Expression of PIWI-interacting RNA-47851 in gastric adenocarcinoma and its influence on proliferation
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摘要:目的
探讨PIWI蛋白相互作用RNA-47851(piR-47851)在胃腺癌中的表达、临床病理意义及其对增殖的影响。
方法通过实时荧光定量聚合酶链反应(qRT-PCR)检查79例胃腺癌组织中piR-47851的表达情况, 分析piR-47851的表达水平与临床特征和生存预后的关系。通过细胞增殖实验观察piR-47851对胃癌细胞增殖活性的影响。应用信息学网站预测piR-47851的下游靶基因。建立MAPK1基因3'非翻译区(UTR)的野生型和突变型质粒，应用双荧光素酶报告系统验证piR-47851与MAPK1基因3'UTR结合从而抑制MAPK1蛋白合成。应用Rescue实验确认piR-47851对MAPK1直接调控作用，并探讨piR-47851/MAPK1对胃癌细胞增殖的影响。通过实验性裸鼠皮下荷瘤实验观察降低piR-47851表达对移植瘤体积和质量的影响; 通过增殖指标Ki-67染色观察降低piR-47851表达对移植瘤细胞增殖活力的影响。
结果qRT-PCR实验结果提示, 79例胃腺癌组织中piR-47851的表达水平显著高于癌旁正常胃组织(P＜0.05)。piR-47851表达水平与肿瘤大小、分化程度和生存预后密切相关(P＜0.05)。生物信息学提示piR-47851与MAPK1基因3'UTR有互补的结合位点。双荧光素酶报告基因实验得出在MAPK1野生型转染组中, piR-47851能显著抑制荧光酶活性，而在MAPK1突变组中无抑制作用。CCK-8增殖活性实验表明，过表达piR-47851后，胃癌细胞增殖活性升高; 降低piR-47851后，细胞增殖活性下降，差异均有统计学意义(P＜0.05)。Rescue实验提示，在改变piR-47851和(或)MAPK1的表达后再通过qRT-PCR检测MAPK1的表达水平, MAPK1都能受到piR-47851的调控。在功能上过度表达MAPK1可以降低piR-47851对胃癌细胞的增殖促进作用，而减低MAPK1表达可进一步提升胃癌细胞的增殖活性。在裸鼠荷瘤实验中，降低piR-47851表达后肿瘤生长体积和质量显著减小(P＜0.05)。增殖指数Ki-67染色表明减低piR-47851表达后增殖活性细胞数目显著低于对照组(P＜0.05), 验证了piR-47851能促进细胞增殖。
结论胃腺癌组织中piR-47851表达水平升高，与肿瘤大小和生存预后密切相关。piR-47851通过与MAPK1的3'UTR结合来下调MAPK1蛋白表达，从而促进胃癌的细胞增殖。
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关键词:
- 胃腺癌 /
- PIWI蛋白相互作用RNA-47851 /
- 细胞增殖 /
- 增殖指数 /
- 生存预后
Abstract:ObjectiveTo investigate the expression and clinical pathological significance of PIWI-interacting RNA-47851 (piR-47851) in gastric adenocarcinoma and its influence on proliferation.
MethodsThe expression of piR-47851 was detected in 79 gastric adenocarcinoma tissues by real time fluorescence quantitative polymerase chain reaction (qRT-PCR), and the correlation of piR-47851 expression level and clinical features with survival and prognosis were analyzed. The effect of piR-47851 on proliferation activity of gastric cancer cells was observed by cell proliferation experiments. Informatics websites were used to predict the downstream target genes of piR-47851. The wild-type and mutant plasmids for the 3'untranslated region (UTR) of MAPK1 gene were established, and a dual luciferase reporting system was used to verify that piR-47851 binded to the 3'UTR of MAPK1 gene, thereby inhibiting MAPK1 protein synthesis. The effect of overexpression/silencing of piR-47851 on MAPK1 expression level was examined through qRT-PCR experiment. Rescue experiment was used to confirm the direct regulatory effect of piR-47851 on MAPK1 and explore the effect of piR-47851/MAPK1 on the proliferation of gastric cancer cells. The effect of reduced piR-47851 expression on the volume and weight of transplanted tumors was observed in nude mice by xenograft tumor experiments; the effect of reduced piR-47851 expression on the cell proliferation activity of xenograft tumors was observed by Ki-67 staining.
ResultsThe qRT-PCR experiment result showed that the expression level of piR-47851 in 79 gastric adenocarcinoma tissues was significantly higher than that in normal gastric tissues adjacent to the cancer (P < 0.05). The expression level of piR-47851 was significantly correlated with tumor size, degree of differentiation, and survival prognosis (P < 0.05). Bioinformatics suggested that there was a complementary binding site between piR-47851 and MAPK1 gene 3'UTR. The dual luciferase reporter gene experiment showed that piR-47851 was able to significantly inhibit fluorescence enzyme activity in the MAPK1 wild-type transfection group, while this inhibitory effect was not observed in the MAPK1 mutant group. The CCK-8 proliferation activity experiment showed that overexpression of piR-47851 was able to significantly increase the proliferation activity of gastric cancer cells; after reducing piR-47851, the proliferation activity of cells decreased significantly (P < 0.05). Rescue experiment indicated that after changing the expression of piR-47851 and (or) MAPK1 and then detecting the expression level of MAPK1 by qRT-PCR, the MAPK1 could be regulated by piR-47851. Overexpression of MAPK1 could functionally reduce the proliferation promoting effect of piR-47851 on gastric cancer cells, while reducing MAPK1 expression could further enhance the proliferation activity of gastric cancer cells. In xenograft tumor experiments, reducing the expression of piR-47851 significantly resulted in smaller tumor growth volume and weight (P < 0.05). The proliferation index Ki-67 staining showed that the number of proliferative active cells in the reduced piR-47851 expression group was significantly lower than that in the control group (P < 0.05), which further validated the promotional effect of piR-47851 on cell proliferation.
ConclusionThe expression level of piR-47851 is increased in gastric adenocarcinoma tissues, which is closely related with tumor size and survival prognosis. The piR-47851 downregulates MAPK1 protein expression by binding to the 3'UTR of MAPK1, thereby promoting the proliferation of gastric cancer cells.
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图 1 不同组织类型及细胞中piR-47851表达水平比较
A: 胃腺癌中piR-47851表达水平高于癌旁正常组织，与癌旁正常组织比较, *P＜0.05; B: SGC7901和AGS细胞中piR-47851表达水平高于正常胃黏膜上皮GES-1细胞，与正常GES-1细胞比较, *P＜0.05。
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图 4 CCK-8实验分析piR-47851对胃腺癌细胞增殖活性的影响
A: 过表达piR-47851后, SGC7901细胞增殖活性升高，与对照组比较, *P＜0.05; B: 降低piR-47851表达后, SGC7901细胞增殖活性下降，与si-NC比较, *P＜0.05。
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图 5 双荧光素酶报告实验检测piR-47851与MAPK1的关系
A: piR-47851与MAPK1 3′UTR的结合位点; B: 野生型MAPK1 3′UTR与piR-47851结合后降低了荧光素酶的活性，与对照组比较, *P＜0.05。
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图 6 Rescue实验确定MAPK1受到piRNA-47851调控
第1组: piRNA-NC+si-NC; 第2组: piRNA-NC+si-MAPK1; 第3组: si-piR-47851+Vector; 第4组: si-piR-47851+MAPK1; 第5组: piR-47851+MAPK1; 第6组: piR-47851+Vector; 第7组: si-piR-47851+si-NC; 第8组: si-piR-47851+si-MAPK1。
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图 7 降低piRNA-47851表达对皮下移植瘤生长的影响
A: 降低piRNA-47851表达后瘤体明显较小; B、C: 降低piRNA-47851表达后可抑制皮下移植瘤的生长体积和质量，与对照组比较, *P＜0.05。
表 1 主要基因的引物序列
引物名称 序列(5′-3′) piR-47851-f TACATGACCCCAGGAGGCG piR-47851-r 试剂盒通用引物 si-piR-47851-1 UGCAACUUCCGCCUCCUGGGG si-piR-47851-2 CCAGGAGGCGGAAGUUGCAGU U6-f CTCGCTTCGGCAGCACA U6-r AACGCTTCACGAATTTGCGT MAPK1-f TAGGGAAGGCTACTACCTAG MAPK1-r TTGCTTCCACTTTACAGCTG 
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表 2 胃腺癌组织中piR-47851表达与临床资料的相关性[n(%)]
临床资料 分类 阳性表达组(n=57) 阴性表达组(n=22) χ2 P 性别 男 36(63.16) 13(59.09) 0.111 0.738 女 21(36.84) 9(40.91) 年龄 ＜50岁 23(40.35) 12(54.55) 1.296 0.255 ≥50岁 34(59.65) 10(45.45) 肿瘤直径 ＜5 cm 24(42.11) 16(72.72) 5.955 0.015 ≥5 cm 33(57.89) 6(27.28) 分化程度 高分化 17(29.82) 14(63.64) 7.611 0.006 中低分化 40(70.18) 8(36.36) 浸润深度 T1~T2期 18(31.58) 10(45.45) 1.336 0.248 T3~T4期 39(68.42) 12(54.55) 淋巴结转移 有 26(45.61) 5(22.73) 3.487 0.062 无 31(54.39) 17(77.27) TNM分期 Ⅰ~Ⅱ期 30(52.63) 12(54.55) 0.023 0.879 Ⅲ期 27(47.37) 10(45.45)
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表 3 Rescue实验对MAPK1和增殖活性的影响
组别 MAPK1 72 h OD450 nm值 第1组 1.00±0 0.78±0.04 第2组 0.62±0.03* 0.89±0.05* 第3组 2.06±0.09 0.57±0.03 第4组 3.28±0.11# 0.32±0.02# 第5组 1.00±0 0.81±0.04 第6组 0.56±0.03△ 0.59±0.04△ 第7组 0.52±0.03 0.61±0.03 第8组 1.01±0.04▲ 0.80±0.05▲ 第1组: piRNA-NC+si-NC; 第2组: piRNA-NC+si-MAPK1;
第3组: si-piR-47851+Vector; 第4组: si-piR-47851+MAPK1;
第5组: piR-47851+ MAPK1; 第6组: piR-47851+Vector;
第7组: si-piR-47851+si-NC; 第8组: si-piR-47851+si-MAPK1。
与第1组比较, *P＜0.05; 与第3组比较, #P＜0.05;
与第5组比较, △P＜0.05; 与第7组比较, ▲P＜0.05。
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