine (DAB), hematoxylin was retained, the tablets were finally sealed and photographed under the microscope (Thermo Fisher Scientific).

Data analysis

All data in this study were analyzed by GraphPad Prism 8.4.3. The mean ± SEM deviation was used to process the data, and Student’s t test and one-way ANOVA were used for analyzing the differences between the data, n = 3. P < 0.05 means significant difference.

Data availability

No datasets were generated or analysed during the current study.

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Funding

This study was supported by the Health Research Project of Hebei Province (No.20242387).

Author information

Authors and Affiliations

  1. Department of General Surgery, The Second Hospital of Hebei Medical University, No. 215, Heping West Road, Shijiazhuang, 050000, China

    Liming Cao & Wei Bi

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  1. Liming Cao
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Contributions

L.C. conducted the experiment, drafted the manuscript, prepared figures, collected and analyzed the data. W.B. designed and supervised the study. All authors reviewed the manuscript.

Corresponding author

Correspondence to Wei Bi.

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Conflict of interest

The authors report no conflicts of interest in this work.

Ethical approval

The process was authorized by the Ethics Committee of the Second Hospital of Hebei Medical University, and was taken with the informed consent of both the patients and their family. The mice experiments of this research were reviewed and approved by the Animal Ethics Committee of the Second Hospital of Hebei Medical University.

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The written informed consents were gotten from all members.

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Supplementary Information

The correlation between METTL16 expression and overall survival of patients. Supplementary material 1

13008_2025_152_MOESM2_ESM.tif

The direct binding between IGF2BP2 and LAMA4 was confirmed by RNA pull down in Hep3B/DDP and Huh- 7/DDP cells. Supplementary material 2

13008_2025_152_MOESM3_ESM.tif

The correlation between genes (A) mRNA expression of COL4A1 in sensitive tissues (n = 29) and resistant tissues (n = 33). (B) Correlation assay of COL4A1 and METTL16. (C) mRNA expression of IGF2BP2 in sensitive tissues (n = 29) and resistant tissues (n = 33). (D) Correlation assay of IGF2BP2 and COL4A1. *P < 0.05. Supplementary material 3

13008_2025_152_MOESM4_ESM.tif

LAMA4 bound COL4A1 for stabilizing COL4A1 (A) Western blot analysis of COL4A1 expression in Hep3B/DDP cells treated with CHX for 0, 5, 10, or 20 h and si-NC or si-LAMA4. (B) Western blot analysis of COL4A1 expression in Huh- 7/DDP cells treated with CHX for 0, 5, 10, or 20 h and si-NC or si-LAMA4. Supplementary material 4

13008_2025_152_MOESM5_ESM.tif

Western blot analysis of Ki67 and MMP9 expression in mouse tumor tissues of sh-NC + PBS, sh-NC + DDP, sh-METTL16 + PBS, and sh-METTL16 + DDP groups *P< 0.05. Supplementary material 5

13008_2025_152_MOESM6_ESM.tif

LAMA4 reversed the inhibitory effects of sh-METTL16 on tumor growth (A) Tumor volume statistics at 9 Day, 14 Days, 19 Days, 24 Days, and 29 Days after treatment with sh-NC, sh-METTL16, sh-METTL16 + OE-LAMA4. (B) Tumor weight analysis after treatment with sh-NC, sh-METTL16, sh-METTL16 + OE-LAMA4. (C) Western blot analysis of LAMA4 in tumor tissues after treatment with sh-NC, sh-METTL16, sh-METTL16 + OE-LAMA4. *P < 0.05. Supplementary material 6

Supplementary material 7

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Cao, L., Bi, W. METTL16/IGF2BP2 axis enhances malignant progression and DDP resistance through up-regulating COL4A1 by mediating the m6A methylation modification of LAMA4 in hepatocellular carcinoma. Cell Div 20, 9 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13008-025-00152-2

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13008-025-00152-2

Keywords

Cell Division

ISSN: 1747-1028

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