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中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (03) : 146 -154. doi: 10.3877/cma.j.issn.2095-1221.2021.03.003

论著

miR-661靶向HTRA1对结肠癌SW620细胞增殖、迁移和侵袭的影响
钟锋1,†(), 张霄程1   
  1. 1. 065000　廊坊，河北省廊坊市人民医院普外一科
  • 收稿日期:2020-12-22 出版日期:2021-06-01
  • 通信作者: 钟锋

Effects of miR-661 on proliferation, migration and invasion of SW620 cells by targeting HRTA1

Feng Zhong1,†(), Xiaocheng Zhang1   

  1. 1. Department of General Surgery, People's Hospital of Langfang City, Hebei 065000, China
  • Received:2020-12-22 Published:2021-06-01
  • Corresponding author: Feng Zhong
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引用本文：

钟锋, 张霄程. miR-661靶向HTRA1对结肠癌SW620细胞增殖、迁移和侵袭的影响[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(03): 146-154.

Feng Zhong, Xiaocheng Zhang. Effects of miR-661 on proliferation, migration and invasion of SW620 cells by targeting HRTA1[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(03): 146-154.

目的

探讨miR-661靶向高温必需蛋白A1 （HTRA1）对结肠癌细胞增殖、迁移和侵袭的影响。

方法

收集2016年3月至2018年3月在廊坊市人民医院确诊的85例结肠癌患者肿瘤组织及其配对的癌旁组织。RT-qPCR检测结肠癌组织、癌旁组织、结肠癌细胞（SW620、SW480、HCT116、LoVo）、结肠上皮细胞（NCM460）中的miR-661及HTRA1 mRNA表达水平，Western blot检测结肠癌组织、癌旁组织、结肠癌细胞（SW620、SW480、HCT116、LoVo）、结肠上皮细胞（NCM460）中HTRA1的蛋白表达水平。根据不同转染方法将SW620细胞分为anti-miR-NC组（转染anti-miR-NC）、anti-miR-661组（转染anti-miR-661）、pcDNA组（转染pcDNA）、pcDNA-HTRA1组（转染pcDNA-HTRA1）、anti-miR-661+si-NC组（转染anti-miR-661和si-NC）、anti-miR-661+ si-HTRA1组（转染anti-miR-661和si-HTRA1）和对照组（正常培养SW620细胞）。利用TargetScan在线预测、荧光素酶报告基因实验以及Western blot验证miR-661和HTRA1的靶向关系。采用细胞计数试剂盒（CCK-8）、Transwell实验检测SW620细胞增殖、迁移和侵袭变化。两组比较采用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用Dunne-t检验。miR-661或HTRA1的表达与各临床病理参数的关系分析采用c2检验。

结果

与癌旁组织比较，结肠癌组织中miR-661表达升高，HTRA1 mRNA和蛋白表达降低（P < 0.05）。与NCM460细胞比较，SW620、SW480、HCT116、LoVo细胞中miR-661表达均升高，HTRA1 mRNA和蛋白表达水平降低（P均< 0.05）。miR-661靶向HTRA1并负性调控其表达。与anti-miR-661组比较，对照组、anti-miR-NC组SW620细胞增殖活力（0.49±0.04比0.76±0.06、0.72±0.07）、迁移[（48.93±5.28）个比（98.67±8.57）个、（95.14±9.36）个]和侵袭数量[（36.48±4.87）个比（88.48±8.64）个、（81.65±8.36）个]升高（P均< 0.05），而对照组与anti-miR-NC组之间上述指标差异无统计学意义（P > 0.05）。与pcDNA-HTRA1组比较，对照组、pcDNA组SW620细胞增殖活力（0.55±0.05比0.76±0.06、0.74±0.07）、迁移[（56.87±5.32）个比（101.65±9.87）个、（98.69±9.33）个]和侵袭数量[（43.28±4.37）个比（87.21±8.98）个、（85.91±8.42）个]升高（P均< 0.05），而对照组与anti-miR-NC组之间上述指标差异无统计学意义（P > 0.05）。与anti-miR-661+si-NC组比较，anti-miR-661+si-HTRA1组SW620细胞增殖活力（0.48±0.04比0.62±0.06）、迁移数量[（47.58±4.72）个比（82.36±8.21）个和侵袭数量（36.98±3.66）个比（71.54±7.16）个]升高（P均< 0.05）。

结论

在结肠癌中miR-661呈高表达，HTRA1呈低表达。抑制miR-661通过上调HTRA1可抑制结肠癌细胞增殖、迁移和侵袭。

关键词: miR-661, HTRA1, 结肠癌, 细胞增殖, 迁移, 侵袭
Objective

To investigate the effect of miR-661 on the proliferation, migration and invasion of colon cancer cells by targeting high temperature requirement A1 (HTRA1) .

Methods

The colon cancer tissue and their paired para-carcinoma colon tissue of 85 patients in People's Hospital of Langfang City from March 2016 to March 2018 were collected. The expression level of miR-661 and HTRA1 mRNA in colon cancer tissue, para-carcinoma colon tissue, colon cancer cells (SW620, SW480, HCT116, LoVo) and colonic epithelial cells (NCM460) were detected by real-time fluorescence quantitative PCR (RT-qPCR) and the expression level of HTRA1 protein was detected by Western blot. The SW620 cells were divided into anti-miR-NC group (transfected with anti-miR-NC) , anti-miR-661 group (transfected with anti-miR-661) , pcDNA group (transfected with pcDNA) , pcDNA-HTRA1 group (transfected with pcDNA-HTRA1) , anti-miR-661+si-NC group (transfected with anti-miR-661 and si-NC) , and anti-miR-661+si-HTRA1 group (transfected with anti-miR-661 and si-HTRA1) and control (Con) group. The relationship between miR-661 and HTRA1 was determined by Target Scan online prediction, luciferase reporter gene experiments, and Western blot. The proliferation, migration and invasion ability of SW620 cells were detected by CCK-8 method and Transwell test. The independent sample t test was used to compare the two groups, and the One-way ANOVA was used to compare multiple groups. The relationship between miR-661 or HTRA1 expression and clinicopathological parameters was analyzed by c2 test.

Results

Compared with para-carcinoma colon tissue, the expression of miR-661 in colon cancer tissues was increased, while the expression of HTRA1 mRNA and protein were significantly decreased (P < 0.05) . Compared with NCM460 cells, the expression of miR-661 in SW620, SW480, HCT116, LoVo cells were increased, while the expression of HTRA1 mRNA and protein in these cells were significantly decreased (P < 0.05) . miR-661 may negatively regulate HTRA1 expression. Compared with the Con group and anti-miR-NC group, cell proliferation activity (0.49±0.04 vs 0.76±0.06, 0.72±0.07, P < 0.05) , migration rate (48.93±5.28 vs 98.67±8.57, 95.14±9.36, P < 0.05) and invasion ability (36.48±4.87 vs 88.48±8.64, 81.65±8.36, P < 0.05) of SW620 cells in the anti-miR-661 group were decreased, but there was no significantly difference between Con group and anti-miR-NC group (P > 0.05) . Compared with the Con group and pcDNA group, cell proliferation activity (0.55±0.05 vs 0.76±0.06, 0.74±0.07, P < 0.05) , migration rate (56.87±5.32 vs 101.65±9.87, 98.69±9.33, P < 0.05) and invasion ability (43.28±4.37 vs 87.21±8.98, 85.91±8.42, P < 0.05) of SW620 cells in the pcDNA-HTRA1 group were also decreased (P < 0.05) , but there was no significantly difference between Con group and anti-miR-NC group (P > 0.05) . Compared with the anti-miR-661+si-NC group, the cell proliferation activity (0.48±0.04 vs 0.62±0.06, P < 0.05) , migration rate (47.58±4.72 vs 82.36±8.21, P < 0.05) and invasion ability (36.98±3.66 vs 71.54±7.16, P < 0.05) of SW620 cells in the anti-miR-661+si-HTRA1 group were increased (P < 0.05) .

Conclusions

miR-661 was highly expressed and HTRA1 was lowly expressed in colon cancer. Inhibiting miR-661 may up-regulate HTRA1 and then inhibit the proliferation, migration and invasion of colon cancer cells.

Key words: miR-661, HTRA1, Colon cancer, Cell proliferation, Migration, Invasion
表1 引物序列信息
基因 序列（5'→3'） 预期扩增产物长度（bp）
miR-661 上游GTGCCTGGGTCTCTGGCCT 72
下游GTGCCTGGGTCTCTGGCCT
U6（内参） 上游CTCGCTTCGGCAGCACA 94
下游AACGCTTCACGAATTTGCGT
HTRA1 上游CAGCACCCAAAGGTCAATGC 113
下游CAGAGTCATCGTGCTCCCAA
GAPDH 上游ACCACAGTCCATGCCATCAC 450
下游TCCACCACCCTGTTGCTGTA
表2 85例患者结肠癌组织中miR-661和HTRA1在的表达（±s）
  癌旁组织 结肠癌组织 ± Sd t值 P值
miR-661 1.00±0.10 3.14±0.36 2.14±0.28 -52.374 < 0.001
HTRA1 mRNA 1.00±0.07 0.55±0.05 0.45±0.06 44.191 < 0.001
HTRA1蛋白 0.71±0.07 0.49±0.04 0.22±0.05 25.499 < 0.001
表3 结肠癌患者临床病理参数
临床病理特征 例数 miR-661 χ2值 P值 HTRA1 χ2值 P值
低表达（n=30） 高表达（n=55） 低表达（n=50） 高表达（n=35）
性别       0.353 0.552     1.586 0.208
  男 49 16 33     26 23    
  女 36 14 22     24 12    
年龄       0.035 0.851     2.209 0.137
  <55 38 13 25     19 19    
  ≥55 47 17 30     31 16    
临床分期       17.872 0.001     43.540 0.000
  Ⅰ 21 14 7     3 18    
  Ⅱ 16 8 8     4 12    
  Ⅲa 19 3 16     17 2    
  Ⅲb 17 3 14     15 2    
  Ⅳ 12 2 10     11 1    
分化程度       22.949 0.000     49.387 0.000
  高 22 19 3     2 20    
  中 24 8 16     10 14    
  低 39 3 36     38 1    
淋巴结转移       44.350 0.000     52.545 0.000
  无 38 28 10     6 32    
  有 47 2 45     44 3    
图1 HTRA1蛋白在结肠癌组织中的表达
表4 miR-661和HTRA1在结肠癌细胞中的表达（±s）
细胞 实验次数 miR-661 HTRA1 mRNA HTRA1蛋白
NCM460 3 1.00±0.07 1.00±0.08 0.86±0.07
SW620 3 3.41±0.32a 0.29±0.03a 0.56±0.05a
SW480 3 2.56±0.23a 0.51±0.05a 0.62±0.04a
HCT116 3 2.14±0.22a 0.59±0.06a 0.68±0.05a
LoVo 3 2.89±0.27a 0.46±0.04a 0.59±0.03a
F值   44.139 69.850 17.202
P值   < 0.001 < 0.001 < 0.001
图2 HTRA1蛋白在结肠癌细胞中的表达
表5 抑制miR-661表达对结肠癌SW620细胞增殖、迁移和侵袭的影响（±s）
分组 实验次数 miR-661 OD值（490 nm） 迁移细胞数（个）
24 h 48 h 72 h
对照 3 1.00±0.07 0.34±0.02 0.76±0.06 1.15±0.07 98.67±8.57
anti-miR-NC 3 1.02±0.06 0.33±0.03 0.72±0.07 1.11±0.09 95.14±9.36
anti-miR-661 3 0.42±0.04ab 0.31±0.03 0.49±0.04ab 0.63±0.06ab 48.93±5.28ab
F值   103.485 0.955 18.921 45.398 36.695
P值   < 0.001 0.437 0.003 < 0.001 < 0.001
分组 实验次数 侵袭细胞数（个） CyclinD1蛋白 p21蛋白 MMP-2蛋白 MMP-9蛋白
对照 3 88.48±8.64 0.67±0.05 0.28±0.03 0.82±0.08 0.69±0.05
anti-miR-NC 3 81.65±8.36 0.64±0.06 0.29±0.03 0.79±0.07 0.68±0.06
anti-miR-661 3 36.48±4.87ab 0.23±0.03ab 0.76±0.07ab 0.33±0.03ab 0.29±0.03ab
F值   42.711 77.700 201.060 55.648 66.900
P值   < 0.001 < 0.001 < 0.001 < 0.001 0.001
图3 抑制miR-661表达对结肠癌SW620细胞增殖、迁移和侵袭相关蛋白表达的影响
图4 光学显微镜下观察抑制miR-661表达对结肠癌SW620细胞的迁移和侵袭的影响（结晶紫染色，×200）
表6 双荧光素酶报告实验（±s）
分组 实验次数 WT-HTRA1 MUT-HTRA1
miR-NC 3 1.02±0.09 1.00±0.08
miR-661 3 0.53±0.05 1.03±0.08
t值   8.243 0.459
P值   0.001 0.670
图5 miR-661靶向调控HTRA1的表达
表7 HTRA1过表达对结肠癌SW620细胞增殖、迁移和侵袭的影响（±s）
分组 实验次数 HTRA1蛋白 OD值（490 nm） 迁移细胞数（个）
24 h 48 h 72 h
对照 3 0.61±0.05 0.35±0.04 0.76±0.06 1.16±0.11 101.65±9.87
pcDNA 3 0.57±0.05 0.34±0.03 0.74±0.07 1.13±0.09 98.69±9.33
pcDNA-HTRA1 3 0.91±0.08ab 0.33±0.03 0.55±0.05ab 0.71±0.07ab 56.87±5.32ab
F值   26.784 0.265 10.991 22.697 26.528
P值   0.001 0.776 0.010 0.002 0.001
分组 实验次数 侵袭细胞数（个） CyclinD1蛋白 p21蛋白 MMP-2蛋白 MMP-9蛋白
对照 3 87.21±8.98 0.64±0.05 0.30±0.03 0.80±0.08 0.71±0.07
pcDNA 3 85.91±8.42 0.62±0.06 0.31±0.03 0.77±0.07 0.69±0.06
pcDNA-HTRA1 3 43.28±4.37ab 0.29±0.03ab 0.70±0.07ab 0.38±0.04ab 0.32±0.03ab
F值   32.955 49.671 69.896 38.302 46.181
P值   0.001 < 0.001 < 0.001 < 0.001 < 0.001
图6 HTRA1过表达对结肠癌SW620细胞增殖、迁移和侵袭相关蛋白表达的影响
图7 HTRA1过表达对结肠癌SW620细胞迁移和侵袭的影响（结晶紫染色，×200）
图8 干扰HTRA1表达逆转了抑制miR-661表达对结肠癌SW620细胞增殖、迁移和侵袭相关蛋白表达的影响
图9 光学显微镜下观察抑制HTRA1表达逆转了干扰miR-661对结肠癌SW620细胞增殖、迁移和侵袭的影响（结晶紫染色，×200）
表8 抑制HTRA1表达逆转了干扰miR-661表达对结肠癌SW620细胞增殖、迁移和侵袭的作用（±s）
分组 实验次数 HTRA1蛋白 OD值（490 nm） 迁移细胞数（个） 侵袭细胞数（个） CyclinD1蛋白 p21蛋白 MMP-2蛋白 MMP-9蛋白
24 h 48 h 72 h
anti-miR-NC 3 0.56±0.05 0.35±0.03 0.73±0.07 1.14±0.09 96.32±9.54 83.44±8.37 0.63±0.06 0.30±0.03 0.78±0.07 0.66±0.06
anti-miR-661 3 0.93±0.08 0.32±0.03 0.51±0.05 0.66±0.06 49.33±4.87 38.44±3.85 0.24±0.03 0.72±0.06 0.33±0.03 0.28±0.03
t值   6.793 1.225 0.430 7.686 7.599 8.460 10.070 10.844 10.234 9.813
P值   0.002 0.288 0.011 0.002 0.002 0.001 0.001 <0.001 0.001 0.001
anti-miR-661+si-NC 3 0.95±0.09 0.31±0.03 0.48±0.04 0.63±0.05 47.58±4.72 36.98±3.66 0.22±0.03 0.73±0.07 0.32±0.03 0.27±0.03
anti-miR-661+si-HTRA1 3 0.62±0.06 0.34±0.04 0.62±0.06 0.95±0.08 82.36±8.21 71.54±7.16 0.52±0.05 0.41±0.04 0.67±0.06 0.55±0.05
t值   5.284 1.039 3.363 5.875 6.361 7.444 8.911 6.875 9.037 8.317
P值   0.006 0.357 0.028 0.004 0.003 0.002 0.001 0.002 0.001 0.001
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