3.1. miR-4651 targets BRD4 in human lung cancer cells
First, the miRNA database, TargetScan (V7.2, http://www.targetscan.org), was consulted to explore possible BRD4-targeting miRNAs binding to its 3’-UTR. The possible miRNAs were further verified through other miRNA databases, including miRBase, miRNAmap and miRTarbase. The bioinformatics analyses identified one particular miRNA, miR-4651, potentially targets the 3’-UTR (at position 1715-1722) of BRD4 (Figure 1A). To study whether miR-4651 can affect BRD4 expression, a lentivirus-packaged pri-miR-4651 construct, LV-pri-miR-4651, was established and transduced to A549 NSCLC cells. With selection by puromycin two stable cell lines, LV-pri-miR-4651-sLine-1/2, were established. qPCR analyses showed that the mature miR-4651 levels increased over 11-12 folds in stable cells with LV-pri-miR-4651 (Figure 1B). Significantly, ectopic overexpression of miR-4651 led to a dramatic reduction of BRD4 3’-UTR activity (Figure 1C), suggesting that miR-4651 directly targets BRD4 in A549 cells. Further results demonstrated that LV-pri-miR-4651 led to downregulation of BRD4 mRNA (Figure 1D) and protein (Figure 1F). As a result, mRNA and protein expression of BRD4-dependent genes, including c-Myc, Bcl-2, cyclin D1 [6, 10, 11, 33], was significantly downregulated (Figure 1E and F). These results imply that miR-4651 possibly targets BRD4 in A549 lung cancer cells.
To support that BRD4 is the direct target of miR-4651, two mutant miR-4651 mimics (“Mut1/2”), containing mutations within the binding sites of BRD4’s 3-UTR (see Figure 1G), as well as the wild-type (WT) miR-4651 mimic, were transfected to A549 cells. As shown, only the WT miR-4651 mimic, but not the mutants, inhibited BRD4’s 3-UTR activity (Figure 1H) and downregulated BRD4 mRNA levels (Figure 1I). We also tested whether miR-4651 can exert similar activity in primary cancer cells. The primary human lung cancer cells, provided by Dr. Jiang [26, 27], were derived from three written-informed consent NSCLC patients: Pri-Ca-1/2/3. As shown, LV-pri-miR-4651 led to significant increase of mature miR-4651 expression in the primary cancer cells (Figure 1J), causing potent BRD4 mRNA downregulation (Figure 1K). Western blotting assay results, Figure 1L, demonstrated that protein expression of BRD4 and its targets, c-Myc, Bcl-2, cyclin D1, was decreased in Pri-Ca-1 cells with miR-4651 overexpression. Similar results were obtained in two other primary cancer cells (Data not shown). To further confirm that miR-4651 directly targets BRD4 mRNA, the Ago2 immunoprecipitation (Ago2-IP) experiments were carried out. In A549 cells and primary human lung cancer cells (Pri-Ca-1/2/3), BRD4 mRNA was indeed enriched in the Ago2-IP (IP with miR-4651) in the tested NSCLC cells (Figure 1M). Collectively, these results show that miR-4651 is a BRD4-targeting miRNA in human lung cancer cells.
3.2. miR-4651 overexpression inhibits lung cancer cell growth, proliferation and migration, while activating cell apoptosis
We next tested whether ectopic overexpression of miR-4651 could affect the function of lung cancer cells. Results of cell counting assay, in Figure 2A, demonstrated that LV-pri-miR-4651-expressing A549 cells (see Figure 1) grew significantly slower than the parental control cells. Furthermore, miR-4651-overexpressed stable cells presented with reduced cell viability (CCK-8 OD, Figure 2B). A549 cell colony formation was potently inhibited as well with miR-4651 overexpression (Figure 2C). Additionally, EdU incorporation in the two stable cell lines with LV-pri-miR-4651 was largely suppressed, as compared to that in the control cells (Figure 2D). Analyzing cell migration, by the “Transwell” assays, showed that miR-4651 overexpression decreased the number of migrated A549 cells (Figure 2E). These results show that ectopic overexpression of miR-4651 inhibited A549 cell growth, proliferation and migration.
Testing the potential function of miR-4651 on cell apoptosis, we show that LV-pri-miR-4651 significantly increased the number of the Annexin V-positive A549 cells (Figure 2F), and induced cleavages of caspase-3 and poly (ADP) ribose polymerase (PARP) (Figure 2G). These results indicated the pro-apoptosis activity by miR-4651 overexpression. The non-sense control miRNA vector (“miRC”) had no significant effect on the functions of A549 cells (Figure 2A-G). In the primary human lung cancer cells, Pri-Ca-1/2/3, LV-pri-miR-4651-induced miR-4651 overexpression (see Figure 1) significantly inhibited CCK-8 viability (Figure 2H), EdU incorporation (Figure 2I) and cell migration (Figure 2J). A significant apoptosis activation, evidenced by an increased Annexin V-staining, was detected in miR-4651-overexpressed primary cancer cells (Figure 2K). Together, we show that miR-4651 overexpression inhibited lung cancer cell growth, proliferation and migration, while activating cell apoptosis.
3.3. miR-4651 inhibition increases BRD4 expression and promotes lung cancer cell growth, proliferation and migration
To inhibit miR-4651 expression, a lentivirus-packed pri-miR-4651 anti-sense sequence, LV-antagomiR-4651, was transduced to A549 NSCLC cells. Followed by puromycin selection, two stable cell lines, sLine-1/-2, were established. As compared to control cells with control anti-sense sequence (“anta-C”), mature miR-4651 levels decreased over 90% in LV-antagomiR-4651-expressing A549 cells (Figure 3A). On the contrary, LV-antagomiR-4651 increased BRD4 3’-UTR activity (Figure 3B) as well as BRD4 mRNA/protein levels (Figure 3C and E). mRNA and protein expression of BRD4-dependent genes, c-Myc, Bcl-2 and cyclin D1, was upregulated as well in LV-antagomiR-4651-expressing A549 cells (Figure 3D and E). Therefore, miR-4651 inhibition increased BRD4 expression in A549 cells.
Functional studies show that in A549 cells LV-antagomiR-4651 increased CCK-8 OD (Figure 3F) and BrdU incorporation (Figure 3G), promoting cell survival and proliferation. Furthermore, A549 cell migration, tested by counting migrated cells in the “Transwell” assays, was significantly boosted with miR-4651 inhibition (Figure 3H). Similarly in Pri-Ca-1/2/3 primary human NSCLC cells, LV-antagomiR-4651 induced 80-90% reduction of miR-4651 expression (Figure 3I), but upregulated BRD4 mRNA (Figure 3J). Protein expression of BRD4 and its targets, c-Myc, Bcl-2, cyclin D1, was elevated (Figure 3K, in Pri-Ca-1 cells). LV-antagomiR-4651-expressing primary cancer cells presented with increased cell growth (Figure 3L), proliferation (Figure 3M) and migration (Figure 3N), when compared to control cells with anta-C (Figure 3L-N). Together, the functional results show that miR-4651 inhibition promoted lung cancer cell growth, proliferation and migration.
3.4. BRD4 is the main target of miR-4651 in lung cancer cells
To test that BRD4 is the main target of miR-4651 in lung cancer cells, an 3’-UTR mutant (at miR-4651-binding sites, 1716C>A) BRD4 lentiviral construct (“mut-BRD4”) was established, which was transduced to LV-pri-miR-4651-expressing stable A549 cells. As shown the mut-BRD4 restored mature BRD4 mRNA (Figure 4A) and BRD4 protein (Figure 4B) expression in miR-4651-overexpressed cells (LV-pri-miR-4651-sLine-1, Figure 4C). LV-pri-miR-4651-induced downregulation of BRD4-tareget genes, c-Myc, Bcl-2, cyclin D1, was reversed by the mut-BRD4 as well (Figure 4B). Importantly, the mutant BRD4 abolished LV-pri-miR-4651-induced inhibitions on A549 cell growth (cell counting results, Figure 4D), proliferation (EdU incorporation, Figure 4E) and migration (Figure 4F). These results show that restoring BRD4 expression, by the 3’-UTR mutant BRD4, reversed miR-4651 overexpression-induced inhibitions on A549 cells, suggesting that BRD4 is the main target of miR-4651 in A549 cells.
Based on the results, we further hypothesized that BRD4 depletion should mimic miR-4651 overexpression-induced actions in NSCLC cells. The CRISPR/Cas9-BRD4-KO construct (provided by Dr. Zhao [31] at Soochow University), containing the small guide RNA (sgRNA) against BRD4, was transduced to A549 NSCLC cells. Following GFP sorting and puromycin selection two stable cell lines, BRD4-KO-sLine-1/2, were established, showing depleted BRD4 mRNA (Figure 4G) and protein (Figure 4H) expression. In the BRD4-KO A549 cells, c-Myc, Bcl-2, cyclin D1 protein expression was significantly downregulated (Figure 4H). BRD4 KO potently inhibited A549 cell proliferation (EdU incorporation, Figure 4I) and migration (Figure 4J), mimicking miR-4651 overexpression-induced activity. Importantly, in the BRD4-KO cells, miR-4651 overexpression (by LV-pri-miR-4651, see Figure 1) or inhibition (by LV-antagomiR-4651, see Figure 3) was completely ineffective on cell proliferation (EdU incorporation, Figure 4K) and migration (Figure 4L). Although both genetic treatments significantly altered miR-4651 expression (Figure 4M). These results show that miR-4651 was completely ineffective in BRD4-KO cells, further supporting that BRD4 is the primary target of miR-4651 in A549 cells.
3.5. miR-4651 overexpression inhibits A549 cell growth in SCID mice
To study the potential role of miR-4651 on NSCLC cell growth in vivo, LV-pri-miR-4651-expressing stable A549 cells (see Figure 1 and 2) and miRC-expressing control A549 cells were inoculated into the right flanks of the SCID mice. Testing tumor growth by recording tumor volumes, Figure 5A, demonstrated that miR-4651-overexpressed A549 xenografts grew significantly slower than control A549 xenografts expressing miRC. Calculating daily tumor growth, by the formula (tumor volume at Day-35-tumor volume at Day-0)/35, showed that A549 xenograft growth was significantly inhibited with miR-4651 overexpression (Figure 5B). The mice body weights between the two groups were not significantly different (Figure 5B), neither did we notice any signs of apparent toxicities.
At Day-7 and Day-14, one tumor of each group (total four xenograft tumors) was individually isolated, and tissue lysates subjected to qPCR and Western blotting assays. As shown, the mature miR-4651 levels were significantly elevated in LV-pri-miR-4651-expressing A549 xenografts (about ten folds vs. control tumors, Figure 5D). Contrarily, BRD4 mRNA (Figure 5E) and protein (Figure 5F) levels were decreased. Expression of BRD4-dependent genes, c-Myc, Bcl-2, cyclin D1, was also significantly downregulated in miR-4651-overexpressed A549 xenograft tissues (Figure 5E and F). These results together show that miR-4651 overexpression inhibited BRD4 expression and A549 xenograft growth in SCID mice.
3.6. miR-4651 is downregulated in human lung cancer tissues, correlating with upregulation of BRD4 and its pathway genes
At last we tested expression of miR-4651 in human lung cancer tissues. A total of ten (10) pairs of NSCLC tissues (“Ca”) and paracancer lung epithelial tissues (“N”) tissues were obtained. Testing miR-4651 expression, by qPCR, demonstrated that miR-4651 levels were significantly downregulated the “Ca” tissues (P <0.05 vs. the “N” tissues, Figure 6A). Contrarily, BRD4 mRNA levels in the “Ca” tissues were significantly higher than those in the “N” tissues (Figure 6B). mRNA levels of BRD4-dependent genes, c-Myc, Bcl-2, cyclin D1, were also significantly elevated in the “Ca” tissues. Western blotting analyzing tissue lysates from three representative patients, Patient-1/-3/-4, showed that BRD4 and its targets (c-Myc, Bcl-2, cyclin D1) were upregulated in NSCLC tissues (Figure 6C). Quantitative analyses integrating all 10 pairs of tissues confirmed that upregulation of BRD4, c-Myc, Bcl-2 and cyclin D1 proteins in “Ca” tissues was significant (P <0.05 vs. the “N” tissues, Figure 6D). These results together show that miR-4651 is downregulated in human NSCLC tissues, correlating with upregulation of BRD4 and its target genes.