目的 探究結(jié)直腸腺癌經(jīng)典序列轉(zhuǎn)化中的差異基因及其與免疫細(xì)胞浸潤(rùn)和患者生存時(shí)間的相關(guān)性，并預(yù)測(cè)具有靶向作用的中藥。方法 從GEO數(shù)據(jù)庫(kù)尋找符合要求的配對(duì)樣本數(shù)據(jù)，使用R語言篩選序列轉(zhuǎn)化過程中始終存在差異表達(dá)的關(guān)鍵基因，并通過基因本體（gene ontology，GO）進(jìn)行功能注釋。利用基因表達(dá)譜互動(dòng)分析數(shù)據(jù)庫(kù)（Gene Expression Profiling Interactive Analysis，GEPIA）和人類蛋白質(zhì)圖譜數(shù)據(jù)庫(kù)驗(yàn)證關(guān)鍵基因在轉(zhuǎn)錄組和蛋白組水平的表達(dá)。通過Kaplan-Meier Plotter和TIMER數(shù)據(jù)庫(kù)分別進(jìn)行基因表達(dá)水平與結(jié)直腸癌患者生存時(shí)間、免疫細(xì)胞浸潤(rùn)的相關(guān)性分析。最后，利用Coremine Medical、BATMAN-TCM和草本植物數(shù)據(jù)庫(kù)進(jìn)行中藥預(yù)測(cè)，使用Cytoscape構(gòu)建“基因-中藥”靶向網(wǎng)絡(luò)，綜合Network Analyzer和Swiss ADME數(shù)據(jù)庫(kù)評(píng)估活性成分類藥性，PyMOL軟件進(jìn)行分子對(duì)接。結(jié)果 獲得結(jié)直腸腺癌經(jīng)典序列轉(zhuǎn)化的關(guān)鍵差異基因48個(gè)，篩選得到9個(gè)核心基因：TAGLN、FN1、ACTA2、DCN、REG4、GUCA2A、SPINK4、DEFA6、ZG16。與正常組織相比，結(jié)直腸癌組織中的核心基因在轉(zhuǎn)錄組和蛋白質(zhì)水平上均具有統(tǒng)計(jì)學(xué)差異。功能性分析提示其主要參與腸道免疫、細(xì)胞外基質(zhì)構(gòu)成等過程。相關(guān)性分析表明核心基因的表達(dá)水平能夠影響結(jié)直腸癌患者的總生存期和無復(fù)發(fā)生存期，CD4⁺T細(xì)胞、巨噬細(xì)胞、中性粒細(xì)胞和樹突細(xì)胞是被核心基因調(diào)節(jié)的主要免疫細(xì)胞群。獲得靶向中藥129味，包括36味藥食同源中藥和27味毒性中藥，以補(bǔ)益、清熱、化痰為主，性味以寒、平、溫、甘、苦、辛居多，多歸肝、肺、脾經(jīng)，其中葡萄、金蕎麥、半夏、地龍、肉蓯蓉的中心度值較高，篩選得到6種關(guān)鍵活性成分纈氨酸、鼠李糖、脯氨酸、橙花醛、蛋氨酸、甘氨酸，分子對(duì)接顯示中藥小分子活性成分與靶向蛋白質(zhì)對(duì)接穩(wěn)定。結(jié)論 篩選出“正常黏膜-腸腺瘤-腸腺癌”序列轉(zhuǎn)化中始終差異表達(dá)的關(guān)鍵疾病基因，這些基因可能通過參與免疫調(diào)節(jié)、細(xì)胞外基質(zhì)等過程影響結(jié)直腸腺癌患者的遠(yuǎn)期預(yù)后，金蕎麥、葡萄、肉蓯蓉等藥食同源中藥有望成為新藥開發(fā)來源，為預(yù)防腸道早癌和癌癥進(jìn)展提供新的思路。

Objective To identify differentially expressed genes (DEGs) in the process of colorectal mucosa-adenoma-adenocarcinoma sequence and their relationships with immune cell infiltration and survival time of patients with colorectal cancer (CRC), and to predict potential targeted traditional Chinese medicines (TCMs). Methods The paired data was downloaded from the GEO database to obtain DEGs using R. Biological pathways were explored through gene ontology (GO). Expression levels of DEGs were validated using GEPIA and the Human Protein Atlas. Kaplan-Meier Plotter and TIMER databases were used to assess the correlation between DEGs and survival time, as well as immune cell infiltration. Finally, Coremine Medical, BATMAN, and Herb databases were used to predict TCMs. A “Gene-TCM” network was constructed with Cytoscape. Combined with Network Analyzer, the drug-likeness of the active compounds was evaluated using Swiss ADME databases. Molecular docking was performed with PyMOL software. Results Forty-eight key DGEs were identified in transformation, with nine hub genes: TAGLN, FN1, ACTA2, DCN, REG4, GUCA2A, SPINK4, DEFA6, and ZG16. At both at the transcriptome and proteome levels, the hub genes exhibited statistically significant differences in expression between tumor and normal tissues. Functional analysis indicated they were involved in intestinal immunity and extracellular matrix composition. Correlation analysis suggested that the expression levels of hub genes can affect the overall survival (OS) and relapse-free survival (RFS) of patients with CRC. CD4⁺ T cells, macrophages, neutrophils, and dendritic cells were primary immune cells regulated by hub genes. There were 130 potential TCMs, including 36 Medicine-food homologous TCMs and 27 toxic TCMs. The efficacy was mainly to tonify, clear heat, and resolve phlegm. Moreover, their four natures and five flavors were primarily cold, neutral, warm, sweet, bitter, and pungent. Most of them belonged to the liver, lung, and spleen meridians. Putao (Vitis vinifera), Jinqiaomai (Fagopyri Dibotryis Rhizoma), Banxia (Pinelliae Rhizoma), Dilong (Pheretima), Roucongrong (Cistanches Herba) had the strongest connections to hub genes. Six key active compounds were valine, L-rhamnose, proline, neral, D-methionine, and glycine. Molecular docking showed that they were stable in docking with markers. Conclusion This study identified genes that were consistently differentially expressed during the sequence. These DEGs may affect the prognosis of patients with CRC by regulating immune cells and extracellular matrix. Medicine-food homologous TCMs such as Fagopyri Dibotryis Rhizoma, Vitis vinifera, and Cistanches Herba could be promising sources for drug development. This study sheds new light on the prevention of colorectal adenoma and cancer.

Q811.4;R285

北京市自然科學(xué)基金項(xiàng)目（7242237）