Restoration of miR-1228* Expression Suppresses Epithelial-Mesenchymal Transition in Gastric Cancer

<div><p>Dysregulated miRNAs play critical roles during carcinogenesis and cancer progression. In the present study, the function of miR-1228* in regulating cancer progression was investigated in gastric cancer. Decreased expression of miR-1228* was observed in human gastric cancer tissues comparing to normal tissues. Subsequently, the role of miR-1228* was evaluated <i>in vivo</i> using the tumor xenograft model. In this model, miR-1228* overexpression suppressed xenograft tumor formation. Furthermore, we demonstrated miR-1228* negatively regulated NF-κB activity in SGC-7901 gastric cancer cells and found that CK2A2 was a target of miR-1228*. Upregulation of miR-1228* decreased the expression of mesenchymal markers and increased the epithelial marker E-cadherin, suggesting its potential role in suppressing epithelial-mesenchymal transition. Collectively, these findings provide the first evidence that miR-1228* plays an important role in regulating gastric cancer growth and suggest that selective restoration of miR-1228* might be beneficial for gastric cancer therapy.</p> </div

Relationship between miR-1228* expression and clinicopathologic features of patients with gastric cancer (n = 50).

<p>Relationship between miR-1228* expression and clinicopathologic features of patients with gastric cancer (n = 50).</p

Downregulation of miR-1228* in gastric cancer tissues and gastric cancer cell lines.

<p>(A) In human gastric cancer tissues compared with paired adjacent noncancerous (normal) gastric tissues, the miR-1228* was down-regulated. The expression of miR-1228* was analyzed by real-time PCR and normalized to RNU6B. The results are displayed on a log scale. The statistical differences between samples were analyzed with the wilcoxon signed-rank test (n = 50). (B) Relative expression of miR-1228* in 50 gastric cancer tissues compared with matched normal tissues. Data are shown as 2^-ΔΔCT values. (C) The expression of miR-1228* in 3 gastric cancer cell lines and one immortalized normal gastric mucosal epithelial cell line (GES-1) was carried out by real-time PCR and normalized to RNU6B. Results are means ± SEM, n  =  3.</p

Regulation of NF-κB activity and CK2A2 expression by miR-1228*.

<p>(A) SGC-7901-miR-1228* and SGC-7901-miR-NC were transfected with the NF-κB reporter construct, respectively. 48 hours after transfection, the luciferase activity was measured. The luciferase activity was normalized to Renilla luciferase activity. Results are means ± SEM, n  =  3. (B) Schematic graph of the putative binding site of miR-1228* in the CK2A2 predicted by miRanda. (C) Western blot analysis revealed that protein level of CK2A2 in miR-1228* stable overexpression SGC-7901 cells was significantly decreased compared with miR-NC. The level of GAPDH was used as a loading control. (D) miR-1228* significantly reduced the luciferase reading when co-transfected with pMIR-CK2A2 3’UTR plasmid, indicating interaction between miR-1228* and CK2A2 3’UTR at this site. The luciferase activity was normalized to Renilla luciferase activity expressed by pRL-TK. Data were expressed as means ± SEM, n  =  3.</p

NF-κB activation is responsible for the lower expression of miR-1228*.

<p>(A) Schematic representations of the miR-1228* promoter region (the black arrowheads is the c-Rel binding domains), the lower is the pGL3-miR-1228*-promoter construct. (B) 24 hours after transfection with pGL3-miR-1228*-promoter or pGL3-control, SGC-7901 cells were stimulated with or without TNF-α and/or PDTC for 8 hours, then relative luciferase activity was measured. The luciferase activity was normalized to Renilla luciferase activity expressed by pRL-TK and then compared with the relative luciferase level of pGL3-control. * <i>P</i> <0.05 as compared with control. ^#<i>P</i> <0.05 as compared with TNF-α-treated group. (C) SGC-7901 cells were co-transfected with pGL3-miR-1228*-promoter/pGL3-control and GFP-c-Rel/GFP-control, and the relative luciferase expression was detected 48 hours later. The luciferase activity was normalized to Renilla luciferase activity expressed by pRL-TK and then compared with the relative luciferase level of GFP-control. Data were expressed as means ± SEM, n  =  3.</p

miR-1228* restoration inhibits xenograft tumor formation of gastric cancer cells.

<p>(A) miR-1228* was more than ten-fold changes in miR-1228* stable transfected SGC-7901 compared with NC. Results are means ± SEM, n  =  3. (B) Increased miR-1228* expression suppresses xenograft tumor growth. Stable transfection of SGC-7901 cells with miR-1228* or miR-NC were injected subcutaneously into nude mice. The volume of each tumor was measured twice each week. The average volume of tumors developed in nude mice is shown as means ± SEM, n  =  6 per treatment group. The statistical differences between samples were determined by the two-way ANOVA. (C) Compared with the control, the xenografts with miR-1228* overexpression were significantly smaller. The mice were sacrificed 5 weeks after inoculation. Two groups’ photograph of tumors is shown. (D) Tumors from each group were weighed immediately after removal. The tumor weight is indicated as means ± SEM, n  =  6.</p

Effect of miR-1228* on EMT in SGC-7901 cells.

<p>(A) Western blot analysis of epithelial marker (E-cadherin) and mesenchymal markers (Vimentin, β-catenin, Snail, Slug, and ZEB1/2) in miR-1228* stable transfected SGC-7901 cells and control. (B) Transwell migration assay showed that SGC-7901 cells stable transfected with miR-1228* had lower migratory potential in compare with miR-NC (×100). (C) The relative level of cell migration is presented as the mean ± SEM, based on three independent experiments. (D) Expression of EMT-related markers in xenograft tumor. Immunohistochemical staining indicated decreased Vimentin and increased E-cadherin expression in miR-1228* xenograft tumor compared to the control (×400).</p

Hh signaling pathway promoting invasion/migration of pancreatic cancer cells through mediating S100A4.

<p>A: The relative transcription levels of Shh, Gli1, S100A4, E-cadherin and vimentin were regulated by L-Gli1i/L-Shh transduction. B: The relative transcription levels of S100A4, E-cadherin and vimentin in L-Shh transfected cells were reversed by siS100A4 transduction. C: The expression levels of Gli1, S100A4, E-cadherin and vimentin proteins regulated by L-Gli1i/L-Shh transduction. D: The expression levels of S100A4, E-cadherin and vimentin proteins were reversed by siS100A4 transduction. E: The invasion/migration of pancreatic cancer cells regulated by L-Gli1i/L-Shh transduction were analyzed by transwell assays. F: The invasion/migration of L-Shh transfected cells were reversed by siS100A4 transduction. * <i>P</i><0.05, ** <i>P</i><0.01.</p

Binding of Gli1 to promoter region of S100A2, 4 and 6 genes in AsPC-1 cells analyzed by XChIP-PCR assay.

<p>M: DNA Marker; PC: RNA polymerase II antibody for XChIP positive control; IP: Gli1 XChIP; IGG: mouse IgG for XChIP random control; NC: β-actin antibody for XChIP negative control.</p

Expression of the S100 gene family.

<p>A: cDNA microarray data about S100 gene family; B: The differential expression levels of partial members of S100 gene family by qRT-PCR. The expression of GAPDH is as a control. * <i>P</i><0.05, ** <i>P</i><0.01.</p