WITHDRAWN: Mitochondrial ribosomal protein L12 potentiates hepatocellular carcinoma by regulating mitochondrial biogenesis and metabolic reprogramming

doi:10.21203/rs.3.rs-1956391/v1

Download PDF

Research Article

WITHDRAWN: Mitochondrial ribosomal protein L12 potentiates hepatocellular carcinoma by regulating mitochondrial biogenesis and metabolic reprogramming

https://doi.org/10.21203/rs.3.rs-1956391/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

The full text of this preprint has been withdrawn by the authors while they make corrections to the work. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author.

Editorial notes are used to provide important context regarding the topic of a preprint or to alert readers to potential issues concerning that preprint or a downstream publication associated with it. For more information on editorial notes, see our Editorial Policies.

Background: Mitochondrial dysfunction and metabolic reprogramming are the key features of hepatocellular carcinoma (HCC); however, the detailed mechanism has not yet been clarified. Mitochondrial ribosomal protein L12 (MRPL12) has been implicated in transcription in human mitochondria. Although the function of MRPL12 has been documented, the role of abnormal MRPL12 expression in HCC remains unknown. Here, we determined the clinical significance, functional implications, and mechanisms underlying the effects of MRPL12 in HCC.

Methods: Human HCC obtain from patients was used to evaluate the role of MRPL12 in HCC. For evaluating tumor behavior, we used cell culture for in vitro experiments and for in vivo experiments we used mouse HCC xenograft model. Further we used tissue microarray, immunohistochemistry, flowcytometry, Transwell assay, and CCK-8 assay, mitochondrial DNA copy number quantification methods, and seahorse assay to clarify our hypothesis.

Results: Significant upregulation of MRPL12 in patients with HCC correlated with aggressive tumor behavior and poor prognosis. MRPL12 knockdown in HCC cells attenuated cell proliferation and migration in vitro, and tumorigenicity in vivo. We observed that MRPL12 is essential for mitochondrial homeostasis. Gain- and loss-of-function of MRPL12 in HCC altered oxidative phosphorylation (OXPHOS), mitochondrial DNA content, and HCC cell proliferation and invasion. Overall, MRPL12 might play oncogenic role by activating mitochondrial OXPHOS and promoting mitochondrial biosynthesis. Yin Yang 1 (YY1) transcriptionally regulated MRPL12 expression, and YY1 knockdown inhibited MRPL12 activity and suppressed HCC cell proliferation and metastasis. The role of the phosphatidylinositol-3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway in regulating MRPL12 was confirmed. We hypothesize that the PI3K/mTOR-YY1-MRPL12 axis orchestrates HCC cell proliferation and metastasis.

Conclusion: Our study provides insights into MRPL12 signaling in HCC and highlights MRPL12 as a potential therapeutic target.

Trial registration: N/A.

Mitochondrial ribosomal protein L12

Metabolic reprogramming

Oxidative phosphorylation

Yin Yang 1

PI3K-AKT- mTOR pathway.

Liver cancer remains a global health challenge, with an estimated incidence of > 1 million cases by 2025 (1). Hepatocellular carcinoma (HCC) is the most common and fatal primary liver cancers. Although several new chemotherapeutic agents and treatment modalities have been clinically implemented, the survival rate of HCC has not improved significantly and yet to show promising results (2). Therefore, elucidating the mechanisms underlying HCC tumorigenesis and progression is required to develop better therapeutic strategies.

Metabolic reprogramming is a critical hallmark of cancer progression. Besides having bioenergetic functions, mitochondrial metabolism influences all steps related to oncogenesis by generating building blocks for tumor anabolism, maintaining redox homeostasis, participating in transcriptional regulation, and governing cell death (3). Hence, mitochondria have garnered significant attention as a potential cancer therapeutic target (4, 5). In particular, metabolic reprogramming is also a key feature of HCC, and mitochondrial defects are well-documented in this cancer (6–8). Metabolites generated during metabolic reprogramming may have cancer-promoting effects and even function as signature biomolecules of tumor cells (6). Moreover, damage or mutation of mitochondrial DNA (mtDNA) may further potentiate HCC. A low mtDNA copy number is associated with liver cancer and may confer resistance to chemotherapy (9, 10). Although there is a strong evidence linking mitochondrial dysfunction to HCC pathogenesis, a more precise understanding of the underlying molecular mechanism needs to be established.

Mitochondrial ribosomal proteins (MRPs) are essential components for the structural and functional integrity of the mitoribosome complex. MRP encoding gene family has descended from a single ancestral gene, and each MRP has a unique and essential role in the mitoribosome complex (11). MRP L12 (MRPL12) was the first mitochondrial ribosomal protein characterized in mammals. MRPL12 is implicated in transcription by coordinating mitochondrial ribosome biogenesis; moreover, its role is involved in transcription in human mitochondria, wherein it is speculated to interact with the mitochondrial RNA polymerase to directly activate mtDNA transcription and modulate mitochondrial gene expression (12–14). MRPL12 mutations lead to growth retardation, neurological deterioration, and mitochondrial translation deficiency (15). In addition, MRPL12 expression and function is inhibited in transformed cells, suggesting that MRPL12 downregulation might either participate in cell transformation or be a consequence of the loss of specialized functions during cell transformation.We initially confirmed that MRPL12 positively controlled mitochondrial oxidative phosphorylation (OXPHOS) and the mtDNA copy number, and MRPL12 knockdown suppressed mitochondrial activity with increased glycolysis and decreased cellular growth in vitro (16). Nevertheless, the biological functions and molecular mechanism of MRPL12 in HCC need to be investigated further.

2.1. Patients and specimens

Human HCC tissues and paired adjacent noncancerous liver tissues were obtained from seven patients with HCC undergoing tumor resection. The study was approved by the Institutional Medical Ethics Committee of Shandong Provincial Hospital. The inclusion criteria were as follows: at least 18 years old and diagnosed with HCC without any local or systemic treatment. The exclusion criteria were as follows: patients with other types of cancer, liver failure, and taking medications that affect liver function. All subjects signed an Institutional Review Board (IRB)-approved informed consent before enrollment in the study.

The human HCC tissue microarray (Shanghai Outdo Biotech Co., Ltd., Shanghai, China) used in this study was constructed with specimens from 94 patients with HCC who underwent curative resection. All the patients did not have adjuvant chemotherapy or radiotherapy. It was approved by the Ethical Committee of Shanghai Outdo Biotech Co., Ltd.

2.2. Immunohistochemistry and scoring

The tissue slides were first incubated with the primary antibody and then with the secondary antibody (Goat anti-rabbit antibody, Zhongshan Biotechnology Co., Beijing, China).The slides were imaged using a microscope slide scanner (Nikon Ti-S, Tokyo, Japan) and analyzed by Image J (version 1.52, NIH, USA). The modified immunoreactive score (IRS) was used to evaluate immunohistochemical staining. The intensity of specific staining was characterized as not present (0), weak but detectable above control (1), moderate (2), and very strong (3). The percentage of positive cells was evaluated as follows: no staining, 0; <10%, 1; 10–50%, 2; 51–80%, 3; >80%, 4. IRS was assessed independently by three pathologists who had no knowledge of clinicopathologic information or patient outcome. The final score (ranging from 0 to 12) was obtained by multiplying the two scores in each case. A score of < 6 was deemed as low expression, whereas a score ≥ 6 was considered to be high expression.

2.3. Cell lines and Cell culture

Human HCC cell line MHCC97H, was obtained from Shanghai Zhongqiaoxinzhou Biotech. The immortalized normal liver cell line LM3, human HCC cell lines HepG2 and Huh7 were purchased from Shanghai FuHeng Biology. Authentication of cell lines used in this study was performed by short tandem repeat (STR) DNA Profiling and no cellular cross contamination was detected. Cells were cultured following the instruction:

The reagents used in the present study were as follows: Oligomycin A (50µM, OXPHOS inhibitor) for 4h, MHY1845(10µM, mTOR inhibitor) for 6h, LY294002 (15µM, PI3K inhibitor), Rapamycin (10µM, mTOR inhibitor) or 740Y-P (30µM, PI3K activator) for 24h, Crizotinib (8µM, C-MET inhibitor) or 2-Deoxy-D-glucose (2-DG) (5mM, Glycolysis inhibitors) for 48h. All these reagents were purchased from Selleck (Shanghai, China).

2.4. Transfection assay

Overexpression plasmids of MRPL12 and YY1 and the control empty vector were constructed by Genomeditech (Shanghai, China). Lentivirus by which to knock down MRPL12 (sense: 5ʹ-TCAACGAGCTCCTGAAGAAA-3ʹ) was obtained from GenePharma (Shanghai, China,). SiRNAs targeting YY1 (sense: 5ʹ-CCAAACAACUGGCAGAAUUTT-3ʹ) were synthesized by RiboBio (Guangzhou, China). Cells in 6-well plates were transfected with plasmids using Lipofectamine 3000 (Invitrogen, Shanghai, China) or with siRNA using riboFECTTM CP Transfection kit (RiboBio, Guangzhou, China) according to the manufacturer's instructions.

2.5. Western blotting

Treated cells were harvested directly in RIPA (Solarbio, Beijing, China) buffer containing protease and phosphatase inhibitor Cocktail (Solarbio, Beijing, China). 25–50µg proteins were electrophoresed, and electroblotted to PVDF membrane (Merck, Nantong, Jiangsu, China). Then the membranes were incubated in primary antibodies overnight at 4˚C. Primary antibodies were detected with HRP-conjugated goat-anti rabbit or goat-anti mouse secondary antibody (Invitrogen, Shanghai, China). The bands were visualized by Tanon 5200 (Molecular Devices, Beijing, China), and were analyzed by Image J (version 1.52, NIH, USA).

The following primary antibodies were used: anti-MRPL12, anti-PCNA, anti-MMP2, anti-E-cadherin, anti-Bcl-2, anti-BAX, anti-ND1, anti-COXII, anti-CYTB, anti-ATP6, anti-YY1, anti-GAPDH (Proteintech, Wuhan, China), anti-ND6 (Absin, Shanghai, China), and anti-Ki67 (Abcam, Shanghai, China) (Supplementary Table S1).

2.6. Real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted from treated cells using TRIzol Reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. 1µg mRNA was reversed-transcribed to cDNA with PrimeScript™ RT reagent Kit (Perfect Real Time) (TAKARA, Beijing, China). Quantitative real-time PCRs were performed in triplicates using ABGENE PCR STRIPS (Thermo, Shanghai, China). Thermal cycling conditions were 30s at 95˚C, then 40 cycles of 95˚C for 5s and 60˚C for 30s, followed by 95˚C for 5s and 60˚C for 1 min on QuantStudioTM Real-Time PCR Instrument (Thermo Fisher Scientific, Shanghai, China). The primers were obtained from BIOSUNE (Shanghai, China) (Supplementary Table S2)..

2.7. CCK-8 cell viability assays

Cells were seeded into 96-well plates (3000 cells/well) with 100 µL medium and incubated overnight. Then, cells were treated with the negative control or the indicated drugs for 12h, 24h, 36h, 48h, 60h and 72h. At the time of analysis, 10 µL CCK-8 reagent (MedChem Express, Monmouth Junction, NJ, USA) was added to each well. After 1 h of incubation, the OD values were determined using a microplate reader (SpectraMax i3x, USA) at a wavelength of 450 nm. Experiments were performed in triplicates.

2.8. 5-Ethynyl-20-deoxyuridine assay

Cells cultured in 96-well plates were incubated with 5-ethynyl-20-deoxyuridine (EdU, RiboBio, Guangzhou, China) for 5 h following the manufacturer's instruction. After three washes with PBS, the cells were treated with Apollo reaction cocktail for 30 minutes. Then, the DNA contents of the cells were stained and visualized under fluorescence microscope (Nikon Ti-S, Tokyo, Japan). Images were obtained by Nikon microscope imaging system (Nikon Ti-S, Tokyo, Japan), and analyzed by image J (version 1.52, NIH, USA).

2.9. Transwell Assays

Transwell assays were performed with BD chambers (8-mm pores; BD Biosciences, Shanghai, China). About 1x105 cells/well were seeded in the upper chamber and cultured in serum-free medium. Medium with 30% serum was placed in the lower chambers. After migration through the Transwell membrane, the cells were fixed with 4% paraformaldehyde and stained with crystal violet (Solarbio, Beijing, China). The difference between the migration and invasion assays was the Transwell chambers for migration assays were not coated with Matrigel. All Transwell treatments were conducted in triplicates.

2.10. Flow Cytometry

Following different treatments, cell apoptosis was detected using BD PharmingenTM PE Annexin V Apoptosis Detection Kit I (BD Biosciences, Shanghai, China) in accordance with the manufacturer’s instructions. Cells were harvested after trypsin digestion, centrifuged, and washed with Annexin V binding buffer for three times. Then, cells were stained with PE Annexin V and 7-AAD and analyzed by flow cytometry (BD FACSAria II, Shanghai, China).The results were analyzed using FlowJo software (version 10, Ashland, OR, USA).

2.11. Immunofluorescence Staining

For mitochondrial staining, cells were incubated with MitoTracker Red CMXRos (Thermo Fisher Scientific, Shanghai, China), and then fixed with 4% paraformaldehyde (Solarbio, Beijing, China). After washing 3 times, cells were permeabilized in 0.1% Triton X-100 (Solarbio, Beijing, China), blocked for 1 h with 1% goat serum (Solarbio, Beijing, China), and incubated with MRPL12 primary antibody (1:200; Proteintech, Wuhan, China) overnight at 4°C. Followed by incubation at 37°C with secondary antibody Alexa Fluor®-488 and secondary antibody Alexa Fluor®-594 (Cell Signaling Technology, Shanghai, China), nuclei were counterstained with Hoechst 33342 (Life technologies, Shanghai, China). Fluorescent images were taken with Nikon microscope imaging system (Nikon, Tokyo, Japan) and analyzed by Image J (version 1.52, NIH, USA).

2.12. Transmission Electron Microscopy

Transmission electron microscopy was used to examine structural changes of mitochondria. Control, MRPL12-OE and MRPL12-KD HepG2 cells were pre-fixed with 4% glutaraldehyde and then fixed with 1% citric acid. Samples from transplanted tumors derived from MRPL12-OE or -KD HepG2 cells were dehydrated with ethanol, embedded with Eponate 12 epoxy resin, and semi-thin sliced by LEICA EM UC7 Ultra-Thin Slicer (Shanghai, China). After double staining with uranyl acetate and lead citrate, slides were observed under transmission electron microscope (JEOL, Tokyo,Japan).

2.13. Mitochondrial DNA copy number Quantification

Total DNA was isolated from treated cells using Genomic DNA Mini Preparation Kit (Beyotime, Shanghai, China). Reactions for quantitative real-time PCR assay (qPCR) were performed with SYBR Premix EX Taq II kit (Takara, Japan). For mtDNA quantification, the primers sequences were used as follow: D-Loop 2: F-GGCTCTCAACTCCAGCATGT; R-AGGACGAGGGAGGCTACAAT; G6PC: F-CTGTCTTTGATTCCTGCCTCAT; R- GTGGCTGTGCAGACATTCAA. G6PC were regarded as the nuclear control.

2.14. Chromatin Immunoprecipitation (ChIP) assay

Cells were cross-linked with 1% formaldehyde for 10 min and quenched in 125mM glycine for 5 min at room temperature. After incubation in cold lysis buffer for 20 min, cells were harvested with cell scraper. DNA was immunoprecipitated from the sonicated cell lysates using 2µg anti-YY1 antibody (Proteintech, Wuhan, China). Purified DNA was quantified by real-time PCR performed with human MRPL12 promoter primers which probably contained a YY1 binding site predicted from ALLGENPROMO3.0.2. (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo)

2.15. Luciferase Reporter Assay

Cells were seeded in twelve-well plates and transfected with overexpression plasmids of YY1 using Lipofectamine® 3000 (Invitrogen, Shanghai, China). In the meantime, the cells were co-transfected with firefly luciferase reporter plasmids containing wild-type or mutant YY1 binding site. After 48 hours, Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System according to the manufacturers’ instructions (Promega, Madison, WI, USA).

2.16. Seahorse Assay

The OCR and ECAR were determined by XF96e machine using Seahorse XF Cell Mito Stress Test and Glycolysis Stress Test assay following the manufacturer’s instructions (Agilent Technologies, Beijing, China). Treated cells were plated in XF96 cell culture microplates (Agilent Technologies, Beijing, China). Cells were incubated at 37˚C with 5% CO2 overnight for 24 h and changed the medium to a pre-warm assay medium. After non-CO2 incubation for 1 h, cells were treated with 2µM oligomycin, 1µM FCCP or 5µM rotenone/antimycin A (Agilent Technologies, Beijing, China).

2.17. HCC tumor-xenograft mouse model

The animal study was approved by Animal Ethics Committee of Shandong Provincial Hospital. Four-to-five-week-old BALB/c nude mice were used in the present study. Cells after different treatments were prepared with 3 × 106 cells/100 µL. The suspensions were administered subcutaneously to nude mice. Two weeks after injection, the tumor-bearing mice were received intraperitoneal injection of 100 mg/kg LY294002 for 21 days.Tumor size was measured weekly and tumor volume was calculated using the formula: volume (mm3) = tumor length × width2/2. After five weeks, all mice were sacrificed to compare the tumor volume and weight. Tumors were fixed in 10% formalin and paraffin embedded for subsequent IHC examination.

2.18. Statistical analysis

Continuous data following a normal distribution was analyzed by Student's t test or Welch's t test; continuous data with a non-normal distribution was assessed by Mann-Whitney U test. Chi-squared test was used for dichotomous variable and categorical data. Correlations between MRPL12 expression and clinicopathologic characteristics wereevaluated using chi-squared test. Univariate Cox regression and survival analyses were also conducted. Data are presented in bar plots as mean ± standard deviation (SD) of at least three independent experiments. A p-value of < 0.05 (two-tailed) was considered statistically significant. All statistical analyses were performed using Prism 8.0 (GraphPad Software, San Diego, CA).

3.1. MRPL12 is upregulated in HCC tissues

An integrative analysis of data from The Cancer Genome Atlas (TCGA) showed that MRPL12 was strongly expressed in various cancers, including HCC (Fig. 1 (T1) A-B). A whole transcriptome sequencing analysis of 50 pairs of human HCC tumors and their corresponding non-tumorous liver (NTL) tissues was performed. As shown in Fig. 1 (T1) C, the mRNA levels of MRPL12 were increased in HCC. Moreover, MRPL12 expression was associated with tumor stages (Fig. 1 (T1) D) instead of differentiation grades. In addition, an analysis of 182 pairs of matched patients with HCC from TCGA database indicated that a higher level of MRPL12 was correlated with a poor survival rate (Fig. 1 (T1) E).

To determine the clinical significance of MRPL12 upregulation in HCC tissues, immunostaining was performed on tissue microarrays from 94 patients with HCC, of which paired HCC and NTL tissues were obtained from 86 patients. Representative images of tissues at stages 1, 2, 3, and 4 are shown in Fig. 1 (T2) A. The average IHC score of MRPL12 in HCC increased significantly; moreover, enhanced expression of MRPL12 in HCC stages 1, 2, 3, and 4 was quantified and compared (Fig. 1 (T2) B). The MRPL12 expression pattern was analyzed in 86 HCC cases that were divided into two groups (high and low expression) based on the IHC score with a cut-off value of 6 (P < 0.001) (Fig. 1 (T2) C). Furthermore, correlations between MRPL12 and clinical variables were assessed (Supplementary Table S3). MRPL12 expression was significantly associated with American Joint Committee on Cancer stage (P = 0.035) and T stage (P = 0.027). Of the 94 HCC patients, the overall survival rate of those with MRPL12 overexpression was remarkably lower than that of patients with low MRPL12 expression (Fig. 1 (T2) D), which was consistent with the results of TCGA data analysis. Moreover, the MRPL12 leveland clinical parameters with overall survival rate were also evaluated on univariate analyses (Supplementary Table S4). HCC and NTL tissues were collected from seven patients who underwent surgical resection, for further validation. Upregulated expression of MRPL12 was evaluated by performing western blot analysis and real-time quantitative polymerase chain reaction (RT-qPCR) (Fig. 1 (T2) F).

Together, these findings indicated that MRPL12 accumulation was closely associated with the clinical outcome in patients with HCC and might be an independent risk factor of HCC mortality.

3.2. MRPL12 potentiates HCC proliferation, migration, and invasion in vitro and in vivo

Specific knockdown and overexpression approaches were used to explore the role of MRPL12 in HCC cells. First, we detected the expression of MRPL12 in four cell lines, namely LM3, Huh-7, MHCC97H, and HepG2. The MHCC97H and HepG2 cell lines were selected for subsequent experiments because of their high MRPL12 levels. The knockdown and overexpression efficiencies were determined by western blotting and RT-qPCR (Fig. 1 (T3) A). MRPL12 significantly potentiated the proliferation of HCC cells as shown by Cell Counting Kit-8 and 5-ethynyl-2 deoxyuridine uptake assays (Fig. 1 (T3) B-C). HCC cells were arrested in the S phase (Fig. 1 (T3) D). As presented in Fig. 1 (T3) E, the anti-apoptotic ability of HCC cells was inhibited with MRPL12 knockdown. Transwell assays showed that MRPL12 depletion suppressed mobility and metastasis of HCC cells compared with control cells (Fig. 1 (T3) F). In contrast, MRPL12 overexpression in HCC cells exhibited opposite effects.

In vivo experiments were performed to verify the effects of MRPL12 knockdown or overexpression on HCC tumorigenicity. MRPL12 knockdown dramatically suppressed tumor formation in nude mice with smaller tumors being detected (Fig. 1 (T4) A-C). In contrast, MRPL12 overexpression yielded opposite results. Western blot or IHC analysis of the transplanted tumor showed corresponding alterations in the expression of MRPL12 together with Ki-67 and Proliferating cell nuclear antigen (PCNA) (proliferation markers), Matrix metalloproteinase 2 (MMP2) and epithelial cadherin (E-cadherin) (metastasis markers), and B-cell lymphoma 2 (Bcl-2) and Bax (apoptosis markers) (Fig. 1 (T4) D-E). These results suggest that MRPL12 is involved in HCC tumorigenesis and progression in vitro and in vivo and might act as a candidate therapeutic target for patients with HCC.

3.3. MRPL12 mediates its effects in HCC via regulating mitochondrial function

MRPL12 acts to coordinate mitochondrial ribosome biogenesis and transcription in human mitochondria, highlighting its role in mitochondrial homeostasis. Therefore, we hypothesized that the oncogenic role of MRPL12 in HCC may be correlated with metabolic reprogramming. To test this hypothesis, abnormal mitochondrial structural changes were monitored in HCC cells in response to over- or under-expression of MRPL12 using transmission electron microscopy. Some differences in mitochondrial morphology were noticed between the two groups. In the knockdown group, mitochondria had an expanded matrix space with less dense staining of the matrix, and destruction of cristae; furthermore, the outer membrane was found ruptured. However, the overexpressing group exhibited less severe changes (Fig. 2 (T1) A). The expression of mtDNA genes encoding respiratory chain complex subunits was evaluated in HCC cells. Both MRPL12 knockdown and overexpression led to corresponding changes in protein and mRNA levels of target genes (Fig. 2 (T1) B). Immunofluorescence and mitochondrial staining for detecting the localization of MRPL12 and mitochondria were performed in viable HCC cells. Our results showed a consistent trend of decreased MRPL12 expression and reduced mitochondria numbers after MRPL12 knockdown. Conversely, MRPL12 overexpression was discovered with enriched mitochondria number. Next, we analyzed mtDNA that is required to maintain normal mitochondrial respiratory functions (Fig. 2 (T1) D). The mtDNA copy number reduction occurred after MRPL12 knockdown. IHC was performed in sections of transplant tumors derived from MRPL12- knockdown or -overexpression HepG2 cells. The expression of mtDNA genes encoding subunits of the respiratory chain complexes was found with corresponding alterations.

In addition, MRPL12 overexpression or knockdown were accompanied by enhanced or decreased oxygen consumption rates (OCR), respectively; However, no significant differences were observed in glycolytic function as assessed by the extracellular acidification rate (ECAR) (Fig. 2 (T2) A), suggesting that MRPL12 might be involved in regulating the aerobic respiration of mitochondria. Of note, HCC cell proliferation and metastasis were suppressed after exposure to an aerobic respiration inhibitor, and partially restored following MRPL12 overexpression; whereas the glycolysis inhibitor did not affect cell proliferation and invasion (Fig. 2 (T2) B-F), supporting the significance of MRPL12 in maintaining mitochondrial function by OXPHOS. Collectively, these results supported our hypothesis that MRPL12 might promote HCC progression by serving as a pivotal regulator of mitochondrial functions.

3.4. MRPL12 is transcriptionally regulated and driven by Yin Yang 1 (YY1) in HCC cells

To determine how MRPL12 expression was transcriptionally regulated, we firstly uncovered that YY1 might physically interact with the MRPL12 promoter region (Fig. 2 (T3) A). Co-immunoprecipitation and dual-luciferase assays were then performed to clarify the specific binding of YY1 with MRPL12 (Fig. 2 (T3) B-C). The knockdown and overexpression efficiencies were evaluated by western blotting and RT-qPCR. YY1 depletion suppressed MRPL12 expression in HCC cells, coupled with reduced proliferation abilities in vitro and in vivo (Fig. 2 (T3) D-F). Furthermore, the effect of YY1 overexpression or knockdown was rescued by MRPL12 knockdown or overexpression, respectively (Fig. 2 (T3) D-E). IHC was carried out in the sections of transplant tumors derived from YY1 -knockdown or -overexpression HepG2 cells. Lower MRPL12 expression was confirmed with smaller tumor formation in YY1-knockdown group (Fig. 2 (T3)). These results shed light that YY1 is a functional upstream modulator of MRPL12 in HCC cells, and targeting MRPL12 may be a novel strategy for controlling YY1-mediated tumor progression.

3.5. The PI3K/mTOR/YY1 axis exhibits an oncogenic effect via MRPL12 in HCC

The oncogenic pathways, PI3K-AKT-mTOR and the mesenchymal-epithelial transition (MET), are reportedly involved in hepatocarcinogenesis and HCC progression. In particular, recent studies have demonstrated that mTOR directly interacts with YY1 (17, 18). We discovered that the PI3K/mTOR signaling pathway was positively correlated with YY1 and MRPL12 expression and might have a role in regulating MRPL12 function in HCC. As shown in Fig. 8A, MRPL12 expression in HCC cells was not affected after exposure to an MET inhibitor (Crizotinib), while PI3K/mTOR inhibitors (LY294002/Rapamycin) or agonists (740Y-P/MHY1485) led to alterations in MRPL12 levels (Fig. 2 (T4) A-B). YY1 expression was also decreased after exposure to PI3K/mTOR inhibitors (Fig. 2 (T4) A). In addition, HCC cell proliferation and metastasis were suppressed following PI3K inhibitor treatment, whereas MRPL12 overexpression rescued these effects of the PI3K inhibitor (Fig. 2 (T4) C). In contrast, PI3K agonist induced HCC cell proliferation and metastasis, which was reversed partially by MRPL12 knockdown (Fig. 2 (T4) D). Furthermore, in vivo data confirmed that HCC cells overexpressing MRPL12 formed larger liver tumors which was inhibited by PI3K inhibitor treatment (Fig. 2 (T4) E-F). Taken together, these findings indicate that MRPL12 mediates the potentiation of HCC progression via the PI3K/mTOR/YY1 axis.

The present study established an innovative link between the metabolic alterations induced by MRPL12 and HCC progression. MRPL12 protein represents the first mitochondrial ribosomal protein hitherto characterized in mammals (19). It is encoded by a gene located on 17q25 and is transported into the mitochondrial matrix to bind with the rRNAs assembling subunit of the mitochondrial ribosome (12, 20). MRPL12 plays critical roles by participating in the translation of mitochondrial proteins, activating mtDNA transcription by directly binding to mitochondrial RNA polymerase, initiating mitochondrial biosynthesis, and regulating mitochondrial OXPHOS (13).The observed effects of MRPL12 on the expression of mtDNA-encoded proteins might involve not only its function in ribosome assembly and translation, but also a role in transcription (12). AA c.542C > T transition of the MRPL12 gene directly results in the abnormal assembly of the mitochondrial OXPHOS complex, accompanied by defects in whole mitochondrial protein translation, and expression and activity of molecules involves in OXPHOS (15).

MRPL12 has been reported as a pathogenic factor in many diseases, including cancer (21). In the present study, we found that MRPL12 expression was remarkably upregulated in HCC; moreover, MRPL12 overexpression was closely associated with more advanced tumor stages and poor prognosis in HCC patients. Functional characterization of MRPL12 by MRPL12-specific knockdown or overexpression in vitro and in vivo revealed that MRPL12 potentiated cell proliferation, stimulated migration and invasion, and increased anti-apoptotic ability in HCC cells, suggesting MRPL12 may be a oncogenic molecule and plays an important role in the progression and metastasis of HCC.

Of note, MRPL12 is involved in maintaining mitochondrial homeostasis, and mitochondrial dysfunction is reportedly associated with HCC carcinogenesis and progression (8). Herein, MRPL12 knockdown induced mitochondrial dysfunction in HCC. Impressively, the biosynthesis and detoxification functions of the liver are highly dependent on ATP production by mitochondria. After MRPL12 overexpression or knockdown, we observed abnormal mitochondrial structural changes in HCC cells. In addition, the mtDNA copy number increased with MRPL12 overexpression, and the expression of mtDNA genes encoding respiratory chain complex subunits was activated by MRPL12 overexpression. Immunofluorescence indicated a consistent trend between increased MRPL12 expression and increased mitochondrial numbers. These results provide evidence for the oncogenic role played by MRPL12 via serving as a pivotal regulator of mitochondrial functions in HCC.

Reprogrammed metabolism is commonly observed in many cancer types (22, 23). During hypoxia, alternating metabolic response between glycolysis and OXPHOS provides metabolic flexibility that is beneficial to cancer (24). The Warburg effect, characterized by aerobic glycolysis, is a metabolic hallmark of most cancer cells (25). However, anti-tumor therapy targeting the Warburg effect has yet to achieve ideal results in clinical settings, since the Warburg effect in the tumor microenvironment remains debatable and has not been fully elucidated. Furthermore, increasing evidence shows that the occurrence of some cancers is highly dependent on OXPHOS (4, 26); and decreased OXPHOS activity in such cancers may be related to mitochondrial gene mutations or a reduction in the mtDNA content required to maintain normal mitochondrial respiratory function. Recent studies have also expanded our understanding of the complex mechanisms underlying the involvement of altered mtDNA in tumorigenesis, tumor progression, and patient prognosis in cancers such as HCC (27). In the present study, MRPL12 was seemingly involved in regulating the aerobic respiration. HCC cells with MRPL12 knockdown exhibited a reduction in four phases of the real-time OCR: basal respiration, ATP-producing capability, maximum respiration, and spare respiratory capacity. In contrast, MRPL12 overexpression potentiated intracellular ATP production and achieving maximal respiratory capacity. However, no significant differences were detected in glycolytic functions, highlighting that MRPL12 is involved in regulating the aerobic respiration. This conclusion was further verified by the use of aerobic respiration and glycolysis inhibitors. HCC cell proliferation was suppressed by inhibiting aerobic respiration but not by inhibiting glycolysis. We, therefore, concluded that MRPL12 might maintain mitochondrial function and play an oncogenic role by activating mitochondrial OXPHOS and promoting mitochondrial biosynthesis.

To elucidate the potential upstream mechanism underlying MRPL12 activation in HCC, a bioinformatics-based analysis was conducted. The results showed that MRPL12 is a common target of the transcriptional factor Yin Yang 1 (YY1), as also evidenced by the chromatin immunoprecipitation and dual-luciferase assays. YY1 is a zinc-finger protein that belongs to the GLI-Krüppel family. In humans, YY1 is encoded by a 23-kb gene located at the chromosomal locus, 14q32 (28, 29). YY1 affects the transcriptional activity of approximately 10% of mammalian genes, and is implicated in various cellular mechanisms (30). Additionally, YY1 is also transcriptionally involved in controlling several mitochondrial genes, thereby facilitating metabolic reprogramming, modulating mitochondrial pathways, and affecting energy balance (28). Moreover, the involvement of YY1 in regulating genes that are directly linked to malignant transformation suggested that it might play a key role in many cancers. Indeed, YY1 activity is upregulated in various tumors (31, 32), and closely related to tumor progression and poor prognosis (33–35). We performed co-immunoprecipitation and dual-luciferase reporter assays to show the specific binding of YY1 with MRPL12. YY1 depletion suppressed MRPL12 expression in HCC cells, resulting in reduced cellular proliferation and migration. Furthermore, enhanced migration and invasion induced by YY1 overexpression were attenuated by MRPL12 knockdown in HCC cells. Overall, our results revealed that YY1 is a functional upstream modulator of MRPL12 in HCC, and that YY1-promoted HCC proliferation and invasion might be mediated via MRPL12.

Accumulating evidence indicated that the activation of oncogenic pathways, such as PI3K-AKT-mTOR and MET, have been well-documented in hepatocarcinogenesis and tumor progression (36, 37). We discovered that the PI3K/mTOR signaling pathway was positively correlated with MRPL12 expression and was involved in MRPL12-mediated regulation of HCC. MRPL12 expression in HCC cells was analyzed after exposure to an MET inhibitor, and PI3K/mTOR inhibitors or agonists; PI3K/mTOR inhibitors and agonists altered MRPL12 expression. Moreover, inducing PI3K activates AKT, and subsequently mTOR kinase, which is tightly correlated with mitochondrial metabolism and biogenesis (38). These findings provide support for the involvement of PI3K/mTOR in regulating MRPL12 expression. Furthermore, cell proliferation and metastasis were suppressed following treatment with PI3K inhibitor, whereas MRPL12 overexpression restored the effects of the PI3K inhibitor on cell motility and metastasis. Additionally, in vivo data confirmed that HCC cells overexpressing MRPL12 formed larger liver tumors, an effect that was mitigated by PI3K inhibitor treatment. In particular, YY1 was shown to be a direct target of mTOR (17, 39–41), and PI3K/mTOR inhibitors suppressed YY1 expression. The in vivo study also revealed consistent changes in the YY1 level following treatment with PI3K inhibitor or with MRPL12 overexpression. Taken together, the oncogenic pathway orchestrated by PI3K/mTOR may mediate its effects via regulating MRPL12 expression to facilitate hepatocarcinogenesis and tumor development. These deliberations suggest that the PI3K/mTOR/YY1/MRPL12 axis could be crucial in controlling cellular functions related to HCC progression and metastasis.

In summary, we investigated the roles of MRPL12 and its underlying mechanisms in HCC. MRPL12 promoted HCC cell proliferation and migration in vitro and in vivo via modulating mitochondrial functions. Importantly, the PI3K/mTOR/YY1/MRPL12 axis appeared to be crucial in controlling cellular functions. Our findings provide new insights into the roles of MRPL12 and lay a foundation for further research studies in evaluating the potential of MRPL12 as a therapeutic target and prognostic biomarker for HCC.

MRPL12, mitochondrial ribosomal protein L12; HCC, hepatocellular carcinoma; OXPHOS, oxidative phosphorylation; YY1, Yin Yang 1; PI3K, phosphatidylinositol-3-kinase; mTOR, mammalian target of rapamycin; mtDNA, mitochondrial DNA; MRPs, mitochondrial ribosomal proteins; IRB, institutional review board; NTL, non-tumorous liver; OS, overall survival; IHC, immunohistochemistry; IRS, immunoreactive score; STR, short tandem repeat; RT-qPCR, real-time quantitative polymerase chain reaction; ChIP, chromatin immunoprecipitation; MET, mesenchymal-epithelial transition.

6.1. Ethical Approval

The study was approved by the Institutional Medical Ethics Committee of Shandong Provincial Hospital. All subjects signed an Institutional Review Board (IRB)-approved informed consent before enrollment in the study. The human HCC tissue microarray (Shanghai Outdo Biotech Co., Ltd., Shanghai, China) used in this study was constructed with specimens from 94 patients with HCC who underwent curative resection. All the patients did not have adjuvant chemotherapy or radiotherapy. It was approved by the Ethical Committee of Shanghai Outdo Biotech Co., Ltd

6.2. Competing interests

The authors declare no competing interests

6.3. Authors' contributions

L.Y., Y.Z. and W.Q. provided the study design. Z.B., H.S.S., Z.S.W., L.T.T., J.X.Z., Z.Y., D.C., S.T., Y.X.L., and S.S.N., analyzed the data. Z.B., H.S.S, and Z.S.W performed the experiments. L.Y., Y.Z. and W.Q. wrote the manuscript. All authors approved the final submitted version of the manuscript.

6.4. Funding

No funding source has been received.

6.5. Availability of data and materials

All data will be immediately available on request to corresponding authors.

Llovet J, Kelley R, Villanueva A et al. Hepatocellular carcinoma. Nat Rev Dis Primers. 2021;7(1):6.
Ingle P, Samsudin S, Chan P et al. Development and novel therapeutics in hepatocellular carcinoma: a review. Ther Clin Risk Manag. 2016;12:445–455.
Porporato P, Filigheddu N, Pedro J, Kroemer G, Galluzzi L. Mitochondrial metabolism and cancer. Cell Res. 2018;28(3):265–280.
Weinberg S, Chandel N. Targeting mitochondria metabolism for cancer therapy. Nat Chem Biol. 2015;11(1):9–15.
Yang Y, Karakhanova S, Hartwig W et al. Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer Therapy. J Cell Physiol. 2016;231(12):2570–2581.
Nakagawa H, Hayata Y, Kawamura S, Yamada T, Fujiwara N, Koike K. Lipid Metabolic Reprogramming in Hepatocellular Carcinoma. Cancers (Basel). 2018;10(11).
Mello T, Simeone I, Galli A. Mito-Nuclear Communication in Hepatocellular Carcinoma Metabolic Rewiring. Cells. 2019;8(5).
Lee H, Nga H, Tian J, Yi H. Mitochondrial Metabolic Signatures in Hepatocellular Carcinoma. Cells. 2021;10(8).
Yu C, Wang X, Huang L et al. Deciphering the Spectrum of Mitochondrial DNA Mutations in Hepatocellular Carcinoma Using High-Throughput Sequencing. Gene Expr. 2018;18(2):125–134.
Hsu C, Lee H, Wei Y. Mitochondrial DNA alterations and mitochondrial dysfunction in the progression of hepatocellular carcinoma. World J Gastroenterol. 2013;19(47):8880–8886.
Cheong A, Lingutla R, Mager J. Expression analysis of mammalian mitochondrial ribosomal protein genes. Gene Expr Patterns. 2020;38:119147.
Surovtseva Y, Shutt T, Cotney J et al. Mitochondrial ribosomal protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription. Proc Natl Acad Sci U S A. 2011;108(44):17921–17926.
Nouws J, Goswami A, Bestwick M, McCann B, Surovtseva Y, Shadel G. Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import. J Biol Chem. 2016;291(2):989–997.
Wang Z, Cotney J, Shadel G. Human mitochondrial ribosomal protein MRPL12 interacts directly with mitochondrial RNA polymerase to modulate mitochondrial gene expression. J Biol Chem. 2007;282(17):12610–12618.
Serre V, Rozanska A, Beinat M et al. Mutations in mitochondrial ribosomal protein MRPL12 leads to growth retardation, neurological deterioration and mitochondrial translation deficiency. Biochim Biophys Acta. 2013;1832(8):1304–1312.
Chen Y, Cairns R, Papandreou I, Koong A, Denko N. Oxygen consumption can regulate the growth of tumors, a new perspective on the Warburg effect. PloS one. 2009;4(9):e7033.
Cunningham J, Rodgers J, Arlow D, Vazquez F, Mootha V, Puigserver P. mTOR controls mitochondrial oxidative function through a YY1-PGC-1alpha transcriptional complex. Nature. 2007;450(7170):736–740.
Lin J, He Y, Wang B et al. Blocking of YY1 reduce neutrophil infiltration by inhibiting IL-8 production via the PI3K-Akt-mTOR signaling pathway in rheumatoid arthritis. Clin Exp Immunol. 2019;195(2):226–236.
Marty L, Taviaux S, Fort P. Expression and human chromosomal localization to 17q25 of the growth-regulated gene encoding the mitochondrial ribosomal protein MRPL12. Genomics. 1997;41(3):453–457.
Kühl I, Miranda M, Posse V et al. POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA. Sci Adv. 2016;2(8):e1600963.
Kim H, Maiti P, Barrientos A. Mitochondrial ribosomes in cancer. Semin Cancer Biol. 2017;47:67–81.
Hanahan D, Weinberg R. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–674.
Pavlova N, Thompson C. The Emerging Hallmarks of Cancer Metabolism. Cell Metab. 2016;23(1):27–47.
Schito L, Rey S. Cell-Autonomous Metabolic Reprogramming in Hypoxia. Trends Cell Biol. 2018;28(2):128–142.
Xie J, Wu H, Dai C et al. Beyond Warburg effect–dual metabolic nature of cancer cells. Sci Rep. 2014;4:4927.
Ashton T, McKenna W, Kunz-Schughart L, Higgins G. Oxidative Phosphorylation as an Emerging Target in Cancer Therapy. Clin Cancer Res. 2018;24(11):2482–2490.
Zhang X, Wu X, Hu Q et al. Mitochondrial DNA in liver inflammation and oxidative stress. Life Sci. 2019;236:116464.
Shi Y, Seto E, Chang L, Shenk T. Transcriptional repression by YY1, a human GLI-Krüppel-related protein, and relief of repression by adenovirus E1A protein. Cell. 1991;67(2):377–388.
Yao Y, Dupont B, Ghosh S, Fang Y, Leach R, Seto E. Cloning, chromosomal localization and promoter analysis of the human transcription factor YY1. Nucleic Acids Res. 1998;26(16):3776–3783.
Khachigian L. The Yin and Yang of YY1 in tumor growth and suppression. Int J Cancer. 2018;143(3):460–465.
Zhang Q, Stovall D, Inoue K, Sui G. The oncogenic role of Yin Yang 1. Crit Rev Oncog. 2011;16:163–197.
Atchison M, Basu A, Zaprazna K, Papasani M. Mechanisms of Yin Yang 1 in oncogenesis: the importance of indirect effects. Crit Rev Oncog. 2011;16:143–161.
Shi J, Hao A, Zhang Q, Sui G. The role of YY1 in oncogenesis and its potential as a drug target in cancer therapies. Curr Cancer Drug Targets. 2015;15(2):145–157.
de Nigris F, Zanella L, Cacciatore F et al. YY1 overexpression is associated with poor prognosis and metastasis-free survival in patients suffering osteosarcoma. BMC cancer. 2011;11:472.
Zheng L, Chen Y, Ye L et al. miRNA-584-3p inhibits gastric cancer progression by repressing Yin Yang 1- facilitated MMP-14 expression. Sci Rep. 2017;7(1):8967.
Zucman-Rossi J, Villanueva A, Nault J, Llovet J. Genetic Landscape and Biomarkers of Hepatocellular Carcinoma. Gastroenterology. 2015;149(5):1226–1239.e1224.
Whittaker S, Marais R, Zhu A. The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Oncogene. 2010;29(36):4989–5005.
Schieke S, Phillips D, McCoy J et al. The mammalian target of rapamycin (mTOR) pathway regulates mitochondrial oxygen consumption and oxidative capacity. J Biol Chem. 2006;281(37):27643–27652.
Ismail H, Myronova O, Tsuchiya Y, Niewiarowski A, Tsaneva I, Gout I. Identification of the general transcription factor Yin Yang 1 as a novel and specific binding partner for S6 kinase 2. Cell Signal. 2013;25(5):1054–1063.
Ji K, Zheng J, Lv J et al. Skeletal muscle increases FGF21 expression in mitochondrial disorders to compensate for energy metabolic insufficiency by activating the mTOR-YY1-PGC1α pathway. Free Radic Biol Med. 2015;84:161–170.
Blättler S, Verdeguer F, Liesa M et al. Defective mitochondrial morphology and bioenergetic function in mice lacking the transcription factor Yin Yang 1 in skeletal muscle. Mol Cell Biol. 2012;32(16):3333–3346.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

WITHDRAWN: Mitochondrial ribosomal protein L12 potentiates hepatocellular carcinoma by regulating mitochondrial biogenesis and metabolic reprogramming

Status:

Version 1

Editorial Note

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Patients and specimens

2.2. Immunohistochemistry and scoring

2.3. Cell lines and Cell culture

2.4. Transfection assay

2.5. Western blotting

2.6. Real-time polymerase chain reaction (RT-PCR)

2.7. CCK-8 cell viability assays

2.8. 5-Ethynyl-20-deoxyuridine assay

2.9. Transwell Assays

2.10. Flow Cytometry

2.11. Immunofluorescence Staining

2.12. Transmission Electron Microscopy

2.13. Mitochondrial DNA copy number Quantification

2.14. Chromatin Immunoprecipitation (ChIP) assay

2.15. Luciferase Reporter Assay

2.16. Seahorse Assay

2.17. HCC tumor-xenograft mouse model

2.18. Statistical analysis

3. Results

3.1. MRPL12 is upregulated in HCC tissues

3.2. MRPL12 potentiates HCC proliferation, migration, and invasion in vitro and in vivo

3.3. MRPL12 mediates its effects in HCC via regulating mitochondrial function

3.4. MRPL12 is transcriptionally regulated and driven by Yin Yang 1 (YY1) in HCC cells

3.5. The PI3K/mTOR/YY1 axis exhibits an oncogenic effect via MRPL12 in HCC

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1