Krüppel-like factor 4 suppresses the proliferation and phenotypic transition of pericardial interstitial cells via PI3K/Akt signaling pathway
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摘要:
目的 通过微阵列分析识别过表达Krüppel样因子4（KLF4）的心包间质细胞（PIC）中mRNA和环状RNA（circRNA）的表达谱，探讨KLF4在心包纤维化中的潜在调节机制。 方法 使用携带KLF4基因的腺病毒（Ad.KLF4）或携带增强型绿色荧光蛋白基因的腺病毒（Ad.EGFP）感染PIC。通过微阵列分析识别差异表达的mRNA和circRNA。利用基因本体（GO）、京都基因与基因组百科全书（KEGG）进行基因功能和信号通路富集分析。采用CCK-8和蛋白质印迹法评估KLF4对PIC增殖和表型转化的影响，并描绘circRNA-miRNA-mRNA调控网络。 结果 与感染Ad.EGFP的PIC相比，在感染Ad.KLF4的PIC中识别出6 197个差异表达的mRNA、393个差异表达的circRNA。GO分析显示，差异表达mRNA主要富集于细胞增殖与分化相关的生物学过程。KEGG通路富集分析提示差异表达mRNA与PI3K/Akt信号通路相关。蛋白质印迹法和CCK-8实验确认KLF4能够抑制TGF-β1诱导的细胞增殖和表型转化，这一作用可能通过PI3K/Akt信号通路实现。生物信息学分析发现，富集于PI3K/Akt通路的circRNA宿主基因包括溶血磷脂酸受体3（LPAR3）、血小板反应蛋白1（THBS1）和蛋白磷酸酶2催化亚基α（PPP2CA），并据此构建KLF4调控的PI3K/Akt通路相关circRNA-miRNA-mRNA网络。 结论 KLF4可能通过调节PI3K/Akt信号通路相关的circRNA-miRNA-mRNA网络抑制PIC的增殖和表型转化，为抗纤维化治疗提供了新靶点。 -
关键词:
- Krüppel样因子4 /
- 心包间质细胞 /
- 调控网络 /
- 细胞增殖 /
- 表型转化
Abstract:Objective To identify the expression profiles of mRNA and circular RNA (circRNA) in pericardial interstitial cells (PICs) that overexpress Krüppel-like factor 4 (KLF4) through microarray analysis, and to explore the potential regulatory mechanism of KLF4 in pericardial fibrosis. Methods PICs were infected with adenovirus carrying KLF4 (Ad.KLF4) or enhanced green fluorescent protein gene (Ad.EGFP). Microarray analysis was used to identify differentially expressed mRNAs and circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze gene functions and signaling-pathway enrichment. The effects of KLF4 on PIC proliferation and phenotypic transformation were evaluated by cell counting kit 8 (CCK-8) and Western blotting, and a circRNA-miRNA-mRNA regulatory network was constructed. Results Compared with PICs infected with Ad.EGFP, significantly differentially expressed mRNA (n＝6 197) and circRNA (n＝393) were identified in PICs infected with Ad.KLF4. GO analysis showed that the differentially expressed genes were significantly enriched in biological processes related to cell proliferation and differentiation. KEGG pathway enrichment analysis showed that the differentially expressed mRNAs were associated with the PI3K/Akt signaling pathway. Western blotting and CCK-8 assays confirmed that KLF4 could inhibit TGF-β1-induced cell proliferation and phenotypic transition, and this effect might be achieved through the PI3K/Akt signaling pathway. Bioinformatics analysis revealed that the circRNA host genes enriched in the PI3K/Akt pathway included lysophosphatidic acid receptor 3 (LPAR3), thrombospondin 1 (THBS1), and protein phosphatase 2 catalytic subunit α (PPP2CA); and based on this, a KLF4-regulated PI3K/Akt pathway-related circRNA-miRNA-mRNA network was constructed. Conclusion KLF4 may inhibit the proliferation and phenotypic transition of PICs by regulating the circRNA-miRNA-mRNA network related to the PI3K/Akt signaling pathway, providing a new target for anti-fibrotic therapy. -
心包纤维化常见于缩窄性心包炎[1]和自体心包瓣衰败[2]等疾病。心包间质细胞（pericardial interstitial cell，PIC）是心包的主要细胞成分，起着维持心包正常结构和功能的作用。它们具有成纤维细胞的特征，在致病因素的作用下，活化的PIC向成纤维样细胞表型转化，这是心包纤维化的细胞学机制[3]。
Krüppel样因子（Krüppel-like factor，KLF）由一组具有保守锌指结构域的DNA结合转录调节因子组成。KLF4是KLF家族的成员，参与增殖[4]、分化[5]和多能性[6]等细胞过程。近年来，越来越多的研究表明，KLF4在多种纤维化疾病的发病机制中发挥着重要的调节作用。KLF4作为多效性转录调控因子，在肺、肾及心血管纤维化进程中的作用呈现矛盾性。例如，在博来霉素诱导的肺纤维化模型中，KLF4通过抑制TGF-β1诱导的上皮间质转化，减轻了肺纤维化的程度[7]；KLF4通过维持平滑肌细胞来源祖细胞的前体细胞表型，抑制其向肌成纤维细胞分化，从而减轻动脉纤维化[8]；KLF4通过结合并激活TGF-β1启动子，促进心脏成纤维细胞向肌成纤维细胞分化及胶原合成，从而增强心肌细胞纤维化的过程[9]；KLF4通过上调Yes相关蛋白（Yes-associated protein，YAP）的表达和核转位，促进肾小管上皮细胞分泌TGF-β和结缔组织生长因子（connective tissue growth factor，CTGF），从而增强缺血再灌注诱导的肾纤维化过程[10]。目前KLF4对PIC的功能及心包纤维化的调控机制尚未明确。本研究通过微阵列分析揭示KLF4过表达对PIC中mRNA和环状RNA（circular RNA，circRNA）表达谱的影响，结合功能验证实验与竞争性内源RNA（competing endogenouse RNA，ceRNA）网络构建，揭示KLF4可能通过调控PI3K/Akt信号通路抑制PIC增殖与肌成纤维细胞表型转化，为心包纤维化的靶向治疗提供新思路。
1 材料和方法
1.1 PIC的分离与培养
采用组织块贴壁的方法分离培养原代PIC。心包组织来源于海军军医大学第一附属医院心脏移植受体捐献，经医院伦理审查委员会批准。首先，将新鲜心包组织剪成约1 mm3大小的组织块，贴壁于24孔板中。每孔加入500 μL含20% FBS的DMEM培养基（美国Gibco公司），然后将其放置于细胞培养箱中，待PIC爬出。当细胞生长至融合度达到90%时进行传代，使用第3代细胞进行后续细胞学实验。
1.2 细胞感染与处理
使用AdEasy腺病毒系统构建重组KLF4腺病毒（Ad.KLF4）。为了进行腺病毒介导的基因转移，PIC在感染率为50%的条件下接触腺病毒载体进行感染。以携带增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）基因的腺病毒（Ad.EGFP）感染的PIC作为对照。在用Ad.KLF4或Ad.EGFP培养PIC过夜后，去除培养基，在有或没有TGF-β1（20 ng/mL，上海碧云天生物技术有限公司）的条件下进一步培养感染的细胞，持续3 d。
1.3 微阵列分析
微阵列分析由上海欧易生物医学科技有限公司完成。使用美国Agilent Technologies公司的人类lncRNA微阵列芯片2018（4×180 k，设计ID：085630）、芯片扫描仪及配套软件进行分析，流程如下：（1）RNA处理。收集PIC，将总RNA转录为双链cDNA，随后合成cRNA，并用Cy3-CTP进行标记。（2）微阵列分析。标记后的cRNA用于微阵列分析。洗涤后，使用Agilent Scanner G2505C芯片扫描仪扫描微阵列，获取阵列图像。（3）数据提取与分析。获取的图像利用Agilent Feature Extraction 10.7.1.1软件进行分析，获得原始数据，然后使用Genespring 14.8软件包进行定量标准化和后续数据处理。（4）数据筛选。在数据分析中，选择在两个条件中至少有一个条件标记为“检测到”的探针进行进一步分析。设置阈值|log2(FC)|≥1（FC为差异倍数，fold change）、P≤0.05，识别两组之间的差异表达circRNA和mRNA。（5）层次聚类。进行层次聚类分析，以显示样本之间可区分的基因表达模式。
1.4 基因功能和通路富集分析
选择差异表达的基因进行靶向预测。使用DAVID（https://david.ncifcrf.gov/）数据库进行基因本体（Gene Ontology，GO）功能富集分析，GO功能分为生物过程、细胞成分和分子功能。使用京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG；http://www.kegg.jp/kegg/）进行通路富集分析，将基因映射到相应的信号通路，计算每个通路的富集伪发现率（false discovery rate，FDR），设置FDR＜0.05作为显著性阈值。
1.5 蛋白质印迹法
收集PIC，用冷PBS洗涤1次，然后在冰上用RIPA缓冲液裂解15 min。通过BCA法测定总蛋白质浓度。蛋白质通过SDS-PAGE分离，并电转移到NC膜上。电转膜在4 ℃下与一抗孵育过夜，并在室温下用适当的HRP标记的二抗孵育1 h。使用的一抗包括KLF4抗体（英国Abcam公司，稀释比例1∶1 000）、Akt抗体（美国Cell Signaling Technology公司，稀释比例1∶1 000）、磷酸化Akt（p-Akt，Ser473）抗体（美国Cell Signaling Technology公司，稀释比例1∶2 000）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）抗体（美国Affinity Biosciences公司，稀释比例1∶1 000）和增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）抗体（美国Affinity Biosciences公司，稀释比例1∶1 000）。检测结果使用ImageJ软件进行定量分析。蛋白条带经8-bit灰度转换后，扣除背景，测量目标条带的平均灰度值。以GAPDH作为内参，计算目的蛋白的相对表达量。
1.6 CCK-8实验
分别用Ad.KLF4和Ad.EGFP感染PIC，以每孔2×103个细胞的密度接种于96孔板中，在细胞培养箱中培养24 h。向每孔加入10 μL的CCK-8溶液（上海碧云天生物技术有限公司），在37 ℃下孵育2 h。使用分光光度计在450 nm波长处检测各孔的光密度。以对照组的光密度值为基准，对相对细胞活力进行标准化处理。
1.7 KLF4启动子结合位点预测
为预测KLF4在circRNA宿主基因启动子区域的潜在结合位点，首先从真核生物启动子数据库（Eukaryotic Promoter Database，EPD；https://epd.epfl.ch/）中获取相关基因的启动子序列。随后，利用JASPAR数据库（https://jaspar.genereg.net/）对上述启动子序列进行转录因子结合位点预测。以KLF4的已知DNA结合基序为参考，对启动子区域中潜在的KLF4结合位点进行筛选和注释，并绘制其在启动子区域中的分布示意图。
1.8 circRNA-miRNA-mRNA调控网络构建
使用CircRNA Interactome数据库（https://circinteractome.nia.nih.gov/）预测感兴趣circRNA的靶向miRNA。使用miRDB数据库（http://mirdb.org/）和Tarbase数据库（https://www.hsls.pitt.edu/obrc/index.php?page=URL1237572545）预测miRNA与mRNA之间的相互作用，随后使用韦恩图（http://bioinformatics.psb.ugent.be/webtools/Venn/）对预测的mRNA与数据库中差异表达的mRNA进行交集分析。基于差异表达的circRNA、预测的miRNA和差异表达的mRNA之间的相互作用，建立circRNA-miRNA-mRNA调控网络。使用Cytoscape 3.40软件可视化该调控网络。
1.9 统计学处理
使用GraphPad Prism 9软件进行统计学分析。计量资料以x±s表示，两组间比较采用独立样本t检验。检验水准（α）为0.05。
2 结果
2.1 差异表达mRNA和circRNA的识别
将新鲜心包组织块剪成小碎片，并将其贴壁于培养板中，大约在贴壁后1周，PIC开始从组织块边缘爬出，大部分细胞呈长梭形，部分细胞呈多角形（图 1A）。分别用Ad.KLF4和Ad.EGFP感染PIC，蛋白质印迹法分析（图 1B）显示，Ad.KLF4组KLF4蛋白表达量（1.891±0.160）约为Ad.EGFP组（0.445±0.047）的4.25倍。对Ad.KLF4和Ad.EGFP感染的PIC之间的mRNA和circRNA表达谱进行比较，共识别出6 197个差异表达的mRNA，其中3 743个mRNA在Ad.KLF4感染的PIC中上调，2 454个mRNA下调；此外，共识别出393个差异表达的circRNA，其中212个circRNA在Ad.KLF4感染的PIC中上调，181个circRNA下调。火山图显示了这些差异表达的mRNA（图 1C）和circRNA（图 1D）的表达分布。
图 1 PIC的分离培养、KLF4过表达及差异表达mRNA和circRNA的识别Fig. 1 Isolation and culture of PICs, KLF4 overexpression, and identification of differentially expressed mRNAs and circRNAsA: Isolated and cultured PICs; B: KLF4 protein expression in adenovirus-infected PICs (**P<0.01. n = 6, x±s); C: Volcano plot showing 3 743 upregulated and 2 454 downregulated mRNAs in Ad.KLF4-infected PICs compared with Ad.EGFP-infected PICs; D: Volcano plot showing 212 upregulated and 181 downregulated circRNAs in Ad.KLF4-infected PICs compared with Ad.EGFP-infected PICs. PIC: Pericardial interstitial cell; KLF4: Krüppel-like factor 4; circRNA: Circular RNA; Ad.KLF4: Adenovirus carrying the KLF4 gene; Ad.EGFP: Adenovirus carrying the enhanced green fluorescent protein gene; FC: Fold change; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
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2.2 差异表达mRNA的功能分类
GO富集分析结果（图 2A）显示，在生物过程维度，差异表达的mRNA主要富集在“p53类介导的内源性凋亡信号通路”“蛋白酪氨酸激酶活性的正调控”“蛋白质重折叠”等方面，其中上调的差异表达mRNA主要富集于“有丝分裂细胞质分裂”“表皮发育”“半黏附体组装”，而下调的差异表达mRNA主要富集于“未折叠蛋白结合”“蛋白质重折叠”“对未折叠蛋白的反应”；在细胞成分维度，差异表达的mRNA主要富集在“轴突初始段”“细胞表面”“质膜的整合成分”等方面，其中上调的差异表达mRNA主要富集于“回收内体”“纺锤微管”“黏附体”，而下调的差异表达mRNA主要富集于“神经丝”“核质”“突触囊泡”；在分子功能维度，差异表达的mRNA主要富集在“神经纤毛蛋白结合”“化学排斥活性”“神经调节素结合”等方面，其中上调的差异表达mRNA主要富集于“3-氯烯丙醛脱氢酶活性”“异构酶活性”“醛脱氢酶（NAD）活性”，而下调的差异表达mRNA主要富集于“未折叠蛋白结合”“钾通道活性”“锌离子结合”。
图 2 Ad.KLF4感染的PIC与Ad.EGFP感染的PIC中差异表达mRNA的GO（A）和KEGG（B）富集分析Fig. 2 GO (A) and KEGG (B) enrichment analyses of differentially expressed mRNAs in Ad.KLF4-infected PICs compared to Ad.EGFP-infected PICsAd.KLF4: Adenovirus carrying the Krüppel-like factor 4 gene; PIC: Pericardial interstitial cell; Ad.EGFP: Adenovirus carrying the enhanced green fluorescent protein gene; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; ListHit: Number of genes enriched in the given term or pathway; Process: Biological process; Function: Molecular function; Component: Cellular component; MAP: Mitogen-activated protein; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; ECM: Extracellular matrix; SNARE: Soluble N-ethylmaleimide-sensitive factor attachment protein receptor; MAPK: Mitogen-activated protein kinase.下载:
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KEGG通路富集分析结果（图 2B）显示，差异表达的mRNA与“色氨酸代谢”“PI3K/Akt信号通路”“ECM-受体相互作用”通路相关。在22个差异KEGG通路中，上调的差异表达mRNA主要富集在“代谢”通路，包括“β-丙氨酸代谢”“组氨酸代谢”“缬氨酸、亮氨酸和异亮氨酸降解”“色氨酸代谢”“花生四烯酸代谢”，而下调的差异表达mRNA则主要富集在“PI3K/Akt信号通路”“癌症通路”“麻疹”等方面。
2.3 差异表达circRNA的功能分类
GO富集分析结果（图 3A）显示，在生物过程维度，差异表达的circRNA宿主基因主要富集在“细胞衰老”和“参与中脑多巴胺能神经元分化的Wnt信号通路”等方面，其中上调的差异表达circRNA宿主基因主要富集于“翻译起始”“线粒体组织”“负调控双链断裂修复”，而下调的差异表达circRNA宿主基因主要富集于“参与中脑多巴胺能神经元分化的Wnt信号通路”“中脑多巴胺能神经元分化”；在细胞成分维度，差异表达的circRNA宿主基因主要富集在“核质”“膜”“细胞质”等方面，其中上调的差异表达circRNA宿主基因主要富集于“核质”“膜”“细胞质”，而下调的差异表达circRNA宿主基因主要富集于“膜”“核质”“核膜”；在分子功能维度，差异表达的circRNA宿主基因主要富集在“蛋白质结合”“聚（A）RNA结合”“泛素-蛋白转移酶活性”等方面，其中上调的差异表达circRNA宿主基因主要富集于“聚（A）RNA结合”“泛素-蛋白转移酶活性”“rRNA结合”，而下调的差异表达circRNA宿主基因主要富集于“蛋白质结合”“克拉霉素重链结合”“参与Wnt信号通路的共受体活性”。
图 3 Ad.KLF4感染的PIC与Ad.EGFP感染的PIC中差异表达circRNA宿主基因的GO（A）和KEGG（B）富集分析Fig. 3 GO (A) and KEGG (B) enrichment analyses of differentially expressed circRNA host genes in Ad.KLF4-infected PICs compared to Ad.EGFP-infected PICsAd.KLF4: Adenovirus carrying the Krüppel-like factor 4 gene; PIC: Pericardial interstitial cell; Ad.EGFP: Adenovirus carrying the enhanced green fluorescent protein gene; circRNA: Circular RNA; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; ListHit: Number of genes enriched in the given term or pathway; Function: Molecular function; Component: Cellular component; Process: Biological process; ATP: Adenosine triphosphate; TGF: Transforming growth factor; AMPK: AMP-activated protein kinase; TCA: Tricarboxylic acid.下载:
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KEGG通路富集分析结果（图 3B）显示，差异表达的circRNA宿主基因与“DNA复制”“内质网中的蛋白质加工”“泛素介导的蛋白质降解”等通路相关。在13条差异KEGG通路中，下调的差异表达circRNA宿主基因主要富集于“胰高血糖素信号通路”“内质网中的蛋白质加工”“紧密连接”，而上调的差异表达circRNA宿主基因则主要富集在“DNA复制”“泛素介导的蛋白质降解”“嘧啶代谢”方面。
2.4 KLF4通过PI3K/Akt信号通路调控PIC增殖和分化
上述生物信息学分析显示，差异表达mRNA主要富集于细胞增殖与分化相关的生物学过程，并与PI3K/Akt信号通路相关。因此，本研究探讨KLF4是否调节了PI3K/Akt信号通路的激活。蛋白质印迹法分析结果显示，TGF-β1处理诱导了PI3K/Akt信号通路的激活，而KLF4能够抑制这种激活，表现为p-Akt表达的降低（图 4A）。此外，KLF4抑制了TGF-β1诱导的PIC增殖和肌成纤维细胞表型转化，表现为PCNA和α-SMA表达的降低（图 4A）。CCK-8实验结果显示，在感染Ad.KLF4的PIC中，TGF-β1诱导的细胞增殖能力降低（图 4B）。
图 4 KLF4对PIC中PI3K/Akt信号通路的影响Fig. 4 Effects of KLF4 on PI3K/Akt signaling pathway in PICsA: PICs were infected by Ad.KLF4 or Ad.EGFP, followed by treatment with or without TGF-β1 (20 ng/mL) for 3 d. Protein expression levels were analyzed by Western blotting. B: The effect of KLF4 on PIC proliferation was evaluated using the cell counting kit 8 assay (*P<0.05. n = 6, x±s). C: Bioinformatics analysis revealed the presence of KLF4 binding sites in the promoter regions of LPAR3, THBS1, and PPP2CA. KLF4: Krüppel-like factor 4; PIC: Pericardial interstitial cell; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; TGF-β1: Transforming growth factor β1; Ad.EGFP: Adenovirus carrying enhanced green fluorescent protein gene; Ad.KLF4: Adenovirus carrying KLF4 gene; p-Akt: Phosphorylated Akt; α-SMA: α-smooth muscle actin; PCNA: Proliferating cell nuclear antigen; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LPAR3: Lysophosphatidic acid receptor 3; THBS1: Thrombospondin 1; PPP2CA: Protein phosphatase 2 catalytic subunit α.下载:
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生物信息学分析表明，富集在PI3K/Akt信号通路中的circRNA宿主基因包括溶血磷脂酸受体3（lysophosphatidic acid receptor 3，LPAR3）、血小板反应蛋白1（thrombospondin 1，THBS1）和蛋白磷酸酶2催化亚基α（protein phosphatase 2 catalytic subunit α，PPP2CA），它们分别为circ_0013058、circ_0034544和circ_0073942的宿主基因。真核生物启动子数据库显示LPAR3、THBS1和PPP2CA的启动子均存在KLF4结合位点，JASPAR数据库显示KLF4的共识结合序列为GGG(T/C)G(G/T)GGC（图 4C），提示KLF4可能通过调控circ_0013058、circ_0034544和circ_0073942的宿主基因影响其产生。
2.5 涉及PI3K/Akt信号通路的circRNA-miRNA-mRNA调控网络
基于miRNA位点预测算法，对3种差异表达circRNA的潜在靶向miRNA进行筛选，并选择上调最显著的5个miRNA进行后续分析。预测结果显示，circ_0013058可能结合miR-2682-5p、miR-6511a-5p、miR-198、miR-4692和miR-6780b-5p，circ_0034544与miR-3613-3p、miR-3919、miR-4668-5p、miR-6830-3p和miR-1253相互作用，而circ_0073942则与miR-593-5p、miR-7855-5p、miR-6776-3p、miR-302c-3p和miR-183-5p相互作用（图 5A）。
图 5 KLF4调节的PI3K/Akt信号通路相关circRNA-miRNA-mRNA调控网络Fig. 5 KLF4-regulated circRNA-miRNA-mRNA network associated with PI3K/Akt signaling pathwayA: Schematic diagram illustrating the construction of the circRNA-miRNA network regulated by KLF4 and associated with the PI3K/Akt signaling pathway; B: Interaction network of differentially expressed circRNAs and their host genes (LPAR3, THBS1, and PPP2CA), predicted miRNAs, and their target mRNAs. KLF4: Krüppel-like factor 4; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; circRNA: Circular RNA; miRNA: MicroRNA; LPAR3: Lysophosphatidic acid receptor 3; THBS1: Thrombospondin 1; PPP2CA: Protein phosphatase 2 catalytic subunit α.下载:
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为深入解析circRNA、miRNA和mRNA间的相互调控关系，构建调控网络明确3种circRNA（circ_0013058、circ_0034544和circ_0073942）及其宿主基因（LPAR3、THBS1和PPP2CA）与15个预测miRNA的相互作用关系（图 5B）。该网络可视化结果显示，与KLF4调控的PI3K/Akt信号通路相关的circRNA、miRNA和mRNA之间存在潜在的ceRNA竞争性调控机制，circ_0013058、circ_0034544和circ_0073942分别通过与miRNA的海绵作用调节了26、68和22个差异表达的mRNA，这些mRNA主要富集于PI3K/Akt通路节点。
3 讨论
KLF4作为KLF家族的核心转录因子，在纤维化疾病中展现出显著的功能矛盾性，这种组织特异性差异本质上是细胞微环境、翻译后修饰及信号通路相互作用共同塑造的结果。在心脏成纤维细胞中，TGF-β1通过Smad2/3通路激活KLF4转录活性，促使KLF4直接结合TGF-β1启动子（－45~－41 bp区域），形成正反馈环驱动纤维化进程[11]。KLF4在血管平滑肌细胞增殖调控中具有“生理性抑制”与“病理性促进”的双重作用[12]：生理状态下，KLF4-p53-p21轴维持细胞周期停滞，抑制增殖；病理条件下，KLF4缺失虽延缓分化基因下调，但因解除p21抑制、促进表型转换及炎症信号，最终加速增殖与纤维化。肾脏纤维化进一步揭示了KLF4功能的时空动态性。在单侧输尿管梗阻小鼠模型中，早期损伤阶段泛素特异性肽酶11介导的KLF4去泛素化显著提升其蛋白稳定性，激活的KLF4直接结合caspase 1和caspase 3启动子，驱动肾小管上皮细胞焦亡并加速急性肾损伤向慢性肾病转化；而在疾病晚期，持续高表达的KLF4通过激活YAP转录，形成KLF4-YAP-CTGF正反馈环，导致不可逆性纤维瘢痕形成[13]。KLF4在肺纤维化中呈现显著的双向调控作用，具体表现为：KLF4通过驱动M2型巨噬细胞极化（如激活精氨酸酶1、CD206等），促进TGF-β分泌和胶原沉积，从而加速纤维化进程[14]；而在肺泡上皮细胞中，KLF4通过阻断上皮间质转化并抑制α-SMA表达，发挥抗纤维化作用[7]。这种矛盾性也体现在间充质细胞亚群中：KLF4在表达血小板源性生长因子受体β的细胞中促进纤维化，但在表达α-SMA的细胞中通过抑制叉头框蛋白M1/CC基序趋化因子配体2轴减轻炎症浸润和胶原沉积[15]。KLF4在皮肤成纤维细胞中发挥抗纤维化作用。KLF4结合骨形态发生蛋白4启动子激活转录，增强Smad1/5/7信号通路，拮抗TGF-β诱导的肌成纤维细胞转化，减少胶原合成[16]。
PI3K/Akt信号通路在调控细胞增殖、分化和纤维化中扮演着重要角色[17-18]。该通路响应多种胞外信号（如促纤维化因子TGF-β1），通过调控下游关键效应分子，广泛参与细胞存活、生长、代谢和分化等过程。在纤维化疾病中，该通路的异常激活已被证实与成纤维细胞的过度增殖、活化及其向肌成纤维细胞的表型转化密切相关[19-20]，且TGF-β1等因子可强烈激活此通路进而驱动纤维化进程[21]，使其成为重要的干预靶点。本研究证实PI3K/Akt信号通路参与了TGF-β1诱导的PIC增殖和肌成纤维细胞表型转化，KLF4过表达可显著抑制上述过程。KLF4在PI3K/Akt通路中具有组织特异性调控特征。在鼻咽癌细胞中，KLF4通过结合PIK3CA启动子激活PI3K/Akt通路[22]。在前列腺癌干细胞中，KLF4通过激活PI3K/Akt/p21通路维持干细胞特性；抑制KLF4可阻断该通路并抑制肿瘤发生[23]。在肝门部胆管癌中，KLF4抑制Akt磷酸化，表现为对疾病表型的负向调控[24]。在肝细胞癌的进展中，KLF4通过转录激活磷酸酶和张力蛋白同源物，促进磷脂酰肌醇-3, 4, 5-三磷酸去磷酸化为磷脂酰肌醇-4, 5-二磷酸，从而降低Akt激活，抑制PI3K/Akt通路活性[25]。本研究的mRNA富集分析及功能验证共同表明，KLF4在PIC中能够抑制PI3K/Akt通路。这种组织特异性的调控差异可能源于PIC独特的表观遗传微环境，KLF4对PI3K/Akt通路的双向调控机制仍需通过组织特异性基因敲除模型进一步验证。
为了进一步研究KLF4对PI3K/Akt信号通路的生物学影响，本研究构建了与KLF4相关的circRNA-miRNA-mRNA调控网络。结果显示，与PI3K/Akt通路相关的circRNA宿主基因（包括LPAR3、THBS1和PPP2CA）均携带KLF4结合位点，提示KLF4可直接调控其转录。LPAR3作为G蛋白偶联受体，通过激活溶血磷脂酸介导PI3K/Akt信号转导，促进细胞迁移与存活[26-27]。THBS1是一种基质糖蛋白，在基质重塑中促进潜在TGF-β的释放，并拮抗血管内皮生长因子驱动的血管生成[28-29]。PPP2CA是蛋白磷酸酶2催化亚基的α亚型，通过去磷酸化Akt（Ser473位点）负反馈抑制PI3K/Akt通路活性[30]。上述宿主基因的KLF4依赖性转录可能通过调控circ_0013058、circ_0034544及circ_0073942的生物合成，影响PI3K/Akt通路的稳态平衡。
circRNA是一类独特的非编码RNA分子，由于缺乏自由的5'和3'末端而具有稳定的环状结构。它可以作为miRNA海绵来调节mRNA的表达，这是circRNA功能的主要机制[31-32]。本研究发现circ_0013058、circ_0034544和circ_0073942分别通过与miRNA的海绵作用调节了26、68和22个差异表达的mRNA。这些miRNA的靶mRNA主要富集于PI3K/Akt通路节点，表明KLF4-circRNA轴可通过circRNA-miRNA-mRNA网络调控PI3K/Akt的信号转导。
综上所述，本研究通过整合组学分析，在KLF4过表达的PIC中鉴定出差异表达的mRNA与circRNA，并证实KLF4调控的mRNA主要富集于PI3K/Akt信号通路。通过构建与KLF4调控的PI3K/Akt信号通路相关的circRNA-miRNA-mRNA网络，加深了对KLF4在PIC分化及肌成纤维细胞表型转化中作用的理解。本研究存在以下不足：（1）PIC在二维培养中可能丧失空间异质性（如细胞间接触、基质刚度），导致KLF4过表达对PI3K/Akt的抑制效应被高估，需结合类器官模型或三维水凝胶培养验证；（2）PIC包含巨噬细胞、成纤维细胞等亚群，KLF4在抗纤维化（抑制肌成纤维细胞转化）与促修复（激活巨噬细胞增殖）中的双向作用可能被掩盖；（3）未检测纤维化不同阶段KLF4对PI3K/Akt的调控差异，可能忽略其早期激活通路的修复功能。未来需通过细胞特异性机制验证与时空动态分析解决上述局限，推动靶向KLF4的纤维化治疗策略转化。
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图 1 PIC的分离培养、KLF4过表达及差异表达mRNA和circRNA的识别
Fig. 1 Isolation and culture of PICs, KLF4 overexpression, and identification of differentially expressed mRNAs and circRNAs
A: Isolated and cultured PICs; B: KLF4 protein expression in adenovirus-infected PICs (**P<0.01. n = 6, x±s); C: Volcano plot showing 3 743 upregulated and 2 454 downregulated mRNAs in Ad.KLF4-infected PICs compared with Ad.EGFP-infected PICs; D: Volcano plot showing 212 upregulated and 181 downregulated circRNAs in Ad.KLF4-infected PICs compared with Ad.EGFP-infected PICs. PIC: Pericardial interstitial cell; KLF4: Krüppel-like factor 4; circRNA: Circular RNA; Ad.KLF4: Adenovirus carrying the KLF4 gene; Ad.EGFP: Adenovirus carrying the enhanced green fluorescent protein gene; FC: Fold change; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
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图 2 Ad.KLF4感染的PIC与Ad.EGFP感染的PIC中差异表达mRNA的GO（A）和KEGG（B）富集分析
Fig. 2 GO (A) and KEGG (B) enrichment analyses of differentially expressed mRNAs in Ad.KLF4-infected PICs compared to Ad.EGFP-infected PICs
Ad.KLF4: Adenovirus carrying the Krüppel-like factor 4 gene; PIC: Pericardial interstitial cell; Ad.EGFP: Adenovirus carrying the enhanced green fluorescent protein gene; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; ListHit: Number of genes enriched in the given term or pathway; Process: Biological process; Function: Molecular function; Component: Cellular component; MAP: Mitogen-activated protein; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; ECM: Extracellular matrix; SNARE: Soluble N-ethylmaleimide-sensitive factor attachment protein receptor; MAPK: Mitogen-activated protein kinase.
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图 3 Ad.KLF4感染的PIC与Ad.EGFP感染的PIC中差异表达circRNA宿主基因的GO（A）和KEGG（B）富集分析
Fig. 3 GO (A) and KEGG (B) enrichment analyses of differentially expressed circRNA host genes in Ad.KLF4-infected PICs compared to Ad.EGFP-infected PICs
Ad.KLF4: Adenovirus carrying the Krüppel-like factor 4 gene; PIC: Pericardial interstitial cell; Ad.EGFP: Adenovirus carrying the enhanced green fluorescent protein gene; circRNA: Circular RNA; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; ListHit: Number of genes enriched in the given term or pathway; Function: Molecular function; Component: Cellular component; Process: Biological process; ATP: Adenosine triphosphate; TGF: Transforming growth factor; AMPK: AMP-activated protein kinase; TCA: Tricarboxylic acid.
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图 4 KLF4对PIC中PI3K/Akt信号通路的影响
Fig. 4 Effects of KLF4 on PI3K/Akt signaling pathway in PICs
A: PICs were infected by Ad.KLF4 or Ad.EGFP, followed by treatment with or without TGF-β1 (20 ng/mL) for 3 d. Protein expression levels were analyzed by Western blotting. B: The effect of KLF4 on PIC proliferation was evaluated using the cell counting kit 8 assay (*P<0.05. n = 6, x±s). C: Bioinformatics analysis revealed the presence of KLF4 binding sites in the promoter regions of LPAR3, THBS1, and PPP2CA. KLF4: Krüppel-like factor 4; PIC: Pericardial interstitial cell; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; TGF-β1: Transforming growth factor β1; Ad.EGFP: Adenovirus carrying enhanced green fluorescent protein gene; Ad.KLF4: Adenovirus carrying KLF4 gene; p-Akt: Phosphorylated Akt; α-SMA: α-smooth muscle actin; PCNA: Proliferating cell nuclear antigen; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LPAR3: Lysophosphatidic acid receptor 3; THBS1: Thrombospondin 1; PPP2CA: Protein phosphatase 2 catalytic subunit α.
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图 5 KLF4调节的PI3K/Akt信号通路相关circRNA-miRNA-mRNA调控网络
Fig. 5 KLF4-regulated circRNA-miRNA-mRNA network associated with PI3K/Akt signaling pathway
A: Schematic diagram illustrating the construction of the circRNA-miRNA network regulated by KLF4 and associated with the PI3K/Akt signaling pathway; B: Interaction network of differentially expressed circRNAs and their host genes (LPAR3, THBS1, and PPP2CA), predicted miRNAs, and their target mRNAs. KLF4: Krüppel-like factor 4; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; circRNA: Circular RNA; miRNA: MicroRNA; LPAR3: Lysophosphatidic acid receptor 3; THBS1: Thrombospondin 1; PPP2CA: Protein phosphatase 2 catalytic subunit α.
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