[MiR-126 inhibits the proliferation and invasion of gastric cancer by downregulation of IGF-1R].

Objective: To investigate the expression levels and the mechanism of miR-126 and insulin like growth factor 1 receptor (IGF1R) in gastric cancer tissues and cells. Methods: The expression levels of miR-126 and IGF1R in 60 gastric cancer tissues and matched normal gastric tissues were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, respectively. The association of miR-126 expression with clinicopathology and prognosis of gastric cancer patients was further analyzed. CCK-8, soft agar assay, transwell assay were used to analyze the proliferation and invasion capacity of gastric cancer cells, respectively, while the dual luciferase reporter assay was used to determine the direct target of miR-126. Results: The expression of miR-126 was obviously correlated with lymphatic metastasis, distant metastasis and TNM stage of gastric cancer (all P<0.05). Cox multivariate analysis revealed that lymphatic metastasis, TNM stage, miR-126 and IGF-1R expression were independent risk factors for prognosis of gastric cancer patients (all P<0.05). The expression level of miR-126 in gastric cancer tissues was 2.01±0.23 significantly lower than 10.12±2.15 of normal gastric tissues (P<0.05). CCK-8 result showed that the absorbance values of MKN28 and BGC823 cells at 72 hours after transfected with miR-126 mimics were 1.06±0.05 and 1.01±0.09, respectively, significantly lower than 1.55±0.12 and 1.36±0.12 of the control group (all P<0.05). The clone numbers of MKN28 and BGC823 cells transfected with miR-126 mimics formed in the soft agar were 33±9 and 29±8, respectively, significantly lower than 76±13 and 71±11 of the control group (all P<0.05). Transwell assay showed that the invasived number of MKN28 and BGC823 cells transfected by miR-126 mimics was 98±12 and 89±8, respectively, significantly lower than 154±18 and 161±17 of the control group (all P<0.05). Double luciferase assay further clarified that miR-126 the 3'-untranslated region (3'-UTR) of IGF-1R, and inhibited its protein expression. CCK-8 results showed that overexpression of IGF-1R partially reversed the miR-126 induced proliferation inhibition in MKN28 (1.65±0.14 v. s. 0.98±0.11, P=0.003) and BGC823 cells (1.44 ±0.15 v. s. 0.89±0.10; P=0.006). Likewise, overexpression of IGF-1R partially reversed the miR-126-inhibited invasion of MKN28 (176±19 v. s. 101±14, P=0.005) and BGC823 cells (186±21 v. s. 92±9, P=0.002). Moreover, the inhibitory effects of miR-126 on proliferation were aggravated by silencing of IGF-1R in MKN28 (0.67±0.09 v. s. 0.99±0.12, P=0.021) and BGC823 cells (0.57±0.07 v. s. 0.92±0.12, P=0.012). Conclusion: miR-126 suppresses the proliferation and invasion of gastric cancer cells through targeting the 3'-UTR of IGF-1R and inhibiting its expression.目的： 探讨miR－126和胰岛素样生长因子1受体(IGF－1R)在胃癌组织和细胞中的表达及其作用机制。 方法： 选取2011年1月至2013年1月就诊于山东省立第三医院、行胃癌根治手术治疗的60例胃癌患者的组织标本，以及永生化的非肿瘤细胞GES－1、胃癌细胞株MKN28、BGC823、MKN45和SGC7901，采用逆转录聚合酶链反应(RT－PCR)和Western blot检测60例胃癌及癌旁组织中miR－126和IGF－1R的表达，分析患者的临床病理特征和预后。采用CCK－8法、软琼脂集落形成实验、Transwell小室实验和双荧光素酶报告实验等检测胃癌细胞的增殖和侵袭能力以及miR－126的直接靶点。 结果： 胃癌组织中miR－126的表达与患者的分化程度、淋巴结转移、远处转移和TNM分期均有关(均P<0.05)。与癌旁组织(10.12±2.15)比较，胃癌组织中miR－126的表达水平降低(2.01±0.23，P<0.05)。Cox多因素分析显示，淋巴结转移、TNM分期、miR－126和IGF－1R的表达与胃癌患者的预后均有关(均P<0.05)。CCK－8结果显示，miR－126模拟物转染MKN28和BGC823细胞72 h后，吸光度(A)值分别为1.06±0.05和1.01±0.09，与miRNA对照组(分别为1.55±0.12和1.36±0.12)比较，差异均有统计学意义(均P<0.05)。软琼脂集落形成实验显示，miR－126模拟物转染组MKN28和BGC823细胞的集落数分别为(33±9)个和(29±8)个，与miRNA对照组[分别为(76±13)个和(71±11)个]比较，差异均有统计学意义(均P<0.05)。Transwell小室实验结果显示，miR－126模拟物转染组MKN28和BGC823细胞的穿膜数为(98±12)个和(89±8)个，与miRNA对照组[分别为(154±18)个和(161±17)个]比较，差异均有统计学意义(均P<0.05)。双荧光素酶实验明确miR－126靶定IGF－1R的3′－UTR抑制了IGF－1R蛋白的表达。CCK－8检测显示，IGF－1R过表达部分逆转了miR－126模拟物抑制的MKN28细胞增殖能力(A值分别为1.65±0.14和0.98±0.11，P＝0.003)；IGF－1R过表达部分逆转了miR－126模拟物抑制的BGC823细胞增殖能力(A值分别为1.44±0.15和0.89±0.10，P＝0.006)。IGF－1R过表达逆转了miR－126模拟物抑制的MKN28细胞侵袭能力[细胞穿膜数分别为(176±19)个和(101±14)个，P＝0.005]；IGF－1R过表达也逆转了miR－126模拟物抑制的BGC823细胞侵袭能力[细胞穿膜数分别为(186±21)个和(92±9)个，P＝0.002]。miR－126模拟物抑制的MKN28细胞增殖被si－IGF－1R进一步抑制(A值分别为0.67±0.09和0.99±0.12，P＝0.021)；miR－126模拟物抑制的BGC823细胞增殖被si－IGF－1R进一步抑制(A值分别为0.57±0.07和0.92±0.12，P＝0.012)。 结论： miR－126靶向IGF－1R的3′－UTR抑制IGF－1R的表达，进而抑制胃癌细胞的增殖与侵袭。.