Objective:To investigate the effects of long non-coding RNA Hox transcript antisense RNA (lncRNA HOTAIR) on proliferation and migration ability of pancreatic cancer cells by targeting microRNA-520g-3p (microRNA-520g, miR-520g-3p).Methods:Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of miR-520g-3p and HOTAIR in pancreatic cancer cells. HOTAIR small interfering RNA was transfected into SW1990 pancreatic cancer cells. The normal control (NC) group, siHOTAIR-1 group and siHOTAIR-2 group were set up. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation ability. The scratch healing experiment was used to detect cell migration ability. The dual luciferase reporter gene experiment was used to verify the relationship between miR-520g-3p and HOTAIR targeting regulation. The quantitative data between the two groups were compared by t test. Results:qRT-PCR showed that the level of HOTAIR mRNA in SW1990 was significantly higher than that in human normal pancreatic ductal epithelial cells human pancreatic duct epithelial (HPDE) (3.08±0.33 vs. 1.57±0.17, t=29.830, P<0.01). The expression of miR-520g-3p in SW1990 was significantly lower than that in HPDE (0.93±0.11 vs. 2.63±0.24, t=22.410, P<0.01). HOTAIR small interfering RNA was transfected into SW1990, and at72 h, A value in the NC group was significantly higher than that in siHOTAIR-1 group and siHOTAIR-2 group (3.52±0.99 vs. 1.92±0.23, 2.36±0.58, t=6.340, 5.150, P<0.01). At 48 h after down-regulating the HOTAIR expression, the percentage of wound healing area in the NC group was significantly higher than that in the siHOTAIR-1 group and the siHOTAIR-2 group [(85.37±9.82)% vs. (50.56±5.82)%, (35.28±6.86)%, t=7.760, 14.750, P<0.01]. The dual luciferase reporter gene indicated that miR-520g-3p could significantly reduce the luciferase activity of wild-type HOTAIR vector. After down-regulating the expression level of miR-520g-3p, the proliferation and migration ability of SW1990 cells was significantly increased as compared with that in the control group ( t=13.190, 12.480, P<0.01). Conclusion:HOTAIR can influence the proliferation and migration ability of pancreatic cancer cells by targeting and negatively regulating miR-520g-3p.

目的 探究长链非编码RNA(LncRNA)锌指结构反义转录本1(ZFAS1)靶向微小RNA(miR)-373导致肝癌细胞顺铂(DDP)耐药的机制.方法 HepG2细胞设正常培养组、si-NC组、si-ZFAS1组,实时荧光定量PCR(qPCR)检测细胞中ZFAS1、miR-373表达水平.在si-NC组、si-ZFAS1组基础上分别添加0、50、100、200、300μmol/L DDP处理细胞12 h,CCK-8检测细胞增殖情况,Transwell检测细胞侵袭情况,蛋白免疫印迹检测细胞中基质金属蛋白酶(MMP)2、MMP9蛋白表达水平.双荧光素酶验证ZFAS1与miR-373的靶向关系.在si-ZFAS1组基础上添加inhibitor miR-373,检测细胞中MMP2、MMP9蛋白表达水平.结果 si-ZFAS1组细胞中ZFAS1表达水平均低于正常培养组和si-NC组,miR-373表达水平均高于正常培养组和si-NC组(P<0.05).si-NC组和si-ZFAS1组经不同浓度顺铂处理后细胞增殖率、侵袭数量、侵袭蛋白MMP2、MMP9表达水平基本呈降低趋势;si-ZFAS1组上述指标分别低于其对应浓度的si-NC组(P<0.05).Starbase分析发现,miR-373与ZFAS1存在互补的结合位点并经双荧光素酶验证.si-ZFAS1+inhibitor miR-373组细胞MMP2、MMP9蛋白表达水平高于si-ZFAS1组和si-ZFAS1+inhibitor NC组(P<0.05).结论 ZFAS1可能降低肝癌细胞DDP耐药,其机制可能与调控miR-373有关.

Objective:To explore the expression and clinicopathological significance of vacuolar protein sorting 72 (VPS72) in liver cancer tissues and corresponding adjacent tissues, and observe its influence on cell proliferation and migration.Methods:From September 2018 to September 2020, 60 cases of liver cancer tissues and corresponding adjacent tissues from Henan University Henan Provincial People′s Hospital were selected. The liver cancer tissues were divided into center group, and the specimens 2 cm away from the edge of the cancer tissue served as the paracancerous group. Western blotting and immunofluorescence methods were used to detect the expression level and distribution of VPS72 protein in center group and paracancerous group, and its relationship with age, gender, tumor size, number, stage, and degree of differentiation, the presence and absence of vascular tumor thrombus, capsule invasion and other clinicopathological parameters was analyzed. human hepatoma cells-7 (Huh-7) cells with VPS72 silencing by small interfering RNA were divided into experimental group and control group. The scratch assay and Transwell assay were used to verify the effect of VPS72 on the ability of migration. Cell counting kit-8 (CCK-8) experiment and plate cloning experiment were done to detect the ability of proliferation in Huh-7 cells.comparisons between groups were performed by paired-sample t and χ2 test. Results:The relative expression of VPS72 in cancer tissues was (0.900±0.511), significantly higher than that in adjacent tissues (0.729±0.643) ( t=2.027, P<0.05). The expression of VPS72 was significantly correlated with tumor differentiation, size, vascular tumor thrombus, cell proliferation antigen-67 (Ki-67) and staging ( χ2=5.621, 4.855, 4.275, 8.418, 7.202, P<0.05). The scratch assay showed that the number of migrating cells at24 and 48 hin the experimental group [(38.330±3.512), (66.670±5.508)] was significantly less than that in the control group [(78.670±2.517), (165.670±5.033)] ( t=14.162, 16.275, P<0.05). Transwell experiment showed that the number of invasive cells in the experimental group (32.333±3.512) was significantly les than that in the control group (52.667±2.082) ( t=7.625, P<0.05). The CCK-8 experiment showed that the absorbance (A) value (0.235±0.047) at 0 h in the experimental group had no significant difference from the control group (0.237±0.068) ( t=0.020, P>0.05). The A values at24 and 48 h in the experimental group [(0.539±0.300), (0.815±0.038)] were significantly lower than those in the control group [(0.683±0.100), (1.415±0.131)] ( t=4.654, 7.373, P<0.05). The plate cloning experiment showed that the number of cell clusters in the experimental group (50.222±6.222) was significantly reduced as compared with the control group (71.560±7.038) ( t=7.875, P<0.01). Conclusion:The expression of VPS72 protein in liver cancer tissue is obviously high, and it is obviously related to clinicopathological parameters. Silencing the expression of VPS72 can significantly inhibit the ability of proliferation and migration of Huh-7 cell lines.