Identification and Verification of Immune Subtype-Related lncRNAs in Clear Cell Renal Cell Carcinoma.

Background: According to clinical study results, immune checkpoint blockade (ICB) treatment enhances the survival outcome of patients with clear cell renal cell carcinoma (ccRCC). Previous research has divided ccRCC patients into immune subtypes with distinct ICB response rates. However, the study on the association between lncRNAs and ccRCC immune subtypes is lacking. Methods: Differentially expressed lncRNAs/mRNAs between two major immune subgroups were calculated. A weighted gene co-expression network analysis (WGCNA) was conducted to establish the lncRNA-mRNA co-expression network and select the key lncRNAs. Then, prognostic lncRNAs were selected from the network by the bioinformatics method. Next, the risk-score was estimated by lncRNA expression and their coefficients. Finally, a nomogram based on lncRNAs and clinical parameters was created to predict the prognosis of ccRCC. Results: LncRNAs and mRNAs associated with ccRCC immune subtypes were identified. The lncRNAs and mRNAs from a gene module closely linked to the immune subtype were used to construct a network. The KEGG pathways enriched in the network were related to immune system activation processes. These 8 lncRNAs (AL365361.1, LINC01934, AC090152.1, PCED1B-AS1, LINC00426, AC007728.2, AC243829.4, and LINC00158) were found to be positively correlated with immune cells of the tumor microenvironment. The C-index of the nomogram was 0.777, and the calibration curve data suggests that the nomogram has a high degree of discriminating capacity. Conclusion: In summary, we discovered core lncRNAs linked with immune subtypes and created corresponding lncRNA-mRNA networks. These lncRNAs are anticipated to have predictive significance for ccRCC and may provide insight into novel biomarkers for the disease.

TLR4/NF-κB/Ceramide signaling contributes to Ox-LDL-induced calcification of human vascular smooth muscle cells.

Vascular calcification is a major feature of advanced atherosclerosis and highly associated with cardiovascular diseases. Oxidized low density lipoprotein (Ox-LDL) has been recognized as a critical risk factor for atherosclerosis and osteogenic differentiation of vascular smooth muscle cells (VSMCs). Previous studies have demonstrated that toll like receptor 4 (TLR4) is highly expressed in atherosclerotic lesions and participates in the progression of atherosclerosis. However, the role of TLR4 in vascular calcification remains unknown. In this study, we investigated whether TLR4 modulates vascular calcification induced by Ox-LDL. TLR4 expression was up-regulated in cultured human VSMCs treated with Ox-LDL. Knockdown of TLR4 by small interfering RNA (siRNA) significantly reduced Ox-LDL-induced calcification, detected by alizarin red staining and calcium content assay. TLR4 siRNA also decreased the mRNA expression of bone-related proteins including Msx2, osterix, BMP2 and KLF4, but increased the expression of VSMC contractile proteins including SMA and SM22α in VSMCs. In addition, Ox-LDL stimulated nuclear translocation of nuclear factor kappa B (NK-κB) p65. These effects of Ox-LDL on VSMCs were reversed by TLR4 siRNA. Furthermore, NK-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated Ox-LDL-induced VSMC calcification, which was rescued by C2-ceramide treatment. In conclusion, these findings suggest that TLR4 regulates VSMC calcification induced by Ox-LDL through activation of NK-κB, highlighting the critical role of TLR4/NK-κB signaling in vascular calcification.

Curcumin attenuates osteogenic differentiation and calcification of rat vascular smooth muscle cells.

Vascular calcification has been considered as a biological process resembling bone formation involving osteogenic differentiation. It is a major risk factor for cardiovascular morbidity and mortality. Previous studies have shown the protective effects of curcumin on cardiovascular diseases. However, whether curcumin has effects on osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) has not been reported. In the present study, we used an in vitro model of VSMC calcification to investigate the role of curcumin in the progression of rat VSMC calcification. Curcumin treatment significantly reduced calcification of VSMCs in a dose-dependent manner, detected by alizarin red staining and calcium content assay. Similarly, ALP activity and expression of bone-related molecules including Runx2, BMP2, and Osterix were also decreased in VSMCs treated with curcumin. In addition, flow cytometry analysis and caspase-3 activity assay revealed that curcumin treatment significantly suppressed apoptosis of VSMCs, which plays an important role during vascular calcification. Furthermore, we found that pro-apoptotic molecules including p-JNK and Bax were up-regulated in VSMCs treated with calcifying medium, but they were reduced in VSMCs after curcumin treatment. However, curcumin treatment has no effect on expression of NF-κB p65. Taken together, these findings suggest that curcumin attenuates apoptosis and calcification of VSMCs, presumably via inhibition of JNK/Bax signaling pathway.

[High glucose promotes vascular smooth muscle cell calcification by activating WNT signaling pathway].

OBJECTIVE: To investigate whether high glucose-induced vascular calcification is associated with WNT signaling pathway. METHODS: An in vitro model of human vascular smooth muscle cell (VSMC) calcification was induced by exposure of the cells to high glucose. The expressions of WNT signal molecules and bone-related proteins including Cbfa1, Osx, OCN and BMP2 were analyzed with qRT-PCR, and the cell calcification was assessed by alizarin red staining. The effect of Dkk1, a WNT signaling inhibitor, on high glucose-induced cell calcification was tested with alizarin red staining and calcium content analysis. RESULTS: High glucose activated WNT signaling pathway in human VSMCs by up-regulating the expressions of WNT signal molecules including Wnt3a, Wnt7a, Fzd4 and Wisp1 mRNA by 1.86, 1.68, 2.1, and 2.3 folds, respectively, and by promoting the phosphorylation of ß-catenin (2.70∓0.22, P<0.05), a key mediator of WNT signaling pathway. Inhibition of WNT signaling pathway by Dkk1 attenuated high glucose-induced VSMC calcification and down-regulated the expression of bone-related proteins Cbfa1, Osx, OCN, and BMP2 by (51∓9)%, (58∓11)%, (56∓10)%, and (62∓10)% (P<0.01). CONCLUSION: WNT signaling pathway is involved in high glucose-induced VSMC calcification.