piR-7472 affects the mechanism by which voltage-regulated potassium channels promote osteogenic differentiation in mice

doi:10.12307/2025.610

Abstract

Abstract: BACKGROUND: Existing studies have made significant progress in PIWI-interacting RNAs (piRNAs) against osteoporosis, but the specific targets and related mechanisms by which piRNAs exert their functions remain to be explored.
OBJECTIVE: To investigate the effects and downstream mechanisms of piR-7472 on the differentiation of mouse osteoblasts (MC3T3-E1 cells).
METHODS: (1) Twelve C57/BL6J mice were randomly divided into a sham-operated and an ovariectomized group, with six mice in each group. Changes in bone mass and the expression of piR-7472 were detected using Micro-CT and RT-qPCR, respectively, at 8 weeks after surgery. (2) MC3T3-E1 cells were divided into NC mimics group, piR-7472 mimics group, NC inhibitor group, and piR-7472 inhibitor group. The mRNA expression of piR-7472, osteopontin, type I collagen, Runt-related transcription factor 2, and potassium voltage-gated channel modifier subfamily F member 1 were detected by RT-qPCR after 7 days of osteogenic induction. The protein expression of osteopontin, Runt-related transcription factor 2, bone morphogenetic protein 2, and potassium voltage-gated channel modifier subfamily F member 1 (KCNF1) was detected using western blot assay. The expression of alkaline phosphatase was detected by alkaline phosphatase staining after 14 days of osteogenic induction, and the number of mineralized nodules was detected by alizarin red staining after 21 days of induction. Whether piR-7472 could bind to KCNF1 was observed by the dual luciferase reporter gene assay.
RESULTS AND CONCLUSION: (1) Bone mineral density, bone volume fraction, bone trabecular thickness, bone trabecular number were significantly decreased and bone trabecular separation was significantly increased in ovariectomized mice, and piR-7472 in bone tissue was significantly down-regulated in osteoporotic mice. (2) Compared with the NC group, the mRNA expression of osteopontin, type I collagen, and Runt-related transcription factor 2 were significantly increased, the protein expression of osteopontin, Runt-related transcription factor 2, and bone morphogenetic protein 2 were significantly elevated, and the levels of mineralized deposition and alkaline phosphatase were increased in the piR-7472 mimics group. Compared with the NC inhibitor group, the mRNA expression of osteopontin, type I collagen, and Runt-related transcription factor 2 was significantly downregulated, the protein expression of osteopontin, Runt-related transcription factor 2, and bone morphogenetic protein 2 were significantly decreased, and the levels of mineralized deposition and alkaline phosphatase were reduced in the piR-7472 inhibitor group. (3) piR-7472 was found to interact with the potassium voltage-gated channel modifier subfamily F member 1 as predicted by the miRanda database. The dual luciferase reporter gene assay revealed that piR-7472 mimics could bind to and promote the expression of KCNF1. To conclude, piR-7472 can promote osteogenic differentiation of osteogenic precursor cells MC3T3-E1, and its mechanism of action may be achieved by promoting the expression of KCNF1.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: MC3T3-E1 osteoblasts, PIWI-interacting RNA, osteogenic differentiation, osteoporosis, voltage-regulated potassium channel, KCNF1

CLC Number:

Long Yubin, Wang Xiangbin, Fan Jigeng, Yang Houzhi, Yang Yang, Li Yong. piR-7472 affects the mechanism by which voltage-regulated potassium channels promote osteogenic differentiation in mice[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(14): 2868-2874.

Figures/Tables 3

2.1 骨质疏松小鼠骨量下降及骨组织中piR-7472的表达下调 8周龄C57/BL6J小鼠实施卵巢切除后8周进行Micro-CT检测，与假手术组相比，卵巢切除组小鼠骨量明显下降(图 1A)，骨密度、骨体积分数、骨小梁厚度、骨小梁数量明显下降，骨小梁分离度明显升高(图1B-F)，说明造模成功。RT-qPCR检测卵巢切除组小鼠骨组织中 piR-7472 的表达明显下降(图1G)。 2.2 piR-7472促进小鼠成骨细胞 MC3T3-E1成骨分化为了探究piR-7472对于成骨细胞成骨性分化的影响，对MC3T3-E1细胞进行转染，通过荧光素修饰piR-7472转染，发现100 pmol piR-7472转染48 h的转染效果最佳(图2A)。转染后成骨诱导7 d检测成骨特异性基因表达，结果显示，与NC组相比，piR-7472 mimics能够促进骨桥蛋白、胶原蛋白Ⅰ、Runt相关转录因子2 mRNA表达(P < 0.05)；与NC inhibitor组相比，piR-7472 inhibitor 能够抑制骨桥蛋白、胶原蛋白Ⅰ、Runt相关转录因子2 mRNA表达(P < 0.05)(图2B-D)。转染后成骨诱导7 d检测成骨分化特异性蛋白表达，结果显示，与 NC 组相比，转染piR-7472 mimics组骨桥蛋白、Runt相关转录因子2、骨形态发生蛋白2蛋白表达显著升高(P < 0.05)；与NC inhibitor组相比，piR-7472 inhibitor 组骨桥蛋白、Runt相关转录因子2、骨形态发生蛋白2 蛋白表达显著下降(P < 0.05)(图2E-H)，与mRNA表达表现出相同的趋势。成骨诱导14 d碱性磷酸酶染色以及成骨诱导21 d茜素红染色结果显示，与 NC 组相比，piR-7472 mimics 组的橘红色沉积(矿化结节)与碱性磷酸酶(紫色)表达明显增多；与NC inhibitor组相比，piR-7472 inhibitor 组橘红色沉积(矿化结节)与碱性磷酸酶(紫色)表达明显减少(图2I-K)，说明piR-7472能够促进MC3T3-E1细胞碱性磷酸酶的表达以及矿化结节形成从而促进成骨分化与矿化。通过上述结果，发现piR-7472能够在成骨的早期和成熟阶段促进MC3T3-E1成骨分化和矿化。 2.3 piR-7472通过影响KCNF1促进成骨分化通过miRanda数据库分析显示，piR-7472与 KCNF1具有结合位点(图3A)。双荧光素酶报告基因实验提示piR-7472能够与KCNF1结合并促进KCNF1的表达(图3B)。piR-7472可能通过与KCNF1结合从而促进成骨细胞成骨分化。为了探究piR-7472是否能通过影响KCNF1的表达发挥其促进骨形成的功能。转染piR-7472后检测KCNF1的表达。与对照组相比，piR-7472能促进KCNF1的mRNA与蛋白表达(P < 0.05) (图3C-E)。上述结果表明，piR-7472可能是通过KCNF1发挥促进成骨细胞成骨分化的作用。"

References

[1] 《中国老年骨质疏松症诊疗指南(2023)》工作组,中国老年学和老年医学学会骨质疏松分会,中国医疗保健国际交流促进会骨质疏松病学分会,等.中国老年骨质疏松症诊疗指南(2023)[J].中华骨与关节外科杂志,2023,16(10):865-885.
[2] BAUTISTA LITARDO N, LÓPEZ GAVILANEZ E. First Ecuadorian consensus statement on the management of osteoporosis. Arch Osteoporos. 2023;18(1):106.
[3] LIU YY, DING YF, SUI HJ, et al. Pilose antler (Cervus elaphus Linnaeus) polysaccharide and polypeptide extract inhibits bone resorption in high turnover type osteoporosis by stimulating the MAKP and MMP-9 signaling pathways. J Ethnopharmacol. 2023;304:116052.
[4] HE X, WANG Y, LIU Z, et al. Osteoporosis treatment using stem cell-derived exosomes: a systematic review and meta-analysis of preclinical studies. Stem Cell Res Ther. 2023;14(1):72.
[5] LI N, CORNELISSEN D, SILVERMAN S, et al. An Updated Systematic Review of Cost-Effectiveness Analyses of Drugs for Osteoporosis. Pharmacoeconomics. 2021;39(2):181-209.
[6] GARCÍA-GARCÍA P, REYES R, ÉVORA C, et al. Osteoprotective effect of the marine alkaloid norzoanthamine on an osteoporosis model in ovariectomized rat. Biomed Pharmacother. 2022;147:112631.
[7] CHEN J, ZHONG K, QIN S, et al. Astragalin: a food-origin flavonoid with therapeutic effect for multiple diseases. Front Pharmacol. 2023; 14:1265960.
[8] HUANG X, WONG G. An old weapon with a new function: PIWI-interacting RNAs in neurodegenerative diseases. Transl Neurodegener. 2021;10(1):9.
[9] LEE EJ, BANERJEE S, ZHOU H, et al. Identification of piRNAs in the central nervous system. RNA. 2011;17(6):1090-1099.
[10] GALTON R, FEJES-TOTH K, BRONNER ME. Co-option of the piRNA pathway to regulate neural crest specification. Sci Adv. 2022;8(32): eabn1441.
[11] BRENNECKE J, ARAVIN AA, STARK A, et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell. 2007;128(6):1089-1103.
[12] JONES BC, WOOD JG, CHANG C, et al. A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan. Nat Commun. 2016;7:13856.
[13] KAMMINGA LM, LUTEIJN MJ, DEN BROEDER MJ, et al. Hen1 is required for oocyte development and piRNA stability in zebrafish. EMBO J. 2010;29(21):3688-3700.
[14] MOORE RS, KALETSKY R, MURPHY CT. Piwi/PRG-1 Argonaute and TGF-β Mediate Transgenerational Learned Pathogenic Avoidance. Cell. 2019;177(7):1827-1841.e12.
[15] GONG S, GACCIOLI F, DOPIERALA J, et al. The RNA landscape of the human placenta in health and disease. Nat Commun. 2021;12(1):2639.
[16] OZATA DM, GAINETDINOV I, ZOCH A, et al. PIWI-interacting RNAs: small RNAs with big functions. Nat Rev Genet. 2019;20(2):89-108.
[17] ISHIZU H, SIOMI H, SIOMI MC. Biology of PIWI-interacting RNAs: new insights into biogenesis and function inside and outside of germlines. Genes Dev. 2012;26(21):2361-2373.
[18] WANG X, RAMAT A, SIMONELIG M, et al. Emerging roles and functional mechanisms of PIWI-interacting RNAs. Nat Rev Mol Cell Biol. 2023; 24(2):123-141.
[19] HAN H, FAN G, SONG S, et al. piRNA-30473 contributes to tumorigenesis and poor prognosis by regulating m6A RNA methylation in DLBCL. Blood. 2021;137(12):1603-1614.
[20] GAO XQ, ZHANG YH, LIU F, et al. The piRNA CHAPIR regulates cardiac hypertrophy by controlling METTL3-dependent N6-methyladenosine methylation of Parp10 mRNA. Nat Cell Biol. 2020;22(11):1319-1331.
[21] NILSSON KH, HENNING P, EL SHAHAWY M, et al. Osteocyte- and late osteoblast-derived NOTUM reduces cortical bone mass in mice. Am J Physiol Endocrinol Metab. 2021;320(5):E967-E975.
[22] LEE WC, GUNTUR AR, LONG F, et al. Energy Metabolism of the Osteoblast: Implications for Osteoporosis. Endocr Rev. 2017;38(3):255-266.
[23] MAI D, ZHENG Y, GUO H, et al. Serum piRNA-54265 is a New Biomarker for early detection and clinical surveillance of Human Colorectal Cancer. Theranostics. 2020;10(19):8468-8478.
[24] HAN YN, LI Y, XIA SQ, et al. PIWI Proteins and PIWI-Interacting RNA: Emerging Roles in Cancer. Cell Physiol Biochem. 2017;44(1):1-20.
[25] PLEŠTILOVÁ L, NEIDHART M, RUSSO G, et al. Expression and Regulation of PIWIL-Proteins and PIWI-Interacting RNAs in Rheumatoid Arthritis. PLoS One. 2016;11(11):e0166920.
[26] LIU Y, DOU M, SONG X, et al. The emerging role of the piRNA/piwi complex in cancer. Mol Cancer. 2019;18(1):123.
[27] FENG J, YANG M, WEI Q, et al. Novel evidence for oncogenic piRNA-823 as a promising prognostic biomarker and a potential therapeutic target in colorectal cancer. J Cell Mol Med. 2020;24(16):9028-9040.
[28] DELLA BELLA E, MENZEL U, BASOLI V, et al. Differential Regulation of circRNA, miRNA, and piRNA during Early Osteogenic and Chondrogenic Differentiation of Human Mesenchymal Stromal Cells. Cells. 2020; 9(2):398.
[29] MANNING D, SANTANA LF. Regulating voltage-gated ion channels with nanobodies. Nat Commun. 2022;13(1):7557.
[30] YANG JE, SONG MS, SHEN Y, et al. The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization. Int J Mol Sci. 2016; 17(3):407.
[31] WU J, ZHONG D, FU X, et al. Silencing of Ether à go-go 1 by shRNA inhibits osteosarcoma growth and cell cycle progression. Int J Mol Sci. 2014;15(4):5570-5581.
[32] WU X, ZHONG D, GAO Q, et al. MicroRNA-34a inhibits human osteosarcoma proliferation by downregulating ether à go-go 1 expression. Int J Med Sci. 2013;10(6):676-682.