长链非编码RNA SOX21-AS1调控miR-31-5p/MMP-16轴对胰腺癌细胞活性与增殖的影响

doi:10.7659/j.issn.1005-6947.2022.03.006

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长链非编码RNA SOX21-AS1调控miR-31-5p/MMP-16轴对胰腺癌细胞活性与增殖的影响
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                        10.7659/j.issn.1005-6947.2022.03.006
                    
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作者单位:中南大学湘雅三医院 肝胆胰II外科，湖南 长沙 410013
作者简介:臧龙军，中南大学湘雅三医院硕士研究生，主要从事胰腺外科方面的研究。
通信作者:余枭， Email: yuxiaoyx4@126.com
基金项目:国家自然科学基金资助项目（81873589）；湖南省自然科学基金资助项目（2020JJ5876）；湖南省长沙市科学技术局科技计划基金资助项目（kq2004146） 。

Effect of long non-coding RNA SOX21-AS1 on viability and proliferation of pancreatic cancer cells via modulating miR-31-5p/MMP-16 axis

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Department of Hepatopancreatobiliary Surgery II, the Third Xiangya Hospital, Central South University, Changsha 410013, China

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摘要:

背景与目的 胰腺癌是一种常见的消化系统恶性肿瘤，也是癌症死亡的常见病因。长链非编码RNA（lncRNA）已在多种恶性肿瘤中被鉴定为调节因子和特异度生物标志物。最新研究表明，lncRNA SOX21-AS1在多种恶性肿瘤的发生、发展中起重要作用。但其在胰腺癌中的作用尚未明确。因此，本研究探讨SOX21-AS1在胰腺癌中的表达及其功能。方法用qRT-PCR检测SOX21-AS1在20对胰腺癌与邻近癌旁正常组织，以及胰腺癌细胞系（PANC-1、CAPAN2、CFPAC-1、BXPC3、SW1990）与人胰管上皮细胞系（HPDE）中的表达。通过GEPIA数据库分析SOX21-AS1表达与胰腺癌患者预后的关系。采用MTT实验与集落形成实验分析SOX21-AS1沉默后胰腺癌细胞活力与增殖能力的变化。通过DIANA数据库预测SOX21-AS1的靶微小RNA（miRNA）及其下游的靶mRNA，随后进一步通过系列功能实验、双荧光素酶实验、拯救实验以及相关性分析明确它们之间的关系。结果 SOX21-AS1在胰腺癌组织和细胞系中表达均上调，其高表达患者预后差（均P<0.05）。SOX21-AS1沉默后，胰腺癌细胞的活力与增殖能力均明显降低（均P<0.05）。生物信息学分析显示，miR-31-5p与SOX21-AS1及MMP-16 mRNA均存在靶向结合序列，功能实验、荧光素酶实验及挽救实验结果均验证了三者之间的互作关系。此外，相关性分析结果显示，SOX21-AS1的表达与miR-31-5p的表达呈负相关（r2=0.257 1，P<0.05），miR-31-5p的表达与MMP-16 mRNA的表达呈负相关（r2=0.311 3，P=0.010 6）。结论 SOX21-AS1的表达上调与胰腺癌细胞活性、增殖能力增强及胰腺癌患者预后不良相关。机制上，SOX21-AS1可能作为竞争性内源RNA（ceRNA）与miR-31-5p发生竞争性结合，调节MMP-16的表达，从而促进胰腺癌的进展。SOX21-AS1可以作为胰腺癌的潜在预后生物标志物和诊治的靶点。

Abstract:

Background and Aims Pancreatic cancer is a common tumor of the digestive system and a frequent cause of cancer-related death. Long non-coding RNAs (lncRNAs) have been demonstrated as regulators and specific biomarkers for multiple cancers. Recent evidence has indicated that the novel lncRNA SOX21-AS1 plays an important role in the initiation and progression of a variety of malignant tumors. However, its function in pancreatic cancer remains unclear. Therefore, this study was conducted to investigate the expression of SOX21-AS1 in pancreatic cancer and its function.Methods The expressions of SOX21-AS1 in 20 pairs of specimens of pancreatic cancer and adjacent normal tissue as well as in a range of pancreatic cancer cell lines (PANC-1, CAPAN2, CFPAC-1, BXPC3, and SW1990) and human pancreatic duct epithelial cell line (HPDE) were detected by qRT-PCR method. The relationship between SOX21-AS1 expression and the prognosis of pancreatic cancer patients were analyzed through GEPIA database. The changes in cell viability and proliferative capacity in pancreatic cancer cells after SOX21-AS1 silencing were determined by MTT assay and colony-forming assay. The targeted microRNAs (miRNAs) of SOX21-AS1 and its downstream targeted mRNAs were predicted using DIANA database, and then the interactions among them were validated by a series of function experiments, dual luciferase reporter assays, rescue experiments and correlation analyses.Results The expressions of SOX21-AS1 were upregulated in both pancreatic cancer tissue and cell lines, and the patients with its high expression had a poor prognosis (all P<0.05). In pancreatic cancer cells after SOX21-AS1 silencing, the cell viability and proliferative ability were significantly decreased (both P<0.05). Bioinformatics analysis indicated that there was a target binding sequence in miR-31-5p for both SOX21-AS1 and MMP-16 mRNA, and the interactions among them were confirmed by function experiments, dual luciferase reporter assays and rescue experiments. In addition, the results of correlation analysis showed that there was a negative correlation either between SOX21-AS1 expression and miR-31-5p expression (r²=0.257 1, P<0.05), or between miR-31-5p expression MMP-16 mRNA expression (r2=0.311 3, P=0.010 6).Conclusion The increased expression of SOX21-AS1 is associated with the enhanced viability and proliferation of pancreatic cancer cells, as well as the poor prognosis of pancreatic cancer patients. In terms of mechanism, SOX21-AS1 may competitively bind to miR-31-5p as a competing endogenous RNA (ceRNA) to regulate the expression of MMP-16 mRNA, thereby promoting the progression of pancreatic cancer. SOX21-AS1 may probably be a potential biomarker and therapeutic target for pancreatic cancer.

表 2 miRNA与siRNA序列Table 2 The miRNA and siRNA sequences

图1 胰腺癌中SOX21-AS1的表达及其对预后的影响 A：qRT-PCR检测20对胰腺癌标本和邻近正常组织中SOX21-AS1的表达量；B：qRT-PCR检测不同胰腺癌细胞系与正常胰腺导管上皮细胞中SOX21-AS1表达量；C：TCGA生物学数据库分析SOX21-AS1表达对胰腺癌患者预后的影响Fig.1 Expression of SOX21-AS1 in pancreatic cancer and its association with the prognosis A: Relative expression of SOX21-AS1 detected by qRT-PCR in 20 cancer tissues and matched normal tissues; B: qRT-PCR analysis of the relative expression of SOX21-AS1 in pancreatic cell lines and normal pancreatic duct epithelial cell line; C: Influence of SOX21-AS1 on prognosis of pancreatic cancer patients by TCGA analysis

图2 SOX21-AS1对胰腺癌细胞活力、增殖的影响 A：qRT-PCR检测SOX21-AS1沉默后胰腺癌BXPC3、SW1990细胞中SOX21-AS1表达量；B：MTT检测BXPC3与SW1990细胞的活力；C：细胞克隆实验检测BXPC3和SW1990细胞的增殖能力Fig.2 Effect of SOX21-AS1 on viability and proliferation of pancreatic cancer cells A: The relative expression of SOX21-AS1 in BXPC3 and SW1990 cells after SOX21-AS1 silencing detected by qRT-PCR; B: Cell viabilities of HPDE and BXPC3 cells determined by MTT assay; C: The colony formations BXPC3 and SW1990 cells measured by colony-forming assay

图3 SOX21-AS1与miR-31-5p的关系分析 A：核质分离实验检测BXPC3与SW1990中SOX21-AS1的分布情况；B：DIANA预测SOX21-AS1与miR-31-5p中结合的靶向序列；C：qRT-PCR分析在BXPC3、SW1990细胞中过表达miR-31-5p后miR-31-5p的表达量；D：过表达miR-31-5p后，转染SOX21-AS1-WT及SOX21-AS1-MT的BXPC3与SW1990细胞的双荧光素酶实验报告；E：qRT-PCR检测干扰SOX21-AS1后BXPC3、SW1990中miR-31-5p的表达；F：qRT-PCR检测20例胰腺癌及邻近正常组织中miR-31-5p的表达；G：SOX21-AS1与miR-31-5p的相关性分析Fig.3 Analysis of the relationship between SOX21-AS1 and miR-31-5p A: The distributions of SOX21-AS1 in BXPC3 and SW1990 cells examined by nuclear cytoplasmic fractionation assay; B: The targeted binding sequence of SOX21-AS1 with miR-31-5P predicted by DIANA; C: The expression levels of miR-31-5p in BXPC3 and SW1990 cells after miR-31-5p overexpression; D: Relative luciferase activities in BXPC3 and SW1990 cells with miR-31-5p overexpression after transfection with SOX21-AS1-WT and SOX21-AS1-MT; E: Relative expression of miR-106a-3p in BXPC3 and SW1990 cells after SOX21-AS1 interference determined by qRT-PCR; F: Relative expression levels of miR-31-5p in 20 pairs of pancreatic cancer and adjacent normal tissues detected by qRT-PCR; G: Correlation analysis between SOX21-AS1 and miR-31-5p expression

图4 SOX21-AS1靶向调控miR-31-5p表达的验证 A：qRT-PCR检测使用miR-31-5p抑制剂后BXPC3、SW1990细胞中miR-31-5p的表达量；B：qRT-PCR检测在BXPC3、SW1990细胞中，在si-SOX21-AS1质粒转染后加入miR-31-5p抑制剂后miR-31-5p的表达量；C：qRT-PCR检测在BXPC3、SW1990细胞系中，在si-SOX21-AS1质粒转染后加入miR-31-5p抑制剂后SOX21-AS1的表达量；D：MTT实验检测si-SOX21-AS1质粒转染后加入miR-31-5p抑制剂的BXPC3与SW1990细胞的细胞活力；E：细胞克隆实验检测在BXPC3、SW1990细胞中，加入si-SOX21-AS1质粒转染和在此基础上加入miR-31-5p抑制剂的细胞增殖能力Fig.4 Verification of targeted regulation of miR-31-5p expression by SOX21-AS1 A: The expression levels of miR-31-5p in BXPC3 and SW1990 cells transfected with miR-31-5p inhibitors detected by qRT-PCR; B: Relative expression levels of miR-31-5p in BXPC3 and SW1990 cells transfected with si-SOX21-AS1 plasmids after addiction of miR-31-5p inhibitors detected by qRT-PCR; C: Relative expression levels of SOX21-AS1 in BXPC3 and SW1990 cells transfected with si-SOX21-AS1 plasmids after addition of miR-31-5p inhibitors detected by qRT-PCR; D: Cell viabilities of BXPC3 and SW1990 cells transfected with si-SOX21-AS1 plasmids after addiction of miR-31-5p inhibitors detected by MTT assay; E: Proliferation abilities of BXPC3 and SW1990 cells after si-SOX21-AS1#1 transfection and miR-31-5p inhibitor addition detected by colony-forming assay

图5 miR-31-5p靶向调节MMP-16 A：DIANA预测MMP-16与miR-31-5p中结合的靶向序列；B：加入miR-31-5p模拟物后，转染MMP-16-WT与MMP-16-MT的BXPC3细胞与SW1990细胞的双荧光素酶实验；C：qRT-PCR检测20例胰腺癌及邻近正常组织中MMP-16的表达量；D：MMP-16与miR-31-5p的相关性分析Fig.5 Target regulation of MMP-16 expression by miR-31-5p A: The targeted binding sequence in miR-31-5P with MMP-16 predicted by DIANA; B: Relative luciferase activities in BXPC3 and SW1990 cells transfected with MMP-16-WT or MMP-16-MT after addition of miR-31-5p mimics; C: Relative expression levels of MMP-16 in 20 pairs of pancreatic cancer and adjacent normal tissues detected by qRT-PCR; D: Correlation analysis between MMP-16 and miR-31-5p expressions

表 1 引物序列Table 1 Primer sequences

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臧龙军,陈东杰,高文哲,黄珲,朱红伟,余枭.长链非编码RNA SOX21-AS1调控miR-31-5p/MMP-16轴对胰腺癌细胞活性与增殖的影响[J].中国普通外科杂志,2022,31(3):329-339.
DOI:10.7659/j. issn.1005-6947.2022.03.006

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收稿日期:2021-09-16
最后修改日期:2022-02-20
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在线发布日期: 2022-04-02

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