Bioengineered miR-148a-3p suppresses glycolysis and amino acid homeostasis in hepatocellular carcinoma cells by regulating multiple solute carrier transporters

Abstract

Background and aims: Hepatocellular carcinoma (HCC) cells are metabolically reprogrammed for excessive uptake and metabolism of many nutrients. The tumor suppressive microRNA-148a-3p (miR-148a-3p) is downregulated in HCC, whereas its function in regulating HCC cell metabolism remains obscure. Herein we aimed to delineate the role of miR-148a-3p in HCC cell metabolism by using novel bioengineered miR-148a-3p (BioRNALeu/miR-148a-3p) agent producedin vivo.

Methods: BioRNALeu/miR-148a-3p was designed by using human leucyl transfer RNA fused hsa-pre-miR-34a carrier, overexpressed inEscherichia coli (E. coli), and purified to high homogeneity. After transfection into HCC cells, the released miR-148a-3p levels were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell proliferation was determined by CellTiter-Glo assays. Targets were validated by dual-luciferase reporter assays, immunoblotting, and immunofluorescence confocal imaging. Glycolysis capacity was evaluated by Seahorse XF assays, and glucose, lactate, and amino acid levels were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods.

Results: BioRNALeu/miR-148a-3p was efficiently processed into target miR-148a-3p in HCC cells to effectively inhibit cell proliferation in a dose- and time-dependent manner. Mechanistically, miR-148a-3p suppressed the protein levels of glucose transporter GLUT1/SLC2A1 and L-type amino acid transporter LAT1/SLC7A5 via acting on their 3′-untranslated regions, as well as amino acid transporter ASCT2/SLC1A5. These, in turn, led to a reduction of glucose uptake, lactate production, and glycolytic flux in HCC cells, and alteration of intracellular amino acid metabolome including glutamine, leucine, phenylalanine, tyrosine, and methionine.

Conclusions: Reintroduction of miR-148a-3p into HCC cells modulates glucose and amino acid metabolism via regulating multiple SLC transporters, thereby suppressing HCC cell viability. These findings highlight the role of miR-148a-3p in HCC cell metabolism and potential of bioengineered miRNA molecules for functional studies and therapeutic development.

Keywords

Hepatocellular carcinoma (HCC) / MicroRNA-148a-3p (miR-148a-3p) / Cell metabolism / Glucose / Amino acids

References

[1] Chan SL, Sun HC, Xu Y, et al. The Lancet Commission on addressing the global hepatocellular carcinoma burden: comprehensive strategies from prevention to treatment. Lancet. 2025; 406:731-778. https://doi.org/10.1016/S0140—6736(25)01042—6.

[2] Hwang SY, Danpanichkul P, Agopian V, et al. Hepatocellular carcinoma: updates on epidemiology, surveillance, diagnosis and treatment. Clin Mol Hepatol. 2025; 31(Suppl):S228-S254. https://doi.org/10.3350/cmh.2024.0824.

[3] European Association for the Study of the Liver. EASL Clinical Practice Guidelines on the management of hepatocellular carcinoma. J Hepatol. 2025; 82:315-374. https://doi.org/10.1016/j.jhep.2024.08.028.

[4] Sugimoto R, Kurokawa M, Tanaka Y, Senju T, Kohjima M, Tanaka M . Advances in systemic therapy for hepatocellular carcinoma and future prospects. Curr Oncol. 2025; 32:490. https://doi.org/10.3390/curroncol32090490.

[5] Liu X, Xia F, Chen Y, et al. Chinese expert consensus on refined diagnosis, treatment, and management of advanced primary liver cancer (2023 edition). Liver Res. 2024; 8:61-71. https://doi.org/10.1016/j.livres.2024.05.001.

[6] An L, Li Z . Molecular network of metabolic reprogramming and precision diagnosis and treatment of hepatocellular carcinoma. Biomark Res. 2025; 13:124. https://doi.org/10.1186/s40364—025—00844—5.

[7] Li X, Zhou L, Xu X, et al. Metabolic reprogramming in hepatocellular carcinoma: a bibliometric and visualized study from 2011 to 2023. Front Pharmacol. 2024; 15:1392241. https://doi.org/10.3389/fphar.2024.1392241.

[8] Park S, Hall MN . Metabolic reprogramming in hepatocellular carcinoma: mechanisms and therapeutic implications. Exp Mol Med. 2025; 57:515-523. https://doi.org/10.1038/s12276—025—01415—2.

[9] Tang Q, Wu S, Zhao B, et al. Reprogramming of glucose metabolism: the hallmark of malignant transformation and target for advanced diagnostics and treatments. Biomed Pharmacother . 2024; 178:117257. https://doi.org/10.1016/j.biopha.2024.117257.

[10] Jakobsen S, Nielsen CU . Exploring amino acid transporters as therapeutic targets for cancer: an examination of inhibitor structures, selectivity issues, and discovery approaches. Pharmaceutics. 2024; 16:197. https://doi.org/10.3390/pharmaceutics16020197.

[11] Di Martino MT, Tagliaferri P, Tassone P . MicroRNA in cancer therapy: breakthroughs and challenges in early clinical applications. J Exp Clin Cancer Res. 2025; 44:126. https://doi.org/10.1186/s13046—025—03391—x.

[12] Liapopoulos D, Sarantis P, Biniari T, et al. MicroRNA landscape in hepatocellular carcinoma: metabolic re—wiring, predictive and diagnostic biomarkers, and emerging therapeutic targets. Biomedicines. 2025; 13:2243. https://doi.org/10.3390/biomedicines13092243.

[13] Guo F, Li H, Wang J, et al. MicroRNAs in hepatocellular carcinoma: insights into regulatory mechanisms, clinical significance, and therapeutic potential. Cancer Manag Res. 2024; 16:1491-1507. https://doi.org/10.2147/Cmar.S477698.

[14] Traber GM, Yu AM . RNAi—based therapeutics and novel RNA bioengineering technologies. J Pharmacol Exp Ther. 2023; 384:133-154. https://doi.org/10.1124/jpet.122.001234.

[15] Yu AM, Jian C, Yu AH, Tu MJ . RNA therapy: are we using the right molecules? Pharmacol Ther. 2019; 196:91-104. https://doi.org/10.1016/j.pharmthera.2018.11.011.

[16] Cronin JM, Yu AM . Recombinant technologies facilitate drug metabolism, pharmacokinetics, and general biomedical research. Drug Metab Dispos. 2023; 51:685-699. https://doi.org/10.1124/dmd.122.001008.

[17] Traber GM, Yi C, Batra N, Tu MJ, Yu AM . Novel RNA molecular bioengineering technology efficiently produces functional miRNA agents. RNA. 2024; 30:680-694. https://doi.org/10.1261/rna.079904.123.

[18] Li PC, Tu MJ, Ho PY, et al. In vivo fermentation production of humanized noncoding RNAs carrying payload miRNAs for targeted anticancer therapy. Theranostics. 2021; 11:4858-4871. https://doi.org/10.7150/thno.56596.

[19] Tu MJ, Ho PY, Zhang QY, et al. Bioengineered miRNA—1291 prodrug therapy in pancreatic cancer cells and patient—derived xenograft mouse models. Cancer Lett. 2019; 442:82-90. https://doi.org/10.1016/j.canlet.2018.10.038.

[20] Traber GM, Tu MJ, Guan S, Batra N, Yu AM . Bioengineered miR—7—5p modulates non—small cell lung cancer cell metabolism to improve therapy. Mol Pharmacol. 2025; 107:100006. https://doi.org/10.1016/j.molpha.2024.100006.

[21] Li X, Tian Y, Tu MJ, Ho PY, Batra N, Yu AM . Bioengineered miR—27b—3p and miR—328—3p modulate drug metabolism and disposition via the regulation of target ADME gene expression. Acta Pharm Sin B. 2019; 9:639-647. https://doi.org/10.1016/j.apsb.2018.12.002.

[22] Petrek H, Yan Ho P, Batra N, et al. Single bioengineered ncRNA molecule for dual—targeting toward the control of non—small cell lung cancer patient—derived xenograft tumor growth. Biochem Pharmacol. 2021; 189:114392. https://doi.org/10.1016/j.bcp.2020.114392.

[23] Jilek JL, Zhang QY, Tu MJ, et al. Bioengineered Let—7c inhibits orthotopic hepatocellular carcinoma and improves overall survival with minimal immunogenicity. Mol Ther Nucleic Acids. 2019; 14:498-508. https://doi.org/10.1016/j.omtn.2019.01.007.

[24] Babu KR, Muckenthaler MU . miR—148a regulates expression of the transferrin receptor 1 in hepatocellular carcinoma. Sci Rep . 2019; 9:1518. https://doi.org/10.1038/s41598—018—35947—7.

[25] Dawood AA, Saleh AA, Elbahr O, Gohar SF, Habieb MS . Inverse relationship between the level of miRNA 148a—3p and both TGF—beta1 and FIB—4 in hepatocellular carcinoma. Biochem Biophys Rep. 2021; 27:101082. https://doi.org/10.1016/j.bbrep.2021.101082.

[26] Huang Z, Wen J, Yu J, et al. MicroRNA—148a—3p inhibits progression of hepatocelluar carcinoma by repressing SMAD2 expression in an Ago2 dependent manner. J Exp Clin Cancer Res. 2020; 39:150. https://doi.org/10.1186/s13046—020—01649—0.

[27] Yuan X, Wen Y, Shi Q, Zhao Y, Ding J . MicroRNA—148a—3p suppresses the glycolysis and cell proliferation by targeting transmembrane protein 54 in liver cancer. Biochem Biophys Res Commun. 2024; 695:149424. https://doi.org/10.1016/j.bbrc.2023.149424.

[28] Foglia B, Beltra M, Sutti S, Cannito S . Metabolic reprogramming of HCC: a new microenvironment for immune responses. Int J Mol Sci. 2023; 24:7463. https://doi.org/10.3390/ijms24087463.

[29] Tu MJ, Wright HK, Batra N, Yu AM . Expression and purification of tRNA/pre—miRNA—based recombinant noncoding RNAs.Methods Mol Biol . 2021; 2323:249-265. https://doi.org/10.1007/978—1—0716—1499—0_18.

[30] Batra N, Tu MJ, Yu AM . Molecular engineering of functional siRNA agents. ACS Synth Biol. 2024; 13:1906-1915. https://doi.org/10.1021/acssynbio.4c00181.

[31] Chen Y, Tu MJ, Han F, et al. Use of recombinant microRNAs as antimetabolites to inhibit human non—small cell lung cancer. Acta Pharm Sin B. 2023; 13:4273-4290. https://doi.org/10.1016/j.apsb.2023.07.011.

[32] Yi W, Tu MJ, Liu Z, et al. Bioengineered miR—328—3p modulates GLUT1—mediated glucose uptake and metabolism to exert synergistic anti—proliferative effects with chemotherapeutics. Acta Pharm Sin B . 2020; 10:159-170. https://doi.org/10.1016/j.apsb.2019.11.001.

[33] Liu Z, Tu MJ, Zhang C, Jilek JL, Zhang QY, Yu AM . A reliable LC—MS/MS method for the quantification of natural amino acids in mouse plasma: method validation and application to a study on amino acid dynamics during hepatocellular carcinoma progression. J Chromatogr B Analyt Technol Biomed Life Sci. 2019; 1124:72-81. https://doi.org/10.1016/j.jchromb.2019.05.039.

[34] Mao FY, Tu MJ, Traber GM, Yu AM . Comparison of three computational tools for the prediction of RNA tertiary structures. Noncoding RNA . 2024; 10:55. https://doi.org/10.3390/ncrna10060055.

[35] Wu L, Qiu W, Sun J . Down regulation of miR—148a is related to enhanced pancreatic cancer pathogenesis through targeting GLUT1. Int J Clin Exp Pathol. 2018; 11:4950-4956.

[36] Ding X, Liu J, Liu T, Ma Z, Wen D, Zhu J . miR—148b inhibits glycolysis in gastric cancer through targeting SLC2A1. Cancer Med . 2017; 6:1301-1310. https://doi.org/10.1002/cam4.1008.

[37] Nicklin P, Bergman P, Zhang B, et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell . 2009; 136:521-534. https://doi.org/10.1016/j.cell.2008.11.044.

[38] van Geldermalsen M, Wang Q, Nagarajah R, et al. ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple—negative basal—like breast cancer. Oncogene. 2016; 35:3201-3208. https://doi.org/10.1038/onc.2015.381.

[39] Deng L, Petrek H, Tu MJ, Batra N, Yu AX, Yu AM . Bioengineered miR—124—3p prodrug selectively alters the proteome of human carcinoma cells to control multiple cellular components and lung metastasis in vivo. Acta Pharm Sin B. 2021; 11:3950-3965. https://doi.org/10.1016/j.apsb.2021.07.027.

[40] Ho PY, Duan Z, Batra N, et al. Bioengineered noncoding RNAs selectively change cellular miRNome profiles for cancer therapy. J Pharmacol Exp Ther. 2018; 365:494-506. https://doi.org/10.1124/jpet.118.247775.

[41] Long XR, He Y, Huang C, Li J . MicroRNA—148a is silenced by hypermethylation and interacts with DNA methyltransferase 1 in hepatocellular carcinogenesis. Int J Oncol. 2014; 44:1915-1922. https://doi.org/10.3892/ijo.2014.2373.

[42] Yao N, Qu G, Shi Y, et al. The SNHG3/miR—148a—3p axis—mediated high expression of DNMT1 is correlated with poor prognosis and tumor immune infiltration in hepatocellular carcinoma. J Gastrointest Oncol. 2025; 16:2357-2376. https://doi.org/10.21037/jgo—2025—641.

[43] Zhang X, Chen F, Huang P, et al. Exosome—depleted miR—148a—3p derived from hepatic stellate cells promotes tumor progression via ITGA5/PI3K/Akt axis in hepatocellular carcinoma. Int J Biol Sci. 2022; 18:2249-2260. https://doi.org/10.7150/ijbs.66184.

[44] Zhao X, Chai W, Wang T, et al. Identification of the prognostic, diagnostic, and biological significance of the miR—148a—3p/cathepsin A axis in hepatocellular carcinoma. J Biochem Mol Toxicol. 2022; 36:e23208. https://doi.org/10.1002/jbt.23208.

[45] Tiemin P, Peng X, Qingfu L, et al. Dysregulation of the miR—148a—GLUT1 axis promotes the progression and chemoresistance of human intrahepatic cholangiocarcinoma. Oncogenesis. 2020; 9:19. https://doi.org/10.1038/s41389—020—0207—2.

[46] Chadt A, Al—Hasani H . Glucose transporters in adipose tissue, liver, and skeletal muscle in metabolic health and disease. Pflügers Arch. 2020; 472:1273-1298. https://doi.org/10.1007/s00424—020—02417—x.

[47] Karim S, Adams DH, Lalor PF . Hepatic expression and cellular distribution of the glucose transporter family. World J Gastroenterol . 2012; 18:6771-6781. https://doi.org/10.3748/wjg.v18.i46.6771.

[48] Semenza GL . HIF—1 and mechanisms of hypoxia sensing. Curr Opin Cell Biol. 2001; 13:167-171. https://doi.org/10.1016/s0955—0674(00)00194—0.

[49] Amann T, Maegdefrau U, Hartmann A, et al. GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis. Am J Pathol. 2009; 174:1544-1552. https://doi.org/10.2353/ajpath.2009.080596.

[50] Ruggieri F, Jonas K, Ferracin M, Dengler M, Jäger V, Pichler M . MicroRNAs as regulators of tumor metabolism. Endocr Relat Cancer. 2023; 30:e220267. https://doi.org/10.1530/ERC—22—0267.

[51] Zhuang Y, Wang XX, He J, He S, Yin Y . Recent advances in understanding of amino acid signaling to mTORC1 activation. Front Biosci (Landmark Ed) . 2019; 24:971-982. https://doi.org/10.2741/4762.

[52] Kanai Y. Amino acid transporter LAT1 (SLC7A5) as a molecular target for cancer diagnosis and therapeutics. Pharmacol Ther. 2022; 230:107964. https://doi.org/10.1016/j.pharmthera.2021.107964.

[53] Yi C, Yu AM . MicroRNAs in the regulation of solute carrier proteins behind xenobiotic and nutrient transport in cells. Front Mol Biosci . 2022; 9:893846. https://doi.org/10.3389/fmolb.2022.893846.

[54] Yang Z, Follett J, Kerr MC, et al. Sorting nexin 27 (SNX27) regulates the trafficking and activity of the glutamine transporter ASCT2. J Biol Chem. 2018; 293:6802-6811. https://doi.org/10.1074/jbc.RA117.000735.

[55] Efeyan A, Comb WC, Sabatini DM . Nutrient—sensing mechanisms and pathways. Nature. 2015; 517:302-310. https://doi.org/10.1038/nature14190.

[56] Nguyen TP, Frank AR, Jewell JL . Amino acid and small GTPase regulation of mTORC1. Cell Logist . 2017; 7:e1378794. https://doi.org/10.1080/21592799.2017.1378794.

[57] DeBerardinis RJ, Chandel NS . Fundamentals of cancer metabolism. Sci Adv . 2016; 2:e1600200. https://doi.org/10.1126/sciadv.1600200.

[58] Tufail M, Jiang CH, Li N . Altered metabolism in cancer: insights into energy pathways and therapeutic targets. Mol Cancer. 2024; 23:203. https://doi.org/10.1186/s12943—024—02119—3.

[59] Dou Q, Grant AK, Coutinto de Souza P, et al. Characterizing metabolic heterogeneity of hepatocellular carcinoma with hyperpolarized 13C pyruvate MRI and mass spectrometry. Radiol Imaging Cancer. 2024; 6:e230056. https://doi.org/10.1148/rycan.230056.

[60] Wang Q, Liu J, Chen Z, Zheng J, Wang Y, Dong J . Targeting metabolic reprogramming in hepatocellular carcinoma to overcome therapeutic resistance: a comprehensive review. Biomed Pharmacother . 2024; 170:116021. https://doi.org/10.1016/j.biopha.2023.116021.