Differential expression of RNA binding protein Lin28A regulates osteoblastic differentiation of periodontal ligament stem cells

doi:10.12307/2023.713

Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (33): 5283-5291.doi: 10.12307/2023.713

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Differential expression of RNA binding protein Lin28A regulates osteoblastic differentiation of periodontal ligament stem cells

He Qin1, 2, 3, Bu Yan1, 2, Lin Guanglei1, 2, Luo Jing1, 2, Yong Min1, 3, Huang Yongqing1, 4

1Department of Orthodontics, School of Stomatology, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 2Key Laboratory of Stem Cell and Regenerative Medicine of Ningxia Hui Autonomous Region, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 3Department of Orthodontics, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 4Department of Oral and Maxillofacial Surgery, School of Stomatology, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China

Received:2022-10-08 Accepted:2022-11-18 Online:2023-11-28 Published:2023-03-30
Contact: He Qin, MD, Associate professor, Associate chief physician, Department of Orthodontics, School of Stomatology, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Key Laboratory of Stem Cell and Regenerative Medicine of Ningxia Hui Autonomous Region, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Department of Orthodontics, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
About author:He Qin, MD, Associate professor, Associate chief physician, Department of Orthodontics, School of Stomatology, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Key Laboratory of Stem Cell and Regenerative Medicine of Ningxia Hui Autonomous Region, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Department of Orthodontics, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Supported by:
Natural Science Foundation of Ningxia Hui Autonomous Region, No. 2019AAC03078 (to HQ); School Level Special Talents Project of Ningxia Medical University, No. XT2018019 (to HQ); the Backup Training Object Project for Academic and Technical Leaders at the School Level of Ningxia Medical University in 2020, No. 20012602201 (to HQ)

Abstract

Abstract:

BACKGROUND: The cellular activity of Lin28A can be influenced in several ways, including the self-renewal of embryonic stem cells, somatic cell reprogramming, individual growth and development, tissue metabolism, and carcinogenic consequences. However, neither domestically nor internationally have its effects on periodontal ligament stem cell ability to differentiate into osteoblasts been well explored; the underlying mechanisms are not known.

OBJECTIVE: To investigate the impact of Lin28A on periodontal ligament stem cell capacity for osteogenic differentiation.

METHODS: By using conventional enzyme digestion, periodontal ligament stem cells were extracted and cultured. The distribution of Lin28A in these cells and the differential expression of Lin28A at different time points of osteogenic induction were detected by qRT-PCR. Lentiviral vectors with Lin28A overexpression and interference expression were constructed and transfected into periodontal ligament stem cells. Fluorescence microscopy was used to observe the transfection effect. qRT-PCR was used to verify the expression difference of Lin28A after lentivirus transfection. The expression levels of osteoblastic genes alkaline phosphatase, Runt-related transcription factor 2 and osteopontin, alkaline phosphatase staining and alizarin red staining results were further detected from positive and negative aspects. The protein expression level of Runt-related transcription factor 2 was detected by western blot assay. Bioinformatics techniques and dual luciferase were used to predict and verify the relationship between Lin28A-related let-7 family members. The periodontal ligament stem cells differentially expressing Lin28A were cultured through osteoblastic induction, and the miRNA with the most obvious expression change during osteoblastic differentiation was determined.
RESULTS AND CONCLUSION: (1) Lin28A was enriched in the nucleus of periodontal ligament stem cells, and the expression of Lin28A increased at different time points after osteogenic induction. (2) In the periodontal ligament stem cell models with Lin28A overexpression, alkaline phosphatase staining and alizarin red staining were significantly deeper than those in the blank control group; the expression levels of alkaline phosphatase, Runt-related transcription factor 2 and osteopontin were increased 3.3, 5.1 and 2.2 times, respectively, compared with the blank ones (P < 0.01). In cell models that interfered with Lin28A expression, alkaline phosphatase staining and alizarin red staining were also decreased compared with the control group. The levels of alkaline phosphatase, Runt-related transcription factor 2 and osteopontin decreased 0.19-fold, 0.61-fold and 0.1-fold, respectively (P < 0.01). (3) The protein level of Runt-related transcription factor 2 was consistent with the mRNA difference tendency. The expression of Runt-related transcription factor 2 was increased in the overexpression group and decreased in the interference group. (4) Bioinformatics predicted that Lin28A was associated with eight let-7 family members, which were let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g and let-7i, respectively. After screening, the expression levels of let-7a and let-7c showed significant changes with Lin28A expression, and let-7a showed the most significant trend. Double luciferase binding assay confirmed that let-7a was directly bound to the 3'-UTR of Lin28A and was involved in the osteogenic differentiation of periodontal ligament stem cells. (5) The results suggested that Lin28A was involved in the regulation of osteogenic differentiation of periodontal ligament stem cells, with a positive correlation. That was, overexpression of Lin28A promoted the osteogenic ability of periodontal ligament stem cells, and the osteogenic ability of periodontal ligament stem cells was weakened after interference of Lin28A expression. The osteogenic differentiation of periodontal ligament stem cells was regulated by Lin28A .

Key words: periodontal ligament stem cell, osteogenic differentiation, Lin28A, lentiviral vector, transfection, genetic interference, let-7a

CLC Number:

He Qin, Bu Yan, Lin Guanglei, Luo Jing, Yong Min, Huang Yongqing. Differential expression of RNA binding protein Lin28A regulates osteoblastic differentiation of periodontal ligament stem cells[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(33): 5283-5291.

Figures/Tables 5

2.4 各组PDLSCs成骨诱导后成骨检测指标的比较经不同慢病毒载体转染的PDLSCs模型给予相应的成骨诱导。成骨诱导7 d，早期指标碱性磷酸酶染色结果显示，过表达Lin28A组的细胞染色较深，而干扰Lin28A表达组碱性磷酸酶染色较少，成骨能力较弱，过表达空载体组和干扰表达空载体组的染色结果与空白对照组基本一致，没有明显的染色差异，见图4A。各组细胞在成骨诱导21 d后，成骨晚期茜素红染色结果显示：过表达Lin28A组细胞红染的钙结节明显、量多；而干扰Lin28A表达组钙结节形成较少，镜下视野呈现出量少或几乎没有的状态，成骨能力显著降低；同期，过表达空载体组和干扰表达空载体组的钙化结节形成量与空白对照组无明显差别，见图4B。收集骨向诱导14 d的各组细胞总RNA，检测每组细胞中成骨标志基因的mRNA表达水平，仍以GAPDH为参照基因。与空白对照组相比较，转染过表达Lin28A载体的PDLSCs成骨基因碱性磷酸酶、Runt相关转录因子2及骨桥蛋白的表达量显著升高，而转染过表达空载体的PDLSCs成骨基因表达水平与空白对照组相比无明显差异，说明过表达Lin28A可以促进PDLSCs成骨向分化各项成骨指标高表达；转染干扰Lin28A表达载体的PDLSCs其成骨基因的表达水平显著下降，转染干扰表达空载体的PDLSCs成骨基因的表达水平与空白对照组比较无明显差异，说明干扰Lin28A表达后PDLSCs的骨向分化能力减弱，得出与过表达Lin28A相反的结果，见图4C，D。经成骨诱导分化，5组细胞的成骨相关蛋白Runt相关转录因子2的表达趋势与成骨基因表现的趋势一样，即过表达Lin28A组的Runt相关转录因子2蛋白表达相较空白对照和空载体对照组显著增高(P < 0.05)，见图4E；而干扰Lin28A表达后，PDLSCs的成骨相关蛋白Runt相关转录因子2的表达量明显降低(P < 0.01)，见图4F。"

References

[1] GAO X, WU Y, LIAO L, et al. Oral organoids: progress and challenges. J Dent Res. 2021;100(5):454-463.
[2] IWASAKI K, PENG Y, KANDA R, et al. Stem cell transplantation and cell-free treatment for periodontal regeneration. Int J Mol Sci. 2022;23(3):1011.
[3] FEI D, XIA Y, ZHAI Q, et al. Exosomes regulate interclonal communication on osteogenic differentiation among heterogeneous osteogenic single-cell clones through PINK1/Parkin-mediated mitophagy. Front Cell Dev Biol. 2021;9:687258.
[4] ZHAO X, SUN W, GUO B, et al. Circular RNA BIRC6 depletion promotes osteogenic differentiation of periodontal ligament stem cells via the miR-543/PTEN/PI3K/AKT/mTOR signaling pathway in the inflammatory microenvironment. Stem Cell Res Ther. 2022;13(1):417.
[5] LUO Y, GOU H, CHEN X, et al. Lactate inhibits osteogenic differentiation of human periodontal ligament stem cells via autophagy through the MCT1-mTOR signaling pathway. Bone. 2022;162:116444.
[6] CORLEY M, BURNS MC, YEO GW. How RNA-binding proteins interact with RNA: molecules and mechanisms. Mol Cell. 2020;78(1):9-29.
[7] VAN NOSTRAND EL, FREESE P, PRATT GA, et al. A large-scale binding and functional map of human RNA-binding proteins. Nature. 2020;583(7818): 711-719.
[8] SHARMA D, ZAGORE LL, BRISTER MM, et al. The kinetic landscape of an RNA-binding protein in cells. Nature. 2021;591(7848):152-156.
[9] AMBROS V, HORVITZ HR. Heterochronic mutants of the nematode caenorhabditis elegans. Science. 1984;226(4673):409-416.
[10] PIEKNELL K, SULISTIO YA, WULANSARI N, et al. LIN28A enhances regenerative capacity of human somatic tissue stem cells via metabolic and mitochondrial reprogramming. Cell Death Differ. 2022;29(3):540-555.
[11] YUKO AE, CARVALHO RIGAUD VO, KURIAN J, et al. LIN28a induced metabolic and redox regulation promotes cardiac cell survival in the heart after ischemic injury. Redox Biol. 2021;47:102162.
[12] LI MA, AMARAL PP, CHEUNG P, et al. A lncRNA fine tunes the dynamics of a cell state transition involving Lin28, let-7 and de novo DNA methylation. Elife. 2017;6:e23468.
[13] HE Q, YANG S, GU X, et al. Long noncoding RNA TUG1 facilitates osteogenic differentiation of periodontal ligament stem cells via interacting with Lin28A. Cell Death Dis. 2018;9(5):455.
[14] PARK JH, PARK BW, KANG YH, et al. Lin28a enhances in vitro osteoblastic differentiation of human periosteum-derived cells. Cell Biochem Funct. 2017; 35(8):497-509.
[15] GUO L, LI L. LIN28A alleviates inflammation, oxidative stress, osteogenic differentiation and mineralization in lipopolysaccharide (LPS)-treated human periodontal ligament stem cells. Exp Ther Med. 2022;23(6):411.
[16] BI R, LYU P, SONG Y, et al. Function of dental follicle progenitor/stem cells and their potential in regenerative medicine: from mechanisms to applications. Biomolecules. 2021;11(7):997.
[17] HONDA M, OHSHIMA H. Biological characteristics of dental pulp stem cells and their potential use in regenerative medicine. J Oral Biosci. 2022;64(1):26-36.
[18] QUEIROZ A, ALBUQUERQUE-SOUZA E, GASPARONI LM, et al. Therapeutic potential of periodontal ligament stem cells. World J Stem Cells. 2021; 13(6):605-618.
[19] LIN L, LI S, HU S, et al. UCHL1 Impairs Periodontal Ligament Stem Cell Osteogenesis in Periodontitis. J Dent Res. 2022;220345221116031. doi: 10.1177/00220345221116031.
[20] MI J, WANG S, LIU P, et al. CUL4B Upregulates RUNX2 to Promote the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Epigenetically Repressing the Expression of miR-320c and miR-372/373-3p. Front Cell Dev Biol. 2022;10:921663.
[21] LEI F, LI M, LIN T, et al.Treatment of inflammatory bone loss in periodontitis by stem cell-derived exosomes. Acta Biomater. 2022;141:333-343.
[22] QI G, YU K, FENG Y, et al. 1α,25-dihydroxyvitamin D3 promotes early osteogenic differentiation of PDLSCs and a 12-year follow-up case of early-onset vitamin D deficiency periodontitis. J Steroid Biochem Mol Biol. 2021;208:105805.
[23] FU L, LI N, YE Y, et al. MicroRNA hsa-Let-7b regulates the osteogenic differentiation of human periodontal ligament stem cells by targeting CTHRC1. Stem Cells Int. 2021;2021:5791181.
[24] XU L, WANG C, LI Y, et al. ANGPTL4 regulates the osteogenic differentiation of periodontal ligament stem cells. Funct Integr Genomics. 2022;22(5):769-781.
[25] XU W, BISWAS J, SINGER RH, et al. Targeted RNA editing: novel tools to study post-transcriptional regulation. Mol Cell. 2022;82(2):389-403.
[26] YOON DS, LEE KM, CHOI Y, et al. TLR4 downregulation by the RNA-binding protein PUM1 alleviates cellular aging and osteoarthritis. Cell Death Differ. 2022;29(7):1364-1378.
[27] LEE MH, WU X, ZHU Y. RNA-binding protein PUM2 regulates mesenchymal stem cell fate via repression of JAK2 and RUNX2 mRNAs. J Cell Physiol. 2020; 235(4):3874-3885.
[28] WU K, ZHANG R, LU Y, et al. Lin28B regulates the fate of grafted mesenchymal stem cells and enhances their protective effects against Alzheimer’s disease by upregulating IGF-2. J Cell Physiol. 2019;234(12):21860-21876.
[29] SHYH-CHANG N, DALEY GQ. Lin28: primal regulator of growth and metabolism in stem cells. Cell Stem Cell. 2013;12(4):395-406.
[30] WU K, AHMAD T, ERI R. LIN28A: A multifunctional versatile molecule with future therapeutic potential. World J Biol Chem. 2022;13(2):35-46.
[31] BROUGHTON K, ESQUER C, ECHEAGARAY O, et al. Surface Lin28A expression consistent with cellular stress parallels indicators of senescence. Cardiovasc Res. 2022:cvac122.
[32] JOUAN Y, BOUCHEMLA Z, BARDÈCHE-TRYSTRAM B, et al. Lin28a induces SOX9 and chondrocyte reprogramming via HMGA2 and blunts cartilage loss in mice. Sci Adv. 2022;8(34):eabn3106.
[33] WU XL, ZHU ZS, XIAO X, et al. LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells. Zool Res. 2021;42(3):377-388.
[34] SANG H, WANG D, ZHAO S, et al. Dppa3 is critical for Lin28a-regulated ES cells naive-primed state conversion. J Mol Cell Biol. 2019;11(6):474-488.
[35] VALIULIENĖ G, ZENTELYTĖ A, BERŽANSKYTĖ E, et al. Metabolic profile and neurogenic potential of human amniotic fluid stem cells from normal vs. fetus-affected gestations. Front Cell Dev Biol. 2021;9: 700634.
[36] OKAWA ER, GUPTA MK, KAHRAMAN S, et al. Essential roles of insulin and IGF-1 receptors during embryonic lineage development. Mol Metab. 2021;47:101164.
[37] TAN FE, SATHE S, WHEELER EC, et al. Non-microRNA binding competitively inhibits LIN28 regulation. Cell Rep. 2021;36(6):109517.
[38] SATO T, KATAOKA K, ITO Y, et al. Lin28a/let-7 pathway modulates the Hox code via Polycomb regulation during axial patterning in vertebrates. Elife. 2020;9:e53608.
[39] DESHMUKH SK, SRIVASTAVA SK, ZUBAIR H, et al. Resistin induces LIN28A-Mediated Let-7a repression in breast cancer cells leading to IL-6 and STAT3 upregulation. Cancers (Basel). 2021;13(18): 4498.
[40] ALI A, BOUMA GJ, ANTHONY RV, et al. The role of LIN28-let-7-ARID3B pathway in placental development. Int J Mol Sci. 2020;21(10):3637.

Differential expression of RNA binding protein Lin28A regulates osteoblastic differentiation of periodontal ligament stem cells

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