Identification of Prognostic Alternative Splicing Signature in Breast Carcinoma

BackgroundIncreasing evidence indicated a close relationship between aberrant splicing variants and carcinoma, whereas comprehensive analysis of prognostic alternative splicing (AS) profiling in breast cancer (BRCA) is lacking and largely unknown.MethodsRNA-seq data and corresponding clinical information of BRCA patients were obtained and integrated from The Cancer Genome Atlas (TCGA). Then SpliceSeq software was used to assess seven AS types and calculate the Percent Spliced In (PSI) value. Univariate followed by stepwise multivariate Cox regression analyses identified survival associated AS events and constructed the AS signature, which were further sent for enrichment analysis, respectively. Besides, the splicing correlation network was constructed. Additionally, nomogram incorporating AS signature and clinicopathological characteristics was developed and its efficacy was evaluated with respect to discrimination, calibration and clinical utility.ResultsA total of 45,421 AS events were detected, among which 3071 events were found associated with overall survival (OS) after strict filtering. Parent genes of these prognostic events were involved in BRCA-related processes including NF-kappaB and HIF-1 signaling pathway. Besides, the final prognostic signature built with 20 AS events performed well with an area under the curve (AUC) of receiver operating characteristic (ROC) curve up to 0.957 for 5 years. And gene set enrichment analysis (GSEA) also confirmed the candidate 20 AS events contributed to progression of BRCA. Moreover, the nomogram that incorporated 20-AS-event-based classifier, age, pathological stage and Her-2 status showed good calibration and moderate discrimination, with C-index of 0.883 (95% CI, 0.844–0.921). Decision curve analysis (DCA) confirmed more benefit was added to survival prediction with our nomogram, especially in 5 or 8 years with threshold probability up to 80%. Finally, splicing correlation network revealed an obvious regulatory pattern of prognostic splicing factors (SF) in BRCA.ConclusionThis study provided a systematic portrait of survival-associated AS events involved in BRCA and further presented a AS-clinicopathological nomogram, which could be conveniently used to assist the individualized prediction of long-term survival probability for BRCA patients. And a series of bioinformatic analysis provided a promising perspective for further uncovering the underlying mechanisms of AS events and validating therapeutic targets for BRCA

The effect of autophagy inhibition on the cytotoxicity of Huaier extract.

<p>MDA-MB-231 (A and B), MDA-MB-468 (C and D) and MCF7 (E and F) were pretreated with 3-MA or CQ before being exposed to Huaier extract for the indicated time. The cell viability was measured using the MTT assay. The experiments were performed in triplicate and data presented as the mean ± SD of three separate experiments. ^*p < 0.05, ^**p < 0.01.</p

Huaier extract inhibited the activity of mTOR/S6K pathway.

<p>(A and B) Cells were treated with various concentrations of Huaier extract for the indicated time. Cell lysates were prepared and detected via Western blotting. The effect of Huaier extract on the levels of mTOR, p70S6K, S6, 4E-BP1 and their phosphorylated forms are shown. (C) AMPK was activated after Huaier treatment in a dose-dependent manner. Results are representative of three independent experiments.</p

Huaier extract induced autophagy in breast cancer cells.

<p>(A) Representative electron micrographs of breast cancer cells treated with or without 4 mg/ml Huaier extract for 48 h. Bars, 100nm. (B) Acidic vesicular organelles induced by Huaier extract were stained with MDC. Bars, 10 μm. (C) Aggregation of LC3B in Huaier-treated cells. Cells were treated with or without 4 mg/ml Huaier extract and stained with the LC3B antibodies using immunofluorescence staining. Puncta represent the autophagosome formation. Bars, 10 μm. (D) Cell lysates were harvested after incubation with different concentrations of Huaier extract for 48h. β-actin was used as a loading control. (E) Cells in suspension were labeled with acridine orange and quantified using flow cytometry. FL1-H indicates green color intensity (cytoplasm and nucleus), whereas FL3-H shows red color intensity (AVO). Cells in up quadrants were considered AVO-positive. Results shown are representative of three independent experiments.</p

LC3B siRNA blocks Huaier-mediated autophagic cell death.

<p>(A) LC3B expression was knocking-down by siRNA. MDA-MB-231 and MCF7 cells were transfected with siLC3B. siControl was used as the negative control. (B) Huaier-mediated autophagy was blocked by LC3B siRNA. MDA-MB-231 cells were treated with siControl, siControl+Huaier, siLC3B, or siLC3B+Huaier. Autophagy was detected by flow cytometry. (C and D) The cell viability was measured using the MTT assay. The experiments were performed in triplicate and data presented as the mean ± SD of three separate experiments. ^*p < 0.05, ^**p < 0.01.</p

Huaier extract reduced cell viability and triggered intracellular vesicular organelles in breast cancer cells.

<p>(A, C and E) The cytotoxic effect of Huaier extract was measured using the MTT assay. Cells were treated with different concentrations of Huaier extract over indicated time periods and subjected to the MTT assay. The viability of untreated cells was considered 100%. The experiments were performed in triplicate and data is presented as the mean ± SD of three separate experiments. ^*p < 0.05, ^**p < 0.01. (B, D and F) Micrographs show the appearance of vesicular organelles in breast cancer cells after 48 h of exposure to Huaier extract. Arrows indicate the intracellular vacuoles. Bars, 10 μm. (G) AO/EB staining of T47D cells was performed to detect apoptosis and necrosis induced by Huaier extract. Bars, 50 μm. (H) Representative TUNEL staining (red fluorescence) of MDA-MB-231 cells treated with or without Huaier extract. Bars, 50 μm.</p