[1]黄亚兰,赖柏安,杨玲,等.微RNA-342-3p/末端尿苷酰转移酶1/凝血酶敏感素-2通路在胰腺导管腺癌中的作用及机制[J].新乡医学院学报,2022,39(12):1119-1126.[doi:10.7683/xxyxyxb.2022.12.004]
　HUANG Yalan,LAI Boan,YANG Ling,et al.Effect and mechanism of microRNA-342-3p/terminal uridylyl transferase 1/thrombospondin-2 pathway in pancreatic ductal adenocarcinoma[J].Journal of Xinxiang Medical University,2022,39(12):1119-1126.[doi:10.7683/xxyxyxb.2022.12.004]
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微RNA-342-3p/末端尿苷酰转移酶1/凝血酶敏感素-2通路在胰腺导管腺癌中的作用及机制
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
39
期数:
2022年12
页码:
1119-1126
栏目:
基础研究
出版日期:
2022-12-05

文章信息/Info

Title:
Effect and mechanism of microRNA-342-3p/terminal uridylyl transferase 1/thrombospondin-2 pathway in pancreatic ductal adenocarcinoma
作者:
黄亚兰; 赖柏安; 杨玲; 张薇珊; 罗婷
（遂宁市中心医院病理科，四川　遂宁　629000）
Author(s):
HUANG Yalan; LAI Bo′an; YANG Ling; ZHANG Weishan; LUO Ting
（Department of Pathology,Suining Central Hospital,Suining 629000,Sichuan Province,China）
关键词:
微RNA-342-3p; 末端尿苷酰转移酶1; 凝血酶敏感素-2; 胰腺导管腺癌; 临床病理
Keywords:
microRNA-342-3p; terminal uridylyl transferase 1; thrombospondin-2; pancreatic ductal adenocarcinoma; clinicopathology
分类号:
R735.9
DOI:
10.7683/xxyxyxb.2022.12.004
文献标志码:
A
摘要:
目的　探讨微RNA(miR)-342-3p/末端尿苷酰转移酶1（TUT1）/凝血酶敏感素-2(THBS2)通路在胰腺导管腺癌中的作用及miR-342-3p、TUT1、THBS2 mRNA表达与患者临床病理学参数的关系。方法　选择2008年5月至2021年3月在遂宁市中心医院行根治性手术切除的胰腺导管腺癌标本(n=80)和对应癌旁正常组织标本(n=20)为研究对象。采用实时荧光定量聚合酶链式反应法检测胰腺导管腺癌组织和癌旁正常组织中miR-342-3p、TUT1、THBS2 mRNA的表达。将对数生长期人胰腺导管腺癌细胞株PANC-1细胞分为对照组(转染miR-NC)、miR-342-3p过表达组(转染miR-342-3p mimic)、miR-342-3p抑制剂组(转染miR-342-3p inhibitor)、Vector组(转染pCS4-HA vectors)、TUT1组(转染pcDNA3-Flag- TUT1)、阴性对照组(转染sh-NC 慢病毒载体)、TUT1沉默组(转染pGCshL-TUT1载体)、miR-342-3p过表达+TUT1组(同时转染miR-342-3p mimic和TUT1)、miR-342-3p过表达+THBS2组(同时转染miR-342-3p mimic和THBS2)、TUT1+THBS2组(同时转染pcDNA3-Flag-TUT1和THBS2)。采用细胞计数试剂盒-8检测10组细胞的增殖能力，采用流式细胞术检测10组细胞的凋亡情况，采用双荧光素酶报告系统检测miR-342-3p与TUT1的靶向关系，采用Western blot法检测对照组、miR-342-3p过表达组和miR-342-3p抑制剂组PANC-1细胞中TUT1、THBS2蛋白的表达。结果　胰腺导管腺癌组织中miR-342-3p TUT1、THBS2 mRNA的相对表达量显著低于癌旁正常组织 (P<0.05)。不同年龄、不同肿瘤大小、淋巴结是否转移和不同肿瘤分期患者癌组织中miR-342-3p及TUT1、THBS2 mRNA的表达比较差异均无统计学意义(P>0.05)；高分化癌组织中miR-342-3p及TUT1、THBS2 mRNA的表达显著高于中+低分化癌组织 (P<0.05)。培养0 h时，各组PANC-1细胞的增殖能力比较差异均无统计学意义（P>0.05）。培养24、48、72 h时，TUT1组细胞的增殖能力均显著低于Vecort组，细胞的凋亡率显著高于Vecort组 (P<0.05)。培养24、48、72 h时，TUT1沉默组细胞的增殖能力显著高于阴性对照组，细胞凋亡率显著低于阴性对照组(P<0.05)。培养24、48、72 h时，miR-342-3p抑制剂组的细胞增殖能力显著高于对照组(P<0.05)，miR-342-3p过表达组的细胞增殖能力显著低于对照组(P<0.05)。培养24 h时，miR-342-3p过表达+TUT1组、miR-342-3p过表达+THBS2组、TUT1+THBS2组与miR-342-3p过表达组的细胞增殖能力比较差异均无统计学意义（P>0.05）。培养48、72 h时，miR-342-3p过表达+TUT1组、miR-342-3p过表达+THBS2组、TUT1+THBS2组的细胞增殖能力均显著低于miR-342-3p过表达组(P<0.05)。miR-342-3p抑制剂组细胞的凋亡率显著低于对照组(P<0.05)，miR-342-3p过表达组细胞的凋亡率显著高于对照组(P<0.05)。miR-342-3p过表达+TUT1组、miR-342-3p过表达+THBS2组、TUT1+THBS2组细胞的凋亡率显著高于miR-342-3p过表达组(P<0.05)。miR-342-3p过表达组细胞中野生型TUT1报告基因的荧光素酶活性显著高于对照组(P<0.05)，突变型TUT1报告基因的荧光素酶活性与对照组比较差异无统计学意义（P>0.05）。miR-342-3p过表达组细胞中TUT1和THBS2蛋白的相对表达量显著高于对照组(P<0.05)，miR-342-3p抑制剂组细胞中TUT1和THBS2蛋白的相对表达量显著低于对照组(P<0.05)。结论　激活miR-342-3p/TUT1/THBS2通路可抑制PANC-1细胞增殖,促进其凋亡，进而抑制胰腺导管腺癌的发展，miR-342-3p/TUT1/THBS2通路有望成为诊治胰腺癌的潜在靶点。
Abstract:
Objective　To investigate the effect of microRNA(miR)-342-3p/terminal uridylyl transferase 1（TUT1）/thrombospondin-2（THBS2） pathway in pancreatic ductal adenocarcinoma and the relationship between miR-342-3p,TUT1,THBS2 mRNA expression and clinical pathological parameters of patients.Methods　The specimens of pancreatic ductal adenocarcinoma (n=80) and the corresponding normal adjacent tissues (n=20) resected in Suining Central Hospital from May 2008 to March 2021 were selected as research objects.The expressions of miR-342-3p,TUT1,THBS2 mRNA in pancreatic ductal adenocarcinoma and normal adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction method.Pancreatic ductal adenocarcinoma cell line PANC-1 cells at logarithmic growth stage were divided into control group (transfected with miR-NC),miR-342-3p overexpression group (transfected with miR-342-3p mimic),miR-342-3p inhibitor group (transfected with miR-342-3p inhibitor)，Vector group (transfected with pCS4-HA vectors),TUT1 group (transfected with pcDNA3-Flag-TUT1),negative control group (transfected with sh-NC lentivirus vector),TUT1 silence group (transfected with pGCshL-TUT1 vector),miR-342-3p overexpression+TUT1 group (transfected with miR-342-3p mimic and TUT1 simultaneously),miR-342-3p overexpression+THBS2 group (transfected with miR-342-3p mimic and THBS2 simultaneously),TUT1+THBS2 group (transfected with pcDNA3-Flag-TUT1 and THBS2 simultaneously).The proliferation ability of cells in the ten groups was detected by cell counting kit-8;the apoptosis of cells in the ten groups was detected by flow cytometry;the targeting relationship between miR-342-3p and TUT1 was verified and analyzed by luciferase reporting assay;the expressions of TUT1 and THBS2 protein in PANC-1 cells in the control group,miR-342-3p overexpression group and miR-342-3p inhibitor group were detected by Western blot.Results　The relative expressions of miR-342-3p,TUT1,THBS2 mRNA in pancreatic ductal adenocarcinoma were significantly lower than those in normal adjacent tissues (P<0.05).There was no significant difference in the expressions of miR-342-3p and TUT1,THBS2 mRNA in cancer tissues of patients with different ages,tumor sizes,lymph node metastasis and tumor stages (P>0.05);the expressions of miR-342-3p and TUT1,THBS2 mRNA in high differentiation cancer tissues were significantly higher than those in medium and low differentiation cancer tissues (P<0.05).There was no significant difference in the proliferation ability of PANC-1 cells in each group at 0 h of culture (P>0.05).At 24,48,72 h of culture,the proliferation ability of cells in the TUT1 group was significantly lower than that in the Vecort group (P<0.05).The apoptosis rate of cells in the TUT1 group was significantly higher than that in Vecort group (P<0.05).At 24,48,72 h of culture,the proliferation ability of cells in the TUT1 silencing group was significantly higher than that in the negative control group (P<0.05).The apoptosis rate of cells in TUT1 silencing group was significantly lower than that in the negative control group (P<0.05).At 24,48,72 h of culture,the proliferation ability of cells in the miR-342-3p inhibitor group was significantly higher than that in the control group(P<0.05);the proliferation ability of cells in the miR-342-3p overexpression group was significantly lower than that in the control group(P<0.05).At 24 h of culture,there was no significant difference in cell proliferation ability among the miR-342-3p overexpression+ TUT1 group，miR-342-3p overexpression+ THBS2 group and the miR-342-3p overexpression group (P>0.05).At 48,72 h of culture,the proliferation ability of cells in the miR-342-3p overexpression+ TUT1 group,miR-342-3p overexpression+ THBS2group,TUT1+THBS2 group were significantly lower than that in the miR-342-3p overexpression group (P<0.05);the apoptosis rate of cells in the miR-342-3p inhibitor group was significantly lower than that in the control group (P<0.05);the apoptosis rate of cells in the miR-342-3p overexpression group was significantly higher than that in the control group (P<0.05).The apoptosis rates of cells in the miR-342-3p overexpression+ TUT1 group,miR-342-3p overexpression+ THBS2 group,TUT1+THBS2 group were significantly higher than that in the miR-342-3p overexpression group (P<0.05).The luciferase activity of wild-type TUT1 reporter gene in the miR-342-3p overexpression group was significantly higher than that in the control group (P<0.05),while the luciferase activity of mutant TUT1 reporter gene had no significant difference compared with that in the control group (P>0.05).The relative expressions of TUT1 and THBS2 protein in the miR-342-3p overexpression group were significantly higher than those in the control group (P<0.05);the relative expressions of TUT1 and THBS2 protein in the miR-342-3p inhibitor group were significantly lower than those in the control group (P<0.05).Conclusion　Activation of miR-342-3p/TUT1/THBS2 pathway can inhibit the proliferation of PANC-1 cells,promote their apoptosis,and then inhibit the development of pancreatic ductal adenocarcinoma.The miR-342-3p/TUT1/THBS2 pathway is expected to become a potential target for the diagnosis and treatment of pancreatic cancer.

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更新日期/Last Update: 2022-12-05