Abstract
目的
探究长链非编码RNA LINC01285在结直肠癌（CRC）中的表达特点及临床意义，阐明潜在的生物学功能及调控机制。
方法
基于Starbase生物数据库检索LINC01285在CRC及癌旁组织中的表达量；收集本单位结直肠癌患者的CRC样本及周围正常样本各70例，分组为肿瘤组与非肿瘤组；利用荧光定量PCR实验（RT-qPCR）验证本中心CRC队列、肿瘤细胞中LINC01285的表达量，并分析其与患者临床病理参数及无瘤生存时间的关系。采用脂质体转染技术构建LINC01285低表达细胞株并分为si-Control（对照组）、si-LINC01285-1（敲低组1）、si-LINC01285-2（敲低组2）3个组，采用CCK-8实验、流式细胞学、Transwell法及蛋白印迹法检测LINC01285对结直肠癌细胞的增殖、凋亡及转移能力的影响及潜在的调控机制。
结果
Starbasev3.0公共数据库获取TCGA-COAD转录组测序数据发现LINC01285在癌组织中的表达量明显高于正常组织（P=0.00016）；RT-qPCR结果显示：与非肿瘤组相比，LINC01285在肿瘤组表达水平显著上调（P=0.0002），其表达量与肿瘤组织学分化程度（P=0.036）、T分期（P=0.000）、淋巴结转移（P=0.001）、TNM分期（P=0.000）、Duke分期（P=0.009）和无瘤生存时间（P=0.0102）密切相关。相比于正常结直肠粘膜细胞，CRC细胞（尤其在SW620和HT-29）内LINC01285的表达水显著高表达（P < 0.001）。相比于si-Control组，脂质体转染的细胞（si-LINC01285-1、si-LINC01285-2）内LINC01285表达量显著下降（P < 0.001）。同时相比于si-Control组，LINC01285敲低CRC细胞组的增殖能力明显降低（P < 0.001）、早期凋亡、晚期凋亡及总凋亡率明显升高（P < 0.05）。相比于si-Control组，LINC01285敲低组的CRC细胞迁移和侵袭能力显著下降（P < 0.001），且上皮- 间质转化相关通路的E-钙粘蛋白表达量显著升高（P < 0.001、N-钙粘蛋白显著下调（P < 0.001）。
结论
LINC01285的表达与CRC的预后高度相关，可调节上皮-间质转化通路影响CRC细胞的增殖、凋亡与转移能力。
Keywords: 结直肠癌, LINC01285, 增殖, 转移, 上皮-间质转化
Abstract
Objective
To clarify the mechanism by which LINC01285 regulates proliferation and migration of colorectal cancer (CRC) cells and the clinical implications.
Methods
We analyzed the expression of LINC01285 in CRC tissues and normal tissues using data from Starbase public database. We also examined the expression levels of LINC01285 in 70 pairs of CRC and adjacent tissue samples collected from our center and in different CRC cell lines using RT-qPCR, and analyzed the correlation of LINC01285 expression with the clinicopathological parameters and tumor-free survival time of the patients. In CRC cell lines (SW620 and HT-29), the changes in cell proliferation, apoptosis, metastasis and epithelial-mesenchymal transition (EMT) phenotype following LINC01285 knockdown were analyzed using CCK-8 assay, flow cytometry, Transwell assay and Western blotting.
Results
The TCGA-COAD transcriptome sequencing data obtained from the Starbasev3.0 public database revealed a significantly higher expression level of LINC01285 in CRC tissues than in adjacent tissues (P=0.00016), which was verified by RT-qPCR results of the clinical samples (P=0.0002). In CRC patients, the expression level of LINC01285 was closely correlated with histological differentiation of the tumor (P=0.036), T classification (P=0.000), lymph node metastasis (P=0.001), TNM stage (P=0.000), Duke stage (P=0.009) and relapse-free survival (P=0.0102). In SW620 and HT-29 cells, which expressed significantly higher levels of LINC01285 than normal colorectal mucosal cells (P < 0.001), LINC01285 knockdown significantly inhibited cell proliferation (P < 0.001), increased early apoptosis, late apoptosis and total apoptosis rates (P < 0.05), suppressed cell migration and invasion (P < 0.001), upregulated the expression of E-cadherin (P < 0.001), and downregulated the expression of N-cadherin (P < 0.001).
Conclusion
The expression level of LINC01285, which modulates the EMT pathway to regulate the proliferation, apoptosis and metastasis of CRC cells, is closely correlated with the prognosis of CRC patients.
Keywords: colorectal cancer, LINC01285, proliferation, metastasis, epithelial-mesenchymal transition
结直肠癌（CRC）是世界上最常见的消化道恶性肿瘤之一[1]。近年来，我国CRC发病率和死亡率呈明显上升趋势[2-4]。目前，结直肠癌的治疗方式主要有外科治疗、术后辅助治疗和转化治疗等多种方式，手术切除仍是治疗CRC的主要方式，肿瘤复发转移对患者长期生存有着极大影响[5]，针对失去手术机会的患者，改进的化疗方案显示出一定疗效[6]，但总的来说并未显著改善患者的生存预后。因此，深入了解结直肠癌发生发展的机制，发现更多相关诊断和预后的生物标志物，对于改善结直肠癌患者的预后具有重要意义。
近年来，分子靶向治疗由于具备特异性高、毒副损伤小等优点而备受临床医生和科研工作者的重视[7-9]，lncRNA作为一类新型的生物学遗传分子，其长度大于200 bp[10-13]，在调节一系列生物学过程包括基因转录与翻译、表观遗传修饰、细胞增殖与分化、细胞周期与凋亡等过程中发挥重要作用[14, 15]。研究显示，恶性肿瘤中存在lncRNA异常表达，包括HOTAIR、MALAT1、PVT1在内的大量lncRNA被证实在肠癌、胃癌及乳腺癌等多种恶性肿瘤发生发展中起着促癌或抑癌作用[16-18]。深入研究lncRNA在CRC发生发展中的生物学作用及其分子机制有助于寻找潜在的肿瘤标志物，开发新型的靶向治疗策略[19, 20]。
LINC01285是一个定位于染色体Xq24，长度为837 bp的lncRNA分子，其在CRC中的功能和作用尚无任何报道。本研究检索公共数据库发现LINC01285在CRC中的表达量远高于癌旁组织，但对于该基因的功能作用相关研究尚缺乏报道。本研究拟手机本中心组织生物样本库探索其基础表达量、与患者临床病理参数及无瘤生存时间的相关性，并探究其对CRC细胞生物学功能的影响，同时探讨潜在的分子机制，为CRC预后判断及靶向治疗提供实验依据。
1. 材料和方法
1.1. 组织标本
组织标本选自行我院行根治性手术治疗的70例结肠癌患者，所有患者术后均经病理学检查确诊，术前未行放化疗等抗肿瘤治疗（伦理号：PYRC-2021-113）。
1.2. 主要试剂和仪器
逆转录试剂盒、荧光定量PCR试剂盒（Takara），引物序列（上海生工公司），细胞培养基、胎牛血清及Matrigel基质胶（Corning），Lipofectamine 3000转染试剂（Invitrogen），LINC01285-siRNA干扰序列（广州锐博公司），CCK-8细胞增殖试剂盒、细胞凋亡检测试剂盒（上海凯基公司），E-cadherin、N-cadherin、Vimentin、GAPDH兔抗人单克隆抗体（CST），荧光定量PCR检测系统（Roche）、蛋白质印迹检测系统（Bio-rad）、流式细胞仪（BD）。
1.3. 方法
1.3.1. 生物信息学分析
在本实验中，我们利用Starbase数据库获取TCGA-COAD转录组测序数据，同时将LINC01285在结直肠癌中的表达水平进行分析。
1.3.2. 荧光定量PCR反应
Trizol法提取总RNA，按照逆转录试剂盒说明书将总RNA拟转录成cDNA，再以cDNA为模板按照SYBR Green I法进行靶基因的荧光定量PCR反应。采用2-ΔΔCT法表示靶基因的相对表达水平，以GAPDH作为内参。LINC01285引物上游：5'-C CTGGATTTCCTCAGCCTAAA-3'，下游5'-CATGGG CTTCCAGTTCTCTT-3'；GAPDH引物上游：5'-CCC TTCATTGACCTCAACTACA-3'，下游5'-ATGACAA GCTTCCCGTTCTC-3'（江苏吉玛公司）。
1.3.3. 细胞培养和转染
FHC细胞均使用含10%胎牛血清的DMEM培养基、置于37 ℃ 5% CO2培养箱中进行培养。
取处于对数生长期的CRC细胞接种于6孔板，密度为5×105/孔。当细胞生长至汇合度70%~80%时，按照Lipofectamine 3000说明书将siRNA分别转染至CRC细胞，设置分组为si-LINC01285-1、si-LINC01285-2和si-Control。
1.3.4. CCK-8细胞增殖实验
取转染24 h的CRC细胞接种于96孔板，密度为1000/孔，分别在第1、2、3、4天加入10 μL CCK-8试剂，然后避光培养2 h，酶联免疫检测仪检测450 nm波长处的吸光度，绘制细胞生长曲线。
1.3.5. 细胞凋亡实验
取转染48 h的CRC细胞，按照凋亡检测试剂盒的方法进行处理，使用流式细胞仪分析细胞的早期、晚期及总凋亡率。
1.3.6. Transwell迁移、侵袭实验
迁移实验：取转染48 h的CRC细胞，用无血清培养基重悬细胞，按2×105/100 μL加入至上室，下室加500 μL含20% FBS的DMEM培养液，培养48 h后取出小室，棉签拭掉上室内侧细胞，4%多聚甲醛固定细胞，0.5%结晶紫溶液染色，显微镜下选取5个视野进行拍照、计数。侵袭实验：小室经10% Matrigel基质胶预处理，按迁徙实验方法进行侵袭实验。
1.3.7. 蛋白印迹法
取转染48 h的CRC细胞，RIPA总蛋白裂解液裂解细胞，提取总蛋白，BCA检测试剂盒测定总蛋白浓度，总蛋白经10%分离胶的琼脂糖凝胶电泳进行分离，蛋白分离后转移至PVDF膜，5%脱脂牛奶进行封闭，E-cadherin（1∶1000）、N-cadherin（1∶1000）、Vimentin（1∶1000）及GAPDH（1∶5000）等一抗孵育过夜，次日孵育二抗，ECL法显色，采集蛋白条带图像后进行半定量分析。
1.3.8. 统计学分析
所有数据均采用IBM SPSS 20.0处理。计数资料比较则采用卡方检验。采用Kaplan-Meier法进行生存分析，并用Log-rank检验比较组间差异。以α=0.05为检验标准，对以上结果均进行双侧检验。
2. 结果
2.1. LINC01285高表达于CRC组织
Starbasev3.0公共数据库获取TCGA-COAD转录组测序数据，检索发现LINC01285在癌组织中的表达量明显高于对照组的癌旁组织（P=0.00016，图 1）；随机选取70对CRC组织和癌旁组织，采用荧光定量PCR验证LINC01285的表达情况，结果显示LINC01285在癌组织中的表达量显著高于癌旁组织（P=0.0002，图 2），表明LINC01285在人CRC组织中异常高表达。
1.
LINC01285在结直肠癌组织中的表达水平（TCGA队列）
Expression of LINC01285 in colorectal cancer tissues (TCGA cohort).
2.
LINC01285在结直肠癌组织中的表达水平（临床队列）
Expression of LINC01285 in colorectal cancer tissues (clinical cohort).
2.2. 结直肠癌中LINC01285的表达量与临床病理参数及无瘤生存时间的关系
取LINC01285的表达量中位数，将CRC患者分为高表达LINC01285（表达量大于中位数）及低表达LINC01285（表达量小于中位数）两个患者组别。统计学分析LINC01285的表达量与CRC患者临床病理参数的关系，结果表明LINC01285的表达量与肿瘤的组织学分化程度（P=0.036），T分期（P=0.000），淋巴结转移（P=0.001），TNM分期（P=0.000）以及Duke分期（P= 0.009）显著相关，而与年龄（P>0.05）或性别（P>0.05）无关（表 1）；生存分析显示，高表达LINC01285的患者无瘤生存时间较低表达者显著缩短（P=0.0102，图 3）。
1.
LINC01285表达水平与结肠癌患者临床病理参数的关系
Correlation between LINC01285 expression and clinicopathologic parameters of patients with colorectal cancer
| Clinicopathologic parameters | n | LINC01285 expression | χ2 value | P | |
| High | Low | ||||
| Age (year) | |||||
| < 60 | 43 | 18 | 25 | 2.954 | 0.086 |
| ≥60 | 27 | 17 | 10 | ||
| Gender | |||||
| Male | 38 | 20 | 18 | 0.230 | 0.631 |
| Female | 32 | 15 | 17 | ||
| Histologic differentiation | |||||
| Well | 30 | 16 | 14 | 6.640 | 0.036 |
| Moderate | 25 | 8 | 17 | ||
| Poor | 15 | 11 | 4 | ||
| T classification | |||||
| T1+T2 | 39 | 11 | 28 | 16.733 | 0.000 |
| T3+T4 | 31 | 24 | 7 | ||
| Lymph node metastasis | |||||
| Negative | 45 | 16 | 29 | 10.516 | 0.001 |
| Positive | 25 | 19 | 6 | ||
| TNM stage | |||||
| I+II | 40 | 12 | 28 | 14.933 | 0.000 |
| III+IV | 30 | 23 | 7 | ||
| Duke stage | |||||
| A | 24 | 8 | 16 | 11.467 | 0.009 |
| B | 10 | 3 | 7 | ||
| C | 30 | 18 | 12 | ||
| D | 6 | 6 | 0 | ||
3.
LINC01285高表达组和低表达组术后无瘤生存时间的比较
Kaplan-Meier relapse-free survival curves of patients with high and low expressions of LINC01285.
2.3. LINC01285低表达CRC细胞的构建与验证
采用荧光定量PCR检测5株CRC细胞和人正常结直肠粘膜细胞中的LINC01285表达水平，结果显示CRC细胞内LINC01285的表达水均高于人正常结直肠粘膜细胞，其中SW620和HT-29细胞的表达水平最高（P < 0.001，图 4A）。
4.
LINC01285在人正常结直肠粘膜细胞（FHC）、结直肠癌细胞（A）及转染siRNA后的结肠癌细胞中的表达情况（B）
Expression of LINC01285 in normal epithelial cell lines (FHC), colorectal cancer cells (A) and colorectal cancer cells with siRNAtransfection (B). ***P < 0.001 vs si-Control.
采用siRNA干扰技术敲低SW620和HT-29细胞内LINC01285的表达量，通过荧光定量PCR验证敲低水平，结果显示，转染siRNA后SW620和HT-29细胞中的LINC01285表达量明显下调（P < 0.001，图 4B），表明LINC01285表达成功敲减，可用于后续实验。
2.4. LINC01285对CRC细胞增殖的影响
采用CCK-8实验分析LINC01285对CRC细胞增殖的影响，结果表明si-LINC01285-1和si-LINC01285-2组SW620和HT-29细胞的增殖能力明显低于si-Control组（P < 0.001，图 5）。
5.
LINC01285对SW620（A）和HT-29（B）细胞增殖能力的影响
Effect of LINC01285 on cell proliferation in SW620 (A) and HT-29 (B) cells. ***P < 0.001 vs si-control.
2.5. LINC01285对CRC细胞凋亡的影响
采用流式细胞仪分析LINC01285对CRC细胞凋亡的影响，结果表明si-LINC01285-1和si-LINC01285-2组SW620和HT-29细胞的早期凋亡率、晚期凋亡率以及总凋亡率均较si-Control组明显增加（P < 0.05，P < 0.01，P < 0.001，图 6）。
6.
LINC01285对SW620（A）和HT-29（B）细胞凋亡比例的影响。
Effect of LINC01285 on apoptosis in SW620 (A) and HT-29(B) cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs si-control.
2.6. LINC01285对CRC细胞迁移、侵袭的影响
采用Transwell法分析LINC01285对CRC细胞迁移、侵袭的影响，结果表明LINC01285-1和siLINC01285-2组SW620和HT-29细胞的迁移数量和侵袭数量均明显低于si-Control组（P < 0.001，图 7）。
7.
LINC01285对SW620（A）和HT-29（B）细胞迁移和侵袭能力的影响
Effect of LINC01285 on migration and invasion in SW620 (A) and HT-29 (B) cells (Original magnification: × 200).***P < 0.001 vs si-control.
2.7. LINC01285对CRC细胞上皮-间质转化（EMT）特性的影响
采用免疫印迹法分析LINC01285对CRC细胞EMT特性的影响，结果显示，LINC01285-1和si-LINC01285- 2组SW620和HT-29细胞与si-Control组相比较，上皮型标志物E-Cadherin表达明显增加，而间质型标志物N-Cadherin表达明显下降（P < 0.001，图 8）。
8.
LINC01285对SW620（A和B）和HT-29（C和D）细胞EMT表型的影响
Effect of LINC01285 on EMT phenotype in SW620 (A and B) and HT-29 (C and D) cells.***P < 0.001 vs si-Control.
3. 讨论
研究发现，包括结直肠癌在内的癌症的肿瘤组织和正常组织中lncRNA表达量存在显著性差异，提示其在肿瘤演变过程中起重要作用，有望成为恶性肿瘤诊断、治疗及预后监测的生物学标志物[21]。经典的CRC肿瘤标志物包括癌胚抗原（CEA）和糖类抗原199（CA199），血清CEA和CA199水平有助于判断CRC疗效、监测病情发展以及评估预后，可以作为术后随访期间参考的生物学指标，但存在特异性不强，灵敏度不高的缺点[22-24]。既往研究发现，包括结直肠癌在内的癌症的肿瘤组织和正常组织中lncRNA表达量存在显著性差异，提示其在肿瘤演变过程中起重要作用，越来越多新型的lncRNA被作为CRC生物学诊断指标和预后影响因素[25]。在结直肠癌中，lncRNA TINCR被发现可调控miR-107/ cdd36信号轴而促进结肠癌细胞的增殖[26]；lncRNA Ccsc1可促进结直肠癌细胞增殖、迁移和侵袭[27]。本研究首次证实LINC01285是CRC潜在的预后标志物，其高表达于CRC组织，并且与组织低分化、进展T分期及淋巴结转移等恶性特征密切相关，而LINC01285高表达的患者较低表达者明显缩短，进一步提示了LINC01285在疾病进展评估和生存预后判断方面具有重要指导意义。
恶性肿瘤的共同特征是细胞生长失控和细胞凋亡受抑，导致肿瘤细胞永生化。异常表达的lncRNA在调控肿瘤细胞增殖、凋亡和转移等恶性特征方面发挥重要作用[6]。本研究首次从细胞学水平探究了LINC01285对CRC细胞生物学特性的影响。功能学实验显示，敲低LINC01285表达能够显著抑制CRC细胞的增殖特性，并同时诱导细胞发生凋亡；另外，敲低LINC01285表达也能够抑制CRC迁移和侵袭能力，证实了LINC01285在CRC转移复发过程中起着关键的调控作用。
EMT是指上皮型细胞转为间质型细胞的生物学行为，EMT机制被认为是肿瘤细胞在获得高迁移和侵袭能力的关键过程，在肿瘤复发转移过程中发挥重要作用。EMT在分子层面通常表现为上皮行标志物表达下调和间质型标志物表达上调，上皮型细胞失去极性，细胞黏附能力下降，迁徙运动能力增强，导致肿瘤的发生发展和复发转移[28]。众多研究已表明lncRNA的异常表达可以促进EMT进程，从而调控肿瘤进展[29]。本研究显示，LINC01285表达敲低后细胞上皮型蛋白E-Cadherin表达上调，间质型蛋白N-Cadherin表达下调，提示LINC01285可能是通过EMT调控机制来增强CRC细胞的迁移和侵袭能力，并且进一步引出猜想：LINC01285可能是有效的防止CRC复发转移的药物作用靶点。
综上所述，本研究表明LINC01285可以作为CRC潜在的预后指标。LINC01285高表达于CRC，并提示患者的疾病进展和不良预后。LINC01285可能通过调节CRC增殖、迁移以及侵袭特性而发挥促癌作用；LINC01285参与调节肿瘤EMT进程，是其促进结肠癌转移和复发的可能机制之一。然而，本研究也存在样本量不足、机制实验深度不够等诸多缺点，LINC01285究竟通过何种机制调控CRC进展，仍有待继续深入研究。
Funding Statement
广州市番禺区科技计划项目（2022-Z04-024）
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