AIFM2 expression is markedly upregulated in HCC and its upregulation is associated with poor patient survival

We first determined the expression of AIFM2 in HCC using the online UALCAN database [9]. A significant upregulation of AIFM2 at both mRNA and protein levels was observed in tumor tissues of HCC as compared with normal liver tissues (Fig. 1A, B). Upregulation of AIFM2 was further confirmed at mRNA level by quantitative reverse transcription PCR (qRT-PCR) analysis in 30-paired tumor and adjacent non-tumor tissue samples (Fig. 1C) and at protein level by immunohistochemistry (IHC) analysis in another cohort of 213-paired tumor and adjacent non-tumor tissue samples (Fig. 1D). Correlation analysis indicated a significant positive correlation between the expression of AIFM2 and clinicopathological feature of tumor metastasis (Table S3), implying that AIFM2 may play an oncogenic role in HCC progression. Kaplan–Meier survival analysis indicated that HCC patients with high AIFM2 expression had a worse overall survival and higher recurrence than patients with low AIFM2 expression (Fig. 1E, F). Similarly, survival analysis using the online UALCAN also revealed that upregulation of AIFM2 is associated with poor survival of HCC patients (Fig. 1G). Moreover, in line with the expression of AIFM2 in tumor tissues of HCC, a significant increase in AIFM2 expression was also observed in HCC cell lines as compared to normal hepatocytes by using qRT-PCR and western blot analysis (Fig. 1H, I).

Fig. 1: AIFM2 expression is markedly upregulated in HCC and its upregulation is associated with poor patient survival.

A and B The expression of AIFM2 in HCC was assessed using the online UALCAN database at mRNA (A) and protein (B) levels. C AIFM2 expression was determined by qRT-PCR analysis in 30-paired tumor and adjacent non-tumor tissue samples. D AIFM2 expression was determined by immunohistochemistry (IHC) analysis in another cohort of 213-paired tumor and adjacent non-tumor tissue samples. Scale bars, 20 μm. E and F Kaplan–Meier curves of overall survival (E) and recurrence-free survival (F) in different AIFM2 expression groups. G Kaplan–Meier survival analysis in different AIFM2 expression groups using the online UALCAN database. H and I qRT-PCR and western blot analysis for AIFM2 expression in HCC and normal hepatocyte cell lines.

Additionally, the expression of AFIM2 was also analyzed in several other human cancer types using the online database Sangerbox (http://vip.sangerbox.com/). Similar to HCC, the expressions of AIFM2 are also significantly increased in LUAD (Lung adenocarcinoma), STES (Stomach and Esophageal carcinoma), KIRP (Kidney renal papillary cell carcinoma), STAD (Stomach adenocarcinoma), UCEC (Uterine Corpus Endometrial Carcinoma), KICH (Kidney Chromophobe) and CHOL (Cholangiocarcinoma), indicating that AIFM2 may play a crucial oncogenic role in multiple human cancer types.

Knockdown of AIFM2 suppressed metastasis of HCC cells in vitro and in vivo

Significant upregulation of AIFM2 promoted us to hypothesize that AIFM2 may function as an oncogene in HCC. To determine the functions of AIFM2 in HCC cells, AIFM2 was knocked down in SNU-423 and HLF cells expressing high levels of AIFM2 (as indicated in Fig. 1H, I). Strongly downregulation of AIFM2 in SNU-423 and HLF cells was observed upon transfection with siRNAs targeting AIFM2 (Fig. 2A, B). MTS cell viability and colony formation assays indicated that AIFM2 knockdown had no significant effect on both short- and long-term cell proliferation of HCC cells (Fig. 2C, D). Similarly, no notable changes in cell apoptosis and cell cycle were also observed upon AIFM2 knockdown in SNU-423 and HLF cells (Fig. 2E, F). Next, we determined the effect of AIFM2 knockdown on cell migration and invasion in SNU-423 and HLF cells. The results showed that AIFM2 knockdown led to markedly decreased cell migration and invasion (Fig. 2G, H), as evidenced by transwell migration and invasion assays. To validate the effect of AIFM2 on the metastasis of HCC cells in vivo, AIFM2 stable knockdown SNU-423 cells (Fig. S2A and S2B) were constructed and intravenously injected into nude mice (6 mice per group) through the tail vein. In concordance with the in vitro results, the number of lung metastases was also significantly lower in the AIFM2 knockdown group as compared with the control group (Fig. 2I). Together, these results suggest that AIFM2 plays a crucial role in the promotion of HCC metastasis.

Fig. 2: Knockdown of AIFM2 suppressed metastasis of HCC cells in vitro and in vivo.

A and B Downregulation of AIFM2 was tested in SNU-423 and HLF cells upon transfection with siRNAs targeting AIFM2 by qRT-PCR and western blot analysis. C and D Short- and long-term cell proliferation was determined by MTS cell viability (C) and colony formation (D) assays in SNU-423 and HLF cells upon transfection with siRNAs targeting AIFM2. E and F Cell apoptosis and cell cycle were analyzed by flow cytometry in SNU-423 and HLF cells upon transfection with siRNAs targeting AIFM2. G and H Transwell migration and invasion assays in SNU-423 and HLF cells upon transfection with siRNAs targeting AIFM2. I Number of lung metastases was evaluated by Hematoxylin-Eosin staining in nude mice injected with AIFM2 knockdown or control SNU-423 cells (6 mice per group). Scale bars, 20 μm.

Forced expression of AIFM2-promoted metastasis of HCC cells in vitro and in vivo

To provide more evidence for the promotive function of AIFM2 on HCC metastasis, we next overexpressed AIFM2 in SNU-449 and Hep3B cells expressing low levels of AIFM2 (as indicated in Fig. 1H, I). Strongly overexpression of AIFM2 was confirmed by qRT-PCR and upon transfection with AIFM2 expression vector by qRT-PCR and western blot analysis in SNU-449 and Hep3B cells (Fig. 3A, B). Both transwell migration and invasion assays showed that forced expression of AIFM2 significantly enhanced the migration and invasion abilities of SNU-449 and Hep3B cells (Fig. 3C, D). Additionally, forced expression of AIFM2 (Fig. S2C, S2D) also markedly elevated the in vivo metastasis of SNU-449 cells, as indicated by the increased number of lung metastases in nude mice injected with AIFM2 overexpression SNU-449 cells, as compared to those injected with control SNU-449 cells (Fig. 3E).

Fig. 3: Forced expression of AIFM2-promoted metastasis of HCC cells in vitro and in vivo.

A and B Overexpression of AIFM2 was tested in SNU-449 and Hep3B cells upon transfection with AIFM2 expression vector by qRT-PCR and western blot analysis. C and D Transwell migration and invasion assays in SNU-449 and Hep3B cells upon transfection with AIFM2 expression vector. E Number of lung metastases was evaluated by Hematoxylin-Eosin staining in nude mice injected with AIFM2 overexpression or control SNU-449 cells (6 mice per group). Scale bars, 20 μm.

Upregulation of AIFM2 is mainly caused by DNA hypomethylation and decreased miR-150-5p expression

Upregulation of AIFM2 in HCC at both mRNA and protein levels suggests that its upregulation may occur at the pre-translational level. UALCAN-based analysis showed a significantly decreased promoter methylation level of AIFM2 in HCC in comparison with normal liver tissues (Fig. 4A). In agreement with this, a significant negative association was also found between the methylation and mRNA expression levels of AIFM2 (Pearson correlation coefficient:-0.52; p < 0.001; Fig. 4B) using the online cBioportal database. We also determined the methylation level of AIFM2 in 8-paired tumor and adjacent non-tumor tissues using methylation-specific PCR. We observed obviously decreased methylation level of AIFM2 in tumor tissues of HCC, as compared with non-tumor tissues (Fig. 4C). These results imply that DNA hypomethylation may contribute to the upregulation of AIFM2 in HCC. We also explored the contribution of microRNAs (miRNAs), the well-known post-transcriptional regulators of gene expression, to the upregulation of AIFM2 in HCC using a target prediction platform mirDIP [10]. We found that only transfection with miR-150-5p, which is among the top three predicted miRNAs targeting AIFM2 (Fig. S3), markedly decreased AIFM2 expression at both mRNA and protein levels in SNU-423 and HLF cells (Fig. 4D, E). As expected, the levels of miR-150-5p were markedly decreased in 30-paired tumor tissues of HCC as compared with normal liver tissues (Fig. 4F). Additionally, a significant negative correlation also exists between the expressions of AIFM2 and miR-150-5p in tissues of HCC (Fig. 4G). Next, we performed a luciferase reporter assay to verify the binding between the AIFM2 3’-UTR and miR-150-5p with a mutated or wild-type AIFM2 3’-UTR coupled luciferase reporter (Fig. 4H). As shown in Fig. 4I, the luciferase activity was significantly decreased in HCC cells with wild-type AIFM2 3’-UTR upon miR-150-5p transfection, while no change in the luciferase activity was observed in HCC cells with mutated AIFM2 3’-UTR upon miR-150-5p transfection. Furthermore, transwell migration and invasion assays revealed that miR-150-5p transfection markedly attenuated AIFM2 upregulation-enhanced HCC metastasis (Fig. 4J, K). The above findings suggest that AIFM2 upregulation in HCC can be attributed to DNA hypomethylation and downregulated miR-150-5p expression.

Fig. 4: Upregulation of AIFM2 is mainly caused by DNA hypomethylation and decreased miR-150-5p expression.

A Promoter methylation of AIFM2 in HCC was analyzed using the online UALCAN database. B Correlation between the DNA methylation and mRNA expression levels of AIFM2 in HCC was analyzed using the online cBioportal database. C Methylation-specific PCR was used to detect the methylation level of AIFM2 in 8-paired tumor and adjacent non-tumor tissues (M, methylated; U, unmethylated). D and E AIFM2 expression was examined by qRT-PCR and western blot analysis in SNU-423 and HLF cells transfected with indicated miRNAs. F miR-150-5p expression was determined by qRT-PCR analysis in 30-paired tumor and adjacent non-tumor tissue samples. G Correlation between the expression of AIFM2 and miR-150-5p in HCC tissues (n = 30). H Wild-type and corresponding mutant type AIFM2 3’-UTR at the binding sites of miR-150-5p. I Luciferase assay of AIFM2 3’-UTR and AIFM2 3’-UTR-mut reporters co-transfected with miR-150-5p in HCC cells. J and K Rescue transwell migration (G) and invasion (H) experiments were carried out in SNU-449 and Hep3B cells.

AIFM2-promoted mitochondrial biogenesis and oxidative phosphorylation in HCC cells

Given that AIFM2 was reported to play a critical role in glucose metabolism regulation, we, therefore, explored the effects of AIFM2 on glucose uptake and lactate production in HCC cells. Unexpectedly, we did not observe any significant changes in glucose uptake and lactate production in AIFM2 knockdown or overexpression HCC cells, as compared with their control cells (Fig. 5A, B). We next determined the effects of AIFM2 on mitochondrial metabolism by evaluating oxygen consumption rate (OCR), OXPHOS complexes activities, and ATP production. The results showed that AIFM2 knockdown markedly suppressed the rate of oxygen consumption, activities of OXPHOS complexes, and production of ATP in SNU-423 and SNU-449 cells, while AIFM2 overexpression significantly increased these mitochondrial oxidative metabolic phenotypes (Fig. 5C–E). In agreement with these mitochondrial respiratory phenotypes, mitochondrial membrane potential was also markedly decreased or increased when AIFM2 was either knocked-down or overexpressed (Fig. S4). Confocal microscopy analysis of mitochondrial morphology showed that AIFM2 knockdown resulted in a significant decrease of mitochondrial mass in SNU-423 cells, while AIFM2 overexpression markedly increased mitochondrial mass in SNU-449 cells (Fig. 5F). In agreement with this, the content of mitochondrial DNA (mtDNA) was also markedly increased or decreased when AIFM2 was either overexpressed or knocked-down in HCC cells (Fig. 5G). The above results indicate that upregulation of AIFM2 increases mitochondrial biogenesis and oxidative phosphorylation in HCC cells.

Fig. 5: AIFM2-promoted mitochondrial biogenesis and oxidative phosphorylation in HCC cells.

A and B Glucose uptake (A) and lactate production (B) were determined in AIFM2 knockdown or overexpression HCC cells. C–E The rate of oxygen consumption (C), activities of OXPHOS complexes (D) and production of ATP (E) were evaluated in AIFM2 knockdown or overexpression HCC cells. (F) Confocal microscopy analysis of mitochondrial mass in AIFM2 knockdown or overexpression HCC cells. Scale bars, 5 μm. (G) The content of mtDNA was measured by qPCR in AIFM2 knockdown or overexpression HCC cells.

AIFM2-promoted mitochondrial biogenesis and oxidative phosphorylation through activation of SIRT1/PGC-1α signaling

To gain insight into the molecular mechanistic basis of the promotive effect of AIFM2 on mitochondrial biogenesis and oxidative phosphorylation, we determined the effect of AIFM2 on the expression of PGC-1α (a major regulator of mitochondrial biogenesis). The results showed that PGC-1α expression at the protein level, but not at the mRNA level, was markedly down- or upregulated when AIFM2 is either knocked down or overexpressed in HCC cells (Fig. 6A, B), suggesting that PGC-1α is post-transcriptionally upregulated by AIFM2. We then determined whether PGC-1α was involved in AIFM2-promoted mitochondrial biogenesis and oxidative phosphorylation. The results showed that overexpression of PGC-1α markedly reversed the inhibitory effects of AIFM2 knockdown on the rate of oxygen consumption, activities of OXPHOS complexes, and production of ATP. By contrast, the knockdown of PGC-1α significantly attenuated the promotive effects of AIFM2 overexpression on the rate of oxygen consumption, activities of OXPHOS complexes, and production of ATP in SNU-449 cells (Fig. 6C–E). As expected, a significant positive correlation was found in tumor tissues of HCC between the expressions of AIFM2 and PGC-1α at the protein level, as evaluated by IHC staining assay (Fig. 6F). The above results suggest that AIFM2 promotes mitochondrial biogenesis and oxidative phosphorylation by upregulating PGC-1α expression.

Fig. 6: AIFM2-promoted mitochondrial biogenesis and oxidative phosphorylation through activation of SIRT1/PGC-1α signaling.

A and B AIFM2 and PGC-1α expressions were examined by qRT-PCR (A) and western blot (B) analysis in AIFM2 knockdown or overexpression HCC cells. C–E The rate of oxygen consumption (C), activities of OXPHOS complexes (D), and production of ATP (E) were evaluated in HCC cells with indicated treatment. (F) Correlation between the expressions of AIFM2 and PGC-1α expression at the protein level was analyzed by IHC staining assay in tumor tissues of HCC. Scale bars, 20 μm. G and H The levels of NAD+ (G) and activity of SIRT1 (H) were determined in AIFM2 knockdown or overexpression HCC cells. I Acetylation of PGC-1α was examined by western blot analysis in HCC cells with the treatment of MG132 (a proteasome inhibitor). J PGC-1α expression was examined by western blot analysis in HCC cells with the treatment of resveratrol or EX-527.

We further determined the molecular mechanism by which AIFM2 upregulated PGC-1α expression in HCC cells. Given that sirtuin 1 (SIRT1), a crucial NAD+-dependent protein deacetylase has been demonstrated to upregulate the protein expression level of PGC-1α through deacetylation [11, 12], we, therefore, explored the involvement of SIRT1 in AIFM2-upregulated PGC-1α. The results showed that the level of NAD+ and activity of SIRT1 were significantly decreased in SNU-423 cells when AIFM2 was knocked, while increased in SNU-449 cells when AIFM2 was overexpressed (Fig. 6G, H). As expected, the acetylation of PGC-1α was also clearly up- or downregulated, when AIFM2 was knocked-down or overexpressed, in the presence of MG132 (a proteasome inhibitor) (Fig. 6I). Meanwhile, the effect of AIFM2 on the activity of SIRT2, which is another cytoplasmic isoform of SIRT, was also explored. Contrary to our original thought that AIFM2 would affect the activity of SIRT2, its activity was only slightly decreased or increased, but not statistically different, upon AIFM2 silencing or overexpression in SNU-423 and SNU-449 cells (Fig. S5). Moreover, we found that the downregulated PGC-1α expression by AIFM2 knockdown was markedly rescued when SIRT1 was activated by resveratrol (an activator of SIRT1) treatment, while the upregulated PGC-1α expression by forced AIFM2 expression was markedly attenuated when SIRT1 was activated by suppressed by EX-527 (an inhibitor of SIRT1) treatment (Fig. 6J). Collectively, these findings suggest that AIFM2 promotes mitochondrial biogenesis and oxidative phosphorylation in HCC cells through activation of SIRT1/PGC-1α signaling.

AIFM2-promoted HCC metastasis through increasing PGC-1α-regulated mitochondrial biogenesis

Previous studies have shown that PGC-1α-regulated mitochondrial biogenesis plays a crucial role in the promotion of metastasis in several types of human cancers [13,14,15,16]. To explore the involvement of PGC-1α-regulated mitochondrial biogenesis in AIFM2-promoted metastasis of HCC, we suppressed mitochondrial biogenesis by knocking of PGC-1α (Fig. 7A, B). The results indicated that activation of mitochondrial biogenesis by forced PGC-1α expression markedly reversed the migration and invasion of SNU-423 and HLF cells suppressed by AIFM2 knockdown (Fig. 7C, D). By contrast, suppression of mitochondrial biogenesis by PGC-1α knocking-down markedly attenuated the migration and invasion of SNU-449 and Hep3B cells promoted by forced AIFM2 expression. These results suggest that AIFM2 promotes HCC metastasis through enhancing PGC-1α-regulated mitochondrial biogenesis.

Fig. 7: AIFM2-promoted HCC metastasis through increasing PGC-1α-regulated mitochondrial biogenesis.

A and B Overexpression or knockdown of PGC-1α was examined by qRT-PCR (A) and western blot (B) analysis in HCC cells with indicated treatment. C and D Transwell migration (C) and invasion (D) assays in HCC cells with indicated treatment.