Characterization of lncRNA APOC1P1-3
Previously, we measured and compared lncRNA APOC1P1-3 expression in breast cancer and its adjacent tissues via microarray chip. In order to identify its protein-noncoding property, we checked the Open Reading Frame (ORF) of APOC1P1-3 by ORF finder (http://www.ncbi.nlm.nih.gov/orffinder/), and found that it has 8 ORFs and none is more than 200nt, suggesting no protein coding potential (Table S1). CSF is the mutation rate of the codon. Codons are relatively conservative in the coding region, but prone to mutations in the non-coding region. Due to the problem of sequence conservation, Michael  proposed a new value to introduce the evolution model PhyloCSF. We also checked the PhyloCSF of APOC1P1-3 on UCSC (http://genome.ucsc.edu/). All values in the exon of APOC1P1-3 are less than 0, indicating no conservative type.
Correlation between lncRNA-APOC1P1-3 and breast cancer
We measured the expression of lncRNA APOC1P1-3 in 3 malignant (T47D, MCF-7, MDA-MB-231) and a normal (MCF-10A) breast cell lines and in breast cancer tissues, by real-time qPCR (Figure. 1A、1C). The expressions in malignant cells upregulated, especially in the MDA-MB-231 cells (increased by 1.5 fold). In addition, lncRNA APOC1P1-3 was found both in cytoplasm and nucleus in MDA-MB-231 cells (Figure. 1B). To evaluate the role of lncRNA APOC1P1-3 in survival, we used the cBioPortal database in TCGA for related bioinformatics analysis. The survival rate is much higher in patients with low expression (blue line) than with high expression (red line) (Figure. 1D; n=816, P<0.01). This may be related to distant metastasis of the tumor. We further explored the role of APOC1P1-3 in the lung metastasis by injecting MDA-MB-231 and MDA-MB-231-APOC1P1-3-knock-down cells into the tail vein, and found that the lung metastases burden in nude mice of APOC1P1-3-knock-down cells significantly decreased (Figure. 1Ea-b).
The capacity of anoikis resistance of breast cancer cells
Anoikis resistance model was established by culturing the cells on a low-adherent culture plate with polyHEMA . Both MDA-MB-231 and MCF-7 cells survived under adherent and anchorage-independent conditions for 72 h by forming micro-tissues (Figure. 2A、2B), indicating the anoikis resistance. MCF-7a aggregated in large clusters with more cells were dead than MDA-MB-231a, which is similar to a previous report . MDA-MB-231a cells formed many small irregular clumps, which may be responsible for detachment from primary tumor tissues.
Under both adherent culture and ultralow attachment culture, we collected cells with or without underwent anoikis resistance for 48h. Aggregation occurred in all cells (MCF-7a, Figure. 2C and MDA-MB-231a, Figure. 2D). The aggregation was much more in the anoikis group than in the normal group (n=7, **P<0.01).
We compared the number of apoptotic cells under adherent culture and low-adherent culture condition Fluorescence-activated cell sorting (FACS). After 48h culture, the numbers of apoptotic cells were significantly fewer in MCF-7a and MDA-MB-231a (Figure. 2E-F) than in MCF-7 and MDA-MB-231 groups (n= 3, **P<0.01), indicating an effective induction of anoikis resistance.
LncRNA APOC1P1-3 suppressing apoptosis via apoptosis-related proteins
In the gain/loss functionality studies, the expression of lncRNA APOC1P1-3 efficiently up-regulated by transfection with pcDNA3.1-sense in MCF-7 cells and down-regulated by transfection with siRNA-196 in MDA-MB-231 cells (Figure. 3A-B). The percentage of early apoptosis (FACS) was significantly decreased in MCF-7a cells (Figure. 3C) (n=3, *P<0.05), and increased in MDA-MB-231a cells (Figure. 3D) (n=3, *P<0.05). The results support that the lncRNA APOC1P1-3 could promote anoikis resistance.
To probe the mechanisms of anoikis resistance, we measured apoptosis related proteins (Western Blot) from MCF-7a transfected with siRNA-196, as well as MDA-MB-231a transfected with pcDNA3.1-sense (Figure. 3E-F) (n=3, **P<0.0). The expressions of activated-caspase 3, 8, 9, and activated-PARP upregulated in MDA-MB-231a cells which were knockdown with siRNA-196. In contrast, the expressions decreased in MCF-7a cells which were overexpressed with pcDNA3.1-sense. These results indicate that upregulation of lncRNA APOC1P1-3 could influence anoikis resistance via caspase-dependent pathway.
Regulation of anoikis resistance via binding to miRNA-188-3p
We used RNA microarrays (provided by Kangcheng Company) to screen related target microRNAs of lncRNA-APOC1P1-3 in breast cancer tissues, and predicted microRNAs in multiple databases like PITA, MIRDB4.0 and Miranda by matching between sequences (Table. S2). There are 84 microRNAs that may bind to APOC1P1-3. With Venn diagram to compare the predicted results of databases and chip, miRNA-188-3p most likely binds to lncRNA APOC1P1-3 (Figure. 4A). The microarray results suggest that the binding site is at 222 bp-248 bp of APOC1P1-3 (Figure. 4B).
The pmirGLO-APOC1P1-3'UTR and the pmirGLO-APOC1P1-Mut vectors were separately constructed with the Dual-Luciferase Report Gene System. We used the BiBiServ-RNAhybrid database to perform thermodynamic analysis of the predicted binding sites before making a point mutation. The mfe value of the predicted site is -28.5kcal/mol (Figure. S1), the highest absolute value among all 11 predicted sites, indicating a good thermodynamic effect. Group 4 mfe random mutation was -25.4kcal/mol (Table. S3), indicating a better thermodynamic effect.
By transfected miRNA 188-3p mimics, we found that the expression of miRNA-188-3P was significantly higher than that in the control group (Figure. 4C). The pmirGLO-APOC1P1-3'UTR plasmid was bound to the co-transfected miRNA-188-3P mimics. Since the transcription of the luciferase reporter gene was blocked, the fluorescence activity was significantly lowered. After mutation (Figure. S2), miRNA-188-3P mimics could no longer bind to it, and the luciferase reporter gene was transcribed normally (Figure. 4D). The fluorescence activity was not significantly different from the control, suggesting that miRNA-188-3P can specifically bind to lncRNA-APOC1P1-3.
The cross-talking between miRNA-188-3p and lncRNA-APOC1P1-3
We further explored the effect of miRNA-188-3p on anoikis resistance. miRNA-188-3p was first overexpressed in MDA-MB-231a cells by transfection with miRNA-188-3p mimics. Early apoptosis was highly increased after the overexpression (Figure. 4E), suggesting an inhibition of anoikis resistance. After co-overexpression of lncRNA APC1P1-3 and miRNA-188-3p, there was no significant difference in early apoptosis between Sense + Mimics and the controls (Figure. 4F, revealing that miRNA-188-3p can reverse the effect of lncRNA-APOC1P1-3 in anoikis resistance.
Moreover, we studied the mutual regulation between lncRNA APOC1P1-3 and miRNA-188-3p. Firstly, we overexpressed APOC1P1-3 in MCF-7a cells by transfection with pcDNA3.1-sense, and evaluated the expression of miRNA-188-3p, and vice versa. Neither overexpression of miRNA-188-3p nor lncRNA-APOC1P1-3 caused a change (Figure. 4G-H). Thus, although lncRNA APOC1P1-3 binds to miRNA-188-3p, the two molecules do not have any mutual impact.
LncRNA-APOC1P1-3 synergizes with miRNA-188-3p to affect Bcl-2
To study the effect of lncRNA APOC1P1-3 and miRNA-188-3p on the anti-apoptotic protein Bcl-2, we examined the Bcl-2 expression in 4 groups (sense+/mimics-, sense-/mimics+, sense+/mimics+, sense-/mimics-) (Figure. 5A-C). Overexpression of lncRNA APC1P1-3 (sense+/mimics-), significantly increased Bcl-2 (n=3, **P <0.01), while overexpression of miRNA-188-3p (sense-/mimics+) decreased it (n=3, **P <0.01). Interestingly, overexpression of both lncRNA APOC1P1-3 and miRNA-188-3p (sense+/mimics+) did not alter Bcl-2 expression of (n=3, **P>0.01). We obtained the same result with Western Blot (Figure. 5D-E). Thus, APOC1P1-3 may enhance the anoikis resistance by blocking the inhibition of miRNA-188-3p against the Bcl-2.