lncRNAs-REG轴调控结直肠腺瘤癌变的研究

Study on the regulation of colorectal adenoma malignant transformation by the lncRNAs-REG axis

摘要

目的：明确lncRNAs在结直肠腺瘤（CRA）向结直肠癌（CRC）进展过程中的表达谱及功能，筛选具有潜在调控作用的lncRNA及相关分子轴，寻找CRC癌前病变的风险评估指标及治疗靶点。方法：通过高通量RNA测序技术（RNA-seq）分析lncRNAs的表达谱，并基于miRanda算法构建lncRNA-mRNA互作网络，筛选具有潜在调控作用的基因。生物信息学技术预测差异表达lncRNA及mRNA的潜在功能，免疫组化半定量法检测REG家族基因的表达，实时荧光定量PCR（RT-qPCR）验证结果与RNA测序数据一致。Pearson相关性分析lncRNA与mRNA的表达关联。结果：共鉴定出461个CRA和CRC差异表达的lncRNAs，其中250个呈上调表达，211个呈下调表达。CRA中NONHSAT071873的表达上调最为显著。生物信息学技术对lncRNA及mRNA的潜在功能进行预测，发现NONHSAT071873可能作为ceRNA参与调控失调的REG3α表达。免疫组化半定量表达发现REG家族中REG3α在进展期CRA中上调最为显著。RT-qPCR验证结果与RNA-seq数据一致，显示NONHSAT071873及REG3α在CRA中均呈高表达。Pearson相关性分析证实，NONHSAT071873与REG3α表达呈显著正相关。结论：NONHSAT071873/REG3α轴可能是CRC癌前病变的评估风险指标及潜在治疗靶点。

Abstract

Objective: To clarify the expression profiles and functions of lncRNAs in the progression of colorectal adenoma(CRA) to colorectal carcinoma(CRC), screen lncRNAs with potential regulatory roles and related molecular axes, and provide a theoretical basis for the risk assessment of CRC precancerous lesions and the research on therapeutic targets. Methods: An evaluation of lncRNAs’ expression profiles was performed using High-throughput RNA sequencing, a network of lncRNA-mRNA interactions was established, utilizing miranda algorithms to explore potential genes and their positive activities. Additionally, an analysis utilizing bioinformatics techniques was performed to forecast the possible functions of lncRNAs and mRNAs, immunohistochemical semi-quantitative method was used to detect the expression of REG family genes, real-time quantitative PCR (RT-qPCR) was used to verify the results consistent with RNA-seq data, and Pearson correlation analysis was used to clarify the expression correlation. Results: A total of 461 lncRNAs exhibiting differentially expression were identified in patients with CRA and CRC, conprising 250 that were up-regulated and 211 that were down-regulated. Among them, the most notable up-regulation was observed for NONHSAT071873 in the context of CRA. The analysis utilizing bioinformatics techniques was performed to forecast the possible functions of lncRNAs and mRNAs, and NONHSAT071873 might be involved as a ceRNA in regulating the dysregulated REG3α expression. Immunohistochemistry semi-quantitative expression revealed that REG3α in the REG family was most significantly up-regulated in advanced CRA. Using real-time PCR, the findings aligned with the results obtained from RNA sequencing, demonstrating NONHSAT071873 and REG3α were highly expressed in CRA. Moreover, a notably relationship between NONHSAT071873 and REG3α was performed via Pearson correlation analysis. Conclusion: The NONHSAT071873/REG3α axis may act as a promising risk biomarker for evaluating CRC precancerous lesions and serve as a potential therapeutic target.

关键词

结肠肿瘤 / 长链非编码RNA / 高通量RNA测序 / NONHSAT071873

Key words

Colorectal neoplasms / Long non-coding RNA / High-throughput RNA sequencing / NONHSAT071873

参考文献

[1] Siegel RL , Wagle NS , Cercek A , et al. Colorectal cancer statistics, 2023[J]. CA Cancer J Clin, 2023, 73(3): 233-254. DOI: 10.3322/caac.21772.

[2] Huang Y , Wang Y , Wu H , et al. Exploring the mechanism of Jianpi Lishi Jiedu Granules against postoperative recurrence of colorectal adenoma based on IL—6/JAK/STAT3 signaling pathway[J]. Cell Signal, 2024, 127: 111535. DOI: 10.1016/j.cellsig.2024.111535.

[3] Vacante M , Ciuni R , Basile F , et al. Gut microbiota and colorectal cancer development: a closer look to the adenoma—carcinoma sequence[J]. Biomedicines, 2020, 8(11): 489. DOI: 10.3390/biomedicines8110489.

[4] Dekker E , Tanis PJ , Vleugels JLA , et al. Colorectal cancer[J]. Lancet, 2019, 394(10207): 1467-1480. DOI: 10.1016/S0140—6736(19)32319—6.

[5] Bao P , Wu C . Epidemiology, control and prevention of cancers in Shanghai[J]. Shanghai J Prev Med, 2020, 32: 955-962. DOI: 10.13668/j.issn.1004—9231.2020.11.001.

[6] National Clinical Research Center for Digestive Diseases (Shanghai), et al. Chinese consensus of early colorectal cancer screening(2019, Shanghai)[J]. Zhonghua Nei Ke Za Zhi, 2019, 58(10): 736-744. DOI: 10.3760/cma.j.issn.0578—1426.2019.10.004.

[7] Benson AB , Venook AP , Al—Hawary MM , et al. NCCN guidelines insights: colon cancer, version 2.2018[J]. J Natl Compr Canc Netw, 2018, 16(4): 359-369. DOI: 10.6004/jnccn.2018.0047.

[8] Gao QY , Chen HM , Sheng JQ , et al. The first year follow—up after colorectal adenoma polypectomy is important[J]. Front Med China, 2010, 4(4): 436-442. DOI: 10.1007/s11684—010—0107—8.

[9] Sahu A , Singhal U , Chinnaiyan AM . Long noncoding RNAs in cancer: from function to translation[J]. Trends Cancer, 2015, 1(2): 93-109. DOI: 10.1016/j.trecan.2015.08.005.

[10] Luo J , Qu J , Wu DK , et al. Long non—coding RNAs: a rising biotarget in colorectal cancer[J]. Oncotarget, 2017, 8(13): 22187-22202. DOI: 10.18632/oncotarget.15685.

[11] Liu JX , Li W , Li JT , et al. Screening key long non—coding RNAs in early—stage colon adenocarcinoma by RNA—sequencing[J]. Epigenomics, 2018, 10(9): 1215-1228. DOI: 10.2217/epi—2018—0076.

[12] Huang X , Cai W , Yuan W , et al. Identification of key lncRNAs as prognostic prediction models for colorectal cancer based on LASSO[J]. Int J Clin Exp Pathol, 2020, 13(4): 675-684. DOI: 10.1186/s12943—020—01123—9.

[13] Nagtegaal ID , Odze RD , Klimstra D , et al. WHO classification of tumours editorial board the 2019 WHO classification of tumours of the digestive system[J]. Histopathology, 2020, 76(2): 182-188. DOI: 10.1111/his.14060.

[14] Bolger AM , Lohse M , Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data[J]. Bioinformatics, 2014, 30(15): 2114-2120. DOI: 10.1093/bioinformatics/btu170.

[15] Kim D , Langmead B , Salzberg SL . HISAT: a fast spliced aligner with low memory requirements[J]. Nat Methods, 2015, 12(4): 357-360. DOI: 10.1038/nmeth.3317.

[16] Pertea M , Pertea GM , Antonescu CM , et al. StringTie enables improved reconstruction of a transcriptome from RNA—seq reads[J]. Nat Biotechnol, 2015, 33(3): 290-295. DOI: 10.1038/nbt.3122.

[17] Kong L , Zhang Y , Ye ZQ , et al. CPC: assess the protein—coding potential of transcripts using sequence features and support vector machine[J]. Nucleic Acids Res, 2007, 35(Web Server issue): W345-W349. DOI: 10.1093/nar/gkm391.

[18] Li A , Zhang J , Zhou Z . PLEK: a tool for predicting long non—coding RNAs and messenger RNAs based on an improved k—mer scheme[J]. BMC Bioinformatics, 2014, 15: 311. DOI: 10.1186/1471—2105—15—311.

[19] Sun L , Luo H , Bu D , et al. Utilizing sequence intrinsic composition to classify protein—coding and long non—coding transcripts[J]. Nucleic Acids Res, 2013, 41(17): e166. DOI: 10.1093/nar/gkt646.

[20] Finn RD , Bateman A , Clements J , et al. Pfam: the protein families database[J]. Nucleic Acids Res, 2014, 42(D1): D222-D230. DOI: 10.1093/nar/gkt1223.

[21] Love MI , Anders S , Kim V , et al. RNA—Seq workflow: gene—level exploratory analysis and differential expression[J]. F1000Res, 2015, 4: 1070. DOI: 10.12688/f1000research.7368.1.

[22] Guttman M , Amit I , Garber M , et al. Chromatin signature reveals over a thousand highly conserved large non—coding RNAs in mammals[J]. Nature, 2009, 458(7235): 223-227. DOI: 10.1038/nature07672.

[23] Qi XL , Zhang DH , Wu N , et al. ceRNA in cancer: possible functions and clinical implications[J]. J Med Genet, 2015, 52(10): 710-718. DOI: 10.1136/jmedgenet—2015—103334.

[24] Guo LL , Song CH , Wang P , et al. Competing endogenous RNA networks and gastric cancer[J]. World J Gastroenterol, 2015, 21(41): 11680-11687. DOI: 10.3748/wjg.v21.i41.11680.

[25] Xu J , Zhou L , Ji L , et al. The REGγ—proteasome forms a regulatory circuit with IκB and NFκB in experimental colitis[J]. Nat Commun, 2016, 7: 10761. DOI: 10.1038/ncomms10761.

[26] Chaleshi V , Irani S , Alebouyeh M , et al. Association of lncRNA—p53 regulatory network and p53 with clinicopathological features of colorectal tumors[J]. Oncol Lett, 2020, 19(6): 3937-3949. DOI: 10.3892/ol.2020.11695.

[27] Tian Y , Xu Y , Wang H , et al. Comprehensive analysis of microarray expression profiles of circRNAs and lncRNAs in human colorectal cancer[J]. Funct Integr Genomics, 2019, 19(2): 311-327. DOI: 10.1007/s10142—018—0645—8.

[28] Qi Y , Qi H , Liu Z , et al. Bioinformatics analysis of key genes and pathways in colorectal cancer[J]. J Comput Biol, 2019, 26(4): 364-375. DOI: 10.1089/cmb.2018.0215.

[29] Bai B , Xie B , Pan Z , et al. Identification of candidate genes and long non—coding RNAs associated with ATP5J in colorectal cancer[J]. Int J Oncol, 2018, 52(4): 1129-1138. DOI: 10.3892/ijo.2018.4293.

[30] Yu Y , Tian X . Analysis of genes associated with prognosis of lung adenocarcinoma based on GEO and TCGA databases[J]. Medicine (Baltimore), 2020, 99(19): e20183. DOI: 10.1097/MD.0000000000020183.

[31] Ye X , Zeng T , Kong W , et al. Integrative analyses of genes associated with fulminant type 1 diabetes[J]. J Immunol Res, 2020, 2020: 1025857. DOI: 10.1155/2020/1025857.

[32] Zhou XG , Huang XL , Liang SY , et al. Identifying miRNA and gene modules of colon cancer associated with pathological stage[J]. Onco Targets Ther, 2018, 11: 2815-2830. DOI: 10.2147/OTT.S164641.

[33] Tutino V , Defrancesco ML , Tolomeo M , et al. The expression of riboflavin transporters in human colorectal cancer[J]. Anticancer Res, 2018, 38(5): 2659-2667. DOI: 10.21873/anticanres.12558.

[34] Queiroz CJS , Song F , Reed KR , et al. NAP1L1: a novel human colorectal cancer biomarker[J]. Front Oncol, 2020, 10: 1565. DOI: 10.3389/fonc.2020.01565.

[35] Zheng HC , Sugawara A , Okamoto H , et al. Expression profile of the REG gene family in colorectal carcinoma[J]. J Histochem Cytochem, 2011, 59(1): 106-115. DOI: 10.1369/0022155410391121.

[36] Kaprio T , Hagström J , Mustonen H , et al. REG4 independently predicts better prognosis in non—mucinous colorectal cancer[J]. PLoS One, 2014, 9(10): e109600. DOI: 10.1371/journal.pone.0109600.

[37] Kawasaki Y , Matsumura K , Miyamoto M , et al. REG4 is a transcriptional target of GATA6 and essential for colorectal tumorigenesis[J]. Sci Rep, 2015, 5: 14291. DOI: 10.1038/srep14291.

[38] Okamoto K , Fujimori T , Yamaguchi T , et al. Overexpression of regenerating gene Iα in sessile serrated adenoma/polyps of the colon[J]. Diagn Pathol, 2013, 8: 187. DOI: 10.1186/1746—1596—8—187.

[39] Ye Y , Xiao L , Wang SJ , et al. Up—regulation of REG3A in colorectal cancer cells confers proliferation[J]. Oncotarget, 2016, 7(4): 3921-3933. DOI: 10.18632/oncotarget.6888.

[40] Denzler R , Agarwal V , Stefano J , et al. Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance[J]. Mol Cell, 2014, 54(5): 766-776. DOI: 10.1016/j.molcel.2014.03.023.

[41] Wang L , Cho KB , Li Y , et al. Long noncoding RNA—mediated competing endogenous RNA networks in colorectal cancer[J]. Int J Mol Sci, 2019, 20(22): 5758. DOI: 10.3390/ijms20225758.

[42] Zhao YH , Du TT , Du LT , et al. Long noncoding RNA LINC02418 regulates MELK expression as a ceRNA in colorectal cancer[J]. Cell Death Dis, 2019, 10(8): 568. DOI: 10.1038/s41419—019—1998—8.

[43] Ma ZF , Han CC , Xia WJ , et al. Circ5615 functions as a ceRNA to promote colorectal cancer progression[J]. Cell Death Dis, 2020, 11(5): 356. DOI: 10.1038/s41419—020—0878—8.

[44] Liu Y , Liu DL , Dong LL , et al. miR—612 suppresses stem cell—like property of hepatocellular carcinoma cells[J]. Cell Death Dis, 2016, 7(9): e2377. DOI: 10.1038/cddis.2016.281.

[45] Sheng L , He P , Yang X , et al. miR—612 negatively regulates colorectal cancer growth and metastasis by targeting AKT2[J]. Cell Death Dis, 2015, 6(7): e1808. DOI: 10.1038/cddis.2015.177.

[46] 赵晓东， 刘洋， 陈志强， 等 . 结直肠腺瘤术后复发风险预测模型构建及临床验证[J]. 中国现代普通外科进展， 2025， 28（3）： 198-202. DOI: 10.3969/j.issn.1009—9905.2025.03.008.

[47] Wang L , Bo X , Zheng Q , et al. Paired box 8 suppresses tumor angiogenesis and metastasis in gastric cancer via miR—612[J]. J Exp Clin Cancer Res, 2018, 37(1): 159. DOI: 10.1186/s13046—018—0817—9.

[48] Liu M , Chen Y , Huang B , et al. Tumor—suppressing effects of microRNA—612 in bladder cancer cells[J]. Int J Oncol, 2018, 52(6): 1923-1933. DOI: 10.3892/ijo.2018.4319.

[49] Zhang L , Wang DL , Yu P . LncRNA H19 regulates HOXA10 in endometrial carcinoma through competing with miR—612[J]. Eur Rev Med Pharmacol Sci, 2018, 22(15): 4820-4827. DOI: 10.1471—2105—18—4820.

[50] Jin Y , Zhou X , Yao X , et al. MicroRNA—612 inhibits cervical cancer progression by targeting NOB1[J]. J Cell Mol Med, 2020, 24(5): 3149-3156. DOI: 10.1111/jcmm.15042.

基金资助

浦东新区科技发展基金(PKJ2022-Y84)