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Title: [LncRNA HULC promots HCC growth by downregulating miR-29].
Author: Zhu LR, Feng JL, Liu XJ, Wang JM.
Journal: Zhonghua Zhong Liu Za Zhi; 2019 Sep 23; 41(9):659-666. PubMed ID: 31550855.
Abstract:
Objective: To explore the effects of lncRNA HULC on hepatocellular carcinoma (HCC) growth by down-regulating miR-29. Methods: The expression levels of HULC and miR-29 in HCC tissues and cells were detected by real-time quantitative PCR (RT-qPCR), and the correlation analysis was performed. After HCC cells were transfected with HULC overexpressed plasmid or siRNA, the expressions of miR-29 and its target gene SETDB1 were determinate by RT-qPCR. According to the bioinformatic prediction of the miR-29 binding site in the HULC sequence, the report gene plasmids were constructed. The HCC cells were co-transfected with miR-29 mimics or miR-29 inhibitor, and the HULC targeted regulation of miR-29 was verified by dual luciferase reporter assay. The effect of miR-29 on the HULC-mediated proliferation in HCC cells was detected by cell count kit 8 (CCK-8) experiment. Expression of tumor proliferation antigen Ki-67 was detected by RT-qPCR.The Hep3B cells were inoculated in mice and miR-29 mimics and miR-29 negative control (NC) further injected into the lesions. The tumor volume was observed, and the expressions of tumor proliferation antigen ki-67 in tumor tissues were detected by immunohistochemical staining. Results: The expression of HULC was significantly up-regulated while the expression of miR-29 was significantly down-regulated in HCC tissues and cells (P<0.01). The level of HULC was negatively correlated with miR-29 in tumor tissues (r=-0.754, P<0.01) and HCC cells (r=-0.865, P<0.05). The in vitro experiments showed that, compared with the blank control group, the expression of miR-29 in HULC overexpressed Huh7 cells was significantly reduced, while the mRNA level of miR-29 target gene SETDB1 was increased (P<0.01). The expression of miR-29 was significantly increased in HULC deleted Hep3B cells, while the mRNA expression of SETDB1 was decreased (P<0.01). Double luciferase reporter gene assay showed that miR-29 mimics significantly inhibited the luciferase activity of Hep3B cells transfected with HULC wide type (psi-HULC-WT) plasmid but had no effect on Hep3B cells transfected with mutant plasmid (psi-HULC-Mut). However, the miR-29 inhibitor antagonized the inhibitory effect of miR-29 mimics on luciferase activity of psi-HULC-WT (P<0.01). Cell proliferation experiments showed that, compared with the control group, the proliferation ability of miR-29 mimics overexpressed Huh7 cells was significantly reduced.After 24, 48 and 72 hours of treatment, the proliferation rates of Huh7 cells in the HULC overexpressed group were (43.87±3.82)%, (83.45±7.46)% and (123.34±8.67)%, respectively, significantly higher than (13.45±1.77)%, (23.54±1.37)% and (38.21±2.09)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of miR-29 mimics transfected Huh7 cells were (57.10±1.94)% and (73.76±3.46)%, respectively, significantly lower than (83.45±7.46)% and (123.34±8.67)% of control group (P<0.05). After treatment for 48 and 72 hours, the proliferation rates of Huh7 cells transfected with miR-29 mimics and miR-29 inhibitor group were (76.45±3.24)% and (89.37±4.37)%, respectively, significant higher than (57.10%±1.94)% and (73.76±3.46)% of the control group (P<0.05). After 48 h transfection, the expression of Ki-67 in Huh7 transfected with miR-29 mimics was significantly inhibited compared with the control group (P<0.01). However, the expression of Ki-67 mRNA was increased in Huh7 cells transfected with miR-29 inhibitor (P<0.01). The results of in vivo experiments showed that the tumor volumes of the control group, miR-29 mimics group and miR-29 mimics + miR-29 inhibitors group were (504.0±19.6) mm(3), (310.0±24.3) mm(3) and (483.7±21.2) mm(3), respectively. Injection of miR-29 mimics reduced while miR-29 inhibitor promoted tumorigenesis ability of Huh7 in nude mice (P<0.01). The immunohistochemical staining showed that the average optical density values of Ki-67 protein in tumor tissues of the control group, miR-29 mimics group and miR-29 analogue+ miR-29 inhibitor group were 0.65±0.08, 0.36±0.07 and 0.56±0.06, respectively. The expression level of Ki-67 protein in miR-29 mimics group was significantly reduced (P<0.01) while increased in the miR-29 mimics+ miR-29 inhibitor group (P<0.01). Conclusion: LncRNA HULC promotes HCC growth by down-regulating miR-29. 目的： 探讨长链非编码RNA(lncRNA)HULC通过下调微小RNA－29(miR－29)的表达促进肝细胞癌(HCC)细胞增殖的作用及分子机制。 方法： 运用实时荧光定量PCR法检测HCC组织和细胞中HULC和miR－29的表达水平，并进行相关性分析。HCC细胞转染HULC过表达质粒或以小干扰RNA(siRNA)敲低HULC的表达，以实时荧光定量PCR检测miR－29和靶基因SET结构域分支1(SETDB1)的表达水平。通过生物信息学预测HULC序列上存在的miR－29结合位点，构建报告基因质粒与miR－29模拟物、miR－29抑制物共转染HCC细胞，通过双荧光素酶报告基因实验验证HULC靶向调控miR－29的分子机制。以细胞计数盒8(CCK－8)法检测miR－29对HULC的促HCC细胞增殖作用的影响。以实时荧光定量PCR法检测Ki－67的表达。建立Hep3B细胞移植瘤模型，瘤内注射miR－29模拟物、miR－29抑制物、miR－29阴性对照，观察肿瘤体积。采用免疫组化法检测小鼠肿瘤组织中肿瘤增殖抗原Ki－67的表达。 结果： 与癌旁组织和正常肝细胞比较，HULC在HCC组织和HCC细胞中均呈高表达(P<0.01)，而miR－29均呈低表达(P<0.01)，HULC和miR－29在HCC组织(r＝－0.754，P<0.01)和HCC细胞(r＝－0.865，P<0.05)中的表达均呈负相关。与空白对照组比较，过表达HULC组Huh7细胞中miR－29表达明显降低，而miR－29靶基因SETDB1 mRNA的表达升高(P<0.01)，si－HULC组Hep3B细胞中miR－29表达明显升高，而miR－29靶基因SETDB1 mRNA的表达降低(P<0.01)。miR－29模拟物可显著抑制转染psi－HULC－WT质粒组但不能抑制转染突变(psi－HULC－Mut)组Hep3B细胞的荧光素酶活性，而miR－29抑制物可拮抗miR－29模拟物对psi－HULC－WT荧光素酶活性的抑制作用(P<0.01)。与对照组比较，miR－29模拟物组Huh7细胞的增殖能力明显减弱。处理24、48和72 h后，HULC过表达组Huh7细胞的增殖率分别为(43.87±3.82)%、(83.45±7.46)%和(123.34±8.67)%，与对照组[分别为(13.45±1.77)%、(23.54±1.37)%和(38.21±2.09)%]比较，差异有统计学意义(P<0.05)。处理48和72 h后，转染miR－29模拟物组Huh7细胞的增殖率分别为(57.10±1.94)%和(73.76±3.46)%，与对照组[分别为(83.45±7.46)%和(123.34±8.67)%]比较，差异有统计学意义(P<0.05)。处理48和72 h后，转染miR－29模拟物＋miR－29抑制物组Huh7细胞的增殖率分别为(76.45±3.24)%和(89.37±4.37)%，与对照组[分别为(57.10±1.94)%和(73.76±3.46)%]比较，差异有统计学意义(P<0.05)。与对照组比较，转染48 h，转染miR－29模拟物Huh7细胞中Ki－67 mRNA的表达明显受到抑制(P<0.01)。转染miR－29抑制物Huh7细胞中Ki－67 mRNA的表达升高(P<0.01)。对照组、miR－29模拟物组和miR－29模拟物＋miR－29抑制物组裸鼠移植瘤的肿瘤体积分别为(504.0±19.6)mm(3)、(310.0±24.3)mm(3)和(483.7±21.2)mm(3)。与对照组比较，miR－29模拟物组的裸鼠成瘤能力明显降低(P<0.01)。与miR－29模拟物组比较，miR－29模拟物＋miR－29抑制物组裸鼠成瘤能力增强(P<0.01)。对照组、miR－29模拟物组、miR－29模拟物＋miR－29抑制物组荷瘤鼠肿瘤组织中Ki－67蛋白的吸光度值分别为0.65±0.08、0.36±0.07、0.56±0.06。与对照组比较，miR－29模拟物组Ki－67蛋白表达量明显降低(P<0.01)。与miR－29模拟物组比较，miR－29模拟物＋miR－29抑制物组Ki－67蛋白表达量升高(P<0.01)。 结论： lncRNA HULC通过分子吸附miR－29、并下调miR－29的表达促进HCC细胞增殖。HULC下调miR－29表达可能是其参与促进HCC生长的新机制。.

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