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. 2020 May 6;11(7):1801–1816. doi: 10.1111/1759-7714.13450

Figure 5.

Figure 5

HOTAIR facilitates the expression of HSPA1A by sequestering miR‐449b‐5P. (a) The correlation between the levels of miR‐449b‐5p and HOTAIR in 20 cases of clinical BRCA tissues were tested by real‐time PCR and assayed by Pearson's correlation (R = −0.5407, P < 0.05). (b and c) The BRCA cell lines MDA‐MB‐231 (b) and MCF‐7 (c) were transfected with pcDNA3.1 or pcDNA3.1‐HOTAIR 12 hours before 10 Gy irradiation treatment. Total RNA was extracted and polyadenylated by poly (A) polymerase 36 hours after the irradiation. The effects of HOTAIR on the expression of miR‐449b‐5p were monitored by real‐time PCR analysis. (d and e) The BRCA cell line MDA‐MB‐231 was transfected with Ctrl/50 nM of miR‐449b‐5p/ 100 nM of miR‐449b‐5p (d) or Ctrl/50 nM of miR‐449b‐5p inhibitor/100 nM of miR‐449b‐5p inhibitor (e) 12 hours before 10 Gy irradiation treatment. Total RNA was extracted 36 hours after the irradiation. The effects of miR‐449b‐5p on the expression of HOTAIR were monitored by real‐time PCR analysis. (f) The BRCA cell line MDA‐MB‐231 was cotransfected with pGL‐HSPA1A, pRL‐TK plus Ctrl/miR‐449b‐5p/miR‐449b‐5p + HOTAIR/miR‐449b‐5p + HOTAIR‐449b‐mut 12 hours before 10 Gy irradiation treatment. The luciferase activities of pGL‐HSPA1A were measured 36 hours later by luciferase reporter gene assays. (g) The BRCA cell line MDA‐MB‐231 was transfected with Ctrl/miR‐449b‐5p/miR‐449b‐5p + HOTAIR/miR‐449b‐5p + HOTAIR‐449b‐mut 12 hours before 10 Gy irradiation treatment. Total proteins were extracted 36 hours later. The protein levels of HSPA1A were detected by western blot analysis, respectively. Data are shown as mean ± SD of three independent experiments. Statistical significant differences are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no significance; Student's t‐test.