A positive feedback between cholesterol synthesis and the pentose phosphate pathway rather than glycolysis promotes hepatocellular carcinoma

A positive feedback between cholesterol synthesis and the pentose phosphate pathway rather than glycolysis promotes hepatocellular carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A positive feedback between cholesterol synthesis and the pentose phosphate pathway rather than glycolysis promotes hepatocellular carcinoma Guisheng Song, junjie hu, Ningning Liu, David Song, Clifford Steer, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2485059/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jun, 2023 Read the published version in Oncogene → Version 1 posted 9 You are reading this latest preprint version Abstract Hepatic cholesterol accumulation and hypercholesterolemia are implicated in hepatocellular carcinoma (HCC). However, the therapeutic effects of cholesterol lowering drugs on HCC are controversial, indicating that the relationship between cholesterol metabolism and HCC is more complex than anticipated. A positive feedback between cholesterol synthesis and the pentose phosphate pathway (PPP) rather than glycolysis was formed in tumors of c-Myc mice. Blocking the PPP prevented inhibited cholesterol synthesis and thereby HCC in c-Myc mice, while ablating glycolysis did not affect cholesterol synthesis and failed to prevent c-Myc-induced HCC. Unexpectedly, HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) and G6PD (glucose-6-phosphate dehydrogenase), the rate-limiting enzymes of cholesterol synthesis and the PPP, were identified as direct targets of microRNA-206. By targeting Hmgcr and G6pd , microRNA-206 disrupted the positive feedback and fully prevented HCC in c-Myc mice, while 100% of control mice dies of HCC. Disrupting the interaction of microRNA-206 with Hmgcr and G6pd restored cholesterol synthesis, the PPP and HCC growth that was inhibited by miR-206. Conclusions : This study identified a previously undescribed positive feedback loop between cholesterol synthesis and the PPP, which drives HCC, while microRNA-206 prevents HCC by disrupting this loop. Cholesterol synthesis as a process rather than cholesterol itself is the major contributor of HCC. Biological sciences/Cancer/Cancer metabolism Biological sciences/Molecular biology/Non-coding RNAs DNA synthesis glycolysis microRNA therapeutic agent Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction HCC is a lethal malignancy without effective therapeutic approaches [ 1 ]. The incidence rate of HCC nearly matched its mortality, demonstrating the aggressiveness of this malignancy and limited therapeutic options [ 1 , 2 ]. Although hepatitis B (HBV) and C (HCV) infection are considered the major causal factors of HCC, NAFLD/MAFLD (non-alcoholic fatty liver disease/metabolic associated fatty liver disease) is associated with an increasing incidence of HCC in the Western world [ 3 , 4 ]. Given limited effects of chemotherapy and the insensitivity of HCC to radiotherapy, tumor extirpation represents the only choice for a long-term cure. Unfortunately, even with successful surgical removal, the presence of NAFLD/MAFLD is associated with an increased recurrence of tumor. Although immunotherapies have recently been approved to treat a variety of cancers, this approach is largely unsuccessful for the treatment of HCC. Further studies are needed to identify new targets for developing new drugs for this malignancy. Cholesterol is an important component of cell membrane and required for cell growth; and the liver is the main organ for its synthesis. In addition, cholesterol plays an important role in modulating membrane trafficking and facilitating signal transduction [ 5 ]. An imbalance in cholesterol homeostasis can contribute to liver injury, which triggers HCC. However, the roles of cholesterol in regulating cancer development and the potential of therapeutically targeting cholesterol homeostasis is controversial [ 6 ]. It is reported that hepatic accumulation of cholesterol drives liver injury and subsequent HCC [ 7 ]. Hypercholesterolemia has been considered as a risk factor of HCC [ 8 ]. Statins, the drug for hypercholesterolemia, show the capacity to protect against the development and recurrence of HCC [ 9 – 11 ]. In contrast, other studies reported that statins failed to reduce the incidence of HCC in NAFLD-associated HCC patients [ 12 ]. In mice, atorvastatin exhibits no effect on N-nitrosodiethylamine-induced HCC [ 13 ]. The potential role of cholesterol lowering drugs in treating HCC remains controversial. Cholesterol homeostasis requires collaboration between various organs, which ensures a balance between cholesterol absorption (in the intestine) and cholesterol synthesis and removal in the liver [ 14 ]. In addition to activation of cholesterol synthesis, enhancement of cholesterol absorption and impaired cholesterol removal also contributes to increased hepatic and serum cholesterol [ 14 ]. However, current studies described above only focused on hepatic and serum levels of cholesterol rather than cholesterol synthesis in HCC patients. In addition, statins function by driving hepatic uptake of cholesterol rather than cholesterol synthesis, which could potentially explain some of the controversy in the field. The pentose phosphate pathway (PPP) is a metabolic pathway parallel to glycolysis [ 15 ]. It is widely accepted that glycolysis is a major energy resource for cancer development, while the PPP produces NADPH (reduced nicotinamide adenine dinucleotide phosphate) and ribose 5-phosphate (R5P). R5P is a key substrate of DNA synthesis that is required for cell proliferation; and NADPH provides reducing power for cholesterol synthesis [ 15 – 17 ], suggesting that cholesterol synthesis is closely connected to the PPP and/or glycolysis. Our study was based on the notion that a positive feedback between cholesterol synthesis and the PPP promotes the development of HCC and cholesterol synthesis as a process rather than cholesterol is the major risk factor of HCC. Amplification and overexpression of the cMYC oncogene is frequently observed in HCC patients and is associated with increased aggressiveness and poor prognosis [ 18 , 19 ]. In addition, cMyc-induced HCC in rodents can recapitulate, in a highly reliable way, the phases of tumor initiation and progression that occur in humans [ 20 ]. Considering the role of cholesterol in HCC, we analyzed c-Myc mice and observed a significant increase in cholesterol and metabolites of the PPP in tumors. In addition, c-Myc also significantly impaired biogenesis of micoRNA-206 (miR-206) that directly targeted HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) and G6DP (glucose-6-phosphate dehydrogenase), the rate-limiting enzymes of cholesterol synthesis and the PPP. In this study, we tested the hypothesis that a positive feedback loop between cholesterol synthesis and the PPP promoted HCC development and the disruption of this loop by miR-206 prevented HCC development. Materials And Methods Establishment of c-Myc HCC mice Eight-week-old wild-type male FVB/N mice maintained on normal diet were hydrodynamically injected with either 5 μg pT3-EF1α-cMyc and 0.2 μg pCMV/ SB ( n =6) or 5 μg pT3-EF1α and 0.2 μg pCMV/ SB (control, n =6), as described previously [ 20 ]. Eight weeks post injection, mice were sacrificed for further analysis. Mice were housed, fed, and monitored in accordance with protocols approved by the committee for animal research at the Hubei University of Chinese Medicine and the University of Minnesota. Metabolomics by UPLC-MS/MS 50 mg of livers (in liquid nitrogen) were thawed in a 2 mL EP tube on ice. After 500 uL of pre-cooled extractant (70% methanol aqueous solution) and small steel balls were added to the EP tube, liver tissues were homogenized at 30 Hz for 30 second for four times. Homogenized livers were shaken at 1500 r/min for 5 min, incubated for15 min on ice, and centrifuged with 12,000 r/min at 4 °C for 10 min. The supernatant were collected for UPLC-MS/MS analysis using an LC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system; MS, AB SCIE QTRAP System). The detailed procedure of UPLC analysis is included in Supplementary Materials and Methods. Cholesterol synthesis assay via incorporation of 14 C-acetate sodium The rate of cholesterol synthesis is high in the morning hours [ 21 ]. Therefore, mice were sacrificed between 10 am and 12 pm to collect liver samples. Liver homogenates were prepared based on the protocol described previously [ 22 ]. Cold liver homogenates (400 mg) were incubated in the presence of 2 mM sodium acetate containing 16.7 µCi of 14 C sodium acetate (PerkinElmer), as reported [ 23 ]. After incubation, the liver homogenates were transferred to new 20 mL glass tubes with cap and further saponified for three hours at 70 ℃ in the presence of 5 mL ethanol and 0.5 mL 90% potassium hydroxide (KOH). Cholesterol was precipitated as digitonide and its radioactivity determined in a liquid scintillation counter. Effects of NAD and NADP on cholesterol synthesis Liver homogenates were prepared as described previously [ 24 ]. After centrifuge at 800 x g for 10 minutes at -1 ℃, the supernatant was collected and supplemented with glucose-6-phosphate (G6P, 20 x 10 -3 M), nicotinamide adenine dinucleotide (NAD, 0.8 x 10 -3 M) or nicotinamide adenine dinucleotide phosphate (NADP, 0.8 x 10 -3 M), potassium acetate (2 x 10 -3 M) and 16.7 µCi of 14 C sodium acetate. After one hour of incubation at 37 ℃, the liver homogenate was saponified by adding 90% KOH. Cholesterol was precipitated as digitonide and its radioactivity was determined. Incorporation of 3 H-thymidine into DNA Primary hepatocytes were plated in collagen-coated 35 mm dishes containing DMEM medium. Forty-eight hours after transfection of pT3-EF1α (control), pT3-EF1α-cMyc or a combination of pT3-EF1α-cMyc and pT3-EF1α-shG6pd or pT3-EF1α-shHmgcr, the medium was replaced with fresh media containing 7.5 µCi 3 H-thymidine. After 24 hours, cells were harvested to analyze incorporation of 3 H-thymidine DNA by standard methods [ 25 , 26 ]. In Vitro analysis of 14 C-acetate incorporation into cholesterol After 4 hours of incubation with 10 μCi [2- 14 C] acetate, murine hepatocytes were washed with cold PBS twice, solubilized with 0.1 M sodium hydroxide, and saponified. Nonsaponifiable lipid was extracted with isohexane. Labeled cholesterol was measured by flow scintillography after HPLC. Expression vectors of miR-206 and c-Myc A mouse DNA fragment containing miR-206 precursor was inserted into pT3-EF1α vector, referred to as pT3-EF1α-miR-206 [ 20 ]. To rule out a non-specific effect of the vector, we generated a miR-206 mis-matched-expression vector by mutating the seed region of miR-206 (pT3-EF1α-miR-206-MM). pCMV/ Sleeping Beauty transposase (pCMV/SB) and pT3-EF1α-c-Myc have been described previously [ 20 ]. CRISPR/Cas9 to ablate miR-206 binding sites within 3’UTRs of Hmgcr and G6pd The locations of sgRNA pairs were selected at the boundary of the miR-206 binding site within the 3’UTRs of Hmgcr and G6pd . Two sgRNAs for each of the miR-206 binding sites were designed by CRISPR and synthesized in IDT (Coralville, IA) [ 27 ]. Four pairs of sgRNAs were further cloned into pX601-AAV8-CMV-SaCas9 (Addgene, Watertown, MA), termed AAV8-sgRNA. The viruses of AAV8-SaCas9 and AAV8-sgRNA were packaged and tittered in the Viral Vector and Cloning Core at the University of Minnesota. To delete the miR-206 binding sites, Group Ⅰ mice ( n =6) received 5 μg pT3-EF1α-c-Myc, 10 μg pT3-EF1α-miR-206-MM and 0.6 μg pCMV/SB; Group Ⅱ mice ( n =6) received 5 μg pT3-EF1α-c-Myc, 10 μg pT3-EF1α-miR-206, and 0.6 μg pCMV/SB, and 5x10 11 GC AAV8-SaCas9 viruses; and Group Ⅲ mice ( n =6) received 5 μg pT3-EF1α-c-Myc, 10 μg pT3-EF1α-miR-206, 0.6 μg pCMV/SB, and 5x10 11 GC AAV8-SaCas9 and AAV8-sgRNA viruses. Eight weeks post-injection, mice were sacrificed for further analysis. Statistical A nalysis . Statistical analysis was performed using GraphPad Prism Software®. Data derived from cell-line experiments were presented as mean ± SEM and assessed by a two-tailed Student T-test. Statistical difference for cell cycle progression analysis was evaluated using Chi-squared test. Mann-Whitney test was used to evaluate the statistical significance for mouse experiments. All the experiments were repeated at least three times. P < 0.05 was considered statistically significant. Results Increased cholesterol synthesis in tumors of HCC patients Both cholesterol synthesis and excretion and absorption of cholesterol contribute to change in hepatic and blood cholesterol [ 14 ]. Hepatic cholesterol accumulation is implicated in HCC patients. However, the effect of cholesterol lowering drugs on HCC is controversial. In HCC patients, compared to adjacent normal livers, cholesterol levels were significantly elevated in tumors ( Fig. 1A ). Hepatocytes are the major site of cholesterol synthesis. We next analyzed mRNA levels of HMGCR , the rate-limiting enzyme of cholesterol synthesis, in hepatocytes isolated from adjacent normal livers and HCC tumors. As expected, increased HMGCR mRNA was observed in malignant hepatocytes compared to normal hepatocytes ( Fig. 1B ). In TCGA database, compared to normal individuals ( n =50), HCC patients ( n =369) exhibited high expression of HMGCR ( Fig. 1C ), which was associated with poor survival ( Fig. 1D ). Increased hepatic cholesterol can be caused by activation of cholesterol synthesis, impaired cholesterol excretion and increased cholesterol absorption from the food [ 14 ]. In addition, cholesterol-lowering drugs such as statins exhibit no effect on HCC, leading us to speculate cholesterol synthesis as a process rather than cholesterol itself is the major contributor of HCC development. 14 C-acetate incorporation into cholesterol was much greater in HCC tumors than in normal livers ( Fig. 1E ), suggesting activation of de novo cholesterol synthesis in HCC tumors. In sum, cholesterol synthesis was activated in tumors of HCC patients, and high levels of HMGCR in malignant hepatocytes correlated with poor survival of HCC patients. c-Myc activated hepatic cholesterol synthesis, the pentose phosphate pathway and glycolysis in mice. Almost 30% of HCC patients show c-MYC gene amplification or overexpression [ 28 ]. A positive correlation between HMGCR and c-MYC was observed in tumors of HCC patients from TCGA database ( Supplementary Fig. 1A ), indicating that c-MYC is a potential driver of cholesterol synthesis. HDI of c-Myc led to c-Myc accumulation and increased expression of Hmgcr in hepatocytes of mice ( Supplementary Figure 1B-C ) and triggered development of HCC ( Fig. 2A ). All c-Myc mice died of HCC within eight weeks post injection of c-Myc, while 100% control mice were healthy at that time point ( Fig. 2B ). As we observed in HCC patients, mRNA levels of Hmgcr , enzyme activity of HMGCR, and hepatic cholesterol were significantly increased in tumors of c-Myc mice ( Fig. 2C-E ). Cholesterol synthesis, via acetyl-CoA, interfaces with de novo lipogenesis (DNL), glycolysis and the PPP [ 24 ], suggesting a possible mechanism of action. In fact, metabolites of glycolysis and the PPP were significantly increased in tumors of c-Myc mice ( Fig. 2F ); and acetyl-CoA, the major precursor of cholesterol synthesis, was increased in c-Myc mice ( Fig. 2F ). Consistent with an increase in the glycolytic and the PPP metabolites, expression of the genes controlling glycolysis and the PPP was significantly increased in malignant hepatocytes of c-Myc mice ( Fig. 2G ). An increase in the glycolytic rate was observed in malignant hepatocytes of c-Myc mice ( Fig. 2H ). In sum, c-Myc signaling promoted cholesterol synthesis, glycolysis and the PPP in hepatocytes. Ablation of the PPP reduced cholesterol synthesis and delayed growth of HCC, while ablation of glycolysis did not affect these processes in c-Myc mice. c-Myc activated the PPP and glycolysis ( Fig. 2F-G ). Glycolysis produces pyruvate that can be converted to acetyl-CoA, a precursor of cholesterol synthesis. We next determined the effect of glycolysis on cholesterol synthesis. NAD is a driver of glycolysis [ 24 , 29 ]. We, therefore, treated liver homogenates of pT3 and c-Myc mice with NAD. Although NAD enhanced glycolysis in both pT3 and c-Myc mice ( Supplementary Fig. 2A ), it did not affect cholesterol synthesis in both pT3 and c-Myc mice ( Fig. 3A ). We next deleted pyruvate kinase (PKM), the rate-limiting enzyme of glycolysis in c-Myc mice, which ablated glycolysis ( Supplementary Fig. 3A-B ). Unexpectedly, ablation of Pkm at the time of c-Myc overexpression in murine livers did not affect cholesterol synthesis in c-Myc mice ( Fig. 3B ). However, cholesterol synthesis is still much higher in c-Myc mice compared to pT3 mice ( Fig. 3A ), indicating that other pathways activated by c-Myc such as the PPP might be able to drive cholesterol synthesis in mice. The PPP is a metabolic pathway parallel to glycolysis, which shares a common starting molecule with glycolysis, glucose-6-phosphate (G6P). Two major products of the PPP are R6P and NADPH. As expected, NADPH and R5P as well as expression of G6pd was significantly increased in malignant hepatocytes from c-Myc mice ( Fig. 3C-D, Supplementary Fig. 4A ). NADPH, as a cofactor of HMGCR, is required for cholesterol synthesis. These established findings led us to speculate that the PPP is potentially involved in enhanced cholesterol synthesis in HCC. To test this speculation, we treated liver homogenates of pT3 and c-Myc mice with NADPH. As expected, NADPH significantly increased enzyme activity of G6PD ( Supplementary Fig. 4B ), which in turn increased incorporation of 14 C-acetate into cholesterol in liver homogenates from pT3 and c-Myc mice ( Fig. 3E) . Since the PPP is activated in c-Myc mice, cholesterol synthesis, as revealed by 14 C-acetate labeling experiment, was much higher in c-Myc mice compared to pT3 mice ( Fig. 3E ). To confirm this speculation, we ablated the PPP via knocking down G6pd in hepatocytes of c-Myc mice ( Supplementary Fig. 5 ). Knocking down G6pd significantly inhibited the PPP, which was reflected by a decrease in NADPH and R5P ( Fig. 3F-G ). Consistent with reduced NADPH that is required for cholesterol synthesis, incorporation of 14 C-acetate into cholesterol was also significantly reduced in liver homogenates of c-Myc/shG6pd mice ( Fig. 4H ). Phenotypically, ablation of glycolysis did not affect growth of HCC, while knockdown of G6pd significantly delayed growth of HCC in c-Myc mice ( Fig. 3I-J ). In sum, the PPP at least in part is required for cholesterol synthesis and hepatocarcinogenesis in c-Myc mice. A positive feedback between the PPP and cholesterol synthesis drove DNA synthesis and cell proliferation. Activation of the PPP produces more NADPH, which provides a cofactor for cholesterol synthesis. Enhancement of cholesterol synthesis should rapidly deplete NADPH, a major production of PPP. Therefore, we hypothesized that cholesterol synthesis and the PPP formed a positive feedback loop, which amplifies production of R5P, the substrate of DNA synthesis, and NADPH, co-factor for HMGCR. To determine if activation of the PPP drives cholesterol synthesis, DNA synthesis, and proliferation, three groups of hepatocytes were treated empty vector (pT3), c-Myc, or a combination of c-Myc and G6pd shRNA to knock down G6pd ( Supplementary Fig. 6A ). c-Myc overexpression enhanced the PPP, cholesterol synthesis, DNA synthesis and proliferation of hepatocytes ( Fig. 4A-E ), while knockdown of G6pd offset the effects of c-Myc overexpression ( Fig. 4A-E ). These findings indicated that activation of the PPP is required for c-Myc to drive cholesterol synthesis, DNA synthesis and hepatocyte proliferation. To test if enhancement of cholesterol synthesis promotes the PPP, DNA synthesis and proliferation, three groups of hepatocytes were treated with pT3 (control), c-Myc or a combination of c-Myc and Hmgcr shRNA ( Supplementary Fig. 6B ). c-Myc activated the PPP, cholesterol synthesis, DNA synthesis and hepatocyte proliferation; and knockdown of Hmgcr counteracted the effects of c-Myc ( Fig. 4F-J) . The significant increase in levels of 14 C-acetate-labeled cholesterol and 3 H-thymidine incorporation into DNA was observed in c-Myc mice ( Fig. 4K-L ). In sum, a positive feedback loop between cholesterol synthesis and the PPP enhanced production of cholesterol and R5P, which meets the needs for rapid growth and proliferation of malignant hepatocytes both in vitro and in vivo . miR-206 repressed expression of HMGCR and G6PD in hepatocytes by binding to their 3’UTRs . HMGCR and G6PD are the rate-limiting enzymes of cholesterol synthesis and the PPP. MicroRNAs (miRNAs) can simultaneously fine tune multiple pathways and exhibit the strong therapeutic potential for cancers and other diseases [ 30 ]. We next attempted to identify those miRNAs that can simultaneously target both HMGCR and G6PD . For this purpose, we analyzed murine and human Ago HITS-CLIP databases (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation from Argonaute protein complex) of HMGCR and G6PD . Unexpectedly, miR-206 was identified as the only miRNA that can target human and mouse HMGCR and G6PD and 3’UTRs of mouse and human HMGCR and G6PD contains two miR-206 binding site [ 31 ] [ 32 ] ( Supplementary Table 1 ). To exclude the false positive peaks of Ago-HITS-CLIP, we further used DIANA-microT-CDS to scan the 3’UTRs of murine and human HMGCR and G6PD , confirming the binding sites of miR-206 within the 3’UTRs of murine and human HMGCR and G6PD . 3' UTRs of both human and mouse HMGCR and G6PD mRNAs are 100% complementary to the miR-206 5' seed region exhibiting the highest prediction scores and binding energy ( Fig. 5A-B ). In addition, levels of miR-206 were significantly reduced in malignant hepatocytes isolate from c-Myc mice ( Supplementary Fig. 7 ). All these findings led us to focus on miR-206. In Fig. 1C-D, high levels of HMGCR predicted poor survival of HCC patients. Similarly, elevated levels of G6PD predicted poor survival of HCC patients TCGA database ( Fig. 5C-D ). Inclusion of the 3’UTRs of Hmgcr or G6pd into the luciferase reporter constructs reduced luciferase activities upon co-transfection with miR-206 into Hepa1-6 cells ( Fig. 5 E-F ). Mutation of the miR-206 binding sites within the 3’UTRs of Hmgcr and G6pd was necessary to completely offset the inhibitory effects of miR-206 on luciferase activities ( Fig. 5 E-F ), suggesting that miR-206 directly recognized the predicted binding site within the 3' UTR of Hmgcr and G6pd . miR-206 also reduced protein and mRNA levels of Hmgcr and G6pd in Hepa1-6 cells ( Fig. 5 G-H ). MiR-206 was able to inhibit expression of HMGCR and G6PD in human hepatocytes by binding to their 3’UTRs ( Supplementary Fig. 8A-C ). In sum, HMGCR and G6PD are direct targets of miR-206 in both human and moues hepatocytes. m iR-206 disrupted the positive feedback loop between cholesterol synthesis and the PPP by targeting Hmgcr and G6pd , which impaired DNA synthesis and proliferation of hepatocytes. Since Hmgcr and G6pd are direct targets of miR-206, we hypothesized that miR-206 is able to disrupt the positive feedback loop between cholesterol synthesis and the PPP, thereby inhibiting DNA synthesis, cholesterol synthesis and proliferation of malignant hepatocytes. To test this, CRISPR/Cas9 technique was used to delete the binding sites of miR-206 within the 3’UTRs of both G6pd and Hmgcr in malignant hepatocytes isolated from c-Myc mice [ 33 , 34 ]. Such a design disrupted the interaction of miR-206 with G6pd and Hmgcr ( Supplementary Fig. 9A ), allowing us to determine if G6pd and Hmgcr are required for miR-206 to inhibit cholesterol synthesis and the PPP. Overexpression of miR-206 inhibited cholesterol synthesis, the PPP and DNA synthesis, which was reflected by a significant reduction in incorporation of 14 C-acetate into cholesterol, the PPP metabolites, 3 H-thymidine incorporation into DNA and proliferation of hepatocytes ( Fig. 6A-E ). In contrast, ablating the miR-206 binding sites within the 3’UTRs of both Hmgcr and G6pd offset the inhibitory effects of miR-206 ( Fig. 6A-E ). We assumed that a positive feedback loop between cholesterol synthesis and the PPP amplifies DNA synthesis and proliferation of malignant hepatocytes. Inhibition of either cholesterol synthesis or the PPP should be able to at least in part disrupt this positive feedback loop and thereby prevent DNA synthesis and proliferation. To test this, we ablated the binding sites of miR-206 within the 3’UTR of Hmgcr or G6pd . Ablation of the miR-206 binding sites within the 3’UTR of Hmgcr was able to recover cholesterol synthesis, the PPP, DNA synthesis and cell proliferation that were inhibited by miR-206 ( Supplementary Fig. 9B , Fig. 6F-J ). Similarly, ablation of the miR-206 binding sites within the 3’UTRs of G6pd also recovered cholesterol synthesis, the PPP, DNA synthesis and proliferation ( Supplementary Fig. 9C, Fig. 6K-O ). These results indicate that miR-206 is able to disrupt the positive feedback loop between cholesterol synthesis and the PPP by targeting either Hmgcr or G6pd , which subsequently inhibits DNA synthesis and cell proliferation. miR-206 inhibited cholesterol synthesis and the PPP in c-Myc mice. We next determined if miR-206 was able to simultaneously inhibit cholesterol synthesis and the PPP in vivo . c-Myc mice were injected with pT3-EF1α-miR-206-MM (control) or pT3-EF1α-miR-206. Eight weeks post injection, all miR-206-treated c-Myc mice were healthy, while 100% of c-Myc mice died of HCC ( Fig. 7A ). Upon dissection, no tumors were observed in c-Myc/miR-206 mice ( Fig. 7C) . The long-term survival experiment revealed that all c-Myc/miR-206 mice were healthy 24 weeks of post injection of miR-206 ( Fig. 7B ). Upon dissection, no tumor nodules were observed in livers of this group of c-Myc/miR-206 mice ( Fig. 7C ). All these findings indicated the long-term effect of miR-206 on preventing HCC. Our hypothesis is that miR-206, by disrupting the positive feedback loop between cholesterol synthesis and the PPP, inhibits HCC. As expected, miR-206 significantly reduced expression of Hmgcr and G6pd in hepatocytes of c-Myc mice ( Fig. 7D ). Consistent with reduced Hmgcr and G6pd , both cholesterol and the metabolites of the PPP were significantly reduced in miR-206-treated c-Myc mice ( Fig. 7E-G ). Incorporation of 14 C-acetate into cholesterol and 3 H-thymidine incorporation into DNA were reduced in miR-206-treated c-Myc mice ( Fig. 7H-I ). These results established that miR-206 disrupted the positive feedback loop between cholesterol synthesis and the PPP, which subsequently inhibited DNA synthesis and growth of malignant hepatocytes. Unexpectedly, miR-206 treatment significantly induced expression of genes encoding PFK (phosphofructokinase) and PKM (pyruvate kinase), two rate-limiting enzymes of glycolysis, in c-Myc mice ( Fig. 7J ). The glycolytic rate was also significantly increased in livers of c-Myc/miR-206 mice ( Fig. 7K ). Although miR-206 induced glycolysis, it still fully prevented c-Myc-induced HCC, further indicating that glycolysis is not required for miR-206 to inhibit HCC. This finding is consistent with our observation that ablation of glycolysis failed to prevent c-Myc-induced HCC. In sum, miR-206 disrupted the positive feedback between cholesterol synthesis and the PPP, which prevented c-Myc-induced HCC. HMGCR and G6PD are required for miR-206 to prevent c-Myc-induced HCC. We next employed an AAV8-based CRISPR/Cas9 technique to ablate the binding sites of miR-206 within the 3’UTRs of Hmgcr and G6pd in the genome of hepatocytes in c-Myc mice, which disrupted the interaction of miR-206 with Hmgcr and G6pd . Ablation of the miR-206 binding sites impaired the ability of miR-206 to inhibit expression of Hmgcr and G6pd in hepatocytes ( Fig. 8A ). Phenotypically, 100% c-Myc mice died of HCC within 8 weeks post injection of c-Myc ( Fig. 8B ). MiR-206 fully prevented c-Myc-induced HCC, while disrupting its interaction with Hmgcr and G6pd resulted in renewed growth of HCC that was fully prevented by miR-206 ( Fig. 8B-C ). Mechanistically, miR-206 markedly reduced hepatic cholesterol and metabolites of the PPP, while disrupting the interaction of miR-206 with Hmgcr and G6pd recovered levels of hepatic cholesterol and the metabolites of the PPP ( Fig. 8D-F ). As revealed by 14 C-acetate- and 3 H-thymidine-labeling experiments, ablating the miR-206 binding sites recovered cholesterol synthesis and DNA synthesis in miR-206-treated c-Myc mice ( Fig. 8G-H ). In sum, by disrupting the positive feedback loop between the cholesterol synthesis and the PPP, miR-206 inhibited growth of HCC. Discussion Amplification of c-MYC has been implicated in ~ 27% HCC patients [ 35 ]. It has been reported that c-MYC drives cholesterol synthesis [ 36 ]; and a positive correlation between c-MYC and HMGCR was observed in HCC patients. Furthermore, we discovered that cholesterol synthesis is activated in tumors of HCC patients and high levels of HMGCR predicted poor survival of HCC patients. However, how cholesterol synthesis drives HCC development is poorly understood. To simulate the observation in HCC patients, we overexpressed c-Myc in livers of mice. Cholesterol synthesis is activated in livers of c-Myc mice. Mechanistically, activation of c-Myc maintained the positive feedback loop between cholesterol synthesis and the PPP. Specifically, activation of the PPP overproduced R5P and NADPH that served as substrates of DNA synthesis and the cofactor of HMGCR. Enhancement of cholesterol synthesis depleted NADPH, thereby driving the PPP. This positive feedback loop maintained rapid production of DNA and cholesterol, which are required for rapid growth and proliferation of malignant hepatocytes. These findings potentially explained the rapid development of HCC in c-Myc mice. In our previous study, we observed that miR-206 is able to suppress immunosuppression by preventing overproduction of TGFβ in malignant hepatocytes, which partially contributed to the prevention of c-Myc-induced HCC [ 37 ]. However, miR-206 was able to fully prevent c-Myc-induced HCC, while 100% of c-Myc mice died of HCC within 8 weeks post injection (Fig. 7 A-C). Such a potent inhibitory effect of miR-206 on HCC led us to speculate that in addition to recovering anti-tumor immunity, other mechanism(s) are involved in miR-206-mediated inhibition of HCC in c-Myc mice. Unexpectedly, miR-206 was identified as the only miRNA that can simultaneously target both Hmgcr and G6pd . By simultaneously targeting Hmgcr and G6pd , miR-206 disrupted the positive feedback loop between cholesterol synthesis and the PPP, thereby mitigating cholesterol and DNA synthesis that are required for growth and proliferation of malignant hepatocytes. Our findings fill the knowledge gap regarding the roles of cholesterol synthesis, the PPP and glycolysis in c-Myc-driven HCC. First, we proposed a new concept that cholesterol synthesis rather than cholesterol is the major contributor of HCC development. It is well-established that dysregulated cholesterol metabolism is involved in HCC development. Most of these studies consider cholesterol as a major causal factor of hepatocarcinogenesis [ 12 , 38 ]. However, the major purpose of these studies is to reduce hepatic cholesterol and the findings are controversial [ 6 , 12 , 38 , 39 ]. In fact, levels of hepatic and blood cholesterol are controlled by cholesterol synthesis and excretion and absorption of cholesterol [ 14 ]. Statins function via driving cholesterol reverse transport (RCT) rather than hepatic cholesterol synthesis [ 14 ], which might be the major reason of these controversial findings. In this manuscript, we for the first time established that cholesterol synthesis rather cholesterol is the major contributor of hepatocarcinogenesis, which potentially provides an explanation of controversial findings regarding the roles of cholesterol metabolism in HCC. Second, we observed that the PPP rather than glycolysis promoted cholesterol synthesis. Activation of the PPP and glycolysis has been observed in c-Myc-induced HCC. Cholesterol synthesis is closely connected glycolysis and the PPP and cholesterol synthesis is activated in HCC, urging us to assess if glycolysis or the PPP affects cholesterol synthesis. As described above, activation of the PPP promoted cholesterol synthesis. However, glycolysis exhibited no effect on this process. We established that cholesterol synthesis is required for c-Myc-induced HCC, which potentially explained that ablation of glycolysis did not affect growth of HCC in c-Myc mice. It is well accepted that glycolysis provides energy for rapid growth of tumors. In fact, studies of ATP production by glycolysis and oxidative phosphorylation (OXPHOS) in various types of cells and organs concluded that OXPHOS is the main contributor of ATP under aerobic conditions [ 16 ], further suggesting that glycolysis is not the major energy supply for HCC growth. This is confirmed by our observation that although miR-206 markedly promoted glycolysis, it fully prevented c-Myc-induced HCC (Fig. 7 G). Another explanation for glycolysis to promote HCC is that the pathway provides important biosynthetic precursors [ 40 ]. In fact, the majority of the biomass of proliferating cells is derived from amino acids rather than glucose [ 40 ]. The PPP supports cancer cell growth by providing NADPH for cholesterol synthesis and generating R5P for DNA synthesis [ 41 ]. It is the PPP rather than glycolysis is a major contributor of c-Myc-induced HCC. The third novel observation is the positive feedback loop between cholesterol synthesis and the PPP in c-Myc-induced HCC. Cholesterol accumulation and activation of the PPP have been observed in HCC patients [ 6 , 42 ]. Cholesterol accumulation in the liver contributes to lipotoxicity, hepatic inflammation, and fibrosis, which have been considered key causal factors of HCC [ 38 ]. Conversely, some clinical studies have revealed that high levels of cholesterol are, in fact, associated with a reduced risk of HCC [ 38 ]. Our findings address the controversy in that cholesterol synthesis rather than cholesterol itself is a major driver of hepatocarcinogenesis. In detail, enhancement of cholesterol synthesis, by depleting NADPH, drives the PPP, which produces R5P for DNA synthesis and NADPH for cholesterol synthesis. It is our speculation that rapid growth of cancer cells depletes cholesterol and R5P, which further enhances the positive feedback loop. Indeed, dietary cholesterol treatment significantly reduced enzyme activity of G6PD [ 43 ], further confirming our findings. Somewhat unexpectedly, miR-206 was identified as the only miRNA that can target both HMGCR or G6PD by interacting with two miR-206 binding sites within the 3’UTR of HMGCR or G6PD . Consistently, levels of HMGCR and G6PD are significantly increased in tumors of HCC patients; and a positive association was observed between HMGCR and G6PD in HCC ( Supplementary Fig. 10 ). More importantly, high levels of their expression predicted poor survival among HCC patients. This unexpected finding indicated the strong inhibition of miR-206 on cholesterol synthesis and the PPP. Considering the potent inhibitory effects of miR-206 on HCC, we speculated that miR-206 fully prevents HCC growth via multiple mechanisms. In addition to restoring antitumor immunity [ 37 ], miR-206-mediated inhibition of cholesterol synthesis and the PPP at least in part contributes to HCC prevention. Our final goal is to develop miR-206 as a therapeutic drug for the treatment of HCC. The novel findings in this study provide additional layer of evidence that miR-206 prevents growth of HCC by cutting off building blocks of hepatocyte proliferation. Abbreviations 3'-UTR, 3'-untranslated regions; ATP: adenosine 5′-triphosphate; CRISPR/Cas9, the clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9; DNL: de novo lipogenesis; ECAR: extracellular acidification rate; G6P: glucose-5-phosphate; HBV: hepatitis B; HCV: hepatitis C; HCC, hepatocellular carcinoma; HDI: hydrodynamic injection; HITS-CLIP: high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; MTT: 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NADPH: reduced nicotinamide adenine dinucleotide phosphate; NAFLD/MAFLD: non-alcoholic fatty liver disease/metabolic associated fatty liver disease; OXPHOS: oxidative phosphorylation; PFKL: phosphofructokinase liver type; PKM: pyruvate kinase; PPP: pentose phosphate pathway; R5P: ribose 5-phosphate; sgRNA: guide RNA; TGFβ: transforming growth factor β. Declarations Acknowledgements This work was supported by the Fund for Distinguished Young Scholar of Hubei University of Chinese Medicine awarded to Hu JJ and the Research Scholar Award (IS-16-210-01-RMC) to Song GS Authors’ Contributions Hu J., Liu N and Song D: acquisition of data, analysis, and interpretation of data, and drafting the manuscript. Steer, C: data analysis and manuscript editing. Song G and Zheng G: obtaining funding, study supervision, study concept, design, and revision and final approval of the manuscript. Competing interests The authors involved in this study declared that they have nothing to disclose regarding funding or conflict of interest with respect to this manuscript. Data availability statement All data in this manuscript is available upon reasonable request. References Slotta JE, Kollmar O, Ellenrieder V, Ghadimi BM, Homayounfar K. Hepatocellular carcinoma: Surgeon's view on latest findings and future perspectives. 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Clinicopathological and prognostic significance of OCT4 in patients with hepatocellular carcinoma: a meta-analysis. OncoTargets and Therapy. 2018;11:47-57. Lewis KF, Blumenthal HJ, Weinrach RS, Weinhouse S. An isotope tracer study of glucose catabolism in Pseudomonas fluorescens. Journal of Biological Chemistry. 1955;216:273-86. Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nature Reviews Drug Discovery. 2017;16:203-22. Paraskevopoulou MD, Georgakilas G, Kostoulas N, Vlachos IS, Vergoulis T, Reczko M, et al. DIANA-microT web server v5. 0: service integration into miRNA functional analysis workflows. Nucleic acids research. 2013;41:W169-W73. Li J-H, Liu S, Zhou H, Qu L-H, Yang J-H. starBase v2. 0: decoding miRNA-ceRNA, miRNA-ncRNA and protein–RNA interaction networks from large-scale CLIP-Seq data. Nucleic Acids Research. 2014;42:D92-D7. Yang Y, Wang L, Bell P, McMenamin D, He Z, White J, et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nature Biotechnology. 2016;34:334-8. Qiupeng Zheng XC, Meng How Tan, Steven Schaffert, Christopher P. Arnold, Xue Gong, Chang-Zheng Chen, and Shenglin Huang. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. Biotechniques. 2014;57:115-24. Ally A, Balasundaram M, Carlsen R, Chuah E, Clarke A, Dhalla N, et al. Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell. 2017;169:1327-41. e23. Yang F, Kou J, Liu Z, Li W, Du W. MYC Enhances cholesterol biosynthesis and supports cell proliferation through SQLE. Frontiers in Cell and Developmental Biology. 2021:520. Liu N, Steer CJ, Song G. MicroRNA‐206 enhances antitumor immunity by disrupting the communication between malignant hepatocytes and regulatory T cells in c‐Myc mice. Hepatology. 2022;76:32-47. Zhou F, Sun X. 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Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 26 Jun, 2023 Read the published version in Oncogene → Version 1 posted Editorial decision: revise 16 Feb, 2023 Review # 2 received at journal 15 Feb, 2023 Review # 1 received at journal 10 Feb, 2023 Reviewer # 2 agreed at journal 07 Feb, 2023 Reviewer # 1 agreed at journal 27 Jan, 2023 Reviewers invited by journal 25 Jan, 2023 Submission checks completed at journal 17 Jan, 2023 Editor assigned by journal 16 Jan, 2023 First submitted to journal 16 Jan, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2485059","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":168410775,"identity":"2b5cfd24-5c70-4739-9c13-4d9eddc3a2e9","order_by":0,"name":"Guisheng Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApUlEQVRIiWNgGAWjYNCCCgglQYKWMyRrYWwjRYu8/+FjD7/OOxxtzsB88DYPMVoMb6SlG8tuO5y7s4Et2Zo4LTN4zKQlgVo2HAAyiNPSfwaoZQ5IC/834rTIM+SYSX5sANvCRpwWA4m0NGmGY+m5Gw6zGVvOIcqW/sPHJH/UWOduON788MYbomw5wMDADHYPMzHKwbY0AGPyB7GqR8EoGAWjYGQCAEBJMOP7NjGqAAAAAElFTkSuQmCC","orcid":"","institution":"University of Minnesota","correspondingAuthor":true,"prefix":"","firstName":"Guisheng","middleName":"","lastName":"Song","suffix":""},{"id":168410776,"identity":"4f33381a-2f60-4ddf-b6d5-3ead819ab92d","order_by":1,"name":"junjie hu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"junjie","middleName":"","lastName":"hu","suffix":""},{"id":168410777,"identity":"dd7abd9e-872d-48ac-9220-895a44f3efa6","order_by":2,"name":"Ningning Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ningning","middleName":"","lastName":"Liu","suffix":""},{"id":168410778,"identity":"2a70f1eb-5060-4ef2-bda6-82ab0c85bd97","order_by":3,"name":"David Song","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Song","suffix":""},{"id":168410779,"identity":"0fa350b2-2142-42e1-b827-af3969d8eecc","order_by":4,"name":"Clifford Steer","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Clifford","middleName":"","lastName":"Steer","suffix":""},{"id":168410780,"identity":"8e3919bc-ce30-4eb5-be29-f897a061d825","order_by":5,"name":"Guohua Zheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Guohua","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2023-01-16 18:46:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2485059/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2485059/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41388-023-02757-9","type":"published","date":"2023-06-26T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":32230248,"identity":"afc47e17-d69c-4abc-b720-46cf40d62b9c","added_by":"auto","created_at":"2023-01-30 20:58:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":42071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of cholesterol synthesis predicted poor survival of HCC patients\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Levels of cholesterol in tumors and adjacent normal livers of HCC patients (\u003cem\u003en\u003c/em\u003e=48). NT: normal liver tissues. (\u003cstrong\u003eB\u003c/strong\u003e) mRNA levels of \u003cem\u003eHMGCR\u003c/em\u003e in tumors and adjacent normal livers of HCC patients (\u003cem\u003en\u003c/em\u003e=48), as revealed by qRT-PCR. (\u003cstrong\u003eC\u003c/strong\u003e) A significant increase in \u003cem\u003eHMGCR\u003c/em\u003e in tumors of HCC patients (\u003cem\u003en\u003c/em\u003e=369) versus normal individuals (\u003cem\u003en\u003c/em\u003e=50) from the TCGA database. NT: liver tissues from normal individuals (\u003cstrong\u003eD\u003c/strong\u003e) High levels of \u003cem\u003eHMGCR\u003c/em\u003e predicted poor survival of HCC patients in TCGA database. (\u003cstrong\u003eE\u003c/strong\u003e) Increased cholesterol synthesis in HCC tumors (\u003cem\u003en\u003c/em\u003e=18) versus normal adjacent liver tissues (\u003cem\u003en\u003c/em\u003e=18), which was reflected by increased incorporation of \u003csup\u003e14\u003c/sup\u003eC acetate into cholesterol. Data represent mean ± SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (Fig. 1A-C, E: two-tailed student’s \u003cem\u003et\u003c/em\u003e test; Fig. 1D: log-rank test).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/7fe45c215ffcabeb98fdef1c.png"},{"id":32231067,"identity":"9e6fc271-438e-4bd9-8f69-15bfafbb2002","added_by":"auto","created_at":"2023-01-30 21:14:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":723020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ec-Myc drove cholesterol synthesis, the PPP and HCC development. (A\u003c/strong\u003e) Representative photos of livers and H\u0026amp;E (10X) staining from FVB/NJ mice injected with pT3-EF1α (pT3, \u003cem\u003en\u003c/em\u003e=6, 8 w.p.i) and pT3-EF1α-c-Myc (\u003cem\u003en\u003c/em\u003e=6, 8 w.p.i). LW: liver weight. w.p.i: weeks post injection. (\u003cstrong\u003eB)\u003c/strong\u003eKaplan-Meier survival curves of pT3 and c-Myc mice. (\u003cstrong\u003eC-D\u003c/strong\u003e) mRNA levels of \u003cem\u003eHmgcr \u003c/em\u003eand HMGCR enzyme activities in livers of pT3 and c-Myc mice. (\u003cstrong\u003eE)\u003c/strong\u003e Hepatic cholesterol levels in pT3 and c-Myc mice. (\u003cstrong\u003eF\u003c/strong\u003e) Levels of the metabolites of the PPP and glycolysis in livers of pT3 (\u003cem\u003en\u003c/em\u003e=3) and c-Myc (\u003cem\u003en\u003c/em\u003e=3) mice. (\u003cstrong\u003eG\u003c/strong\u003e) Upregulated genes controlling the PPP and glycolysis in livers of c-Myc mice compared to pT3 mice. (\u003cstrong\u003eH\u003c/strong\u003e) The extracellular acidification rate (ECAR) in hepatocytes isolated from pT3 and c-Myc mice after sequential additions of 10 mM glucose, 2 μg/mL oligomycin, and 100 mM 2-deoxyglucose. Data represent mean ± SEM. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and *** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 (Fig. 2A, C-H: two-tailed student’s \u003cem\u003et\u003c/em\u003etest; Fig. 2B: log-rank test)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/ae3d94daea4d09c87e8c449e.png"},{"id":32230773,"identity":"6d63f050-b3e4-455f-b731-6246a418be98","added_by":"auto","created_at":"2023-01-30 21:06:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1001477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAblation of the PPP reduced cholesterol synthesis and delayed growth of HCC, while ablation of glycolysis failed to affect this processes in c-Myc mice.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) The rates of \u003csup\u003e14\u003c/sup\u003eC acetate incorporation into cholesterol in livers of pT3 (\u003cem\u003en\u003c/em\u003e=6) and c-Myc mice (\u003cem\u003en\u003c/em\u003e=6) after treatment with diphosphopyridine nucleotide (NAD, a driver of glycolysis). (\u003cstrong\u003eB\u003c/strong\u003e) The rates of \u003csup\u003e14\u003c/sup\u003eC acetate incorporation into cholesterol in livers of pT3 (\u003cem\u003en\u003c/em\u003e=6), c-Myc (\u003cem\u003en\u003c/em\u003e=6) or c-Myc and \u003cem\u003ePkm \u003c/em\u003eshRNA (shG6pd, \u003cem\u003en\u003c/em\u003e=6) mouse cohort. (\u003cstrong\u003eC-D\u003c/strong\u003e) Levels of NADPH and ribose-5-phosphate (R5P) in livers of pT3 and c-Myc mice. (\u003cstrong\u003eE\u003c/strong\u003e) The rates of \u003csup\u003e14\u003c/sup\u003eC acetate incorporation into cholesterol in livers of pT3 and c-Myc mice after treatment with triphosphopyridine nucleotide (NADP, a promoter of the PPP). (\u003cstrong\u003eF-G\u003c/strong\u003e) Levels of NADPH and ribose-5-phosphate (R5P) in livers of c-Myc mice treated with scramble (\u003cem\u003en\u003c/em\u003e=6) and \u003cem\u003eG6pd\u003c/em\u003e shRNA (\u003cem\u003en\u003c/em\u003e=6). (\u003cstrong\u003eH\u003c/strong\u003e) The rates of \u003csup\u003e14\u003c/sup\u003eC acetate incorporation into cholesterol in livers of c-Myc mice injected with scramble (\u003cem\u003en\u003c/em\u003e=6) and \u003cem\u003eG6pd \u003c/em\u003eshRNA (\u003cem\u003en\u003c/em\u003e=6). (\u003cstrong\u003eI\u003c/strong\u003e) Representative photos of livers and H\u0026amp;E (10X) staining from FVB/NJ mice injected with pT3-EF1α (pT3, \u003cem\u003en\u003c/em\u003e=6, 8 w.p.i), c-Myc (\u003cem\u003en\u003c/em\u003e=6, 8 w.p.i), c-Myc and \u003cem\u003ePkm\u003c/em\u003e shRNA (c-Myc/shPKM, \u003cem\u003en\u003c/em\u003e=6, 8 w.p.i) or c-Myc and \u003cem\u003eG6pd\u003c/em\u003e shNRA (c-Myc/shG6pd, \u003cem\u003en\u003c/em\u003e=6). LW: liver weight. w.p.i: weeks post injection. (\u003cstrong\u003eJ\u003c/strong\u003e) Kaplan-Meier survival curves of c-Myc, c-Myc/shPkm and c-Myc/shG6pd mouse cohorts. Data represent mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 and ns: no significance (Fig. 3A, B, E, I: two-way ANOVA test; Fig. 3J: log-rank test; and Fig. 3C-D, F-H: two-tailed student’s \u003cem\u003et\u003c/em\u003e test)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/7c92716cb0d2b336192834e3.png"},{"id":32230249,"identity":"91e01b71-c05d-444f-9a70-610e30302c4e","added_by":"auto","created_at":"2023-01-30 20:58:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":72993,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ec-Myc promoted a positive feedback loop between cholesterol synthesis and the PPP, which drove DNA synthesis and cell proliferation. (A-D) \u003c/strong\u003eLevels of R5P and NADPH, the rates of \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol, and the rates of \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA in primary hepatocytes treated with pT3, c-Myc or a combination of c-Myc and \u003cem\u003eG6pd\u003c/em\u003e shRNA. (\u003cstrong\u003eE\u003c/strong\u003e) Proliferation of hepatocytes transfected with pT3, c-Myc or a combination of c-Myc and \u003cem\u003eG6pd\u003c/em\u003e shRNA, as revealed by MTT assay. (\u003cstrong\u003eF-I\u003c/strong\u003e) The rates of \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol, levels of R5P and NADPH, and the rates of \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA in hepatocytes treated with pT3, c-Myc or a combination of c-Myc and \u003cem\u003eHmgcr\u003c/em\u003e shRNA. (\u003cstrong\u003eJ\u003c/strong\u003e) Proliferation of hepatocytes transfected with pT3, c-Myc or a combination of c-Myc and \u003cem\u003eHmgcr \u003c/em\u003eshRNA, as per the MTT assay. (\u003cstrong\u003eK-L\u003c/strong\u003e) The rates of \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol and the rates of \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA in livers of pT3 and c-Myc mice. Data represent mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (Fig. 4A-J: two-way ANOVA test; Fig. 4K-L: two-tailed student’s \u003cem\u003et\u003c/em\u003e test)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/ea900c511ba36ef68a67a7b1.png"},{"id":32230252,"identity":"135ac6d2-6ced-495c-b3da-cbf3de5b9d17","added_by":"auto","created_at":"2023-01-30 20:58:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":80199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eHMGCR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eG6PD\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eare the direct targets of miR-206\u003c/strong\u003e.\u003cstrong\u003e (A-B) \u003c/strong\u003eGraphic representation of the conserved miR-206 binding sites within the 3’UTRs of \u003cem\u003eHMGCR \u003c/em\u003eor \u003cem\u003eG6PD\u003c/em\u003e between human and mouse. (\u003cstrong\u003eC\u003c/strong\u003e) Levels of \u003cem\u003eG6PD\u003c/em\u003e in tumors of HCC patients (\u003cem\u003en\u003c/em\u003e=369) and livers of normal individuals (\u003cem\u003en\u003c/em\u003e=50) from the TCGA database. (\u003cstrong\u003eD\u003c/strong\u003e) High levels of \u003cem\u003eG6PD\u003c/em\u003e predicted poor survival of HCC patients from the TCGA database. (\u003cstrong\u003eE-F\u003c/strong\u003e) miR-206 markedly reduced luciferase activity of the reporter construct containing wild-type 3’UTR of murine \u003cem\u003eHmgcr\u003c/em\u003e or \u003cem\u003eG6pd\u003c/em\u003e. Mutation of two miR-206 sites within the 3’UTR of \u003cem\u003eHmgcr\u003c/em\u003eor \u003cem\u003eG6pd\u003c/em\u003e nullified the ability of miR-206 to inhibit luciferase activity. (\u003cstrong\u003eG-H\u003c/strong\u003e) mRNA and protein levels of \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e in hepatocytes transfected with pT3-EF1α-miR-206-MM (control) or pT3-EF1α-miR-206. Data represent mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01,\u003cem\u003e \u003c/em\u003e***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ns: no significance (Fig. 5C, E-H: two-tailed student’s \u003cem\u003et\u003c/em\u003e test; Fig. 5D: log-rank test)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/70f7bf39408db9ae95743a3e.png"},{"id":32230775,"identity":"143db1a8-5f7f-4193-bf13-f1d39f720801","added_by":"auto","created_at":"2023-01-30 21:06:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-206 inhibited DNA synthesis and cell proliferation by disrupting the positive feedback loop between cholesterol synthesis and the PPP. (A-E) \u003c/strong\u003eThe rates of \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol, levels of R5P and NADPH, the rates of \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA, and cellular proliferation in hepatocytes transfected with pT3, miR-206 or a combination of miR-206 and sgRNAs of both \u003cem\u003eHmgcr \u003c/em\u003eand \u003cem\u003eG6pd\u003c/em\u003e (sgRNAs). Cellular proliferation was evaluated via MTT. (\u003cstrong\u003eF-J\u003c/strong\u003e) The rates of \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol, levels of NADPH and R5P, the rates of \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA, and cellular proliferation in hepatocytes transfected with pT3, miR-206 or a combination of miR-206 and \u003cem\u003eHmgcr\u003c/em\u003e sgRNAs. (\u003cstrong\u003eK-O\u003c/strong\u003e) Levels of R5P and NADPH, the rates of \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol, the rates of \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA, and cellular proliferation in hepatocytes transfected with pT3, miR-206 or a combination of miR-206 and \u003cem\u003eG6pd\u003c/em\u003e sgRNAs. Data represent mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (Fig. 6: two-way ANOVA test)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/e721a5c9a54afe7de8f77cca.png"},{"id":32230255,"identity":"282e7090-e2bf-4168-9d3b-0092b3ecce07","added_by":"auto","created_at":"2023-01-30 20:58:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1332187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-206 inhibited cholesterol synthesis and the PPP but promoted glycolysis in c-Myc mice.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Kaplan-Meier survival curves of c-Myc/miR-206-MM (control, \u003cem\u003en\u003c/em\u003e=6) and c-Myc/miR-206 mice (\u003cem\u003en\u003c/em\u003e=6). Eight-week-old wild-type FVB/NJ mice were hydrodynamically injected with c-Myc and pT3-EF1α-miR-206-MM or c-Myc and pT3-EF1α-miR-206. Eight weeks post injection; mice were sacrificed for further analysis. (\u003cstrong\u003eB\u003c/strong\u003e) Kaplan-Meier survival curves of c-Myc/miR-206-MM (\u003cem\u003en\u003c/em\u003e=6) and c-Myc/miR-206 mice (\u003cem\u003en\u003c/em\u003e=6). Eight-week-old wild-type FVB/NJ mice were hydrodynamically injected with -Myc and pT3-EF1α-miR-206-MM or c-Myc and pT3-EF1α-miR-206. Twenty-four weeks post injection, mice were sacrificed for further analysis. (\u003cstrong\u003eC\u003c/strong\u003e) Macroscopic (upper panel) and microscopic (lower panel) appearance of livers from c-Myc/miR-206-MM (control, \u003cem\u003en\u003c/em\u003e=6, 8 w.p.i), c-Myc/miR-206 mice (\u003cem\u003en\u003c/em\u003e=6, 8 w.p.i) or c-Myc/miR-206 mice (\u003cem\u003en\u003c/em\u003e=6, 24 w.p.i) stained with H\u0026amp;E (10X). LW: liver weight. (\u003cstrong\u003eD\u003c/strong\u003e) Western blot analysis of HMGCR and G6PD in pooled hepatocytes of c-Myc/miR-206-MM (\u003cem\u003en\u003c/em\u003e=3, 8 w.p.i), c-Myc/miR-206 mice (\u003cem\u003en\u003c/em\u003e=3, 8 w.p.i) or c-Myc/miR-206 (\u003cem\u003en\u003c/em\u003e=3, 24 w.p.i) mouse cohort. (\u003cstrong\u003eE\u003c/strong\u003e) Hepatic cholesterol levels in three groups of mice. (\u003cstrong\u003eF-G\u003c/strong\u003e) Levels of NADPH and R5P in livers of three groups of mice. (\u003cstrong\u003eH-I\u003c/strong\u003e) The rates of \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol and the rates of \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA in livers of three groups of mice. (\u003cstrong\u003eJ\u003c/strong\u003e) Protein levels of PKM and PFK in pooled hepatocytes (\u003cem\u003en\u003c/em\u003e=3) isolated from three groups of mice. (\u003cstrong\u003eK\u003c/strong\u003e) The extracellular acidification rate (ECAR) in hepatocytes isolated from three groups of mice after sequential additions of 10 mM glucose, 2 μg/mL oligomycin, and 100 mM 2-deoxyglucose. Data represent mean ± SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (Fig. 7A-B: log-rank test; Fig. 7C-K: two-way ANOVA test)\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/5993b27347e9e2ffe79d9296.png"},{"id":32230777,"identity":"e80c207b-23df-49ee-84b2-59cccee8f1fb","added_by":"auto","created_at":"2023-01-30 21:06:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1190275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisrupting the interaction of miR-206 with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHmgcr \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eG6pd\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e impaired the ability of miR-206 to inhibit cholesterol synthesis, the PPP and HCC. (A) \u003c/strong\u003eProtein levels of HMGCR and G6PD\u003cstrong\u003e \u003c/strong\u003ein pooled hepatocytes isolated from c-Myc/miR-206-MM (\u003cem\u003en\u003c/em\u003e=3, 8 w.p.i), c-Myc/miR-206 (\u003cem\u003en\u003c/em\u003e=3, 8 w.p.i) or c-Myc/miR-206/sgRNA (\u003cem\u003en\u003c/em\u003e=3, 8 w.p.i). Eight week-old wild-type FVB/NJ mice were hydrodynamically injected with\u0026nbsp;pT3-EF1α-cMyc and\u0026nbsp;pT3-EF1α-miR-206-MM, pT3-EF1α-c-Myc and\u0026nbsp;pT3-EF1α-miR-206, or a combination of pT3-EF1α-cMyc,\u0026nbsp;pT3-EF1α-miR-206, and sgRNAs of \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e. (\u003cstrong\u003eB\u003c/strong\u003e) Kaplan-Meier survival curves of c-Myc/miR-206-MM (\u003cem\u003en\u003c/em\u003e=6), c-Myc/miR-206 (\u003cem\u003en\u003c/em\u003e=6) and c-Myc/miR-206/sgRNA mouse cohorts. (\u003cstrong\u003eC\u003c/strong\u003e) Macroscopic (upper panel) and microscopic (lower panel) appearance of livers from c-Myc/miR-206-MM (\u003cem\u003en\u003c/em\u003e=6), c-Myc/miR-206 (\u003cem\u003en\u003c/em\u003e=6) and c-Myc/miR-206/sgRNA mouse cohorts stained with H\u0026amp;E (10X). LW: liver weight. (\u003cstrong\u003eD\u003c/strong\u003e) Relative levels of hepatic cholesterol in three groups of mice. (\u003cstrong\u003eE-F\u003c/strong\u003e) Levels of R5P and NADPH in livers of three groups of mice. (\u003cstrong\u003eG-H\u003c/strong\u003e) The rates of \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol and the rates of \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA in livers of three groups of mice. Data represent mean ± SEM. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (Fig. 8B: log-rank test; Fig. 8C-H: two-way ANOVA test)\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/acade6d2fe001e2115495469.png"},{"id":41802320,"identity":"58761acf-af4e-4294-b21a-dd6d9b589ebc","added_by":"auto","created_at":"2023-08-19 07:05:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4652174,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/e2a4e2c6-e85e-4c10-9636-d1d15cff972f.pdf"},{"id":32231370,"identity":"a28a692c-6e35-4533-b7e8-a9e7170ff4fe","added_by":"auto","created_at":"2023-01-30 21:22:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":399070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-2485059/v1/ce08aeff8f6aa4f5303e6441.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"A positive feedback between cholesterol synthesis and the pentose phosphate pathway rather than glycolysis promotes hepatocellular carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHCC is a lethal malignancy without effective therapeutic approaches [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The incidence rate of HCC nearly matched its mortality, demonstrating the aggressiveness of this malignancy and limited therapeutic options [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although hepatitis B (HBV) and C (HCV) infection are considered the major causal factors of HCC, NAFLD/MAFLD (non-alcoholic fatty liver disease/metabolic associated fatty liver disease) is associated with an increasing incidence of HCC in the Western world [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Given limited effects of chemotherapy and the insensitivity of HCC to radiotherapy, tumor extirpation represents the only choice for a long-term cure. Unfortunately, even with successful surgical removal, the presence of NAFLD/MAFLD is associated with an increased recurrence of tumor. Although immunotherapies have recently been approved to treat a variety of cancers, this approach is largely unsuccessful for the treatment of HCC. Further studies are needed to identify new targets for developing new drugs for this malignancy.\u003c/p\u003e \u003cp\u003eCholesterol is an important component of cell membrane and required for cell growth; and the liver is the main organ for its synthesis. In addition, cholesterol plays an important role in modulating membrane trafficking and facilitating signal transduction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. An imbalance in cholesterol homeostasis can contribute to liver injury, which triggers HCC. However, the roles of cholesterol in regulating cancer development and the potential of therapeutically targeting cholesterol homeostasis is controversial [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is reported that hepatic accumulation of cholesterol drives liver injury and subsequent HCC [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Hypercholesterolemia has been considered as a risk factor of HCC [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Statins, the drug for hypercholesterolemia, show the capacity to protect against the development and recurrence of HCC [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast, other studies reported that statins failed to reduce the incidence of HCC in NAFLD-associated HCC patients [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In mice, atorvastatin exhibits no effect on N-nitrosodiethylamine-induced HCC [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The potential role of cholesterol lowering drugs in treating HCC remains controversial. Cholesterol homeostasis requires collaboration between various organs, which ensures a balance between cholesterol absorption (in the intestine) and cholesterol synthesis and removal in the liver [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition to activation of cholesterol synthesis, enhancement of cholesterol absorption and impaired cholesterol removal also contributes to increased hepatic and serum cholesterol [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, current studies described above only focused on hepatic and serum levels of cholesterol rather than cholesterol synthesis in HCC patients. In addition, statins function by driving hepatic uptake of cholesterol rather than cholesterol synthesis, which could potentially explain some of the controversy in the field. The pentose phosphate pathway (PPP) is a metabolic pathway parallel to glycolysis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It is widely accepted that glycolysis is a major energy resource for cancer development, while the PPP produces NADPH (reduced nicotinamide adenine dinucleotide phosphate) and ribose 5-phosphate (R5P). R5P is a key substrate of DNA synthesis that is required for cell proliferation; and NADPH provides reducing power for cholesterol synthesis [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], suggesting that cholesterol synthesis is closely connected to the PPP and/or glycolysis. Our study was based on the notion that a positive feedback between cholesterol synthesis and the PPP promotes the development of HCC and cholesterol synthesis as a process rather than cholesterol is the major risk factor of HCC.\u003c/p\u003e \u003cp\u003eAmplification and overexpression of the \u003cem\u003ecMYC\u003c/em\u003e oncogene is frequently observed in HCC patients and is associated with increased aggressiveness and poor prognosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, cMyc-induced HCC in rodents can recapitulate, in a highly reliable way, the phases of tumor initiation and progression that occur in humans [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Considering the role of cholesterol in HCC, we analyzed c-Myc mice and observed a significant increase in cholesterol and metabolites of the PPP in tumors. In addition, c-Myc also significantly impaired biogenesis of micoRNA-206 (miR-206) that directly targeted \u003cem\u003eHMGCR\u003c/em\u003e (3-hydroxy-3-methylglutaryl-CoA reductase) and \u003cem\u003eG6DP\u003c/em\u003e (glucose-6-phosphate dehydrogenase), the rate-limiting enzymes of cholesterol synthesis and the PPP. In this study, we tested the hypothesis that a positive feedback loop between cholesterol synthesis and the PPP promoted HCC development and the disruption of this loop by miR-206 prevented HCC development.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eEstablishment of c-Myc HCC mice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old wild-type male FVB/N mice maintained on normal diet were hydrodynamically injected with either 5 \u0026mu;g pT3-EF1\u0026alpha;-cMyc and 0.2 \u0026mu;g pCMV/\u003cem\u003eSB\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e=6) or 5 \u0026mu;g pT3-EF1\u0026alpha; and 0.2 \u0026mu;g pCMV/\u003cem\u003eSB\u003c/em\u003e (control, \u003cem\u003en\u003c/em\u003e=6), as described previously\u0026nbsp;[\u003ca href=\"#_ENREF_20\" title=\"Tao, 2015 #20\"\u003e20\u003c/a\u003e]. Eight weeks post injection, mice were sacrificed for further analysis. Mice were housed, fed, and monitored in accordance with protocols approved by the committee for animal research at the Hubei University of Chinese Medicine and the University of Minnesota.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolomics by UPLC-MS/MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e50 mg of livers (in liquid nitrogen) were thawed in a 2 mL EP tube on ice. After 500 uL of pre-cooled extractant (70% methanol aqueous solution) and small steel balls were added to the EP tube, liver tissues were homogenized at 30 Hz for 30 second for four times. Homogenized livers were shaken at 1500 r/min for 5 min, incubated for15 min on ice, and centrifuged with 12,000 r/min at 4 \u0026deg;C for 10 min. The supernatant were collected for UPLC-MS/MS analysis using an LC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system; MS, AB SCIE QTRAP System). The detailed procedure of UPLC analysis is included in Supplementary Materials and Methods.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCholesterol synthesis assay via incorporation of \u003csup\u003e14\u003c/sup\u003eC-acetate sodium\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe rate of cholesterol synthesis is high in the morning hours\u0026nbsp;[\u003ca href=\"#_ENREF_21\" title=\"Kandutsch, 1970 #21\"\u003e21\u003c/a\u003e]. Therefore, mice were sacrificed between 10 am and 12 pm to collect liver samples. Liver homogenates were prepared based on the protocol described previously\u0026nbsp;[\u003ca href=\"#_ENREF_22\" title=\"Erickson, 1982 #22\"\u003e22\u003c/a\u003e]. Cold liver homogenates (400 mg) were incubated in the presence of 2 mM sodium acetate containing 16.7 \u0026micro;Ci of \u003csup\u003e14\u003c/sup\u003eC sodium acetate (PerkinElmer), as reported\u0026nbsp;[\u003ca href=\"#_ENREF_23\" title=\"Rao, 1984 #23\"\u003e23\u003c/a\u003e]. After incubation, the liver homogenates were transferred to new 20 mL glass tubes with cap and further saponified for three hours at 70 ℃ in the presence of 5 mL ethanol and 0.5 mL 90% potassium hydroxide (KOH). Cholesterol was precipitated as digitonide and its radioactivity determined in a liquid scintillation counter.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNAD and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNADP on cholesterol synthesis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver homogenates were prepared as described previously\u0026nbsp;[\u003ca href=\"#_ENREF_24\" title=\"Siperstein, 1958 #24\"\u003e24\u003c/a\u003e]. After centrifuge at 800 x g for 10 minutes at -1\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e℃, the supernatant was collected and supplemented with glucose-6-phosphate (G6P, 20 x 10\u003csup\u003e-3\u003c/sup\u003e M), nicotinamide adenine dinucleotide (NAD, 0.8 x 10\u003csup\u003e-3\u003c/sup\u003e M) or nicotinamide adenine dinucleotide phosphate (NADP, 0.8 x 10\u003csup\u003e-3\u003c/sup\u003e M), potassium acetate (2 x 10\u003csup\u003e-3\u003c/sup\u003e M) and 16.7 \u0026micro;Ci of \u003csup\u003e14\u003c/sup\u003eC sodium acetate. After one hour of incubation at 37 ℃, the liver homogenate was saponified by adding 90% KOH. Cholesterol was precipitated as digitonide and its radioactivity was determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncorporation of \u003csup\u003e3\u003c/sup\u003eH-thymidine\u003c/strong\u003e \u003cstrong\u003einto DNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary hepatocytes were plated in\u0026nbsp;collagen-coated 35 mm dishes containing DMEM medium. Forty-eight hours after transfection of\u0026nbsp;pT3-EF1\u0026alpha; (control), pT3-EF1\u0026alpha;-cMyc or a combination of pT3-EF1\u0026alpha;-cMyc and pT3-EF1\u0026alpha;-shG6pd or pT3-EF1\u0026alpha;-shHmgcr, the medium was replaced with fresh media containing 7.5 \u0026micro;Ci \u003csup\u003e3\u003c/sup\u003eH-thymidine. After 24 hours, cells were harvested to analyze incorporation of \u003csup\u003e3\u003c/sup\u003eH-thymidine DNA by standard methods\u0026nbsp;[\u003ca href=\"#_ENREF_25\" title=\"Francavilla, 1986 #25\"\u003e25\u003c/a\u003e, \u003ca href=\"#_ENREF_26\" title=\"Mitaka, 1991 #26\"\u003e26\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eIn Vitro\u003c/em\u003e analysis of \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 4 hours of incubation with 10 \u0026mu;Ci [2-\u003csup\u003e14\u003c/sup\u003eC] acetate, murine hepatocytes were washed with cold PBS twice, solubilized with 0.1 M sodium hydroxide, and saponified. Nonsaponifiable lipid was extracted with isohexane. Labeled cholesterol was measured by flow scintillography after HPLC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression vectors of miR-206 and c-Myc\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA mouse DNA fragment containing miR-206 precursor was inserted into pT3-EF1\u0026alpha; vector, referred to as pT3-EF1\u0026alpha;-miR-206\u0026nbsp;[\u003ca href=\"#_ENREF_20\" title=\"Tao, 2015 #20\"\u003e20\u003c/a\u003e].\u0026nbsp;To rule out a non-specific effect of the vector, we generated a miR-206 mis-matched-expression vector by mutating the seed region of miR-206 (pT3-EF1\u0026alpha;-miR-206-MM).\u0026nbsp;pCMV/\u003cem\u003eSleeping Beauty\u003c/em\u003e transposase (pCMV/SB) and pT3-EF1\u0026alpha;-c-Myc have been described previously\u0026nbsp;[\u003ca href=\"#_ENREF_20\" title=\"Tao, 2015 #20\"\u003e20\u003c/a\u003e].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRISPR/Cas9 to ablate miR-206 binding sites within 3\u0026rsquo;UTRs of \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe locations of sgRNA pairs were selected at the boundary of the miR-206 binding site within the 3\u0026rsquo;UTRs of\u0026nbsp;\u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e.\u0026nbsp;Two sgRNAs for each of the miR-206 binding sites were designed by CRISPR and synthesized in IDT (Coralville, IA)\u0026nbsp;[\u003ca href=\"#_ENREF_27\" title=\"Concordet, 2018 #27\"\u003e27\u003c/a\u003e]. Four pairs of sgRNAs were further cloned into pX601-AAV8-CMV-SaCas9 (Addgene, Watertown, MA), termed AAV8-sgRNA.\u0026nbsp;The viruses of AAV8-SaCas9 and AAV8-sgRNA were packaged and tittered in the Viral Vector and Cloning Core at the University of Minnesota. To delete the miR-206 binding sites, Group Ⅰ mice (\u003cem\u003en\u003c/em\u003e=6) received 5 \u0026mu;g pT3-EF1\u0026alpha;-c-Myc, 10 \u0026mu;g pT3-EF1\u0026alpha;-miR-206-MM and 0.6 \u0026mu;g pCMV/SB; Group Ⅱ mice (\u003cem\u003en\u003c/em\u003e=6) received 5 \u0026mu;g pT3-EF1\u0026alpha;-c-Myc, 10 \u0026mu;g pT3-EF1\u0026alpha;-miR-206, and 0.6 \u0026mu;g pCMV/SB, and 5x10\u003csup\u003e11\u0026nbsp;\u003c/sup\u003eGC AAV8-SaCas9 viruses; and Group Ⅲ mice (\u003cem\u003en\u003c/em\u003e=6) received 5 \u0026mu;g pT3-EF1\u0026alpha;-c-Myc, 10 \u0026mu;g pT3-EF1\u0026alpha;-miR-206, 0.6 \u0026mu;g pCMV/SB, and 5x10\u003csup\u003e11\u0026nbsp;\u003c/sup\u003eGC AAV8-SaCas9 and AAV8-sgRNA viruses.\u0026nbsp;Eight weeks post-injection, mice were sacrificed for\u0026nbsp;further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003enalysis\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eStatistical analysis was performed using GraphPad Prism Software\u0026reg;. Data derived from cell-line experiments were presented as mean \u0026plusmn; SEM and assessed by a two-tailed Student T-test.\u0026nbsp;Statistical difference for cell cycle progression analysis was evaluated using Chi-squared test. Mann-Whitney test was used to evaluate the statistical significance for mouse experiments.\u0026nbsp;All the experiments were repeated at least three times. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIncreased cholesterol synthesis\u0026nbsp;in tumors of HCC patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth cholesterol synthesis and excretion and absorption of cholesterol contribute to change in hepatic and blood cholesterol\u0026nbsp;[\u003ca href=\"#_ENREF_14\" title=\"Zhao, 2009 #14\"\u003e14\u003c/a\u003e].\u0026nbsp;Hepatic cholesterol accumulation is implicated in HCC patients. However, the effect of cholesterol lowering drugs on HCC is controversial. In HCC patients, compared to adjacent normal livers, cholesterol levels were significantly elevated in tumors (\u003cstrong\u003eFig. 1A\u003c/strong\u003e). Hepatocytes are the major site of cholesterol synthesis. We next analyzed mRNA levels of \u003cem\u003eHMGCR\u003c/em\u003e, the rate-limiting enzyme of cholesterol synthesis, in hepatocytes isolated from adjacent normal livers and HCC tumors. As expected, increased \u003cem\u003eHMGCR\u003c/em\u003e mRNA was observed in malignant hepatocytes compared to normal hepatocytes (\u003cstrong\u003eFig. 1B\u003c/strong\u003e). In TCGA database, compared to normal individuals (\u003cem\u003en\u003c/em\u003e=50), HCC patients (\u003cem\u003en\u003c/em\u003e=369) exhibited high expression of \u003cem\u003eHMGCR\u003c/em\u003e (\u003cstrong\u003eFig. 1C\u003c/strong\u003e), which was associated with poor survival (\u003cstrong\u003eFig. 1D\u003c/strong\u003e). Increased hepatic cholesterol can be caused by activation of cholesterol synthesis, impaired cholesterol excretion and increased cholesterol absorption from the food\u0026nbsp;[\u003ca href=\"#_ENREF_14\" title=\"Zhao, 2009 #14\"\u003e14\u003c/a\u003e]. In addition, cholesterol-lowering drugs such as statins exhibit no effect on HCC, leading us to speculate cholesterol synthesis as a process rather than cholesterol itself is the major contributor of HCC development. \u003csup\u003e14\u003c/sup\u003eC-acetate incorporation into cholesterol was much greater in HCC tumors than in normal livers (\u003cstrong\u003eFig. 1E\u003c/strong\u003e), suggesting activation of \u003cem\u003ede novo\u003c/em\u003e cholesterol synthesis in HCC tumors. In sum, cholesterol synthesis was activated in tumors of HCC patients, and high levels of \u003cem\u003eHMGCR\u003c/em\u003e in malignant hepatocytes correlated with poor survival of HCC patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec-Myc activated hepatic cholesterol synthesis, the pentose phosphate pathway and glycolysis in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlmost 30% of HCC patients show \u003cem\u003ec-MYC\u003c/em\u003e gene amplification or overexpression\u0026nbsp;[\u003ca href=\"#_ENREF_28\" title=\"Liang, 2018 #28\"\u003e28\u003c/a\u003e]. A positive correlation between \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003ec-MYC\u003c/em\u003e was observed in tumors of HCC patients from TCGA database (\u003cstrong\u003eSupplementary Fig. 1A\u003c/strong\u003e), indicating that c-MYC is a potential driver of cholesterol synthesis.\u0026nbsp;HDI of\u0026nbsp;c-Myc led to c-Myc accumulation and increased expression of \u003cem\u003eHmgcr\u003c/em\u003e in hepatocytes of mice (\u003cstrong\u003eSupplementary Figure 1B-C\u003c/strong\u003e) and triggered development of HCC (\u003cstrong\u003eFig. 2A\u003c/strong\u003e). All c-Myc mice died of HCC within eight weeks post injection of c-Myc, while 100%\u0026nbsp;control mice were healthy at that time point (\u003cstrong\u003eFig. 2B\u003c/strong\u003e).\u0026nbsp;As we observed in HCC patients, mRNA levels of \u003cem\u003eHmgcr\u003c/em\u003e, enzyme activity of HMGCR, and hepatic cholesterol were significantly increased in tumors of c-Myc mice (\u003cstrong\u003eFig. 2C-E\u003c/strong\u003e). Cholesterol synthesis, via acetyl-CoA, interfaces with \u003cem\u003ede novo\u003c/em\u003e lipogenesis (DNL), glycolysis and the PPP\u0026nbsp;[\u003ca href=\"#_ENREF_24\" title=\"Siperstein, 1958 #24\"\u003e24\u003c/a\u003e], suggesting a possible mechanism of action. In fact, metabolites of glycolysis and the PPP were significantly increased in tumors of c-Myc mice (\u003cstrong\u003eFig. 2F\u003c/strong\u003e); and acetyl-CoA,\u0026nbsp;the major precursor\u0026nbsp;of cholesterol synthesis, was increased in c-Myc mice (\u003cstrong\u003eFig. 2F\u003c/strong\u003e). Consistent with an increase in the glycolytic and the PPP metabolites, expression of the genes controlling glycolysis and the PPP was significantly increased in malignant hepatocytes of c-Myc mice (\u003cstrong\u003eFig. 2G\u003c/strong\u003e). An increase in the glycolytic rate was observed in malignant hepatocytes of c-Myc mice (\u003cstrong\u003eFig. 2H\u003c/strong\u003e). In sum, c-Myc signaling promoted cholesterol synthesis, glycolysis and the PPP in hepatocytes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAblation of the PPP reduced cholesterol synthesis and delayed growth of HCC, while ablation of glycolysis did not affect these processes in c-Myc mice.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ec-Myc activated the PPP and glycolysis (\u003cstrong\u003eFig. 2F-G\u003c/strong\u003e). Glycolysis produces pyruvate that can be converted to acetyl-CoA, a precursor of cholesterol synthesis. We next determined the effect of glycolysis on cholesterol synthesis. NAD is a driver of glycolysis\u0026nbsp;[\u003ca href=\"#_ENREF_24\" title=\"Siperstein, 1958 #24\"\u003e24\u003c/a\u003e, \u003ca href=\"#_ENREF_29\" title=\"Lewis, 1955 #29\"\u003e29\u003c/a\u003e]. We, therefore, treated liver homogenates of pT3 and c-Myc mice with NAD. Although NAD enhanced glycolysis in both pT3 and c-Myc mice\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. 2A\u003c/strong\u003e), it did not affect cholesterol synthesis in both pT3 and c-Myc mice (\u003cstrong\u003eFig. 3A\u003c/strong\u003e).\u0026nbsp;We next deleted pyruvate kinase (PKM), the rate-limiting enzyme of glycolysis in c-Myc mice, which ablated glycolysis (\u003cstrong\u003eSupplementary Fig. 3A-B\u003c/strong\u003e). Unexpectedly, ablation of \u003cem\u003ePkm\u003c/em\u003e at the time of c-Myc overexpression in murine livers did not affect cholesterol synthesis in c-Myc mice (\u003cstrong\u003eFig. 3B\u003c/strong\u003e).\u0026nbsp;However, cholesterol synthesis is still much higher in c-Myc mice compared to pT3 mice (\u003cstrong\u003eFig. 3A\u003c/strong\u003e), indicating that other pathways activated by c-Myc such as the PPP might be able to drive cholesterol synthesis in mice.\u003c/p\u003e\n\u003cp\u003eThe PPP is a metabolic pathway parallel to glycolysis, which shares a common starting molecule with glycolysis, glucose-6-phosphate (G6P). Two major products of the PPP are R6P and NADPH. As expected, NADPH and R5P as well as expression of \u003cem\u003eG6pd\u003c/em\u003e was significantly increased in malignant hepatocytes from c-Myc mice (\u003cstrong\u003eFig. 3C-D, Supplementary Fig. 4A\u003c/strong\u003e). NADPH, as a cofactor of HMGCR, is required for cholesterol synthesis. These established findings led us to speculate that the PPP is potentially involved in enhanced cholesterol synthesis in HCC. To test this speculation, we treated liver homogenates of pT3 and c-Myc mice with NADPH. As expected, NADPH significantly increased enzyme activity of G6PD\u0026nbsp;(\u003cstrong\u003eSupplementary Fig. 4B\u003c/strong\u003e), which in turn increased incorporation of \u003csup\u003e14\u003c/sup\u003eC-acetate into cholesterol in liver homogenates from pT3 and c-Myc mice (\u003cstrong\u003eFig. 3E)\u003c/strong\u003e. Since the PPP is activated in c-Myc mice, cholesterol synthesis, as revealed by \u003csup\u003e14\u003c/sup\u003eC-acetate labeling experiment, was much higher in c-Myc mice compared to pT3 mice (\u003cstrong\u003eFig. 3E\u003c/strong\u003e). To confirm this speculation, we ablated the PPP via knocking down \u003cem\u003eG6pd\u0026nbsp;\u003c/em\u003ein hepatocytes of c-Myc mice (\u003cstrong\u003eSupplementary Fig. 5\u003c/strong\u003e). Knocking down \u003cem\u003eG6pd\u003c/em\u003e significantly inhibited the PPP, which was reflected by a decrease in NADPH and R5P (\u003cstrong\u003eFig. 3F-G\u003c/strong\u003e). Consistent with reduced NADPH that is required for cholesterol synthesis, incorporation of \u003csup\u003e14\u003c/sup\u003eC-acetate into cholesterol was also significantly reduced in liver homogenates of c-Myc/shG6pd mice (\u003cstrong\u003eFig. 4H\u003c/strong\u003e). Phenotypically, ablation of glycolysis did not affect growth of HCC, while knockdown of \u003cem\u003eG6pd\u003c/em\u003e significantly delayed growth of HCC in c-Myc mice (\u003cstrong\u003eFig. 3I-J\u003c/strong\u003e). In sum, the PPP at least in part is required for cholesterol synthesis and hepatocarcinogenesis in c-Myc mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA positive feedback between the PPP and cholesterol synthesis drove DNA synthesis and cell proliferation.\u003c/strong\u003e Activation of the PPP produces more NADPH, which provides a cofactor for cholesterol synthesis. Enhancement of cholesterol synthesis should rapidly deplete NADPH, a major production of PPP. Therefore,\u0026nbsp;we hypothesized that cholesterol synthesis and the PPP formed a positive feedback loop, which amplifies production of R5P, the substrate of DNA synthesis, and NADPH, co-factor for HMGCR. To determine if activation of the PPP drives cholesterol synthesis, DNA synthesis, and proliferation, three groups of hepatocytes were treated empty vector (pT3), c-Myc, or a combination of c-Myc and \u003cem\u003eG6pd\u003c/em\u003e shRNA to knock down \u003cem\u003eG6pd\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eSupplementary Fig. 6A\u003c/strong\u003e). c-Myc overexpression enhanced the PPP, cholesterol synthesis, DNA synthesis and proliferation of hepatocytes (\u003cstrong\u003eFig. 4A-E\u003c/strong\u003e), while knockdown of \u003cem\u003eG6pd\u003c/em\u003e offset the effects of c-Myc overexpression (\u003cstrong\u003eFig. 4A-E\u003c/strong\u003e). These findings indicated that activation of the PPP is required for c-Myc to drive cholesterol synthesis, DNA synthesis and hepatocyte proliferation. To test if enhancement of cholesterol synthesis promotes the PPP, DNA synthesis and proliferation, three groups of hepatocytes were treated with pT3 (control), c-Myc or a combination of c-Myc and \u003cem\u003eHmgcr\u003c/em\u003e shRNA (\u003cstrong\u003eSupplementary Fig. 6B\u003c/strong\u003e). c-Myc activated the PPP, cholesterol synthesis, DNA synthesis and hepatocyte proliferation; and knockdown of \u003cem\u003eHmgcr\u0026nbsp;\u003c/em\u003ecounteracted the effects of c-Myc (\u003cstrong\u003eFig. 4F-J)\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe significant increase in levels of \u003csup\u003e14\u003c/sup\u003eC-acetate-labeled cholesterol and \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA was observed in c-Myc mice (\u003cstrong\u003eFig. 4K-L\u003c/strong\u003e). In sum, a positive feedback loop between cholesterol synthesis and the PPP enhanced production of cholesterol and R5P, which meets the needs for rapid growth and proliferation of malignant hepatocytes both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-206 repressed expression of \u003cem\u003eHMGCR\u0026nbsp;\u003c/em\u003eand \u003cem\u003eG6PD\u003c/em\u003e in hepatocytes by binding to their 3\u0026rsquo;UTRs\u003c/strong\u003e. HMGCR and G6PD are the rate-limiting enzymes of cholesterol synthesis and the PPP. MicroRNAs (miRNAs) can simultaneously fine tune multiple pathways and exhibit the strong therapeutic potential for cancers and other diseases\u0026nbsp;[\u003ca href=\"#_ENREF_30\" title=\"Rupaimoole, 2017 #30\"\u003e30\u003c/a\u003e]. We next attempted to identify those miRNAs that can simultaneously target both \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e. For this purpose, we analyzed murine and human Ago HITS-CLIP databases (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation from Argonaute protein complex) of \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e. Unexpectedly, miR-206 was identified as the only miRNA that can target human and mouse \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e and 3\u0026rsquo;UTRs of mouse and human \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e contains two miR-206 binding site\u0026nbsp;[\u003ca href=\"#_ENREF_31\" title=\"Paraskevopoulou, 2013 #31\"\u003e31\u003c/a\u003e]\u0026nbsp;[\u003ca href=\"#_ENREF_32\" title=\"Li, 2014 #32\"\u003e32\u003c/a\u003e]\u0026nbsp;(\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e). To exclude the false positive peaks of Ago-HITS-CLIP, we further used DIANA-microT-CDS to scan the 3\u0026rsquo;UTRs of murine and human \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e, confirming the binding sites of miR-206 within the 3\u0026rsquo;UTRs of murine and human \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e. 3\u0026apos; UTRs of both human and mouse \u003cem\u003eHMGCR\u0026nbsp;\u003c/em\u003eand \u003cem\u003eG6PD\u003c/em\u003e mRNAs are 100% complementary to the miR-206 5\u0026apos; seed region exhibiting the highest prediction scores and binding energy (\u003cstrong\u003eFig. 5A-B\u003c/strong\u003e). In addition, levels of miR-206 were significantly reduced in malignant hepatocytes isolate from c-Myc mice (\u003cstrong\u003eSupplementary Fig. 7\u003c/strong\u003e). All these findings led us to focus on miR-206.\u0026nbsp;In Fig. 1C-D, high levels of \u003cem\u003eHMGCR\u003c/em\u003e predicted poor survival of HCC patients. Similarly, elevated levels of \u003cem\u003eG6PD\u003c/em\u003e predicted poor survival of HCC patients TCGA database (\u003cstrong\u003eFig. 5C-D\u003c/strong\u003e).\u0026nbsp;Inclusion of the 3\u0026rsquo;UTRs of \u003cem\u003eHmgcr\u003c/em\u003e or\u0026nbsp;\u003cem\u003eG6pd\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003einto the luciferase reporter constructs reduced luciferase activities upon co-transfection with miR-206 into Hepa1-6 cells (\u003cstrong\u003eFig. 5\u003c/strong\u003e\u003cstrong\u003eE-F\u003c/strong\u003e). Mutation of the miR-206 binding sites within the 3\u0026rsquo;UTRs of \u003cem\u003eHmgcr\u003c/em\u003e and\u0026nbsp;\u003cem\u003eG6pd\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas necessary to completely offset the inhibitory effects of miR-206 on luciferase activities (\u003cstrong\u003eFig. 5\u003c/strong\u003e\u003cstrong\u003eE-F\u003c/strong\u003e), suggesting that\u0026nbsp;miR-206\u0026nbsp;directly recognized the predicted binding site within the 3\u0026apos; UTR of \u003cem\u003eHmgcr\u003c/em\u003e and\u0026nbsp;\u003cem\u003eG6pd\u003c/em\u003e. miR-206 also reduced protein and mRNA levels of \u003cem\u003eHmgcr and G6pd \u0026nbsp;\u003c/em\u003ein Hepa1-6 cells (\u003cstrong\u003eFig. 5\u003c/strong\u003e\u003cstrong\u003eG-H\u003c/strong\u003e). MiR-206 was able to inhibit expression of \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e in human hepatocytes by binding to their 3\u0026rsquo;UTRs (\u003cstrong\u003eSupplementary Fig. 8A-C\u003c/strong\u003e). In sum, \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e are direct targets of miR-206 in both human and moues hepatocytes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;m\u003c/strong\u003e\u003cstrong\u003eiR-206 disrupted the positive feedback loop between cholesterol synthesis and the PPP by targeting \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e, which impaired DNA synthesis and proliferation of hepatocytes.\u0026nbsp;\u003c/strong\u003eSince\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eare direct targets of miR-206,\u0026nbsp;we hypothesized that miR-206 is able to disrupt the positive feedback loop between cholesterol synthesis and the PPP, thereby inhibiting DNA synthesis, cholesterol synthesis and proliferation of malignant hepatocytes. To test this, CRISPR/Cas9 technique was used to delete the binding sites of miR-206 within the 3\u0026rsquo;UTRs of both \u003cem\u003eG6pd\u003c/em\u003e and \u003cem\u003eHmgcr\u003c/em\u003e in malignant hepatocytes isolated from c-Myc mice\u0026nbsp;[\u003ca href=\"#_ENREF_33\" title=\"Yang, 2016 #33\"\u003e33\u003c/a\u003e, \u003ca href=\"#_ENREF_34\" title=\"Qiupeng Zheng, 2014 #34\"\u003e34\u003c/a\u003e]. Such a design disrupted the interaction of miR-206 with \u003cem\u003eG6pd\u003c/em\u003e and \u003cem\u003eHmgcr\u003c/em\u003e (\u003cstrong\u003eSupplementary Fig. 9A\u003c/strong\u003e), allowing us to determine if \u003cem\u003eG6pd\u003c/em\u003e and \u003cem\u003eHmgcr\u003c/em\u003e are required for miR-206 to inhibit cholesterol synthesis and the PPP.\u0026nbsp;Overexpression of miR-206 inhibited cholesterol synthesis, the PPP and DNA synthesis, which was reflected by a significant reduction in incorporation of \u003csup\u003e14\u003c/sup\u003eC-acetate into cholesterol, the PPP metabolites, \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA and proliferation of hepatocytes (\u003cstrong\u003eFig. 6A-E\u003c/strong\u003e). In contrast, ablating the miR-206 binding sites within the 3\u0026rsquo;UTRs of both \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e offset the inhibitory effects of miR-206 (\u003cstrong\u003eFig. 6A-E\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe assumed that a positive feedback loop between cholesterol synthesis and the PPP amplifies DNA synthesis and proliferation of malignant hepatocytes. Inhibition of either cholesterol synthesis or the PPP should be able to at least in part disrupt this positive feedback loop and thereby prevent DNA synthesis and proliferation. To test this, we ablated the binding sites of miR-206 within the 3\u0026rsquo;UTR of \u003cem\u003eHmgcr\u003c/em\u003e or \u003cem\u003eG6pd\u003c/em\u003e. Ablation of the miR-206 binding sites within the 3\u0026rsquo;UTR of \u003cem\u003eHmgcr\u003c/em\u003e was able to recover cholesterol synthesis, the PPP, DNA synthesis and cell proliferation that were inhibited by miR-206 (\u003cstrong\u003eSupplementary Fig. 9B\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eFig. 6F-J\u003c/strong\u003e). Similarly, ablation of the miR-206 binding sites within the 3\u0026rsquo;UTRs of \u003cem\u003eG6pd\u0026nbsp;\u003c/em\u003ealso recovered cholesterol synthesis, the PPP, DNA synthesis and proliferation (\u003cstrong\u003eSupplementary Fig. 9C,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFig. 6K-O\u003c/strong\u003e). These results indicate that miR-206 is able to disrupt the positive feedback loop between cholesterol synthesis and the PPP by targeting either \u003cem\u003eHmgcr\u003c/em\u003e or \u003cem\u003eG6pd\u003c/em\u003e, which subsequently inhibits DNA synthesis and cell proliferation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-206 inhibited cholesterol synthesis and the PPP in c-Myc mice.\u0026nbsp;\u003c/strong\u003eWe next\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edetermined if miR-206 was able to simultaneously inhibit cholesterol synthesis and the PPP \u003cem\u003ein vivo\u003c/em\u003e. c-Myc mice were injected with pT3-EF1\u0026alpha;-miR-206-MM (control) or pT3-EF1\u0026alpha;-miR-206. Eight weeks post injection, all miR-206-treated c-Myc mice were healthy, while 100% of c-Myc mice died of HCC (\u003cstrong\u003eFig. 7A\u003c/strong\u003e). Upon dissection, no tumors were observed in c-Myc/miR-206 mice (\u003cstrong\u003eFig. 7C)\u003c/strong\u003e. The long-term survival experiment revealed that all c-Myc/miR-206 mice were healthy 24 weeks of post injection of miR-206 (\u003cstrong\u003eFig. 7B\u003c/strong\u003e). Upon dissection, no tumor nodules were observed in livers of this group of c-Myc/miR-206 mice (\u003cstrong\u003eFig. 7C\u003c/strong\u003e). All these findings indicated the long-term effect of miR-206 on preventing HCC. Our hypothesis is that miR-206, by disrupting the positive feedback loop between cholesterol synthesis and the PPP, inhibits HCC. As expected, miR-206 significantly reduced expression of \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e in hepatocytes of c-Myc mice (\u003cstrong\u003eFig. 7D\u003c/strong\u003e). Consistent with reduced \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e, both cholesterol and the metabolites of the PPP were significantly reduced in miR-206-treated c-Myc mice (\u003cstrong\u003eFig. 7E-G\u003c/strong\u003e). Incorporation of \u003csup\u003e14\u003c/sup\u003eC-acetate into cholesterol and \u003csup\u003e3\u003c/sup\u003eH-thymidine incorporation into DNA were reduced in miR-206-treated c-Myc mice (\u003cstrong\u003eFig. 7H-I\u003c/strong\u003e). These results established that miR-206 disrupted the positive feedback loop between cholesterol synthesis and the PPP, which subsequently inhibited DNA synthesis and growth of malignant hepatocytes. Unexpectedly, miR-206 treatment significantly induced expression of genes encoding PFK\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(phosphofructokinase)\u0026nbsp;and PKM (pyruvate kinase), two rate-limiting enzymes of glycolysis, in c-Myc mice (\u003cstrong\u003eFig. 7J\u003c/strong\u003e). The glycolytic rate was also significantly increased in livers of c-Myc/miR-206 mice (\u003cstrong\u003eFig. 7K\u003c/strong\u003e). Although miR-206 induced glycolysis, it still fully prevented c-Myc-induced HCC, further indicating that glycolysis is not required for miR-206 to inhibit HCC. This finding is consistent with our observation that ablation of glycolysis failed to prevent c-Myc-induced HCC. In sum, miR-206 disrupted the positive feedback between cholesterol synthesis and the PPP, which prevented c-Myc-induced HCC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHMGCR and G6PD are required for miR-206 to prevent c-Myc-induced HCC.\u0026nbsp;\u003c/strong\u003eWe next\u0026nbsp;employed an AAV8-based CRISPR/Cas9 technique to ablate the binding sites of miR-206 within\u0026nbsp;the 3\u0026rsquo;UTRs of\u0026nbsp;\u003cem\u003eHmgcr\u0026nbsp;\u003c/em\u003eand \u003cem\u003eG6pd\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ein the genome of hepatocytes in c-Myc mice, which disrupted the interaction of miR-206 with \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e. Ablation of the miR-206 binding sites impaired the ability of miR-206 to inhibit expression of\u0026nbsp;\u003cem\u003eHmgcr\u0026nbsp;\u003c/em\u003eand \u003cem\u003eG6pd\u003c/em\u003e in hepatocytes (\u003cstrong\u003eFig. 8A\u003c/strong\u003e). Phenotypically, 100% c-Myc mice died of HCC within 8 weeks post injection of c-Myc (\u003cstrong\u003eFig. 8B\u003c/strong\u003e). MiR-206 fully prevented c-Myc-induced HCC, while disrupting its interaction with\u0026nbsp;\u003cem\u003eHmgcr\u0026nbsp;\u003c/em\u003eand \u003cem\u003eG6pd\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eresulted in renewed\u0026nbsp;growth of HCC that was fully prevented by miR-206 (\u003cstrong\u003eFig. 8B-C\u003c/strong\u003e).\u0026nbsp;Mechanistically, miR-206 markedly reduced hepatic cholesterol and metabolites of the PPP, while disrupting the interaction of miR-206 with \u003cem\u003eHmgcr\u0026nbsp;\u003c/em\u003eand \u003cem\u003eG6pd\u003c/em\u003e recovered levels of hepatic cholesterol and the metabolites of the PPP (\u003cstrong\u003eFig. 8D-F\u003c/strong\u003e).\u0026nbsp;As revealed by \u003csup\u003e14\u003c/sup\u003eC-acetate- and\u003csup\u003e\u0026nbsp;3\u003c/sup\u003eH-thymidine-labeling experiments, ablating the miR-206 binding sites recovered cholesterol synthesis and DNA synthesis in miR-206-treated c-Myc mice (\u003cstrong\u003eFig. 8G-H\u003c/strong\u003e). In sum, by disrupting the positive feedback loop between the cholesterol synthesis and the PPP, miR-206 inhibited growth of HCC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAmplification of c-MYC has been implicated in ~\u0026thinsp;27% HCC patients [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It has been reported that c-MYC drives cholesterol synthesis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]; and a positive correlation between \u003cem\u003ec-MYC\u003c/em\u003e and \u003cem\u003eHMGCR\u003c/em\u003e was observed in HCC patients. Furthermore, we discovered that cholesterol synthesis is activated in tumors of HCC patients and high levels of \u003cem\u003eHMGCR\u003c/em\u003e predicted poor survival of HCC patients. However, how cholesterol synthesis drives HCC development is poorly understood. To simulate the observation in HCC patients, we overexpressed c-Myc in livers of mice. Cholesterol synthesis is activated in livers of c-Myc mice. Mechanistically, activation of c-Myc maintained the positive feedback loop between cholesterol synthesis and the PPP. Specifically, activation of the PPP overproduced R5P and NADPH that served as substrates of DNA synthesis and the cofactor of HMGCR. Enhancement of cholesterol synthesis depleted NADPH, thereby driving the PPP. This positive feedback loop maintained rapid production of DNA and cholesterol, which are required for rapid growth and proliferation of malignant hepatocytes. These findings potentially explained the rapid development of HCC in c-Myc mice. In our previous study, we observed that miR-206 is able to suppress immunosuppression by preventing overproduction of TGFβ in malignant hepatocytes, which partially contributed to the prevention of c-Myc-induced HCC [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, miR-206 was able to fully prevent c-Myc-induced HCC, while 100% of c-Myc mice died of HCC within 8 weeks post injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). Such a potent inhibitory effect of miR-206 on HCC led us to speculate that in addition to recovering anti-tumor immunity, other mechanism(s) are involved in miR-206-mediated inhibition of HCC in c-Myc mice. Unexpectedly, miR-206 was identified as the only miRNA that can simultaneously target both \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e. By simultaneously targeting \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e, miR-206 disrupted the positive feedback loop between cholesterol synthesis and the PPP, thereby mitigating cholesterol and DNA synthesis that are required for growth and proliferation of malignant hepatocytes. Our findings fill the knowledge gap regarding the roles of cholesterol synthesis, the PPP and glycolysis in c-Myc-driven HCC.\u003c/p\u003e \u003cp\u003eFirst, we proposed a new concept that cholesterol synthesis rather than cholesterol is the major contributor of HCC development. It is well-established that dysregulated cholesterol metabolism is involved in HCC development. Most of these studies consider cholesterol as a major causal factor of hepatocarcinogenesis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, the major purpose of these studies is to reduce hepatic cholesterol and the findings are controversial [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In fact, levels of hepatic and blood cholesterol are controlled by cholesterol synthesis and excretion and absorption of cholesterol [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Statins function via driving cholesterol reverse transport (RCT) rather than hepatic cholesterol synthesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which might be the major reason of these controversial findings. In this manuscript, we for the first time established that cholesterol synthesis rather cholesterol is the major contributor of hepatocarcinogenesis, which potentially provides an explanation of controversial findings regarding the roles of cholesterol metabolism in HCC.\u003c/p\u003e \u003cp\u003eSecond, we observed that the PPP rather than glycolysis promoted cholesterol synthesis. Activation of the PPP and glycolysis has been observed in c-Myc-induced HCC. Cholesterol synthesis is closely connected glycolysis and the PPP and cholesterol synthesis is activated in HCC, urging us to assess if glycolysis or the PPP affects cholesterol synthesis. As described above, activation of the PPP promoted cholesterol synthesis. However, glycolysis exhibited no effect on this process. We established that cholesterol synthesis is required for c-Myc-induced HCC, which potentially explained that ablation of glycolysis did not affect growth of HCC in c-Myc mice. It is well accepted that glycolysis provides energy for rapid growth of tumors. In fact, studies of ATP production by glycolysis and oxidative phosphorylation (OXPHOS) in various types of cells and organs concluded that OXPHOS is the main contributor of ATP under aerobic conditions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], further suggesting that glycolysis is not the major energy supply for HCC growth. This is confirmed by our observation that although miR-206 markedly promoted glycolysis, it fully prevented c-Myc-induced HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Another explanation for glycolysis to promote HCC is that the pathway provides important biosynthetic precursors [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In fact, the majority of the biomass of proliferating cells is derived from amino acids rather than glucose [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The PPP supports cancer cell growth by providing NADPH for cholesterol synthesis and generating R5P for DNA synthesis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It is the PPP rather than glycolysis is a major contributor of c-Myc-induced HCC.\u003c/p\u003e \u003cp\u003eThe third novel observation is the positive feedback loop between cholesterol synthesis and the PPP in c-Myc-induced HCC. Cholesterol accumulation and activation of the PPP have been observed in HCC patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Cholesterol accumulation in the liver contributes to lipotoxicity, hepatic inflammation, and fibrosis, which have been considered key causal factors of HCC [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Conversely, some clinical studies have revealed that high levels of cholesterol are, in fact, associated with a reduced risk of HCC [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our findings address the controversy in that cholesterol synthesis rather than cholesterol itself is a major driver of hepatocarcinogenesis. In detail, enhancement of cholesterol synthesis, by depleting NADPH, drives the PPP, which produces R5P for DNA synthesis and NADPH for cholesterol synthesis. It is our speculation that rapid growth of cancer cells depletes cholesterol and R5P, which further enhances the positive feedback loop. Indeed, dietary cholesterol treatment significantly reduced enzyme activity of G6PD [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], further confirming our findings.\u003c/p\u003e \u003cp\u003eSomewhat unexpectedly, miR-206 was identified as the only miRNA that can target both \u003cem\u003eHMGCR\u003c/em\u003e or \u003cem\u003eG6PD\u003c/em\u003e by interacting with two miR-206 binding sites within the 3\u0026rsquo;UTR of \u003cem\u003eHMGCR\u003c/em\u003e or \u003cem\u003eG6PD\u003c/em\u003e. Consistently, levels of \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e are significantly increased in tumors of HCC patients; and a positive association was observed between \u003cem\u003eHMGCR\u003c/em\u003e and \u003cem\u003eG6PD\u003c/em\u003e in HCC (\u003cb\u003eSupplementary Fig.\u0026nbsp;10\u003c/b\u003e). More importantly, high levels of their expression predicted poor survival among HCC patients. This unexpected finding indicated the strong inhibition of miR-206 on cholesterol synthesis and the PPP. Considering the potent inhibitory effects of miR-206 on HCC, we speculated that miR-206 fully prevents HCC growth via multiple mechanisms. In addition to restoring antitumor immunity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], miR-206-mediated inhibition of cholesterol synthesis and the PPP at least in part contributes to HCC prevention. Our final goal is to develop miR-206 as a therapeutic drug for the treatment of HCC. The novel findings in this study provide additional layer of evidence that miR-206 prevents growth of HCC by cutting off building blocks of hepatocyte proliferation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e3\u0026apos;-UTR, 3\u0026apos;-untranslated regions; ATP:\u0026nbsp;adenosine 5\u0026prime;-triphosphate; CRISPR/Cas9, the clustered\u0026nbsp;regularly interspaced short palindromic repeats/CRISPR associated protein 9; DNL: \u003cem\u003ede novo\u003c/em\u003e lipogenesis; ECAR: extracellular acidification rate; G6P: glucose-5-phosphate; HBV: hepatitis B; HCV: hepatitis C; HCC, hepatocellular carcinoma; HDI: hydrodynamic injection; HITS-CLIP: high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; MTT: 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NADPH: reduced nicotinamide adenine dinucleotide phosphate; NAFLD/MAFLD: non-alcoholic fatty liver disease/metabolic associated fatty liver disease; OXPHOS: oxidative phosphorylation; PFKL: phosphofructokinase liver type; PKM: pyruvate kinase; PPP: pentose phosphate pathway; R5P: ribose 5-phosphate; sgRNA: guide RNA; TGF\u0026beta;: transforming growth factor \u0026beta;.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Fund for Distinguished Young Scholar of Hubei University of Chinese Medicine awarded to Hu JJ and the Research Scholar Award (IS-16-210-01-RMC) to Song GS\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHu J., Liu N and Song D: acquisition of data, analysis, and interpretation of data, and drafting the manuscript. Steer, C: data analysis and manuscript editing. Song G\u0026nbsp;and Zheng G: obtaining funding, study supervision, study concept, design, and revision and final approval of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors involved in this study declared that they have nothing to disclose regarding funding or conflict of interest with respect to this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data in this manuscript is available upon reasonable request. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSlotta JE, Kollmar O, Ellenrieder V, Ghadimi BM, Homayounfar K. Hepatocellular carcinoma: Surgeon\u0026apos;s view on latest findings and future perspectives. World Journal of Hepatology. 2015;7:1168.\u003c/li\u003e\n\u003cli\u003eYang JD, Roberts LR. 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The Journal of Nutrition. 1973;103:93-101. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"DNA synthesis, glycolysis, microRNA, therapeutic agent","lastPublishedDoi":"10.21203/rs.3.rs-2485059/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2485059/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHepatic cholesterol accumulation and hypercholesterolemia are implicated in hepatocellular carcinoma (HCC).\u003cstrong\u003e \u003c/strong\u003eHowever,\u003cstrong\u003e \u003c/strong\u003ethe therapeutic effects of cholesterol lowering drugs on HCC are controversial, indicating that the relationship between cholesterol metabolism and HCC is more complex than anticipated. A positive feedback between cholesterol synthesis and the pentose phosphate pathway (PPP) rather than glycolysis was formed in tumors of c-Myc mice. Blocking the PPP prevented inhibited cholesterol synthesis and thereby HCC in c-Myc mice, while ablating glycolysis did not affect cholesterol synthesis and failed to prevent c-Myc-induced HCC. Unexpectedly, \u003cem\u003eHMGCR \u003c/em\u003e(3-hydroxy-3-methylglutaryl-CoA reductase) and \u003cem\u003eG6PD\u003c/em\u003e (glucose-6-phosphate dehydrogenase), the rate-limiting enzymes of cholesterol synthesis and the PPP, were identified as direct targets of microRNA-206. By targeting \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e, microRNA-206 disrupted the positive feedback and fully prevented HCC in c-Myc mice, while 100% of control mice dies of HCC. Disrupting the interaction of microRNA-206 with \u003cem\u003eHmgcr\u003c/em\u003e and \u003cem\u003eG6pd\u003c/em\u003e restored cholesterol synthesis, the PPP and HCC growth that was inhibited by miR-206.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: This study identified a previously undescribed positive feedback loop between cholesterol synthesis and the PPP, which drives HCC, while microRNA-206 prevents HCC by disrupting this loop. Cholesterol synthesis as a process rather than cholesterol itself is the major contributor of HCC.\u003c/p\u003e","manuscriptTitle":"A positive feedback between cholesterol synthesis and the pentose phosphate pathway rather than glycolysis promotes hepatocellular carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-30 20:58:22","doi":"10.21203/rs.3.rs-2485059/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-02-16T15:46:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-02-15T18:35:46+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-02-10T22:18:09+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-02-07T15:07:04+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-01-27T10:58:30+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-01-26T02:36:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-01-17T10:40:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-01-16T18:45:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2023-01-16T18:44:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"158dffa4-8c36-4590-a4c2-2e82e2839f92","owner":[],"postedDate":"January 30th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":18471243,"name":"Biological sciences/Cancer/Cancer metabolism"},{"id":18471244,"name":"Biological sciences/Molecular biology/Non-coding RNAs"}],"tags":[],"updatedAt":"2023-08-19T07:05:34+00:00","versionOfRecord":{"articleIdentity":"rs-2485059","link":"https://doi.org/10.1038/s41388-023-02757-9","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2023-06-26 04:00:00","publishedOnDateReadable":"June 26th, 2023"},"versionCreatedAt":"2023-01-30 20:58:22","video":"","vorDoi":"10.1038/s41388-023-02757-9","vorDoiUrl":"https://doi.org/10.1038/s41388-023-02757-9","workflowStages":[]},"version":"v1","identity":"rs-2485059","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2485059","identity":"rs-2485059","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}