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. 2021 Jan 1;18(4):902-910.
doi: 10.7150/ijms.50984. eCollection 2021.

Effect of lentivirus-mediated miR-182 targeting FGF9 on hallux valgus

Wei-Lin Zhang  1 , Duo-Yi Zhao  1 , Wei Zhao  1 , Yan Cui  1 , Qin Li  2 , Zhi-Yu Zhang  1
Affiliations

Effect of lentivirus-mediated miR-182 targeting FGF9 on hallux valgus

Wei-Lin Zhang et al. Int J Med Sci. .

Abstract

The pathogenesis of hallux valgus is not clearly understood. However, genetics research about hallux valgus is rare. Therefore, the present study aimed to explore the pathogeny of hallux valgus from the perspective of genetics. Human samples were collected from normal bone tissue and hallux valgus region bone tissue. The bone samples were studied using real time-PCR, western blot and immunohistochemical. Lentivirus-mediated miR-182 transfected osteoblasts and tested the expression of FGF9 mRNA with real time-PCR. To test alkaline phosphatase activity, number of calcium nodules and proliferation of osteoblast with enzymatic activity analysis, calcium nodules stained and MTT assay. We found that (1) FGF9 expressed in hallux valgus region bone tissue was significantly higher than normal bone tissue. (2) miR-182 expression levels in hallux valgus region bone tissue were notably lower than those of normal bone tissue. (3) miR-182 could negatively regulate the expression of FGF9 in osteoblasts. (4) FGF9 may enhance osteoblasts proliferation. We have demonstrated that miR-182 promotes the formation of bone by targeting FGF9, implicating an essential role of miR-182 in the etiology of hallux valgus. Moreover, miR-182 might potentially be a therapeutic target for hallux valgus treatment.

Keywords: FGF9; alkaline phosphatase; hallux valgus; miR-182; osteoblast.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Correlation between FGF9 and miR-182 in hallux valgus. (A)RT-PCR analysis of Collagen-I, ALP, OPG, OCN, and Runx-2. (B) RT-PCR analysis of FGF1, FGF4, FGF7, FGF8, FGF9. (C) The situation of FGF9 expression in the first metatarsal using immunohistochemical method. Hallux valgus group was significantly stronger than cadaver group. Scale bars, 20μm. FGF9 semi-quantitative immunohistochemical analysis showed that Hallux valgus group was significantly stronger than cadaver group. (B) real time-PCR for the expression of miR-182 in Hallux valgus patients and cadaver. (C) Western blot analysis shows expression of FGF9 in Hallux valgus group is stronger than cadaver group. n=40 Hallux valgus group, n=20 cadaver group. Data are means ± SD. *P < 0.05, **P < 0.01.
Figure 2
Figure 2
miR-182 can inhibit FGF9 via directly targeting his 3'-UTRs. Complementarity between miR-182 and the putative hFGF9 3'-UTR target site. hFGF9 mut indicate the FGF9 mRNA 3'-UTRs with three consecutive mutation sites in miR-182 binding sites. The relative luciferase activities show hFGF9 3'-UTR 104-111 bp site is binding site of miR-182. n=3 per group. Data are means ± SD. *P < 0.05, **P < 0.01.
Figure 3
Figure 3
miR-182 regulates FGF9 in osteoblasts. (A) Lentivirus expressing miR-182 could be stably transfected into osteoblasts. real time-PCR analysis showed that miR-182 was down-regulated while FGF9 mRNA is down-regulated; in addition miR-182 is up-regulated while FGF9 mRNA was up-regulated. (B) miR-182 regulates FGF9 in osteoblasts. Western blot analysis shows expression of FGF9 protein in ant-LV-pre-miR-182 group is stronger than LV-pre-miR-182 group. No statistically significant difference between the other groups. (C) Immunofluorescence of FGF9 in hFOB1.19 cells. (D)Western blot analysis of FGF9 expression in transfected hFOB1.19 cells. n=5 per group. Data are means ± SD. *P < 0.05, **P < 0.01.
Figure 4
Figure 4
The influence of FGF9 on alkaline phosphatase and osteoblast proliferation. (A) Alkaline phosphatase activity analysis shows that the activity in LV-pre-miR-182 group is significantly lower than the anti- LV-pre-miR-182 group. n=5 per group. Data are means ± SD. *P < 0.05, **P < 0.01. (B) The MTT assay showed that osteoblast proliferation in LV-pre-miR-182 group is significantly lower than the anti- LV-pre-miR-182 group. And (C), in staining calcium nodules, the phenomenon has been verified. n=5 per group. Data are means ± SD. *P < 0.05, **P < 0.01.
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