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肝细胞癌(hepatocellular carcinoma, HCC)是一种常见的消化道恶性肿瘤之一,其死亡率高居恶性肿瘤第3位[1]。但因其起病隐匿,早期多无明显临床症状,大部分病人在初次诊断时已为晚期,失去了手术机会。近年来,尽管在手术治疗、放化疗以及靶向治疗等方面取得很大进展,但由于其高侵袭性、转移、复发等因素,HCC病人预后仍相对较差[2-3]。因此,探究肝癌侵袭转移的机制,确定有效的作用靶点或预后标志物成为目前HCC研究的重要方向。长链非编码RNA(long-chain non-coding RNA, LncRNA)是一类长度超过200个核苷酸的不编码蛋白质的RNA[4]。LncRNA可以通过调控肿瘤细胞的增殖、迁移、凋亡、血管生成等方面参与肿瘤的发生、发展过程[5-6]。LINC00319是肿瘤相关lncRNA的一种[7-8]。LINC00319通过分子海绵吸附miR-335-5p上调ADCY3从而促进胃癌细胞的肿瘤生长和远处转移[9],通过调控miR-32的表达促进肺癌的增殖和侵袭[10],通过调控miR-3127-5p/RPP25轴促进宫颈癌细胞迁移、侵袭和上皮间质转化(epithelial-mesenchymal transition, EMT)[11]。但是,LINC00319在肝癌中的表达水平及其临床意义仍不清楚。因此,本研究旨在探讨LINC00319在肝细胞癌组织中的表达及临床意义,及其对肝癌细胞侵袭迁移能力的影响。
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肝癌组织中的LINC00319在肝癌组织中的表达(0.57±0.02)明显高于癌旁组织(0.27±0.01)(t=15.02,P < 0.01)。
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将LINC00319按中位数分为LINC高表达组(n=49)和低表达组(n=48)。结果显示LINC00319高表达与TNM分期(Ⅲ+Ⅳ)和血管侵犯均相关(P < 0.01和P < 0.05)(见表 1)。
临床病理 n LINC00319 χ2 P 高表达组 低表达组 年龄/岁 < 60
≥6064
3331(48.44)
18(54.54)33(51.56)
15(45.46)0.33 >0.05 性别 男
女80
1740(50.00)
9(52.94)40(50.00)
8(47.06)0.05 >0.05 肿瘤大小/cm < 5
≥568
2930(44.12)
19(65.52)38(55.88)
10(34.48)3.72 >0.05 肿瘤数量 单发
多发84
1340(47.62)
9(69.23)44(52.38)
4(30.77)2.10 >0.05 病理分级 Ⅰ+Ⅱ
Ⅲ+Ⅳ23
749(39.13)
40(54.05)14(60.87)
34(45.95)1.56 >0.05 TNM分期 Ⅰ+Ⅱ
Ⅲ+Ⅳ72
2530(41.67)
19(76.00)42(58.33)
6(24.00)8.75 < 0.01 血管侵犯 是
否16
8112(75.00)
37(45.68)4(25.00)
44(54.32)4.60 < 0.05 甲胎蛋白/(ng/mL) < 400
≥40022
7513(59.09)
36(48.00)9(40.91)
39(52.00)0.84 >0.05 乙肝表面抗原 阳性
阴性89
845(50.56)
4(50.00)44(49.44)
4(50.00)0.01 >0.05 表 1 LINC00319的表达与肝癌临床病理特征关系[n;构成比(%)]
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随访发现,LINC00319高表达病人的5年生存率(20.41%)明显低于LINC00319低表达组病人(52.08%)(χ2=11.59,P < 0.01)(见图 1)。
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LINC00319在肝癌细胞系中的表达均高于L02正常肝细胞系(P < 0.05~P < 0.01),且在MHCC-97H和HCCLM3细胞系中表达最高(P < 0.01)(见表 2)。基于以上结果,选择MHCC-97H和HCCLM3细胞系为后续敲低实验研究对象,研究LINC00319对肝癌细胞系生物学功能的影响。
细胞系 n LINC00319表达水平 F P MS组内 L02 3 1.00±0.02 22.34 < 0.01 0.001 Hep3B 3 1.89±0.12* SMMC-7721
Huh73
31.96±0.11*
2.56 ±0.15**△#MHCC-97H 3 3.23±0.12**△△##▲ HCCLM3 3 3.65±0.13**△△##▲▲ q检验:与L02比较*P < 0.05,**P < 0.01;与Hep3B比较△P < 0.05,△△P < 0.01;与SMMC-7721比较#P < 0.05,##P < 0.01;与Huh7比较▲P < 0.05,▲▲P < 0.01 表 2 LINC00319在不同肝癌细胞系中的表达水平(x±s)
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将si-Control、siRNA#1、siRNA#2分别转染MHCC-97H和HCCLM3细胞系干扰LINC00319的表达48 h后,用qRT-PCR检测干扰效率。结果显示,与si-Control对照组相比,siRNA#1、siRNA#2均能抑制MHCC-97H和HCCLM3细胞中LINC00319的相对表达量(P < 0.01)(见表 3)。提示该两条siRNA可用于后续实验对LINC00319的敲低。
分组 n LINC00319/MHCC-97H LINC00319/HCCLM3 si-Control 3 1.00±0.05 1.00±0.03 siRNA#1 3 0.32±0.05** 0.30±0.05** siRNA#2 3 0.23±0.04** 0.28±0.04** F — 52.26 35.67 P — < 0.01 < 0.01 MS组内 — 0.003 0.001 q检验:与si-Control组比较**P < 0.01 表 3 各组细胞中LINC00319表达水平(x±s)
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利用未铺胶的Transwell实验证实,敲低LINC00319能抑制MHCC-97H和HCCLM3细胞的迁移能力和侵袭能力(P < 0.01)(见表 4)。
分组 n MHCC-97H HCCLM3 迁移细胞数 侵袭细胞 迁移细胞数 侵袭细胞数 si-Control 3 155.23±14.35 95.63±9.56 172.39±14.83 89.64±8.37 siRNA#1 3 65.43±8.25** 42.38±5.26** 58.43±8.75** 41.35±5.59** siRNA#2 3 43.23±7.14** 38.65±4.15** 45.28±7.24** 35.62±4.25** F — 54.08 59.64 93.45 55.04 P — < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 0.002 0.001 0.001 0.001 q检验:与si-Control组比较** P < 0.01 表 4 siRNA干扰LINC00319表达水平后细胞迁移及侵袭能力(x±s)
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敲低LINC00319能够促进上皮表型标志物E-cadherin的表达(P < 0.01),而抑制间质表型标志物N-cadherin和Vimentin的表达(P < 0.01)(见表 5)。说明LINC00319能够调控肝癌细胞EMT的发生。
分组 n MHCC-97H HCCLM3 E-cadherin N-cadherin Vimentin E-cadherin N-cadherin Vimentin si-Control 3 1.00±0.02 1.00±0.03 1.00±0.02 1.00±0.02 1.00±0.04 1.00±0.02 siRNA#1 3 2.53±0.12** 0.37±0.05** 0.29±0.05** 2.60±0.12** 0.42±0.05** 0.31±0.05** siRNA#2 3 2.10±0.11** 0.26±0.04** 0.23±0.04** 2.21±0.11** 0.31±0.04** 0.38±0.04** F — 54.08 65.34 89.36 67.38 46.35 56.89 P — < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 0.001 0.001 0.001 0.001 0.001 0.001 q检验:与si-Control组比较**P < 0.01 表 5 siRNA干扰LINC00319表达水平后EMT相关蛋白的表达量(x±s)
长链非编码RNA LINC00319在肝细胞癌中表达上调并促进肝癌细胞迁移和侵袭
Observation of the high-expression of long-chain non-coding RNA LINC00319 and promotion of cell migration and invasion in hepatocellular carcinoma
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摘要:
目的探讨LINC00319在肝细胞癌(HCC)组织中的表达、临床意义及可能的分子机制。 方法实时定量PCR检测LINC00319在HCC及癌旁组织中的表达水平,并分析LINC00319表达水平与临床病理特征的关系及生存率。Transwell实验检测LINC00319对HCC细胞的侵袭及迁移作用,Western blotting法检测LINC00319对HCC细胞EMT的影响。 结果LINC00319在肝癌组织中表达水平明显高于相应癌旁组织(P < 0.01);肝癌组织LINC00319高表达与TNM分期及血管侵犯相关(P < 0.01和P < 0.05);高表达LINC00319病人5年生存率明显高于低表达组(P < 0.01);LINC00319下调可显著抑制HCC细胞的侵袭及迁移(P < 0.01);LINC00319下调抑制HCC细胞EMT的发生(P < 0.01)。 结论LINC00319在肝癌组织中表达升高,其高表达与肝癌恶性临床病理特征有关,LINC00319能够通过调控HCC细胞EMT促进其侵袭和迁移。 Abstract:ObjectiveTo investigate the level and clinical significance of LINC00319 expression in human hepatocellular carcinoma(HCC) and analyze the potential underlying mechanisms. MethodsThe LINC00319 expression level in HCC was checked by real time quantitative PCR method, and the relationship among LINC00319 expression level, clinical pathological features and survival rate were analyzed.Transwell assay was performed to measure the invasion and migration of effects of LINC00319 on HCC cells.The expression of EMT-related protein was determined by Western blotting. ResultsThe expression level of LINC00319 in HCC tissues was significantly higher than that in the adjacent normal tissues(P < 0.01), and it was significantly associated with TNM stage and vascular infiltration(P < 0.01 and P < 0.05).Patients in the higher LINC00319 group had a worse 5-year survival than those in the lower LINC00319 group(P < 0.01).Down-expression of LINC00319 significantly inhibited the invasion and migration of HCC cells(P < 0.01).Down-expression of LINC00319 inhibited the EMT of HCC(P < 0.01). ConclusionsLINC00319 is up-regulated in HCC tissues.Its high expression is correlated with the malignant clinical pathological features.LINC00319 high-expression regulates EMT and subsequently promotes invasion and migration of HCC cells. -
Key words:
- hepatocellular carcinoma /
- LINC00319 /
- invasion /
- migration /
- epithelial-mesenchymal transition
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表 1 LINC00319的表达与肝癌临床病理特征关系[n;构成比(%)]
临床病理 n LINC00319 χ2 P 高表达组 低表达组 年龄/岁 < 60
≥6064
3331(48.44)
18(54.54)33(51.56)
15(45.46)0.33 >0.05 性别 男
女80
1740(50.00)
9(52.94)40(50.00)
8(47.06)0.05 >0.05 肿瘤大小/cm < 5
≥568
2930(44.12)
19(65.52)38(55.88)
10(34.48)3.72 >0.05 肿瘤数量 单发
多发84
1340(47.62)
9(69.23)44(52.38)
4(30.77)2.10 >0.05 病理分级 Ⅰ+Ⅱ
Ⅲ+Ⅳ23
749(39.13)
40(54.05)14(60.87)
34(45.95)1.56 >0.05 TNM分期 Ⅰ+Ⅱ
Ⅲ+Ⅳ72
2530(41.67)
19(76.00)42(58.33)
6(24.00)8.75 < 0.01 血管侵犯 是
否16
8112(75.00)
37(45.68)4(25.00)
44(54.32)4.60 < 0.05 甲胎蛋白/(ng/mL) < 400
≥40022
7513(59.09)
36(48.00)9(40.91)
39(52.00)0.84 >0.05 乙肝表面抗原 阳性
阴性89
845(50.56)
4(50.00)44(49.44)
4(50.00)0.01 >0.05 表 2 LINC00319在不同肝癌细胞系中的表达水平(x±s)
细胞系 n LINC00319表达水平 F P MS组内 L02 3 1.00±0.02 22.34 < 0.01 0.001 Hep3B 3 1.89±0.12* SMMC-7721
Huh73
31.96±0.11*
2.56 ±0.15**△#MHCC-97H 3 3.23±0.12**△△##▲ HCCLM3 3 3.65±0.13**△△##▲▲ q检验:与L02比较*P < 0.05,**P < 0.01;与Hep3B比较△P < 0.05,△△P < 0.01;与SMMC-7721比较#P < 0.05,##P < 0.01;与Huh7比较▲P < 0.05,▲▲P < 0.01 表 3 各组细胞中LINC00319表达水平(x±s)
分组 n LINC00319/MHCC-97H LINC00319/HCCLM3 si-Control 3 1.00±0.05 1.00±0.03 siRNA#1 3 0.32±0.05** 0.30±0.05** siRNA#2 3 0.23±0.04** 0.28±0.04** F — 52.26 35.67 P — < 0.01 < 0.01 MS组内 — 0.003 0.001 q检验:与si-Control组比较**P < 0.01 表 4 siRNA干扰LINC00319表达水平后细胞迁移及侵袭能力(x±s)
分组 n MHCC-97H HCCLM3 迁移细胞数 侵袭细胞 迁移细胞数 侵袭细胞数 si-Control 3 155.23±14.35 95.63±9.56 172.39±14.83 89.64±8.37 siRNA#1 3 65.43±8.25** 42.38±5.26** 58.43±8.75** 41.35±5.59** siRNA#2 3 43.23±7.14** 38.65±4.15** 45.28±7.24** 35.62±4.25** F — 54.08 59.64 93.45 55.04 P — < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 0.002 0.001 0.001 0.001 q检验:与si-Control组比较** P < 0.01 表 5 siRNA干扰LINC00319表达水平后EMT相关蛋白的表达量(x±s)
分组 n MHCC-97H HCCLM3 E-cadherin N-cadherin Vimentin E-cadherin N-cadherin Vimentin si-Control 3 1.00±0.02 1.00±0.03 1.00±0.02 1.00±0.02 1.00±0.04 1.00±0.02 siRNA#1 3 2.53±0.12** 0.37±0.05** 0.29±0.05** 2.60±0.12** 0.42±0.05** 0.31±0.05** siRNA#2 3 2.10±0.11** 0.26±0.04** 0.23±0.04** 2.21±0.11** 0.31±0.04** 0.38±0.04** F — 54.08 65.34 89.36 67.38 46.35 56.89 P — < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 0.001 0.001 0.001 0.001 0.001 0.001 q检验:与si-Control组比较**P < 0.01 -
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