let-7b/g silencing activates AKT signaling to promote gastric carcinogenesis

The information of human gastric cancer cell lines (MKN1, MKN7, MKN28, MKN45, SNU1, SNU16, AGS, KatoIII, NCI-N87) were previously described [19]. Cells were maintained in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY). Among them, AGS (poorly differentiated and p53-wide type), NCI-N87 (well differentiated and p53-mutation) and MKN45 (with xenograft formation ability) were representatively chosen for functional studies.

The primary paired samples from gastric cancer patients were randomly selected from Prince of Wales Hospital (Year 1999–2010). All participants provided written consent for the research experiment. Ethical approval was obtained from the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (CREC Ref. No.2009.521).

The normal tissue corresponds to Human Stomach Total RNA commercially available from Ambion (AM7996, Grand Island, NY). A total of 67 paired (tumor and corresponding adjacent non-tumorous tissue) RNA samples were achieved from frozen tissues and 9 paired samples were got from micro-dissection of paraffin embedded tissues. RNA was extracted using TRIzol reagent (Invitrogen, Grand Island, NY). High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Grand Island, NY) were used for cDNA synthesis. qRT-PCR was used to quantitative differences in mRNA expression of AKT2 and primers were as following (sense-Exon 9: CAA AGA TGG CCA CAT CAA GA; anti-sense-Exon 10: GTC ATT GTC CTC CAG CAC CT). AKT1 expression were detected using the following primers (sense-Exon 9: GAG ATT GTG TCA GCC CTG GA; anti-sense-Exon 10: AGC CCG AAG TCT GTG ATC TT). The relative expression level was normalized by RPL29 (sense-Exon 3: GGA CCC CAA GTT CCT GAG G; anti-sense-Exon 4: GCA TTG TTG GCC TGC ATC TT) in gastric tissues and B2M (sense-Exon 1: ACT CTC TCT TTC TGG CCT GG; anti-sense-Exon 2: ATG TCG GAT GGA TGA AAC CC) in gastric cancer cell lines [20]. PCR was performed using SYBR Green PCR reagents (Applied Biosystems) according to the manufacturer's instructions. The reactions were incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

For miRNA expression detection, Taqman miRNA assays were used to quantify the expression levels of mature let-7b and let-7g (KIT, 002619 and 002282, Applied Biosystems). The relative expression level of microRNAs was normalized by RNU6B (KIT, 001093, Applied Biosystems). The reactions were performed in 7500 Fast Real-Time System (Applied Biosystems) and the reaction mix was incubated at 95°C for 30 seconds, followed by 40 cycles of 95°C for 8 seconds and 60°C for 30 seconds.

Protein was extracted from gastric cancer cell lines and paired primary tissues using RIPA lysis buffer with proteinase inhibitor. Protein concentration was measured by the method of Bradford (Bio-Rad, Hercules, CA) and 20 μg of protein mixed with 2 × SDS loading buffer was loaded per lane, separated by 12% SDS-polyacrylamide gel electrophoresis. Protein expression was detected using primary monoclonal anti-AKT2 antibody (1:1000 dilution, #3063, Cell Signaling, Danvers, MA), anti-phospho-AKT (S473) (1:1000 dilution, #9271, Cell Signaling) and anti-pS6 antibody (1:1000, #4858, Cell Signaling). The secondary antibodies were anti-Mouse IgG-HRP (1:30000, 00049039, Dako, Glostrup, Denmark) and anti-Rabbit IgG-HRP (1:10000, 00028856, Dako). The Western blot bands were quantified by ImageJ.

The miRNA precursors, let-7b (PM11050), let-7g (PM11758), scramble control (AM17110) were purchased from Applied Biosystems. And siAKT2 (SI00287672 and SI00287679) were obtained from Qiagen (Valencia, CA). All transfection were performed using Lipofectamine 2000 Transfection Reagent (Invitrogen) in a 20 nM concentration for 48 hours followed with functional study and RNA/protein analysis. Cell proliferation was assessed using CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI) according to manufacturer's instruction. For colony formation assays in monolayer cultures, the transfected cells were cultured in 6-well plates for 10 days. Cells were fixed with 70% ethanol for 15 minutes and stained with 2% crystal violet. The cell invasion assays using BD Biocoat Matrigel Invasion Chambers (BD Biosciences, Franklin Lakes, NJ) has been described previously by W. Kang [21]. The experiment was performed in triplicate and the mean including SDs was plotted.

The annealed oligonucleotides containing the putative let-7b/g binding site was cloned into pMIR-REPORT vector (Ambion) via HindIII and SpeI. The sequences of oligonucleotides were (sense) AGA CAC TAG TTA GCA CTT CAC ACC CAT TGA and (antisense) GAG AAA GCT TCT AGA GAT TAC AGG CAT GAG CCA CT). Mutations located on the predicted seed binding region were introduced into the luciferase reporter vector using QuickChange® II Site-Directed Mutagenesis Kit according to the manufacturer's instruction. The primer sequences for mutagenesis were 5'-GTG TGG GCA CAG GCC TGG GTT CGT GAT CTT TTT AGT GCC TCT C-3' and 5'-GAG AGG CAC TAA AAA GAT CAC GAA CCC AGG CCT GTG CCC ACA C-3'. The firefly luciferase reporter construct and the control Renilla luciferase construct were co-transfected into NCI-N87 cells in 24-well plates. Cells were harvested after 36 hours for reporter activity analysis using Dual Luciferase Reporter assays (Promega, Madison, WI) as described previously [22].

Total three batches of animal models were used in this study. For let-7b/g or siAKT2 functional study, MKN45 cells were transfected with let-7b/g precursors or siAKT2 together with their control counterparts. The cells (5 × 10⁶ cells suspended in 0.1 ml PBS) were injected subcutaneously into the dorsal flank of eight 4-week-old male Balb/c nude mice. For the rescue in vivo study, MKN45 was transfected with let-7b/g for 24 hours following by AKT2 or empty vector transfection for another 24 hours. Then the cells were injected subcutaneously into the dorsal flank of eight Balb/c nude mice. Tumor diameter was measured and documented every 4 days until the tumor reached 10 mm in diameter. Tumor volume (mm³) was estimated by measuring the longest and shortest diameter of the tumor and calculated using the following formula: volume = (shortest diameter)² × (longest diameter) × 0.5 [23]. All animal handling and experimental procedures were approved by Department of Health, Hong Kong (Reference No: 12–241 in DH/HA&P/8/2/1).

The Student T test was employed to compare the functional effect between the target transfectants and the controls. Expression of let-7b/g and AKT2 (mRNA and protein) in primary cancerous tissues and their corresponding paired noncancerous tissues were compared by paired T test. Correlations between let-7b/g expression and clinicopathologic parameters were assessed by Pearson correlation analysis. The let-7b/g expression in gastric cancer cell lines was compared with it copy number change by non-parametric Spearman's rho rank test. The Kaplan-Meier method was employed to estimate the survival rates for each variable. The equivalences of the survival curves were tested by log-rank statistics. All statistical analysis was performed by SPSS software (version 16.0; SPSS Inc). A two-tailed P value of less than 0.05 was considered statistically significant.

The expression of let-7b/g was measured in gastric cancer cell lines and normal stomach epithelium tissue by miRNA qRT-PCR. let-7b and let-7g were observed down-regulated in 6 and 7 gastric cancer cell lines respectively compared with normal gastric epithelial sample (Figure 1A). In a total of 76 paired primary RNA samples, the expression of let-7b and let-7g showed a decreased level in tumors compared with the corresponding non-tumorous gastric mucosal samples (let-7b, P = 0.013; let-7g, P = 0.003; Figure 1B).

In 76 paired primary samples, let-7b was found down-regulated in 53 (69.7%) tumor tissues which normalized by its expression in adjacent normal gastric tissue. Then two groups were stratified according to the median expression of let-7b and each group had the same 38 cases. The low-expression of let-7b group showed a poor survival compared with high-expression group (P = 0.001, up panel of Figure 1C). Meanwhile let-7g showed decreased expression in 48 (63.2%) tumor tissues when compared with adjacent normal tissues and its downregulation also predicted poorer prognosis in gastric cancer with a marginal significant P-value (P = 0.051, lower panel of Figure 1C). We further investigated the clinicopathologic correlation of let-7b and let-7g. Additional file 2: Table S2 summarized the correlation of let-7b/g with other clinicopathologic parameters in gastric cancer patients. The decreased expression of let-7b was correlated with lymph node metastasis (P = 0.021). The let-7g downregulation was also associated with N-stage (P = 0.044) and lymph node metastasis (P = 0.009), suggesting that these two miRNAs might be involved in gastric cancer cell metastasis.

To investigate the functional role of let-7b/g, the precursors of these two miRNAs were transfected into AGS, NCI-N87 and MKN45 cells. let-7b/g suppressed the growth of gastric cancer cells AGS, NCI-N87 and MKN45 as demonstrated by a 5-day MTT assay (P < 0.001, Figure 2A). The tumor suppressor function of let-7b/g was further validated by monolayer colony formation. A significant reduction of colony number was observed in let-7b/g transfectants compared with scramble miRNA groups (P < 0.001, Figure 2B). Ectopic expression of let-7b or let-7g was further revealed to inhibit the invasive capacity of gastric cancer cells as demonstrated by Matrigel invasion assays (P < 0.05, Figure 2C). The MKN45 cells with let-7b/g transfection were injected subcutaneously into the dorsal flank of nude mice. let-7b/g transfectants formed smaller xenografts than those scramble transfectants after 22 days (let-7b, P < 0.001; let-7g, P < 0.05; Figure 2D).

AKT2 was predicted to be a putative target of let-7b/g by TargetScan (www.​targetscan.​org). The putative binding sites of let-7b/g within the 3'UTR of AKT2 were shown in Figure 3A. We transfected let-7b/g into gastric cancer cells and decreased AKT2 mRNA expression level was observed in AGS, NCI-N87 and MKN45 cells (P < 0.05; left bar chart of Figure 3B). However, ectopic expression of let-7b/g did not change the AKT1 mRNA expression (right bar chart of Figure 3B), indicating let-7b/g only exerts its inhibitory effect on AKT2 but not on AKT1. AKT2 protein showed a decrease expression after ectopic let-7b/g expression (Figure 3C), suggesting that let-7b/g triggered a silencing effect on the endogenous AKT2 expression. Meanwhile, the phosphorylated AKT (S473) and its direct downstream effector of AKT-mTOR pathway, pS6, showed decreased expression upon let-7b/g overexpression. A luciferase reporter assay was performed to test whether AKT2 is a direct target of let-7b/g. As shown in Figure 3D, let-7b/g decreased the relative luciferase activity of constructs encompassing AKT2 3'UTR binding sites in NCI-N87 cells (P < 0.05). Meanwhile, let-7b/g did not suppress the luciferase activity in the constructs containing the mutated binding site. The results in this part revealed that let-7b/g specifically recognized the binding sites in AKT2 3'UTR and directly repressed AKT2 expression through mRNA degradation and protein synthesis inhibition.

Expression of AKT2 mRNA was up-regulated in 8 gastric cancer cell lines except SNU1 compared with normal gastric mucosal sample (Figure 4A). In 28 pairs of primary gastric cancers, AKT2 mRNA expression showed upregulation in 19 (67.9%) tumor samples by normalization with its expression in adjacent non-tumorous tissues. The expression of AKT2 in the cancer tissues showed higher expression than it expression in adjacent normal gastric tissues (P = 0.034, Figure 4B). Strong AKT2 protein expression can be strongly detected in all 9 gastric cancer cell lines but only one normal gastric mucosal tissue showed week AKT2 protein expression (Figure 4C). In 28 paired primary protein samples, AKT2 protein expression showed up-regulated in the cancer tissues compared with adjacent non-tumorous tissues after quantification (P = 0.001, Figure 4D). We then examined the AKT2 mRNA and let-7b/g expression in 28 paired primary gastric cancer samples. The AKT2 mRNA expression was negatively correlated with let-7b/g expression (let-7b, P = 0.001; let-7g, P = 0.021; Figure 4E), suggesting let-7b/g downregulation might be, at least partly, responsible for the aberrant activation of AKT2 in gastric cancers.

We have demonstrated that let-7b/g targeted AKT2 and exerted tumor suppressor function in gastric cancer. siRNA-mediated AKT2 knockdown was therefore performed to investigate if AKT2 downregulation phenocopied the tumor-suppressive effect of let-7b/g. We transfected siAKT2 into gastric cancer cell lines AGS, NCI-N87 and MKN45. Successful AKT2 knockdown was confirmed by Western blot analysis. p-AKT (S473) and pS6 protein showed decreased expression upon siAKT2 transfection (Figure 5A). AKT2 knockdown suppressed AGS, NCI-N87 and MKN45 cell proliferation in a 5-day MTT assays (P < 0.001, Figure 5B). AKT2 knockdown also decreased monolayer colony formation in gastric cancer cells to a significant level (Figure 5C). Moreover, siAKT2 inhibited cell invasion of gastric cancer cells (P < 0.001, Figure 5D). The effect of siAKT2 on tumor growth in vivo was also investigated. The siAKT2-MKN45 and vector control cells were injected into nude mice subcutaneously. The tumor growth in siAKT2 groups was significantly decreased compared with the vector control after 22 days (P < 0.05, Figure 5E).

Since AKT2 is a predicted target of let-7b/g, we further investigated if AKT2 re-expression diminished the suppressive phenotype change caused by let-7b and let-7g. Interestingly, we found that the growth inhibitory effect of let-7b/g were partially abrogated by AKT2 re-expression (MTT proliferation assays, Figure 6A; monolayer colony formation assays, Figure 6B), suggesting AKT2 was functionally involved in let-7b/g-inducing suppression of gastric cancer cell growth. In vivo experiments yielded similar results. Cells co-transfected with let-7b/g and AKT2 formed bigger xenografts than those with let-7b/g alone (P < 0.001, Figure 6C).