The in vivo effects of knockdown of long non-coding RNA XIST on fibroid growth and gene expression

Abstract

The role of long non-coding RNAs in fibroid pathogenesis remains largely unexplored. In a previous study, we found elevated XIST (X-inactive specific transcript) levels in fibroids, which sponged miR-29c and miR-200c, leading to the overexpression of their target genes. This study aimed to assess the therapeutic potential of XIST downregulation in fibroid treatment. Ovariectomized SCID (severe combined immunodeficiency) mice were implanted with fibroid tumors transduced with XIST siRNA or a control via lentivirus. After 1 month, animals were sacrificed and the xenografts were removed for further analysis. XIST knockdown reduced tumor weight by 15% and increased miR-29c and miR-200c expression by 3.9-fold and 2.2-fold, respectively. The mRNA expression of miR-29c targets (COL3A1, TGF-β3, CDK2, SPARC) and miR-200c targets (CDK2, FN1, TDO2), as well as PRL, E2F1, and EZH2, was significantly decreased. Protein abundance of collagen, COL3A1, FN1, CDK2, SPARC, and EZH2 was also reduced. IHC analysis of xenograft sections using the markers of Ki67 for cell proliferation and cleaved caspase 3 for apoptosis showed decreased cell proliferation and no changes in apoptosis in the XIST knockdown xenografts. This analysis also revealed decreased collagen and E2F1 staining nuclei in the XIST knockdown xenografts. These results indicate that downregulation of XIST in fibroids has beneficial therapeutic effects, by reducing tumor growth and the expression of genes involved in cell proliferation, inflammation, and extracellular matrix regulation.

Keywords: ECM; XIST; cell proliferation; fibroid; inflammation; miR‐200c; miR‐29c.

Figure 1.

(A) XIST levels in xenografts of control (siNC) or XIST knockdown (siXIST) groups (n=8). (B) The tumor weight of explants 4 weeks after implantation. (C) Relative expression of miR-29c and miR-200c in the xenografts after 4 weeks of implantation. The results are presented as mean ± SEM with p values indicated at the corresponding line. *p < 0.05; **p < 0.01.

Figure 2.

Relative mRNA levels of CDK2, SPARC, COL3A1, FN1, TGF-β3, TDO2, CYP1B1, E2F1, PRL, and EZH2 in xenografts following 4 weeks of implantation (n=8). The results are presented as mean ± SEM with P values indicated at the corresponding line. *p < 0.05; **P < 0.01.

Figure 3.

(A) Total collagen levels assessed by ELISA assay in 8 xenografts. (B) Representative Western blot analysis, with bar graphs (C) illustrating the relative band densities in the xenografts (n=6). The results are presented as mean ± SEM with P values indicated at the corresponding line. *P < 0.05; **P < 0.01.

Figure 4.

Representative IHC staining images of fibroid xenografts from the control (siNC) and XIST knockdown treatment groups (n=5) are shown, along with the quantification of staining intensity using Halo software for Ki67 (A-B), E2F1 (C-D), COL3A1 (E-F), and Cleaved Caspase 3 (G-H). (I) Representative histopathological images of fibroid xenografts from the control (siNC) and XIST knockdown treatment groups, determined by Masson’s trichrome staining, where blue indicates collagen fibers and red denotes smooth muscle cells. (J) Quantification of staining intensity by Halo software. Data are presented as mean ± SEM, with p-values indicated at the corresponding lines. *p < 0.05.

Figure 4.

Representative IHC staining images of fibroid xenografts from the control (siNC) and XIST knockdown treatment groups (n=5) are shown, along with the quantification of staining intensity using Halo software for Ki67 (A-B), E2F1 (C-D), COL3A1 (E-F), and Cleaved Caspase 3 (G-H). (I) Representative histopathological images of fibroid xenografts from the control (siNC) and XIST knockdown treatment groups, determined by Masson’s trichrome staining, where blue indicates collagen fibers and red denotes smooth muscle cells. (J) Quantification of staining intensity by Halo software. Data are presented as mean ± SEM, with p-values indicated at the corresponding lines. *p < 0.05.

Figure 5.

Correlation analysis of the fold change (siXIST/siNC) in tumor weight with XIST levels and with miR-200c levels in the xenografts, as well as the relationship between XIST and miR-200c levels in the xenografts.