Effect of Lentiviral Vector-Mediated Knockdown of miR-195 on Osteogenic and Angiogenic Differentiation of BMMSCs

doi:10.3870/j.issn.1672-8009.2026.05.006

Abstract

Abstract: Objective To investigate the regulatory effect and potential mechanism of lentiviral vector-mediated microRNA-195 （miR-195） downregulation on osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells （BMMSCs）. Methods Human BMMSCs were divided into three groups： blank control group, inhibitor-negative control （NC） group （transfected with lentiviral NC sequence）, and miR-195-inhibitor group （transfected with lentiviral miR-195 inhibitor sequence）. The expression and localization of miR-195 were detected by RNA fluorescence in situ hybridization. Cell proliferation was measured by CCK-8 assay. Osteogenic differentiation was evaluated by alkaline phosphatase （ALP） staining, alizarin red staining, and Runt-related transcription factor 2 （Runx2） immunofluorescence. Tube formation assay was performed for angiogenesis assessment. The mRNA levels of vascular endothelial growth factor （VEGF）, ALP, Runx2, VEGF receptor 2 （VEGFR2）, angiopoietin-1 （Ang-1）, and Wnt/β-catenin pathway-related molecules （Wnt3a, Wnt10b, β-catenin, Osterix） were determined by qRT-PCR. The protein expression of the above pathway and osteogenesis-related molecules was detected by Western blotting. Results Compared with the control groups, the miR-195-inhibitor group showed significantly decreased miR-195 expression （all P<0.05）, and markedly increased cell proliferation, ALP activity （4.15±0.52 vs 1.00±0.15）, alizarin red positive rate （64.67%±7.78% vs 31.86%±2.24%）, Runx2 fluorescence intensity （852.38±122.70 vs 121.31±12.16 RFU）, and tubular structure length （218.60±24.75 vs 49.80±5.25 μm/field）. The mRNA levels of VEGF, ALP, Runx2, VEGFR2, Ang-1, Wnt3a, Wnt10b, β-catenin, and Osterix, as well as the protein levels of Wnt3a, Wnt10b, β-catenin, and Osterix were all significantly upregulated （all P<0.05）. There were no significant differences in the above indices between the inhibitor-NC group and the blank control group （P>0.05）. Conclusion Lentiviral-mediated miR-195 downregulation synergistically promotes osteogenic and angiogenic differentiation of BMMSCs via activating the Wnt/β-catenin signaling pathway.

Key words: lentivirus, microRNA-195, bone marrow mesenchymal stem cell, osteogenic differentiation, angiogenic differentiation

CLC Number:

R329.2

DING Liuchuang, PEI Pei, JI Xiaowei. Effect of Lentiviral Vector-Mediated Knockdown of miR-195 on Osteogenic and Angiogenic Differentiation of BMMSCs[J]. Journal of Medical Molecular Biology, 2026, 23(5): 523-530.

References

[1] YUAN X,ZHU W,YANG Z,et al.Recent advances in 3D printing of smart scaffolds for bone tissue engineering and regeneration[J]. Adv Mater,2024,36(34):e2403641.
[2] WONG S K,YEE M M F,CHIN K Y,et al. A review of the application of natural and synthetic scaffolds in bone regeneration[J]. J Funct Biomater,2023,14(5):286.
[3] ANDALIB N,KEHTARI M,SEYEDJAFARI E,et al.In vivo bone regeneration using a bioactive nanocomposite scaffold and human mesenchymal stem cells[J]. Cell Tissue Bank,2021,22(3):467-477.
[4] CHAO C,LI F,TAN Z,et al.miR-195 inhibited abnormal activation of osteoblast differentiation in MC3T3-E1 cells via targeting RAF-1[J]. Exp Cell Res,2018,362(2):293-301.
[5] HARIMI S,KHANSARINEJAD B,FESAHAT F,et al.Hsa-miR-15b-5p/miR-195-5p controls osteogenic differentiation of human adipose-derived mesenchymal stem cells through regulating Indian hedgehog expression[J]. Appl Biochem Biotechnol,2024,196(8):4793-4806.
[6] DIOGUARDI M,SPIRITO F,CALORO G A,et al.Is the non-coding RNA miR-195 a biodynamic marker in the pathogenesis of head and neck squamous cell carcinoma? a prognostic meta-analysis[J]. J Pers Med,2023,13(2):275.
[7] 邓岱,田东丽,白雨,等. SFRP5去甲基化对β-catenin/NLRP3焦亡介导血管平滑肌细胞钙化的调节机制[J]. 遵义医科大学学报,2025,48(10):993-1001.
DENG D,TIAN D L,BAI Y,et al.SFRP5 demethylation protects against vascular smooth muscle cell calcification by inhibiting β-catenin/NLRP3-mediated pyroptosis[J]. J Zunyi Med Univ,2025,48(10):993-1001.
[8] 王贺贺,李姣. 骨髓间充质干细胞衰老与骨髓微环境失衡在老年性骨质疏松中的作用机制[J]. 遵义医科大学学报,2025,48(8):860-872.
WANG H H,LI J.Mechanisms of bone marrow mesenchymal stem cell senescence and bone marrow microenvironmental imbalance in senile osteoporosis[J]. J Zunyi Med Univ,2025,48(8):860-872.
[9] ZHENG S,HU G,ZHENG J,et al.Osthole accelerates osteoporotic fracture healing by inducing the osteogenesis-angiogenesis coupling of BMSCs via the Wnt/β-catenin pathway[J]. Phytother Res,2024,38(8):4022-4035.
[10] LIN W,HOU L,TANG J,et al.miR-195-5p targets Smad7 regulation of the Wnt/β-catenin pathway to promote osteogenic differentiation of vascular smooth muscle cells[J]. BMC Cardiovasc Disord,2024,24(1):221.
[11] GAO Z,ZHANG R,JIANG L,et al.Administration of miR-195 inhibitor enhances memory function through improving synaptic degradation and mitochondrial dysfunction of the hippocampal neurons in SAMP8 mice[J]. J Alzheimers Dis,2022,85(4):1495-1509.
[12] WANG G,WANG H,ZHANG L,et al.miR-195-5p inhibits proliferation and invasion of nerve cells in Hirschsprung disease by targeting GFRA4[J]. Mol Cell Biochem,2021,476(5):2061-2073.
[13] LIN X L,ZHENG Z Y,ZHANG Q S,et al.Expression of miR-195 and its target gene Bcl-2 in human intervertebral disc degeneration and their effects on nucleus pulposus cell apoptosis[J]. J Orthop Surg Res,2021,16(1):412.
[14] YANG L,FANG C,SONG C,et al.Mesenchymal stem cell-derived exosomes are effective for radiation enteritis and essential for the proliferation and differentiation of Lgr5+ intestinal epithelial stem cells by regulating mir-195/Akt/β-catenin pathway[J]. Tissue Eng Regen Med,2023,20(5):739-751.
[15] KIM H,LEE Y Y,KIM V N.The biogenesis and regulation of animal microRNAs[J]. Nat Rev Mol Cell Biol,2025,26(4):276-296.
[16] HU X,XING H,WANG X,et al.Knockdown of LncRNA SNHG1 suppresses corneal angiogenesis by the regulation of miR-195-5p/VEGF-A[J]. J Ophthalmol,2021,2021:6646512.
[17] LIN W,CHOW S K H,CUI C,et al. Wnt/β-catenin signaling pathway as an important mediator in muscle and bone crosstalk: A systematic review[J]. J Orthop Translat,2024,47:63-73.
[18] HUANG K,CAI S,FU T,et al.Wnt10b regulates osteogenesis of adipose-derived stem cells through Wnt/β-catenin signalling pathway in osteoporosis[J]. Cell Prolif,2024,57(1):e13522.
[19] LI J,HAN Q,CHEN H,et al.Carbon monoxide-releasing molecule-3 enhances osteogenic differentiation of rat bone marrow mesenchymal stem cells via miR-195-5p/Wnt3a pathway[J]. Drug Des Devel Ther,2022,16:2101-2117.
[20] YANG L,BI T,ZHOU S,et al.CircRASSF₂ facilitates the proliferation and metastasis of colorectal cancer by mediating the activity of Wnt/β-catenin signaling pathway by regulating the miR-195-5p/FZD4 axis[J]. Anticancer Drugs,2021,32(9):919-929.