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. 2025 Jun 20;16:1167. doi: 10.1007/s12672-025-03028-7

Effect and mechanism of PYCR1 on biological function of hepatocellular carcinoma cells under hypoxia

Jiayi Guo 1, Xinxin Jin 1, Junli Zhang 3, Yuming Zhang 4, Wenjuan Wu 1,2,✉
PMCID: PMC12181541  PMID: 40542176

Abstract

Background

Pyrrole-5-carboxylate reductase 1 (PYCR1) is a key enzyme involved in proline synthesis, which is closely related to the occurrence and development of liver cancer. In this study, we aimed to investigate the relationship and mechanism of PYCR1 on proline metabolism in hepatocellular carcinoma cells under hypoxic conditions.

Methods

GO and KEGG enrichment analyses were used to predict the biological function and possible mechanism of PYCR1 in hypoxic microenvironments. The energy metabolism kit and Western blot were used to detect the metabolic changes of SUN449 and Hep3B liver cancer cells under hypoxic conditions. The proliferative capacity of cells was evaluated using EDU incorporation assay and the Ki67 staining protocol. The apoptotic rates and Western blot were measured using flow cytometry. Additionally, Western blot analysis was used to examine the levels of proteins associated with relevant signaling pathways and pathway inhibitors.

Results

GO enrichment analysis showed that hypoxic PYCR1 might be related to amino acid metabolism. The 1% hypoxia model promoted proline synthesis and lactate dehydrogenase synthesis in hepatocellular carcinoma cells. Knockdown of PYCR1 reverses hypoxia-induced proline synthesis and NAD+/NADH ratio. PYCR1 promoted the proliferation of hepatocellular carcinoma cells under hypoxic conditions. PYCR1 knockdown reduces proliferation and increases apoptosis. Hypoxia can activate the MAPK/ERK/STAT3 pathway; knockdown of PYCR1 can inhibit the levels of ERK and STAT3 phosphorylated proteins, inhibit the proliferation of hepatocellular carcinoma cells, and the ERK inhibitor U0126 inhibits the expression of P-STAT3 in the downstream.

Conclusion

In summary, we report that hypoxia-mediated PYCR1 promotes proline synthesis in HCC, cell proliferation, inhibits apoptosis, and ultimately promotes tumor progression through the MAPK/ERK/STAT3 signaling pathway, suggesting that PYCR1 is a potential therapeutic target for HCC.

Keywords: Liver cancer, PYCR1, Hypoxia, Metabolism, Proliferation

Introduction

Hepatocellular carcinoma (HCC) is among the most prevalent types of cancer globally, with the sixth highest rate of occurrence and third highest rate of fatality [1]. Tumor growth is significantly influenced by the tumor microenvironment. The tumor microenvironment (TME) includes immunity, mesenchymal and hypoxic microenvironments, and angiogenesis [2]. Solid tumors can cause local hypoxia due to rapid proliferation of tumor tissue [3]. Hepatocellular carcinoma cells can use this shared TME to promote the aggregation of cancer cells, and at the same time produce chemokines and metabolites that are immunosuppressive to inhibit anti-tumor immune cells, hypoxia is one of the key factors causing this immunosuppression [4]. Therefore, hypoxia is closely related to the degree of tumor differentiation, distant metastasis, and tolerance to radiotherapy and chemotherapy [5]. The relationship between hypoxia and the occurrence and development of liver cancer should be further explored to provide a reliable strategy for the clinical diagnosis and treatment of HCC.

Accumulating evidence suggests that metabolism of other nutrients, in addition to glucose and glutamine, the two most abundant nutrients, is critical for cell survival and growth [6]. Proline is a unique non-essential amino acid that plays a key role in both protein structure and cellular stress response and promotes tumorigenesis in many types of cancer, including liver cancer [7]. Among the metabolic traits of cancer cells, proline metabolism is particularly distinctive and tends to be up-regulated in numerous forms of cancer [8]. At the same time, it is also involved in the regulation of cell biological behaviors such as cell proliferation, apoptosis and invasion, and is closely related to the occurrence and development of diseases such as skin laxity, breast cancer and lung cancer [9]. Metabolic changes are important drivers of tumor development [10]. Tumor metabolic changes establish “metabolic reprogramming” [11]. Based on this view, some studies have shown that lactic acid produced by cancer cells can be reused by cancer cells or send special signals to specific cells [12]. This metabolic reprogramming has been shown to play a key role in cellular energy requirements in the presence of acute hypoxic stress [13], And there are studies that show that human cancer cells cultured under hypoxic conditions convert glucose into lactate in large quantities and excrete it out of the cell [14].

PYCR1, a pivotal enzyme in proline biosynthesis, plays a significant role in the etiology and progression of numerous diseases [15]. The mitochondrial isoenzymes PYCR1 and PYCR2 are responsible for the second step in the synthesis of proline from glutamate. While proline is generated,PYCR1 and PYCR2 oxidize reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide (NAD+) in the mitochondria [16]. Mitochondrial proline synthesis may be an important mechanism for buffering changes in the mitochondrial NAD+/NADH ratios [9]. PYCR1 plays an important role in mitochondrial amino acid metabolism, intracellular redox potential balance, and mitochondrial integrity through the enzymatic cycle between 1-pyrrolidine-5-carboxylic acid (Pyrroline-5-carboxylate, P5C) and proline [17]. At present, the potential mechanism of PYCR1 involvement in HCC progression in hypoxic environments is remains unclear. Therefore, we evaluated the potential pro-tumor effect of PYCR1 by constructing an in vitro cell hypoxia model and explored the effect and significance of PYCR1 in the metabolism and proliferation of hepatocellular carcinoma cells.

Materials and methods

GO and KEGG enrichment analysis predicted the biological function and mechanism of PYCR1

Transcriptome data GSE15366 of hepatoma cells after hypoxia were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The difference between the normoxia group and hypoxia groups was analyzed using LIMMA package, and the threshold was as follows:|logFC|> 0.5, FDR < 0.05. Based on the results of differential analysis, GO and KEGG enrichment analysis of 1821 differential genes was performed using the R package “clusterProfiler” (pvalueCutoff = 0.05, qvalueCutoff = 0.05). The results were visualized using the R package “ggplot2”.

Cell culture, transfection, and treatment

The human HCC cells lines Hep3B were purchased from the Procell (Wuhan, China), SUN449 were purchased from the Chinese Academy of Sciences (Shanghai, China).Cells were grown in RPMI-1640 medium (Gibco, USA) and DMEM (Gibco, USA) with high glucose, supplemented with heat-inactivated 10% fetal bovine serum (FBS) (Gibco, USA) and 1% Penicillin/streptomycin (Gibco, USA). The cells were incubated at 37 °C in a 5% CO2 atmosphere and grown to 90% confluence prior to passaging or experimental use.

The lentiviral interference vector (shPYCR1-1, shPYCR1-2, shPYCR1-3), and the negative control viral vector (shPYCR1-NC) were designed, constructed, and packaged by Shanghai HANBI. Lentivirus transfection was carried out using shRNA recombinant lentiviral vectors to infect Hep3B and SUN449 cells, puromycin was screened 48H after transfection. Western blotting and RT-PCR were used to detect knockdown. In addition, the ERK inhibitor U0126 (MCE, USA) was added under hypoxic conditions for further analysis, the concentration of U0126 is selected as 10 μM, and the action time is 12 H.

Hypoxia model

Using a tri-gas incubator (Memmert, Germany), the oxygen concentration in the box was maintained at 1%, and the carbon dioxide concentration was maintained at 5%. After each opening of the box door, nitrogen gas was filled through an automatic inflation system until the oxygen concentration returned to the preset 1% level. After the liver cancer cells SUN449 and Hep3B were sub-cultured for 24 h, the freshly prepared complete medium was changed under sterile conditions, and then the cells were transferred from the carbon dioxide incubator to the hypoxia incubator for continued incubation. During the culture period, the medium was changed every 24 h. After 48 h of incubation, the cells were taken out in a sterile environment for subsequent experiments. The experiments were divided into 4 groups: Normoxia control group (NC), PYCR1 knockdown group(shPYCR1), Hypoxia + NC group(hypoxia-NC), Hypoxia + shPYCR1(hypoxia-shPYCR1).

RT-PCR

Total cellular RNA was extracted using the TRIZOL (Invitrogen, USA) method and cDNA was synthesized following the protocol provided by the Vazyme reverse transcription kit. Subsequently, a fluorescence quantitative PCR (qPCR) reaction mix was prepared using cDNA as a template. The primer sequences utilized are listed below: Human PYCR1 primers Forward:5‘-CCTGGCACCCAGCACAAT-3’, Reverse:5’-GGGCCGGACTCGTCATAC-3’; Human β-actin primers Forward:5-TCCATTGAGAAGAAGCTGTCAG-3’, Reverse: 5’-CATCAATCAGGTCCTCTTCCAC-3’.

Western blotting

After extracting the total protein of hepatocellular carcinoma cells and quantifying BCA, the stacking gel was run at 80 V and 30 min at 120 V in the SDS-PAGE procedure, and the separation gel was run for 60 min. The subsequent transfer to the PVDF membrane was performed at 400 mA for 50 min. The membrane was blocked with 5% skim milk for more than 2 H, followed by incubation with the primary antibody at 4 °C overnight. The secondary antibodies were incubated at room temperature for 2 H. The cells were then washed and exposed. ImageJ software was used to analyze the relative expression of the proteins.

EDU stain

EDU Kits (Beyotime, China) A2X working solution was prepared and incubated at room temperature for approximately 2 H. For cell fixation, a 4% paraformaldehyde solution was applied for 15 min. The cells were then washed three times with PBS containing 3% BSA, with each wash lasting 3 min. The cells were then permeabilized with PBS containing 0.3% Triton X-100 for 15 min at room temperature. Wash the cells again. Finally, the click reaction solution was prepared in accordance with the Biyotian kit’s recommended ratios. DAPI staining was performed for 6 min and the cells were washed (same as above). Images were captured using a confocal microscope was followed by photographs. The excitation and emission wavelengths of DAPI were 346 and 460 nm, respectively, and are blue fluorescence.

Immunofluorescence

They The membranes were washed three times in PBS at room temperature, 4% paraformaldehyde was added to fix for 20 min, washed three times in PBS for 3 min each time, 0.5% TritonX-100 was added to penetrate the membrane, permeabilize for 20 min, wash three times in PBS for 3 min each time, and block BSA at room temperature for 1 h, BSA diluted primary antibody and 4 °C overnight (1:500), PBST wash three times the next day for 3 min each time, dropwise fluorescent secondary antibody (1:400). The samples were incubated in the dark at room temperature for 2-h incubation were rinsed three times with PBST, with 2 min per rinse. Subsequently, DAPI was added and the cells were incubated in the dark at room temperature for 6 min, followed by three additional rinses. An anti-fluorescence embrittlement agent was added dropwise to the mount and photographed using a Zeiss fluorescence microscope.

Proline detection

Cells were seeded into at a density of 2 × 106 cells 6 cm Petri dish. After 48 H of hypoxia intervention, 100 µl of hepatoma cell supernatant were added to 90 µl of extract mix well, shake and set in metal bath for extraction for 10 min, 10,000g, centrifuge at room temperature for 10 min, take the supernatant, cool it to be measured, adjust the wavelength to 520 nm with microplate reader, add 0.25 ml to cell supernatant, 0.25 ml for reagent 1, 0.25 ml for reagent 2, After mixing, close the lid tightly, place it in a metal bath for 30 min, and after cooling, draw 0.2 mL into a 96-well plate at 520 nm wavelength, record the absorbance value, and calculate △A = A assay tube-A blank tube, the regression equation y = kx + b. Substituting ΔA into the equation to obtain x and calculating Pro content (μg/mL) = 10*x.

NADH detection

Cells were seeded into at a density of 2 × 106 cells 6 cm Petri dish, Hypoxia intervention was used after 48 H. After rinsing the cells with ice-cold PBS (0.1 M, pH 7.4), approximately 1.5 × 10^6 cells were collected and homogenized in 0.4 mL of pre-chilled reagent. The mixture was centrifuged at 4 °C, 12,000g for 10 min. The supernatant was then separated, with a portion reserved for protein concentration analysis. It is important to note that tissue and cell homogenates contain enzymes which can degrade NAD + . Thus, it is suggested that following sample extraction and centrifugation, the supernatant undergoes ultrafiltration using a 10 KD tube at 4 °C, 10,000g for an additional 10 min to remove these degrading enzymes. When measuring total NAD + and NADH levels, the ultrafiltered sample’s supernatant is measured directly. To measure NADH separately, a suitable quantity of the ultrafiltered sample is transferred to an EP tube and incubated in a 60 °C water bath for half an hour. After cooling in running water, the sample is mixed and prepared for measurement. The procedure is as follows: ① Add 20 μL of the test sample to the corresponding wells of the enzyme plate. ② Add 120 μL of the reaction working solution to the test wells from step ① and to the standard wells. ③ Add 40 μL of reagent three to the test wells and standard wells from step ②. ④ Shake the plate for 5 s and incubate precisely for 30 min in a 37 °C incubator. Then, measure each well’s optical density (OD) values at 450 nm using an enzyme plate reader. Standard curve fitting: y = ax + b; Formula for calculating the total amount of NAD + and NADH in the sample:

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Formula for calculating the NADH content in the sample:

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The calculation formula for the content of NAD + in the sample:

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The formula for calculating the ratio of NAD + to NADH in the sample:

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LDH detection

Cells were seeded into at a density of 2 × 106 cells 6 cm Petri dish, Hypoxia intervention was used after 48 H. Remove the cell culture fluid from each group and create blank wells, standard wells, test wells, and control wells on a 96-well plate. In this setup, introduce 25 μL of double-distilled water to the blank wells, 5 μL of double-distilled water to the standard and control wells, 20 μL of a 0.2 μmol/mL pyruvic acid standard solution to the standard wells, and 20 μL of the cell culture fluid from each group into the test and control wells. After adding 25 μL of matrix buffer to each well, include an additional 5 μL of coenzyme I to the standard wells, mix, and incubate at 37 °C for 15 min. Then, add 25 μL of 2,4-dinitrophenylhydrazine to each well, mix, and incubate at 37 °C for 15 min; incorporate 250 μL of a 0.4 mol/L NaOH solution to each well, mix, and let stand at room temperature for 5 mi. Subsequently, measure the absorbance values of each group at a wavelength of 450 nm using an enzyme-linked immunosorbent assay instrument. Finally, calculate the activity of LDH (U/L) in the cell culture fluid of each group using the formula: (A test – A control) / (A standard – A blank) × 200.

Flow cytometry assay

After 48 H of cell intervention, trypsinization without EDTA, and centrifugation PBS was washed twice at 1500 r/5 min, stained with fluorescein isothiocyanate (5 μL)for 15 min, and then stained with propidium iodide (PI) (4 μL) in a dark place for 6 min, and the apoptosis experiment was carried out immediately. Cells were analyzed using a CytoFLEX flow cytometer (Beckman, China). The data were analyzed using the FlowJo software.

Statistical analysis

SPSS16.0 and Graph Pad software were used for statistical analysis. All experiments were repeated three times, and data are presented as mean (mean) standard deviation (SD). The independent samples t-test was used for comparison between the two groups. Statistical significance was set at P < 0.05.

Results

The 1% hypoxia model promotes proline and lactate dehydrogenase synthesis in hepatocellular carcinoma cells

GO enrichment analysis showed that hypoxic PYCR1 might be related to the intrinsic apoptosis signaling pathway, organic acid biosynthesis, amino acid metabolism, and other functions (Fig. 1A). Given the role of GO bioinformatics analysis, we evaluated the potential function of PYCR1 in vitro. By constructing a 1% hypoxia model to explore the effects of hypoxia on proline metabolism and cell biology function in hepatocellular carcinoma cells. Our findings indicate that the synthesis of proline and lactate dehydrogenase (LDH) is temporally dependent. Specifically, the production of both proline and LDH was observed to escalate in correlation with extended periods of hypoxia (12 H, 24 H, 48 H) when compared to the baseline at 0 H (Fig. 1B, C). The key proline biosynthetic enzyme PYCR1 was then detected, and interestingly, Western blot results showed that PYCR1 did not change with the hypoxic time gradient compared to 0 H (Fig. 1D, E), suggesting that the normoxic expression of the enzyme may be able to support the flux observed in excess of normoxic conditions. We found that this hypoxic model promotes proline synthesis and glycolysis in hepatocellular carcinoma cells.

Fig. 1.

Fig. 1

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Proline and lactate dehydrogenase synthesis in HCC cells in the 1% hypoxia model. A GO enrichment analysis of biological functions of hypoxic PYCR1. B Effects of different hypoxia durations on proline synthesis in HCC (n = 3 independent experiments). C The effect of different hypoxia times on LDH activity in HCC was detected (n = 6 independent experiments). D The effect of hypoxia on the expression of PYCR1 protein was detected by western blotting (n = 3 independent experiments). E Quantification graph of PYCR1 protein expression. Data are presented as mean ± SD (* P < 0.05, ** P < 0.01 vs 0 H)

A PYCR1 knockdown model was constructed

Hypoxia promotes proline synthesis, but the key enzyme PYCR1 is not altered, so we considered whether the deletion of this gene would affect the synthesis of proline and thus affect cell function. We used transfection assays to construct stable knockdown PYCR1 cells from SUN449 cells and Hep3B cells, Puromycin was stably screened for one week (2 μg/ml), and verified the knockdown efficiency by measuring protein levels (Fig. 2A) and mRNA levels (Fig. 2B). Western blotting and RT-PCR results indicated no marked change in the expression levels of shPYCR1-1 and shPYCR1-2 in the knockdown groups compared to the control group, and shPYCR1-3 knockdown had the best effect. Although the mitochondrial isoenzymes PYCR1 and PYCR2 showed 80% similarity, the knockdown sequence had no effect on PYCR2 protein expression. Therefore, the stable transfection of shPYCR1-3 was chosen for subsequent experiments (Fig. 2A, B). The immunofluorescence results showed that compared with the NC group, the fluorescence signal of PYCR1 in the shPYCR1 group decreased significantly, the knockdown effect was significant, the expression of PYCR1 fluorescence signal increased slightly after hypoxia, and the catalytic activity increased but was not statistically significant (Fig. 2C, D). The immunofluorescence of the Normoxic NC group and the hypoxic 48-h group was consistent with the previous WB data, indicating that PYCR1 was able to synthesize sufficient proline in the presence of Normoxia to support the hypoxic stress environment.

Fig. 2.

Fig. 2

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Construction of a PYCR1 lentivirus-silenced hepatocellular carcinoma cell model. A Western blotting was used to detect PYCR1 and PYCR2 knockdowns in hepatocellular carcinoma cells (n = 3 independent experiments). B RT-PCR was used to detect the expression level of PYCR1 in hepatocellular carcinoma cells (n = 3 independent experiments). C, D Immunofluorescence to detect the expression level of PYCR1 in hepatocellular carcinoma cells (n = 5 independent experiments). Data are presented as mean ± SD. (** P < 0.01, ***P < 0.001 vs NC group; ###P < 0.001 vs hypoxia-NC group)

PYCR1 promotes proline synthesis and cell proliferation in hepatocellular carcinoma cells under hypoxic conditions.

By intervening in PYCR1 to explore the function of hypoxia-mediated PYCR1 in hepatocellular carcinoma cells, we found that proline synthesis decreased in the shPYCR1 group and significantly increased in the hypoxia-NC group compared to the NC group. Compared to hypoxia NC, the proline to NAD + /NADH ratio was notably reduced in the hypoxia-shPYCR1 group. (Fig. 3A, B). The experiments outlined above indicate that the downregulation of PYCR1 can inhibit proline synthesis under hypoxic conditions. Concurrently, evidence was provided demonstrating that PYCR1 can function as an integral part of the mitochondrial NAD + regeneration system. These findings suggest that PYCR1 plays a significant role in the mitochondrial redox shuttling process and may be implicated in the progression of liver cancer. Therefore, we conducted in vitro cell function experiments, which indicated a reduction in both the proliferation rate and fluorescence intensity within the shPYCR1 group when compared with the NC group. Conversely, a notable increase in these parameters was observed in the hypoxia NC group. Furthermore, compared to the hypoxia-NC group, the hypoxia-shPYCR1 group exhibited a significant decline in proliferation rate and a decrease in fluorescence intensity. (Fig. 3C–F). In conclusion, our study highlights the ability of hypoxia to foster hepatocellular carcinoma cell growth, but this effect is mitigated by the knockdown of PYCR1 in cells experiencing both normal and reduced oxygen levels.

Fig. 3.

Fig. 3

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PYCR1 promotes proline synthesis and cell proliferation in hepatocellular carcinoma cells under hypoxic conditions. A Effect of hypoxia-induced PYCR1 on proline (n = 3 independent experiments). B Effect of hypoxia-induced PYCR1 on NAD+/NADH ratio (n = 3 independent experiments). C, D Effect of hypoxia-induced PYCR1 on hepatocellular carcinoma cell proliferation detected by EDU (n = 3 independent experiments). E, F Immunofluorescence Ki67 detection to detect the effect of hypoxia-induced PYCR1 on hepatocellular carcinoma cell proliferation (n = 4 independent experiments). Data is presented as mean ± SD. (*P < 0.05, **P < 0.01 vs NC group; ##P < 0.01, ###P < 0.001vs hypoxia-NC group.)

PYCR1 mediates anti-apoptotic effect of hepatocellular carcinoma cells under hypoxic conditions

We conducted an investigation to ascertain whether PYCR1 could trigger apoptosis in hepatocellular carcinoma cells under hypoxic conditions, we performed flow cytometry. The results showed that hepatocellular carcinoma cells hypoxia-NC group reduced the percentage of cell apoptosis from 13.76% and 9.9% to 5.82% and 4.58% in the NC group, respectively, and hepatocellular carcinoma cells hypoxia-shPYCR1 group increased the percentage of cell apoptosis from 5.82% and 4.58% to 21.55% and 24.83% in hypoxia-NC group, respectively (Fig. 4A, B). In tandem, western blot analyses demonstrated that hypoxia induced an upregulation of the anti-apoptotic protein Bcl2 and a concurrent decrease in pro-apoptotic BAX. The expression of Bcl2 decreased and that of BAX increased when PYCR1 was knocked down (Fig. 4C). In summary, PYCR1 facilitates the anti-apoptosis of hepatoma cells under both normoxic and hypoxic conditions. Consequently, it can be postulated that PYCR1 contributes to the proliferation of hepatoma cells by inhibiting apoptsis.

Fig. 4.

Fig. 4

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Anti-apoptotic effect of PYCR1 in hepatocellular carcinoma cells under hypoxic conditions. A, B Flow cytometry to detect the effect of PYCR1 on apoptosis of hepatocellular carcinoma cells (n = 3 independent experiments). C Western blot was used to detect the expression of apoptosis-related proteins such as Bcl2 and BAX (n = 3 independent experiments). Data are presented as mean ± SD. (*P < 0.05, **P < 0.01 vs NC group; #P < 0.05, ##P < 0.01vs hypoxia-NC group)

PYCR1 promotes the proliferation and survival of hepatocellular carcinoma cells through the MAPK/ERK/STAT3 pathway under hypoxic conditions

To elucidate the molecular mechanism of PYCR1 in hypoxic conditions, we conducted a KEGG enrichment analysis. The results suggest that PYCR1 influences the development and progression of hepatocellular carcinoma cells via the MAPK pathway in such environments (Fig. 5A). In the case of liver cancer cells SUN449 and Hep3B, our research indicated that the protein expression levels of P-ERK/ERK, P-STAT3/STAT3, and PYCR1 were significantly reduced in the shPYCR1 group when compared to the NC group. Interestingly, these proteins exhibited an upregulation in their expression levels following a hypoxic condition, which suggests that hypoxia might activate this particular pathway. Simultaneously, when compared to the hypoxia-NC group, the protein expression levels of P-ERK/ERK, P-STAT3/STAT3, and PYCR1 in the hypoxia-shPYCR1 group were significantly downregulated (Fig. 5B). This suggests that the knockdown of PYCR1 can reverse the activation of pathway protein expression induced by hypoxia. To further ascertain if hypoxia operates through the MAPK/ERK/STAT3 pathway, We conducted an intervention using ERK inhibitor U0126 for 12H under hypoxic conditions. Subsequently, we compared the expression levels of P-ERK protein in the hypoxia-NC + U0126 group and observed a significant decrease. Additionally, there was a notable downregulation in the expression level of P-STAT3 protein in the downstream group (Fig. 5C). We then performed Ki67 immunofluorescence staining and discovered that cell proliferation in the inhibitor group had diminished in comparison to the hypoxia-NC group (Fig. 5D, E). PYCR1 enhances the proliferation and differentiation of hepatocellular carcinoma cells under hypoxic conditions through the MAPK/ERK/STAT3 signaling pathway. (Fig. 6).

Fig. 5.

Fig. 5

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PYCR1 promotes the proliferation and survival of hepatocellular carcinoma cells through the MAPK/ERK/STAT3 pathway under hypoxic conditions. A KEGG enrichment analysis showed that PYCR1 may affect the occurrence and progression of hepatocellular carcinoma cells through the MAPK pathway in the hypoxic environment. B Western blot was used to detect the expression levels of P-ERK, ERK, P-STAT3, STAT3 and PYCR1 in hypoxic HCC (n = 3 independent experiments). C Western blot was used to detect the protein expression levels of P-ERK, ERK, P = STAT3 and STAT3 after U0126 intervention under hypoxic conditions (n = 3 independent experiments). D, E Immunofluorescence Ki67 was used to detect the effect of U0126 on the proliferation of hepatocellular carcinoma cells in hypoxia (n = 5 independent experiments). Data are presented as mean ± SD. (*P < 0.05, **P < 0.01 vs NC group; ##P < 0.01, ###P < 0.001vs hypoxia-NC group)

Fig. 6.

Fig. 6

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This mechanistic diagram illustrates the schematic representation of the molecular pathways by which hypoxia induces survival in liver cancer cells. Under hypoxic conditions, PYCR1 plays a dual role: (1) catalyzing the synthesis of proline, leading to a significant increase in intracellular proline levels; (2) regenerating NAD⁺ by consuming NADH, enhancing the critical NAD⁺/NADH ratio. The accumulation of proline and the increase in the NAD⁺/NADH ratio synergistically activate key pro-survival signaling pathways. Under hypoxia, PYCR1 activates the MAPK/ERK signaling pathway and the downstream STAT3 signaling pathway, leading to the enhancement of ERK phosphorylation activation (p-ERK) and STAT3 phosphorylation activation (p-STAT3). p-ERK and p-STAT3 serve as key effector molecules, ultimately converging and driving the enhanced survival capability of liver cancer cells, enabling them to survive under hypoxic stress

Discussion

Hepatocellular carcinoma ranks among the leading malignancies globally [18], and surgery remains the key modality for curative intent [19]. Despite significant advancements in liver cancer therapies, the overall prognosis is still unsatisfactory owing to its complex clinical manifestations and large heterogeneity [20]; Therefore, it is necessary to study the mechanism of HCC progression in depth and explore new key targets for improvement.

Cancer progresses too fast, and improving the tumor microenvironment is considered critical [21]. Tumor hypoxia is a widely known phenomenon that leads to decreased oxygenation within the tumor as cancer cells continue to proliferate abnormally and demand oxygen is high [22]. Abnormal vascular production leads to spatiotemporal heterogeneity of tumor blood flow and oxygenation, and these abnormal microenvironments accelerate the progression of tumor cells and also lead to reduced efficacy of chemotherapy, radiotherapy, and immunotherapy [23]. The hypoxia signaling pathway has been shown to be closely linked to tumor progression, which not only regulates the survival and proliferation of cancer cells, but also participates in key biological processes such as angiogenesis, immune escape, Epithelial-Mesenchymal transition (EMT), and distant metastasis of tumors [24]. Liver cancer cells can use this shared tumor microenvironment to promote the aggregation of cancer cells, so hypoxia represented by liver cancer is a feature of all solid tumors, contributing to tumor progression and treatment resistance [25].

In the mitochondrial tricarboxylic acid cycle, PYCR1 is a key mitochondrial enzyme that promotes glutamate to proline conversion [26]. PYCR1 and PYCR2 oxidize NADH to NAD+ in mitochondria, while PYCR3(L) oxidizes NADPH to NADP+ in the cytoplasm, with PYCR1 playing a key role in proline biosynthesis [27]. Functionally, the expression of mitochondrial PYCRs is required for cancer cell survival and proliferation [28], Therefore, proline biosynthesis is one of the key factors in maintaining protein synthesis and supporting mitochondrial function and nucleotide biosynthesis [29]. Studies have substantiated that, within hypoxic environments, PYCR1 in cancer cells is capable of regenerating NAD⁺ via the proline cycle, thereby preserving the redox equilibrium of hypoxic cells [30, 31]. Previous studies have concluded that the expression of PYCR1 in hepatocellular carcinoma cells is correlated with clinical prognosis, and the differential expression of PYCR1 in hepatocellular carcinoma has been verified to be clinically significant, but the correlation between hepatocellular carcinoma cells and PYCR1, a key enzyme for proline synthesis, in hypoxia is still unclear. In view of the increasing research on tumor metabolic abnormalities, the effects of PYCR1 on the biological behavior and metabolism of tumor cells have potential clinical application value.

Our hypothesis suggests that an increase in proline synthesis within cells occurs when cellular redox homeostasis is disrupted under hypoxic conditions, or when there is a demand for enhanced oxidation of mitochondrial NADH. To examine this proposition, we subjected human hepatocellular carcinoma cells to either normooxygen (21% O2) or hypoxia (1% O2) conditions. Our findings indicate that hypoxia amplifies the synthesis of proline and its efflux into the medium (Fig. 1B), while also increasing Lactate Dehydrogenase (LDH) activity (Fig. 1C). Notably, a significant rise in proline synthesis was observed at 48 h of hypoxia exposure. Furthermore, the knockdown of PYCR1, a crucial enzyme in proline synthesis, resulted in a notable inhibition of this process (Fig. 3A), which is consistent with the results of the Rebecca L. Westbrook study [9], Our findings indicate that the expression level of this critical enzyme under normoxic conditions is adequate to provide the necessary metabolic support for cells in a hypoxic state. This mechanism allows tumor cells to sustain their survival and proliferation capabilities in an environment with limited oxygen supply. We also examined the alterations in the NAD+/NADH ratio in this model. The knockdown of PYCR1 led to a decrease in this ratio (Fig. 3A, B), further emphasizing the role of PYCR1 in the mitochondrial NAD+ regeneration system under hypoxic conditions in hepatocellular carcinoma cells [32], These findings suggest that PYCR1 may play a crucial role in mitochondrial redox shuttling, particularly in maintaining the malate-aspartate shuttle, which significantly influences cellular anabolism.

Given the significance of PYCR1 in tumor metabolism, we conducted in vitro cell function experiments to ascertain the impact of PYCR1 gene knockdown on hepatocellular carcinoma cell proliferation under hypoxic conditions. The EDU and Ki67 immunofluorescence analyses revealed that a 1% hypoxic atmosphere significantly augmented the proliferation of hepatocellular carcinoma cells compared to the normoxic condition (Fig. 3C–F). Knockdown of PYCR1 was diminished in both normoxic and hypoxic settings, indicating that PYCR1 plays a pivotal role in modulating the proliferation and survival of hepatocellular carcinoma cells. Through flow cytometry analysis, we observed that while hypoxia inhibits apoptosis, PYCR1 knockdown reverses this effect, leading to an enhanced rate of apoptosis (Fig. 4A, B). Consistent with this, Western blot data confirm a hypoxia-induced increase in Bcl2 and a decrease in BAX expression. And the expression of Bcl2 decreases and BAX increases when knocking down PYCR1 (Fig. 4C).

In our gene enrichment study using KEGG, we discovered that PYCR1 potentially influences the incidence and progression of hepatocellular carcinoma cells via the MAPK pathway under hypoxic conditions. (Fig. 5A). The pivotal role of MAPK signaling pathways in coordinating a spectrum of vital cellular physiological and pathological processes, inclusive of cell growth and inflammatory response, is well-established [33]. Hypoxia-induced activation of the mitogen-activated protein kinase (MAPK) signaling pathway is a pivotal mechanism in cellular adaptation to hypoxic conditions [34].Among them, the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway mainly mediates cell proliferation, inflammation, and anti-apoptotic effects [35]. Tao Chen et al. studied the activation of the ERK signaling pathway by hypoxia in colorectal cancer, and inhibiting PYCR1 can suppress tumor growth by blocking the ERK pathway and metabolic reprogramming [32]. In the low-oxygen environment characteristic of hepatocellular carcinoma cells, STAT3, a member of the mammalian STAT family, is engaged in numerous biological functions such as cell growth, survival, differentiation, and the formation of new blood vessels [36]. Consequently, STAT3 is a key factor in oncogenesis and is widely regarded as a potential therapeutic target in cancer treatment [37]. A large number of studies have shown that knocking down PYCR1 can inhibit the growth of lung adenocarcinoma and bladder cancer and affect the JAK/STAT signaling pathway [38, 39]. To clarify the relationship between PYCR1 and the MAPK signaling pathway under hypoxic conditions, further mechanistic studies are warranted. A Western blot assay revealed a significant increase in the phosphorylation levels of Erk1-2 and STAT3 at 48 h of hypoxia (Fig. 5B). Moreover, knocking down PYCR1 reversed the hypoxia-induced increase in P-ERK and P-STAT3 protein expression. The ERK inhibitor U0126 confirmed that inhibiting P-ERK led to a decrease in the level of P-STATS protein (Fig. 5C), as well as a suppression in hepatoma cell proliferation (Fig. 5D, E). These findings suggest that under hypoxic conditions, PYCR1 activates the MAPK/ERK/STAT3 signaling pathway, thereby promoting the proliferation and differentiation of hepatoma cells.

In conclusion, our findings indicate that proline synthesis and LDH activity are enhanced in response to long-term hypoxia in hepatocellular carcinoma SUN449 and Hep3B cells. Furthermore, the elevation in proline synthesis and reduction in the NAD+/NADH ratio were reversed after PYCR1 knockdown. These findings reinforce the significance of PYCR1 in stimulating proline synthesis in hepatocellular carcinoma cells via NADH oxidation. Additionally, they illuminate its potential role as a crucial component of the mitochondrial NAD+ regeneration system. The findings from the EDU and immunofluorescence Ki67 in vitro cell proliferation assays reveal that, under hypoxic conditions, PYCR1 is instrumental in driving the proliferation of liver cancer cells. Notably, PYCR1 activity facilitates both the proliferation and survival of these cells in hypoxic regions via the MAPK/ERK/STAT3 signaling pathway. Notably, the expression of PYCR1 experienced a slight increase under hypoxic conditions, though this rise was not statistically significant. These findings imply that the PYCR enzyme can synthesize sufficient proline to maintain the proliferation rate under stress conditions. The major limitation of this study is the absence of an in vivo validation in animal models. As such, the translation of the in vitro findings to the physiological context remains incomplete. We recognize that the insights obtained from two cellular models are valuable to decipher the underlying mechanism; however, the controlled cellular microenvironments cannot mimic the complexity of the pathophysiology in vivo, such as neuro-humoral regulations and inter-organ communications in hypoxia. Future studies should focus on establishing in situ animal models of clinical hypoxia to determine the dynamic changes of physiological parameters, tissue remodeling, and systemic adaptive responses. This will help to define the spatial and temporal regulation of the discovered mechanisms within a whole body context. In addition, gain-of-function and loss-of-function studies using in vivo models of hypoxia will be performed to establish the role of PYCR1 genes in regulating hepatocarcinogenic cells under pressure. These in vivo studies will bridge the translational gap between cell and organism levels and provide pre-clinical data for developing targeted therapeutic interventions against hypoxia-associated diseases.

Conclusions

In summary, we reported that hypoxia-mediated PYCR1 promotes proline synthesis in HCC, promotes HCC cell proliferation, inhibits apoptosis, and ultimately tumor progression through the MAPK/ERK/STAT3 signaling pathway, suggesting that PYCR1 is a potential therapeutic target for HCC.

Author contributions

Guo. wrote the main manuscript text Jin. verification result Zhang and Zhang. technical and methodological support Wu. paper revision review.

Funding

This work is supported by the Key Project of Natural Science Foundation of Anhui Provincial Department of Education (P. R. China) (2022AH051452, 2022AH051485) and the Program for Science and Technology Research of Bengbu Medical University (2022Byycx23061).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

All authors have provided their consent for publication.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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