Long Non-coding RNA PCED1B Antisense RNA 1 Promotes Cell Proliferation and Invasion in Hepatocellular Carcinoma by Regulating the MicroRNA-34a/CD44 Axis

Objective This study aimed to examine the role of long non-coding RNA PCED1B antisense RNA 1(PCED1B-AS1)in the development of hepatocellular carcinoma(HCC).Methods A total of 62 pairs of HCC tissues and adjacent non-tumor tissues were obtained from 62 HCC patients.The interactions of PCED1B-AS1 and microRNA-34a(miR-34a)were detected by dual luciferase activity assay and RNA pull-down assay.The RNA expression levels of PCED1B-AS1,miR-34a and CD44 were detected by RT-qPCR,and the protein expression level of CD44 was determined by Western blotting.The cell proliferation was detected by cell proliferation assay,and the cell invasion and migration by transwell invasion assay.The HCC tumor growth after PCED1B-AS1 was downregulated was determined by in vivo animal study.Results PCED1B-AS1 was highly expressed in HCC tissues,which was associated with poor survival of HCC patients.Furthermore,PCED1B-AS1 interacted with miR-34a in HCC cells,but they did not regulate the expression of each other.Additionally,PCED1B-AS1 increased the expression level of CD44,which was targeted by miR-34a.The cell proliferation and invasion assay revealed that miR-34a inhibited the proliferation and invasion of HCC in vitro,while CD44 exhibited the opposite effects.Furthermore,PCED1B-AS1 suppressed the role of miR-34a.Moreover,the knockdown of PCED1B-AS1 repressed the HCC tumor growth in nude mice in vivo.Conclusion PCED1B-AS1 may play an oncogenic role by regulating the miR-34a/CD44 axis in HCC.

LncRNA GATA3-AS1通过调控miR-362-3p/FABP5轴抑制宫颈癌细胞增殖、迁移及侵袭

目的:探究长链非编码RNA GATA3反义RNA 1(lncRNA GATA3-AS1)调控微小RNA-362-3p(miR-362-3p)表达对宫颈癌细胞恶性生物学行为的影响。方法:qRT-PCR检测宫颈癌细胞中lncRNA GATA3-AS1、miR-362-3p、FABP5表达;双荧光素酶报告基因实验验证lncRNA GATA3-AS1和miR-362-3p的靶向关系、miR-362-3p和FABP5的靶向关系;将细胞分为pcDNA-NC组、pcDNA-GATA3-AS1组、si-NC组、si-GATA3-AS1组、si-GATA3-AS1+inhibitor-NC组、si-GATA3-AS1+miR-362-3p inhibitor组、miR-NC组、miR-362-3p mimics组、miR-362-3p mimics+pcDNA-NC组、miR-362-3p mimics+pcDNA FABP5组;Western blot检测蛋白表达;EdU法检测细胞增殖;Transwell检测细胞迁移侵袭。结果:在宫颈癌细胞系中,GATA3-AS1、FABP5均为高表达,miR-362-3p均为低表达,选择HeLa细胞进行后续实验;双荧光素酶报告基因实验表明,lncRNA GATA3-AS1和miR-802、miR-362-3p和FABP5具有靶向关系;与pcDNA-NC组比较,pcDNA-GATA3-AS1组Hela细胞EdU阳性率、迁移侵袭及MMP-2、MMP-9表达明显上升(P<0.05);与si-NC组比较,si-GATA3-AS1组HeLa细胞EdU阳性率、迁移侵袭及MMP-2、MMP-9表达明显下降(P<0.05);抑制miR-362-3p表达或过表达FABP5均可以明显逆转沉默GATA3-AS1或过表达miR-362-3p对于HeLa细胞增殖、迁移、侵袭的抑制作用。结论:沉默GATA3-AS1可以靶向上调miR-362-3p表达,抑制FABP5表达,抑制宫颈癌HeLa细胞增殖迁移及侵袭。

LncRNA ZEB1-AS1和LncRNA SOX2OT在糖尿病肾病患者中的表达及与肾功能的相关性研究

目的探究长链非编码RNA锌指E盒结合同源盒蛋白1反义链1(LncRNA ZEB1-AS1)和长链非编码RNA性别决定相关基因簇2重叠转录本(LncRNA SOX2OT)在糖尿病肾病(DN)患者中的表达及与肾功能的相关性。方法选取于2021年11月—2023年12月在武汉大学人民医院肾内科收治的DN患者106例为DN组,并根据24 h尿蛋白定量(24 h Upro)水平分为正常蛋白尿亚组43例(<30 mg)、微量蛋白尿亚组39例(30~<300 mg)、大量蛋白尿亚组24例(≥300 mg),另选取同期医院单纯糖尿病患者106例作对照组,检测患者血清LncRNA ZEB1-AS1、LncRNA SOX2OT水平;Pearson法分析LncRNA ZEB1-AS1和LncRNA SOX2OT与肾功能指标的相关性;Logistic分析影响DN患者肾功能损伤的因素。结果DN组血清LncRNA ZEB1-AS1、LncRNA SOX2OT水平低于对照组(t=11.471、10.257,P均<0.001)。血清LncRNA ZEB1-AS1、LncRNA SOX2OT比较,正常尿蛋白亚组>微量尿蛋白亚组>大量尿蛋白亚组(F=58.720、117.722,P均<0.001),BUN、SCr、UA水平比较,正常尿蛋白亚组<微量尿蛋白亚组<大量尿蛋白亚组,差异均有统计学意义(F=122.493、595.589、53.178,P均<0.001);LncRNA ZEB1-AS1、LncRNA SOX2OT分别与BUN、SCr、UA呈负相关(r=-0.487、-0.498、-0.521,-0.527、-0.515、-0.534,P均<0.001);Logistic回归分析显示,糖尿病病程长及高BUN、SCr、UA水平是影响DN患者肾功能损伤的危险因素[OR(95%CI)=1.672(1.128~2.479)、2.839(1.534~5.253)、2.754(1.512~5.017)、2.693(1.464~4.954)],高LncRNA ZEB1-AS1、LncRNA SOX2OT是保护因素[OR(95%CI)=0.875(0.798~0.959)、0.898(0.832~0.969)]。结论血清LncRNA ZEB1-AS1、LncRNA SOX2OT水平与DN患者肾功能有关,可能是评估DN患者肾功能的潜在指标。

血清长链非编码RNA肌动蛋白纤维相关蛋白1-反义RNA1水平与钙化性主动脉瓣狭窄病人左心室功能的相关性研究

目的 分析血清长链非编码RNA(lncRNA)肌动蛋白纤维相关蛋白1-反义RNA1(AFAP1-AS1)表达水平与钙化性主动脉瓣狭窄(CAS)病人左心室收缩及舒张功能的相关性。方法 于2020年1月至2021年12月,选取中国中医科学院广安门医院就诊的CAS病人129例作为CAS组[左心室射血分数(LVEF)≥50%],同期该院健康志愿者130例作为对照组。收集病人人口学资料、超声及实验室生化指标,检测血清lncRNA AFAP1-AS1表达。受试者操作特征曲线(ROC曲线)分析血清lncRNA AFAP1-AS1诊断CAS效能。结果 对照组血清lncRNA AFAP1-AS1表达水平(1.15±0.18)低于CAS组(1.58±0.30)(P<0.001)。轻度狭窄者血清lncRNA AFAP1-AS1表达水平(1.37±0.26)低于中、重度狭窄者,而中度狭窄者lncRNA AFAP1-AS1表达水平(1.59±0.30)低于重度狭窄者(1.79±0.34)(P<0.001)。ROC结果显示,血清lncRNA AFAP1-AS1诊断CAS、重度狭窄的曲线下面积分别为0.86[95%CI:(0.82,0.91)]、0.88[95%CI:(0.82,0.94)]。CAS组AVA水平低于对照组(P<0.001),左室舒张末期内径(LVEDD)、左室舒张末期容积(LVEDV)、室间隔厚度(IVST)、左室后壁厚度(LVPWT)、左房前后径(LAD)、主动脉瓣平均压差(PGmean)、主动脉瓣峰值流速(Vmax)水平高于对照组(均P<0.001)。相关性分析显示,血清lncRNA AFAP1-AS1与LVEDD、Vmax、二尖瓣口舒张早期血流速度峰值(E峰)、二尖瓣口舒张晚期血流速度峰值(A峰)、LVEDV、PGmean、LVESD呈正相关(r=0.60、0.66、0.72、0.68、0.56、0.57、0.50,均P<0.001),与LVEF、AVA呈负相关(r=-0.78、-0.62,均P<0.001)。结论 CAS病人血清lncRNA AFAP1-AS1表达水平升高,与CAS病情严重程度以及左心室舒张、收缩功能有关,并可作为无创血清标志物辅助临床诊断CAS。

膀胱癌组织LncRNA SPINT1-AS1表达及临床意义

目的检测膀胱癌组织长链非编码核糖核酸(LncRNA)丝氨酸肽酶抑制剂Kunitz 1型反义核糖核酸1(SPINT1-AS1)表达并探讨其临床意义。方法选取安阳市第三人民医院泌尿外科2018年1月至2023年2月收治的328例行腹腔镜下根治术膀胱癌患者作为研究对象,RT-qPCR检测膀胱癌组织和切缘正常组织LncRNA SPINT1-AS1的表达。比较不同临床病理特征患者癌组织LncRNA SPINT1-AS1的表达;随访至2023年5月,分析膀胱癌组织LncRNA SPINT1-AS1的表达与腹腔镜下根治术后复发的关系;Cox回归分析探讨影响膀胱癌患者腹腔镜下根治术后复发的危险因素。结果LncRNA SPINT1-AS1在膀胱癌组织的表达高于切缘正常组织(P<0.05);肌层浸润、TNM分期≥Ⅱb期、最大肿瘤直径≥3 cm、多发病灶、有淋巴结转移患者膀胱癌组织LncRNA SPINT1-AS1的表达分别高于非肌层浸润性、TNM分期<Ⅱb期、最大肿瘤直径<3 cm、单发病灶、无淋巴结转移患者(P<0.05);随访期间患者腹腔镜下根治术后复发率为15.88%(47/296);肌层浸润(HR=1.692,95%CI:1.157~2.475)、TNM分期≥Ⅱb期(HR=1.784,95%CI:1.187~2.682)、多发病灶(HR=1.837,95%CI:1.200~2.810)、膀胱癌组织LncRNA SPINT1-AS1表达(HR=1.557,95%CI:1.195~2.029)均为腹腔镜下根治术后复发的危险因素(P<0.05)。结论膀胱癌组织LncRNA SPINT1-AS1表达高于切缘正常组织,肌层浸润、TNM分期≥Ⅱb期、多发病灶及癌组织LncRNA SPINT1-AS1表达均为膀胱癌腹腔镜下根治术后复发的危险因素。

血清lncRNA ANRIL、lncRNA PVT1水平与急性呼吸窘迫综合征患儿病情及预后的关系

目的探究血清长链非编码RNA INK4位点反义非编码RNA(lncRNA ANRIL)、长链非编码RNA浆细胞瘤变异易位基因1(lncRNA PVT1)水平与急性呼吸窘迫综合征(ARDS)患儿病情及预后的关系。方法选取124例确诊的ARDS患儿为患病组,另选取124例同期体检健康者为对照组。ARDS患儿根据病情严重程度分为重度组(34例)、中度组(42例)和轻度组(48例);ARDS患儿根据预后情况分为预后不良组(55例)和预后良好组(69例)。采用荧光定量聚合酶链反应测定血清中lncRNA ANRIL、lncRNA PVT1水平。采用Logistic回归分析法分析ARDS患儿发生预后不良的影响因素;采用受试者工作特征(ROC)曲线分析血清lncRNA ANRIL、lncRNA PVT1水平对ARDS患儿发生预后不良的预测价值。结果患病组的血清lncRNA ANRIL、lncRNA PVT1水平高于对照组,差异有统计学意义(P<0.05)。轻度组、中度组和重度组的血清lncRNA ANRIL、lncRNA PVT1水平随病情严重程度升高(P<0.05)。预后不良组的血清lncRNA ANRIL、lncRNA PVT1水平、氧合指数(OI)、呼吸频率、急性生理与慢性健康状况评分系统Ⅱ(APACHEⅡ)评分高于预后良好组,差异有统计学意义(P<0.05);OI、呼吸频率、lncRNA ANRIL、lncRNA PVT1为患儿发生预后不良的影响因素(P<0.05)。血清lncRNA ANRIL、lncRNA PVT1水平预测ARDS患儿发生预后不良的曲线下面积(AUC)分别为0.827、0.737,截断值分别是11.35、4.36,二者联合预测的AUC为0.876。二者联合预测的AUC优于血清lncRNA ANRIL、lncRNA PVT1水平单独预测(P<0.05)。结论ARDS患儿的血清lncRNA ANRIL、lncRNA PVT1水平均上调,且lncRNA ANRIL、lncRNA PVT1为患儿发生预后不良的影响因素,二者联合预测的价值较高。

Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.

Antisense to cyclin D1 reverses the transformed phenotype of human gastric cancer cells

AIM To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor. METHODS A cyclin D1 subcloning plasmid termed BKSD1 was constructed by subcloning the human cyclin D1 cDNA into Bluescript KS, a plasmid vector with a pair of T7 and T3 promoters, with recombinant DNA technology of molecular biology. So, it is easy to generate digoxigenin (DIG) labeled RNA probes of antisense and sense to cyclin D1 using RKSD1 as a template vector. PDORD1AS, an eukaryotic expression vector containing the full length human cyclin D1 cDNA in its antisense orientation cloned into the retroviral vector pDOR neo, was successfully constructed with BKSD1 to change restriction sites. A gastric cancer cell line, SGC7901/VCR, was transfected with pDORD1AS by Lipofect Amine mediated introduction and a subline termed SGC7901/VCRD1AS, which had stable overexpression of antisense RNA to cyclin D1, was obtained by selection in G418. The subline, control subline transfected pDOR neo and SGC7901/VCR were evaluated by methods of immunohistochemistry, flow cytometry, molecular hybridization, morphology and cell biology. RESULTS Compared with control cell lines, SGC7901/VCRD1AS had a reduced expression of cyclin D1 (inhibition rate was about 36%), increased cell size and cytoplasm to nucleus ratio, increased doubling time (42 2h to 26 8h and 26 4h), decreased saturation density (18 9×10 4 to 4 8×10 5 and 4 8×10 5), increased percentage of cells in the G1/G0 phase (80 9%-64 6% and 63 8%), reacquired serum dependence, and a loss of tumorigenicity in nude mice (0/4 to 4/4 and 4/4). CONCLUSION Stable overexpression of antisense RNA to cyclin D1 can reverse the transformed phenotype of human gastric cancer cells and may provide an approach of gene therapy for gastric cancer.

Effect of Antisense RNA Targeting Polo-like Kinase 1 on Cell Growth in A549 Lung Cancer Cells

In order to investigate the effect of Polo-like kinase-1 （Plk1） depletion on cell cycle progression and cell growth in lung cancer cells, a recombinant plasmid containing antisense RNA targeting Plk1 （pcDNA3-Plk1） was transfected into A549 cells by lipofectine. RT-PCR and Western-blot were used to detect the Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine （BrdU） labeling. Cell cycle distribution and apoptosis were examined by flow cytometry, and the inhibition rate （IR） by vinorebline （NVB） was determined by MTF assay. The results showed that after transfection of pcDNA3-Plk1 into A549 cells, the expression levels of Plk1 mRNA and protein were greatly decreased. In pcDNA3-Plk1 transfected groups, abnormal morphological changes of cells and growth inhibition were observed, and the BrdU labeling index was significantly lower than in the control groups （P〈0.05）. Cells in pcDNA3-Plk1 transfected groups were arresed in G2/M phase and apoptosis was detectable 72 h post transfection. IR induced by vinorebline in pcDNA3-Plk1 transfected groups was significantly higher than in other groups. These data suggested that antisense RNA targeting Plk1 could suppress the Plk1 expression, and therefore, significantly inhibit cell proliferation and induce cell cycle arrest and apoptosis. Moreover, it sensitized lung cancer cells to chemotherapy.

Apoptosis induction with polo-like kinase-1 antisense phosph-orothioate oligodeoxynucleotide of colon cancer cell line SW480

AIM： To investigate the effects of polo-like kinase-1 （PLK1） antisense phosphorothioate oligodeoxynucleotide （ASODN） on apoptosis and cell cycle of human colon cancer cell line SW480. METHODS： After SW480 colon cancer cells were transfected with PLK1 ASODN, Northern and Western blot analyses were used to examine PLK1 gene expression in cancer cells. We studied apoptosis using terminal uridine deoxynucleotidyl nick end labeling. Apoptosis and cell cycle of SW480 cells were examined by fluorescence-activated cell sorter scan. RESULTS： The levels of PLK1 mRNA and protein were greatly inhibited by PLK1 ASODN in SW480 cancer cells transfected with PLK1 ASODN. Apoptosis index （AI） induced PLK1 ASODN in a time- and dose-dependent manner. Results from FLM showed that sub-2N DNA content of transfected cancer cells was significantly increased and arrested at G2/M compared with control groups. CONCLUSION： PLK1 ASODN can induce apoptosis of human colon cancer cell line SW480.

LncRNA FEZF1-AS1通过调控EZH2对肺间质细胞增殖、迁移及侵袭的作用

目的研究长链非编码RNA FEZ家族锌指1-反义RNA 1(lncRNA FEZF1-AS1)调控zeste同源物增强子2(EZH2)对肺间质细胞增殖、迁移、侵袭能力及上皮细胞-间质转化(EMT)的影响及其作用机制。方法将人肺腺癌细胞系A549分为对照组(control)和模型组[model,用转化生长因子β1(TGF-β1)20 ng/mL作用48 h,诱导成为肺间质细胞]。用Western blot检测细胞中E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)及波形蛋白(vimentin)的蛋白表达。RT-qPCR检测细胞中lncRNA FEZF1-AS1和EZH2基因表达。转染组细胞分为转染si NC组、si lncRNA FEZF1-AS1+OE vector组和si lncRNA FEZF1-AS1+OE EZH2组。CCK-8法检测细胞增殖、细胞划痕检测细胞迁移、Transwell小室法检测细胞侵袭;用Western blot检测细胞中E-cadherin、N-cadherin、vimentin及EZH2的蛋白表达,用RNA免疫沉淀(RIP)测定FEZF1-AS1与EZH2的直接结合作用。结果与对照组比较,模型组E-cadherin的蛋白表达水平减少(P<0.05);N-cadherin及vimentin的蛋白表达水平升高(P<0.05);与对照组比较,模型组lncRNA FEZF1-AS1与EZH2基因的表达水平明显升高(P<0.05);与si NC组相比,si lncRNA FEZF1-AS1+OE vector组细胞增殖、迁移、侵袭能力降低,E-cadherin蛋白表达升高,N-cadherin、vimentin、EZH2蛋白表达降低(P<0.05);与si lncRNA FEZF1-AS1+OE vector组比较,si lncRNA FEZF1-AS1+OE EZHZ组细胞增殖、侵袭、迁移能力升高,E-cadherin蛋白表达降低,N-cadherin、vimentin、EZH2蛋白表达升高(P<0.05);RIP实验进一步证实了lncRNA FEZF1-AS1与EZH2具有结合作用。结论LncRNA FEZF1-AS1通过调控EZH2促进肺间质细胞增殖、侵袭、转移和EMT过程。

Effect of NHE1 antisense gene transfection on the biological behavior of SGC-7901 human gastric carcinoma cells

AIM: To study the effect of type 1 Na+/H+ exchanger (NHE1 ) antisense human gene transfection on the biological behavior of gastric carcinoma cell line SGC-7901. METHODS: Antisense NHE1 eukaryotic expression on vector pcDNA3.1 was constructed by recombinant DNA technique and transfected into gastric carcinoma cell line SGC-7901 with DOTAP liposome transfection method. Morphological changes of cells were observed with optic and electron microscopes. Changes in cell proliferative capacity, apoptosis, intracellular pH (pHi), cell cycle, clone formation in two-layer soft agar, and tumorigenicity in nude mice were examined. RESULTS: Antisense eukaryotic expressing vectors were successfully constructed and transfected into SGC-7901. The transfectant obtained named 7901 -antisense (7901-AS) stablely produced antisense NHE1. There was a significant difference between the pHi of 7901-AS cells (6.77 ± 0.05) and that of 7901-zeo cells and SGC-7901 cells (7.24 ± 0.03 and 7.26 ± 0.03, P < 0.01). Compared with SGC-7901 and 7901-zeo cells, 7901-AS cells mostly showed cell proliferation inhibition, G1/G0 phase arrest, increased cell apoptotic rate, recovery of contact inhibition, and density contact. The tumorigenicity in nude mice and cloning efficiency in the two-layer soft agar were clearly inhibited. CONCLUSION: NHE1 antisense gene significantly restrains the malignant behavior of human gastric carcinoma cells, suppresses cell growth and induces cell apoptosis, and partially reverses the malignant phenotypes of SGC-7901 . These results suggest a potential role for human tumor gene therapy.

Effects of Cyclin D1 Antisense Oligodeoxyneucleotides on the Growth and Expression of G_1 Phase Regulators in Gastric Carcinoma Cells

To investigate the effects of Cyclin D1 antisense oligodeoxyneucleotides (ASODN) on the growth, cell cycle progression and expression of G 1 phase regulators in human gastric carcinoma cell lines SGC7901 and HS746T, phosphorothioate modified Cyclin D1 ASODN were encapsulated by LipofectAMINE2000 and transfected into gastric carcinoma cells. Dose dependent inhibitory effects were induced by Cyclin D1 ASODN in two gastric carcinoma cell lines. Treatment of gastric carcinoma cells with 0.2 μmol/L Cyclin D1 ASODN for 24 h could significantly inhibit their growth in vitro and in vivo , reduce expression of Cyclin D1mRNA to 26.3 % (SGC7901) and 17.3 % (HS746T) respectively. The percentage of cells in G 0/G 1 phase was increased as revealed by flow cytometry. Immunohistochemical staining showed that the expression of p21 was increased and the expression of Cyclin D1 and pRb was decreased in the two cell lines; the expression of p27 was increased in HS746T, but unchanged in SGC7901. Cyclin D1 ASODN could inhibit the growth and the expression of Cyclin D1 mRNA in gastric carcinoma cells, influence the cell cycle and expression of its regulators.

ENHANCEMENT OF RADIATION-INDUCED APOPTOSIS IN RAJI CELL LINE BY BC1-2 ANTISENSE OLIGODEOXYNUCLEOTIDE

Objective: To investigate whether the Bc1-2 antisense oligonucleotide(ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression level of bc1-2 protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results: It was found that Bc1-2 ASODN combined with radiation had significantly reduced the number of viable cells (P<0.05). There was no difference on cell survival between mismatch Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone. Bc1-2 ASODN combined with radiation could significantly inhibit expression of Bc1-2 protein in Raji cells (P<0.05). Cells treated with Bc1-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptosis rates of Raji cells treated with Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone, respectively. Conclusion: Bc1-2 antisense oligonucleotide can enhance radiation-induced apoptosis in Raji cell line.

Effects of long non-coding RNA Opa-interacting protein 5 antisense RNA 1 on colon cancer cell resistance to oxaliplatin and its regulation of micro RNA-137

BACKGROUND The incidence of colon cancer(CC)is currently high,and is mainly treated with chemotherapy.Oxaliplatin(L-OHP)is a commonly used drug in chemotherapy;however,long-term use can induce drug resistance and seriously affect the prognosis of patients.Therefore,this study investigated the mechanism of Opainteracting protein 5 antisense RNA 1(OIP5-AS1)on L-OHP resistance by determining the expression of OIP5-AS1 and micro RNA-137(miR-137)in CC cells and the effects on L-OHP resistance,with the goal of identifying new targets for the treatment of CC.AIM To study the effects of long non-coding RNA OIP5-AS1 on L-OHP resistance in CC cell lines and its regulation of miR-137.METHODS A total of 114 CC patients admitted to China-Japan Union Hospital of Jilin University were enrolled,and the expression of miR-137 and OIP5-AS1 in tumor tissues and corresponding normal tumor-adjacent tissues was determined.The influence of OIP5-AS1 and miR-137 on the biological behavior of CC cells was evaluated.Resistance to L-OHP was induced in CC cells,and their activity was determined and evaluated using cell counting kit-8.Flow cytometry was used to analyze the apoptosis rate,Western blot to determine the levels of apoptosisrelated proteins,and dual luciferase reporter assay combined with RNA-binding protein immunoprecipitation to analyze the relationship between OIP5-AS1 and miR-137.RESULTS OIP5-AS1 was up-regulated in CC tissues and cells,while miR-137 was downregulated in CC tissues and cells.OIP5-AS1 was inversely correlated with miR-137(P<0.001).Silencing OIP5-AS1 expression significantly hindered the proliferation,invasion and migration abilities of CC cells and markedly increased the apoptosis rate.Up-regulation of miR-137 expression also suppressed these abilities in CC cells and increased the apoptosis rate.Moreover,silencing OIP5-AS1 and up-regulating miR-137 expression significantly intensified growth inhibition of drug-resistant CC cells and improved the sensitivity of CC cells to LOHP.OIP5-AS1 targetedly inhibited miR-137 expression,and silencing OIP5-AS1 reversed the resistance of CC cells to L-OHP by promoting the expression of miR-137.CONCLUSION Highly expressed in CC,OIP5-AS1 can affect the biological behavior of CC cells,and can also regulate the resistance of CC cells to L-OHP by mediating miR-137 expression.

LncRNA FEZF1-AS1靶向调控miR-200c-3p对人肺成纤维细胞生物学行为的影响

目的探讨FEZ家族锌指1-反义RNA1(LncRNA FEZF1-AS1)靶向调控miR-200c-3p对人肺成纤维细胞HLF生物学行为的影响。方法采用转化生长因子β1(TGF-β1)诱导HLF向肌成纤维细胞转化,分为空白对照组(Blank组)和造模组(HLF+TGF-β1组),另根据转染质粒不同将细胞分为Blank组、TGF-β1+Si LncRNA FEZF1-AS1 NC组和TGF-β1+Si LncRNA FEZF1-AS1组。采用Western blot法检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(CollagenⅠ)和波形蛋白(Vimentin)蛋白的表达。采用实时荧光定量PCR(qRT-PCR)检测LncRNA FEZF1-AS1和miR-200c-3p的表达。采用CCK-8法检测细胞增殖,细胞划痕实验检测迁移能力,Transwell实验检测侵袭能力;采用双萤光素酶实验检测FEZF1-AS1与miR-200c-3p的靶向作用关系。结果与Blank组比较,HLF+TGF-β1组α-SMA、CollagenⅠ、Vimentin蛋白表达及LncRNA FEZF1-AS1表达水平升高,miR-200c-3p表达水平降低(P<0.05);与TGF-β1+Si LncRNA FEZF1-AS1 NC组比较,TGF-β1+Si LncRNA FEZF1-AS1组细胞增殖、迁移、侵袭能力下降,LncRNA FEZF1-AS1表达及α-SMA、CollagenⅠ、Vimentin蛋白表达水平降低,miR-200c-3p表达水平升高(P<0.05);FEZF1-AS1与miR-200c-3p基因序列上存在结合位点。结论LncRNA FEZF1-AS1通过抑制miR-200c-3p促进特发性肺间质纤维化的发生、发展。

Antisense Oligonucleotide Targeting TGF-β1 Abrogates Tumorigenicity of Rhabdomyosarcoma in vivo

OBJECTIVE Over-expression of transforming growth factor β1 （TGF-β1） has been observed in many advanced cancers. The present study was aimed at developing potential antisense oligonucleotides （ASONs） to repress TGF-β1 expression in rhabdomyosarcoma （RMS） RD cells, and to examine their effect on tumorigenicity of RD cells in vivo. METHODS ASONs targeting the region surrounding the start codon of TGF-β1 were synthesized and transferred into cells in the form of complexes with Lipofectamine 2000. The TGF-β1 protein was determined by immunofluorescence and ELISA. The cell viability and cell cycle were examined by MTT and flow cytometry. The RD cells, with or without TGF-β1ASON, in 50 μl of serum-free EMDM medium were injected subcutaneously into the right flank of nude mice. The tumors were then measured and weighed. RESULTS The ASON sequence targeting the first start site at bases 841-855 of the human TGF-β1 gene had the greatest effect on attenuating the expression of TGF-β1 （P 〈 0.05）. The ASONs induced a decrease in OD values after 6 d （P 〈 0.05）. Analysis of the cell cycle revealed that the ASON induced a significant decrease in cells in the S phase and an increase in cells in the G1 phase （P 〈 0,05）. In the nude mice model, the mean tumor volume, after 2 weeks of treatment with Lipofectamine or ASON, decreased to 88.5% or 55% respectively, compared to the control tumor size, resulting in a significant difference （P 〈 0.01）. CONCLUSION The sequence of the ASON, which targeted the start condon at the bases 841-855 of the human TGF-β1 gene, was demonstrated to be a useful agent for studying the regulation of TGF-β1 over-expression in RD cells, and has important therapeutic potential for suppressing the tumorigenicity of human RMS in vivo.

血清lncRNA LOXL1-AS1、miR-3614-5p水平对急性心肌梗死后心律失常的预测价值

目的探讨长链非编码RNA(lncRNA)赖氨酰氧化酶样1-反义RNA1(LOXL1-AS1)、微小RNA(miR)-3614-5p在急性心肌梗死患者血清中的表达水平,以及对心律失常的预测价值。方法选择2021年1月—2023年1月西安交通大学第一附属医院心内科住院治疗急性心肌梗死患者148例作为研究对象(急性心肌梗死组),根据患者是否发生心律失常,分为非心律失常亚组(n=96)和心律失常亚组(n=52),另选取同期与急性心肌梗死患者一般资料相匹配的健康体检者148例为健康对照组。比较各组血清lncRNA LOXL1-AS1、miR-3614-5p水平;多因素Logistic回归分析急性心肌梗死后心律失常的影响因素;绘制受试者工作特征曲线(ROC)并计算曲线下面积(AUC)分析血清lncRNA LOXL1-AS1、miR-3614-5p水平对急性心肌梗死后心律失常的预测价值。结果与健康对照组比较,急性心肌梗死组lncRNA LOXL1-AS1水平升高,miR-3614-5p水平降低(t/P=16.248/<0.001、8.397/<0.001);心律失常亚组病变血管支数、lncRNA LOXL1-AS1水平高于非心律失常亚组,左心室射血分数(LVEF)、miR-3614-5p水平低于非心律失常亚组[χ^(2)(t)/P=14.315/<0.001、7.312/<0.001、3.706/<0.001、7.656/<0.001];Target Scan Human网站预测结果显示,lncRNA LOXL1-AS1与miR-3614-5p有结合位点,可能存在靶向关系;多因素Logistic回归分析结果显示,lncRNA LOXL1-AS1高、病变血管支数多是急性心肌梗死后心律失常的危险因素,miR-3614-5p、LVEF高是保护因素[OR(95%CI)=3.542(1.589~7.896)、1.527(1.081~2.156)、0.721(0.601~0.865)、0.789(0.664~0.938)];lncRNA LOXL1-AS1、miR-3614-5p及二者联合预测急性心肌梗死后心律失常的AUC为0.820、0.890、0.932,二者联合优于各自单独预测(Z/P=3.470/0.001、2.293/0.022)。结论急性心肌梗死后心律失常患者血清lncRNA LOXL1-AS1水平显著升高,miR-3614-5p水平显著降低,两者联合对急性心肌梗死后心律失常有较好的预测价值。

术前lncRNA TPT1-AS1和整合素β3水平与结直肠癌根治性切除术后复发转移的相关性

目的探讨术前血清长链非编码RNA肿瘤蛋白翻译调节因子1-反义RNA1(lncRNA TPT1-AS1)、整合素β3(ITGB3)水平与结直肠癌病人根治性切除术后复发转移的相关性。方法选择2019年3月至2020年3月在海南省第二人民医院进行结直肠癌根治性切除术的90例病人为结直肠癌组,选择同期在海南省第二人民医院进行健康体检的人员90例作为对照组,实时荧光定量逆转录聚合酶链式反应(qRT-PCR)检测血清lncRNA TPT1-AS1表达水平,酶联免疫吸附测定(ELISA)检测血清ITGB3水平,搜集病人资料,Pearson法分析lncRNA TPT1-AS1与ITGB3水平相关性,Cox回归模型分析影响结直肠癌根治性切除术后复发转移的危险因素。结果与对照组比较,结直肠癌组病人术前血清lncRNA TPT1-AS1表达水平(2.62±0.35比1.05±0.12)、ITGB3水平[(236.24±30.62)ng/L比(185.47±25.38)ng/L]显著升高(P<0.05);结直肠癌病人血清lncRNA TPT1-AS1和ITGB3水平呈正相关性(r=0.65,P<0.05);不同分化程度、TNM分期、浸润深度、淋巴结转移及血管侵犯的病人血清lncRNA TPT1-AS1、ITGB3水平比较差异有统计学意义(P<0.05);lncRNA TPT1-AS1、ITGB3高表达组结直肠癌根治术后复发转移率显著高于低表达组(P<0.05);TNM分期、淋巴结转移、血管侵犯、lncRNA TPT1-AS1、ITGB3是影响结直肠癌病人术后复发转移的危险因素(P<0.05)。结论结直肠癌病人根治性切除术前血清lncRNA TPT1-AS1、ITGB3水平升高,与术后复发转移相关,可能成为结直肠癌预后判断的血清标志物。

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.