Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: MIIP facilitates HIF-2α binding to RACK1 rather than HSP90, and promotes HSP90 acetylation. (A) Cells stably overexpressing MIIP 786-O and control cells were transfected with siRACK1 1#, siRACK1 2#, or control (siNC) siRNA. The expression of RACK1, HIF-2α, and HSP90 was detected by Western blot. (B) HIF-2α binding to RACK1 and HSP90 was detected with co-immunoprecipitation assays. The 786-O-MIIP and 786-O-Vector cell lysates were immunoprecipitated with anti-HIF-2α antibody, then immunoblotted for RACK1 and HSP90. (C) The acetylation of HSP90, and HSP90 binding to HDAC6 and HIF-2α in 786-O-MIIP and 786-O-Vector cells was detected by immunoprecipitation with anti-HSP90 antibody followed by immunoblotting with anti-Acetyl Lysine, anti-HDAC6, and anti-HIF-2α antibodies.

Article Snippet: The precipitates were resolved with SDS-PAGE and subjected to Western blot with anti-HIF-2α (1:1000, Proteintech/CST), anti-HSP90 (1:1000, Proteintech), anti-RACK1 (1:1000, Proteintech), anti-MIIP, or anti-acetyl lysine (1:1000, Abcam).

Techniques: Binding Assay, Stable Transfection, Transfection, Expressing, Western Blot, Immunoprecipitation, Plasmid Preparation

Journal: Cancer Biology & Medicine

Article Title: MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

doi: 10.20892/j.issn.2095-3941.2020.0296

Figure Lengend Snippet: MIIP is weakly expressed in RCC and associated with progression, prognosis, and the expression of CYR61 and HIF-2α. (A) MIIP, CYR61, and HIF-2α expression in 13 pairs of RCC tissues vs. adjacent non-tumor tissues was detected by Western blot. C: RCC tissue; N: adjacent non-tumor tissue. (B) MIIP, CYR61, and HIF-2α expression in an RCC tissue microarray was detected with immunohistochemistry (scale bar: 50 μm). (C) MIIP, CYR61, and HIF-2α expression levels were quantified with a scoring system. The staining score was calculated by multiplying the stained area (%) score and the intensity score. Expression group: low, score < 9; high, score ≥ 9. Stage refers to WHO histological grade. * P < 0.05; ** P < 0.01, χ 2 test. (D) The Kaplan-Meier curve depicts the relationship between MIIP expression level and OS of patients with RCC, and the P -value was calculated with a stratified log rank test ( P = 0.026). (E) Schematic representation of the MIIP/HIF-2α/CYR61 axis in ccRCC. In normal cells, MIIP promotes HSP90 acetylation through inhibiting HDAC6 activity, thus impairing HSP90’s chaperone function and its binding to HIF-2α, which in turn causes RACK1 binding and subsequent HIF-2α degradation. Meanwhile, under normoxia, VHL promotes HIF-2α ubiquitination and degradation in an oxygen-dependent manner. In RCC, VHL deficiency and MIIP downregulation together cause HIF-2α accumulation, thereby leading to overexpression of CYR61, which in turn contributes to RCC progression.

Article Snippet: The precipitates were resolved with SDS-PAGE and subjected to Western blot with anti-HIF-2α (1:1000, Proteintech/CST), anti-HSP90 (1:1000, Proteintech), anti-RACK1 (1:1000, Proteintech), anti-MIIP, or anti-acetyl lysine (1:1000, Abcam).

Techniques: Expressing, Western Blot, Microarray, Immunohistochemistry, Staining, Activity Assay, Binding Assay, Over Expression