A double-negative feedback loop between E2F3b and miR- 200b regulates docetaxel chemosensitivity of human lung adenocarcinoma cells

Abstract

MicroRNAs (miRNAs) are non-coding small RNAs which negatively regulate gene expressions mainly through 3'-untranslated region (3'-UTR) binding of target mRNAs. Recent studies have highlighted the feedback loops between miRNAs and their target genes in physiological and pathological processes including chemoresistance of cancers. Our previous study identified miR-200b/E2F3 axis as a chemosensitivity restorer of human lung adenocarcinoma (LAD) cells. Moreover, E2F3b was bioinformatically proved to be a potential transcriptional regulator of pre-miR-200b gene promoter. The existance of this double-negative feedback minicircuitry comprising E2F3b and miR-200b was confirmed by chromatin immunoprecipitation (ChIP) assay, site-specific mutation and luciferase reporter assay. And the underlying regulatory mechanisms of this feedback loop on docetaxel resistance of LAD cells were further investigated by applying in vitro chemosensitivity assay, colony formation assay, flow cytometric analysis of cell cycle and apoptosis, as well as mice xenograft model. In conclusion, our results suggest that the double-negative feedback loop between E2F3b and miR-200b regulates docetaxel chemosensitivity of human LAD cells mainly through cell proliferation, cell cycle distribution and apoptosis.

Keywords: E2F3b; chemoresistance; feedback loop; lung adenocarcinoma (LAD); miR-200b.

Figure 1. Bioinformatical evidence of the direct binding of E2F3 upon miR-200b gene

A. CoreBoost_HM (http://rulai.cshl.edu/tools/CoreBoost_HM/) on-line analysis was used to identify the promoter regions of miR-200b (named as P1 and P2). B. TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) and C. CONSITE (http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite) on-line softwares were performed to find the potential E2F3 binding sites in miR-200b promoter.

Figure 2. Functional evidence of the direct binding of E2F3b upon miR-200b gene

Quantifications of miR-200b expression in docetaxel-resistant LAD cells (SPC-A1/DTX and H1299/DTX) and their parental cells (SPC-A1 and H1299) before A. and after B. manipulated E2F3a and E2F3b expressions were achieved by qRT-PCR. MiRNA abundance was normalized to U6 RNA. C. ChIP assays were performed in SPC-A1 and SPC-A1/DTX cells with antibodies against E2F3 or IgG control. D. Schematic representation of miR-200b gene promoter with the putative E2F3b-binding sites and the sequences of the point mutations. E. Luciferase reporter assays in SPC-A1 and SPC-A1/DTX cells with co-transfection of the pGL4 basic firefly luciferase reporters containing wild or mutated miR-200b promoter sequences and E2F3b or pSil/shE2F3 plasmid vectors as indicated. Data were normalized to luciferase activity and determined relative to empty vector promoter activity and presented as mean±SEM of three independent experiments each performed in triplicate. *p< 0.05, **p < 0.01 vs. control group.

Figure 3. The regulation of E2F3a/b on miR-200b expression and chemosensitivity of LAD cells

E2F3a/b mRNA and protein expression levels in SPCA1/DTX, H1299/DTX cells and the parental SPC-A1, H1299 cells before A. and after B. manipulated E2F3a and E2F3b expressions were detected by qRT-PCR and Western Blot analysis, respectively. Abundance of mRNA and protein was normalized to U6 RNA and GAPDH, respectively. C. Quantifications of miR-200b expression in different manipulated LAD cell lines as indicated was also achieved by qRT-PCR. Abundance of miRNA was normalized to U6 RNA. Data are representative of at least three independent experiments and are shown as mean±SEM. *p < 0.05, **p < 0.01 vs. control group.

Figure 4. The in vitro effects of E2F3a/b on cell proliferation, apoptosis, cell cycle distribution, and response to docetaxel of LAD cells

In SPCA1/DTX, H1299/DTX cells and the parental SPC-A1, H1299 cells, ectopic up- or down-regulation of E2F3a/b was achieved by transfection of pcDNA/E2F3a/b or pSil/shE2F3. A. IC50 values for docetaxel were measured by MTT assay. B. Cell proliferating ability was detected by colony formation assay. C. Cell apoptosis and D. cell cycle distribution data all came from flow cytometric analysis. Results are obtained in three independent experiments and are shown as mean±SEM. *p < 0.05, **p < 0.01 vs. control group.

Figure 5. The role of miR-200b in the in vitro regulation of E2F3b on LAD cells

PcDNA-NC, pcDNA/E2F3b vectors were transfected into SPC-A1 and H1299 cells without (or with) previous transfection of miR-200b mimics; sh-NC, pSil/shE2F3 vectors were transfected into SPC-A1/DTX and H1299/DTX cells without (or with) previous transfection of miR-200b inhibitors. A. Quantification of miR-200b expression was achieved by qRT-PCR. MiRNA abundance was normalized to U6 RNA. B. IC50 values for docetaxel were measured by MTT assay. C. Cell proliferating ability was detected by colony formation assay. D. Cell apoptosis and E. cell cycle distribution data all came from flow cytometric analysis. Error bars represent the mean±SEM of at least three independent experiments. *p < 0.05, **p < 0.01 vs. control group.

Figure 6. The in vivo effects of E2F3b on miR-200b expression and chemosensitivity of LAD cells

SPC-A1 and SPC-A1/DTX cells were transfected with pSil/shE2F3 or pSil/shE2F3-NC and injected subcutaneously into nude mice. When the average tumor size reached about 50 mm³, docetaxel was given through intraperitoneal injection with a dose of 1 mg/kg, one dose every other day with 3 doses in total. A. Growth curve of tumor volumes and representative photographs of tumors formed 35 days after the first administration of docetaxel. B. Quantification of E2F3a/b protein in the transplanted tumor tissues by Western Blot analysis. C. Quantification of miR-200b in the transplanted tumor tissues by qRT-PCR. Abundance of protein and miRNA was normalized to GAPDH and U6 RNA, respectively. D. Immunostaining of E2F3, Ki-67 and PCNA protein and TUNEL assay stained sections of the transplanted tumors as indicated (original magnification, ×400). *p < 0.05, **p < 0.01 vs. control group.