第一部分

食管癌（esophageal cancer, EC）是中国高发且预后差的消化道恶性肿瘤。根据国家癌症中心2022年公布的数据，在全部肿瘤类型中，食管癌的发病率和死亡率分列于第七位和第五位。食管癌存在两种主要的病理亚型：食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）和食管腺癌（esophageal adenocarcinoma, EAC）。在两种组织学亚型中，食管鳞状细胞癌在中国更为高发，占85.79%。虽然筛查技术、外科手术和新治疗方式的发展使中国食管鳞癌的发病率、死亡率呈下降趋势，但由于食管鳞癌发病隐匿、恶性程度以及异质性高，患者就诊时多为中晚期且预后差。因此，阐明食管鳞癌发生、发展的分子机制，寻找食管鳞癌早期诊断标志物与有效治疗靶标具有重要的临床价值。

乳酸曾被认为是一种“代谢废物”，乳酰化修饰（lysine lactylation, Kla）的首次发现在代谢和基因表达调控之间建立了桥梁。乳酰化修饰是一种由乳酸（lactate, LA）或乳酰辅酶A（lactyl-coenzyme A, Lactyl-CoA）提供乳酰基团（lactyl group, Lactyl-），由丙氨酰tRNA合成酶（alanyl-tRNA synthetases, AARSs）或组蛋白乙酰转移酶（histone acetyltransferases, HATs）介导，将乳酰基团共价结合至组蛋白或非组蛋白赖氨酸残基的蛋白翻译后修饰（post-translational modifications, PTMs），并可由去乙酰化酶（HDAC1-3或SIRT1-3）介导修饰的可逆“擦除”。肿瘤中的“Warburg效应”促进乳酸积累，肿瘤中的高乳酸水平驱动高乳酰化修饰水平。乳酰化修饰在多种肿瘤类型中发挥重要作用，促进肿瘤发生、发展、放化疗抵抗、免疫逃逸和代谢重编程等恶性特征。乳酰化修饰作为新发现的蛋白翻译后修饰与肿瘤代谢特征关联紧密，但介导修饰的识别、修饰、去修饰酶以及底物选择尚待进一步探索。研究乳酰化修饰在肿瘤发生、发展不同阶段发挥的作用及分子机制，为肿瘤诊疗提供新思路，是肿瘤研究的新兴热点。

长链非编码RNA（long noncoding RNA, LncRNA）作为非编码RNA曾被认为不具有编码能力，近年来随着蛋白组学和翻译组学技术的不断发展，越来越多的研究表明长链非编码RNA序列中包含短开放阅读框（short open reading frames, sORFs），具有编码功能性短肽（peptide）的能力。LncRNA编码的小肽通过DNA损伤修复调控、转录调控、转录后调控、翻译调控和代谢调控等参与肿瘤发生发展的各个阶段。探索lncRNA编码小肽在肿瘤中发挥作用的分子机制，为肿瘤的诊疗提供了新的可能方案。

LncRNA-J23（lnc-J23）是实验室前期筛选到的在食管鳞癌中发挥关键作用且与干性相关的长链非编码RNA，lncRNA-J23的非编码功能研究由实验室范新义同学完成（详见范新义同学毕业论文）。本论文第一部分以在食管鳞癌中发挥关键促癌作用的长链非编码RNA lncRNA-J23编码的小肽JF2为研究对象，探究小肽JF2在食管鳞癌中独立于lncRNA-J23作为非编码RNA的功能，探索其促进肿瘤发生发展的分子机制。首先，我们通过ORFfinder预测lncRNA-J23的编码潜力，发现lncRNA-J23具有两个开放阅读框，提示lncRNA-J23可能具有编码能力。通过构建小肽-GFP与小肽-flag融合质粒，在食管鳞癌细胞中转染小肽-GFP或小肽-flag的融合质粒，并通过免疫荧光或Western blot实验鉴定小肽的外源表达情况，鉴定小肽JF2的外源表达，选择小肽JF2作为研究对象进行后续研究。

因此，我们定制了小肽JF2的多克隆抗体，从六株抗体中筛选并鉴定到抗体anti-JF2-R3可以通过Western blot特异性识别小肽JF2。通过定制抗体在食管鳞癌细胞系中验证了小肽JF2的内源表达情况，结果提示在食管鳞癌细胞中lncRNA-J23可以编码小肽JF2，并且与永生化食管上皮细胞相比，小肽JF2在食管鳞癌细胞系中高表达。通过核浆分提实验表明，小肽JF2主要分布于细胞浆中。肿瘤细胞功能实验表明，小肽JF2促进食管鳞癌细胞的增殖、克隆形成、侵袭及迁移能力。在5对食管鳞癌组织和配对癌旁组织中利用定制抗体通过Western blot检测了小肽JF2的表达水平，结果表明与配对癌旁组织相比小肽JF2在食管鳞癌组织中显著高表达。

为了探索小肽JF2独立于lncRNA-J23作为非编码RNA的功能，我们通过CRISPR/CAS9单载体慢病毒，构建并筛选得到稳转敲降小肽JF2的单克隆食管鳞癌细胞株，在不影响lncRNA-J23表达的情况下，显著抑制了小肽JF2的表达水平。通过转录组测序和质谱，筛选小肽JF2在食管鳞癌中可能参与的生物学过程以及可能的结合蛋白，结果提示小肽JF2与糖酵解和乳酸合成转运密切相关，进而提出假设小肽JF2可能参与调控食管鳞癌的乳酰化修饰。为了进一步筛选小肽JF2调控乳酰化修饰的下游靶蛋白，通过乳酰化修饰组学筛选CRISPR/CAS9稳转敲降小肽JF2的单克隆细胞株中乳酰化水平下调的蛋白，并对这些蛋白进行GO富集分析，提示与RNA剪接和mRNA加工密切相关。这些蛋白中与可变剪接相关的蛋白包括RBMX、BRDX和BRDY，通过对稳转单克隆细胞株进行乳酰化抗体的Co-IP，筛选小肽JF2正调控乳酰化修饰水平的下游靶蛋白，结果表明小肽JF2可以正调控可变剪接因子RBMX的乳酰化修饰。通过外源添加乳酸钠（Sodium L-lactate, NaLc）提供充足乳酰化修饰供体，发现乳酰基团促进lncRNA-J23编码小肽JF2，且不影响lncRNA-J23表达水平。

综上，lncRNA-J23编码的小肽JF2在食管鳞癌中的表达显著上调，可通过促进可变剪接因子RBMX的乳酰化修饰水平调控可变剪切，促进食管鳞癌的发生发展。我们后续仍需进一步探究小肽JF2在体内对食管鳞癌细胞功能的影响，并探索小肽JF2促进RBMX乳酰化修饰进而调控下游可变剪接的分子机制，为食管鳞癌患者提供诊断标志物或治疗靶点的新方案。

第二部分

非编码RNA（non-coding RNA, ncRNA）被定义为一类不编码蛋白质的RNA分子。微小RNA（microRNA, miRNA）是一类长度约为21个核苷酸的单链非编码小RNA分子，具有高度保守、可在组织、血中稳定存在、易于检测、参与肿瘤发生发展的调控等特征，作为肿瘤诊断标志物具有广阔的前景。在本论文第一部分中，我们探索了长链非编码RNA（long non-coding RNA, lncRNA）编码小肽在肿瘤中的非经典功能；在第二部分中，我们以非编码RNA中的microRNA为研究对象，探究其在卵巢癌化疗耐药中的经典非编码功能。

卵巢癌（ovarian cancer, OC）是死亡率最高的妇科恶性肿瘤，上皮性卵巢癌（epithelial ovarian cancer, EOC）是卵巢癌中最常见的病理类型，占卵巢癌的90%以上。但由于缺乏有效的早期筛查方法，约70%的卵巢癌患者在确诊时已处于III或IV期，目前治疗方式主要包括肿瘤细胞减灭术和以铂类为主的化疗，其中顺铂（cisplatin, DDP）是晚期上皮性卵巢癌患者的一线化疗药物。化疗耐药是卵巢癌患者不良预后的重要原因，尽管初治反应率较高，可达70%-75%，但几乎所有患者最终都会复发并发展为铂类耐药。然而卵巢癌化疗耐药的机制尚不完善，因此，本论文第二部分旨在阐明卵巢癌顺铂耐药的分子机制，探寻有效预测铂类化疗敏感性的分子标志物，探索新治疗靶点以拓展卵巢癌患者化疗增敏的可能性。

卵巢癌患者顺铂耐药与干性特征上调密切相关。在本研究发表前，已有非编码RNA参与调控肿瘤化疗耐药机制的研究，但研究对象的筛选条件多基于TCGA数据库分析或肿瘤细胞耐药株、敏感株测序，鲜有来源于化疗耐药、敏感卵巢癌患者组织测序的研究对象。考虑到卵巢癌化疗耐药与干性特征的密切关联，我们联合了卵巢癌细胞干性成球模型，筛选得到的耐药患者组织来源且与干性特征相关的microRNA：miR-6836。与卵巢癌耐药细胞株测序相比，来源于耐药卵巢癌患者的肿瘤组织测序可以更直接地反应卵巢癌患者体内真实病理状态下的microRNA表达谱，组织测序得到的microRNA数据更具临床意义。

我们在57例敏感和30例耐药卵巢癌患者组织中检测miR-6836的表达，结果验证了miR-6836在耐药卵巢癌患者组织中的表达显著高于敏感患者组织；在65例卵巢癌患者血清和39例健康人或良性卵巢肿瘤患者血清中检测miR-6836的表达，结果提示miR-6836在卵巢癌患者血清中的表达显著高于健康人或良性肿瘤患者血清；KMplot在线数据库分析结果显示，miR-6836的表达水平与卵巢癌患者不良预后相关。提示miR-6836具有作为卵巢癌诊断、化疗敏感性预测的分子标志物的潜力。

miR-6836在卵巢癌细胞中显著促进干性特征、顺铂耐药性、增殖能力、克隆形成能力、侵袭和迁移能力。此外，miR-6836可被外泌体包裹，并通过外泌体介导的细胞间传递，将顺铂耐药特征传递给其他肿瘤细胞。动物实验进一步证实，miR-6836在体内能够增强卵巢癌细胞的干性致瘤能力、皮下成瘤能力以及腹腔转移能力。机制研究发现，miR-6836直接靶向并抑制DLG2（Discs Large Homolog 2）的表达。DLG2的下调促进了YAP1的核转位，进而激活Hippo-YAP信号通路。YAP1的核转位是其发挥转录共激活功能的关键步骤。YAP1与转录因子TEAD1形成复合物，而TEAD1是调控miR-6836表达的关键转录因子。在耐药卵巢癌细胞中，miR-6836通过抑制DLG2激活YAP1，YAP1-TEAD1复合物又进一步促进miR-6836的表达，从而形成miR-6836-DLG2-YAP1-TEAD1正反馈环路，持续驱动肿瘤的干性、耐药和转移。

由此，miR-6836具有作为卵巢癌化疗增敏的治疗靶点的可能性。我们定制了经特殊化学修饰的miR-6836拮抗剂（antagomir），antagomir-miR-6836作为寡核苷酸药物，具有卵巢癌化疗耐药增敏的潜力。我们构建了卵巢癌皮下成瘤模型，通过antagomir-miR-6836与顺铂联用治疗，显著抑制铂耐药卵巢癌皮下瘤增殖。miR-6836拮抗剂在体内通过特异性、竞争性结合miR-6836，促进DLG2抑制YAP1核转位，促进卵巢癌细胞对顺铂的敏感性。我们揭示了卵巢癌化疗耐药的新机制，并提示miR-6836可作为卵巢癌化疗耐药的潜在分子标志物及治疗靶点，为卵巢癌化疗耐药的诊断和治疗提供了新思路。

综上所述，本研究揭示了miR-6836在EOC化疗耐药中的关键作用，并提出了外泌体介导的耐药表型传播的新机制。这些发现为开发新的诊断标志物和治疗策略提供了实验依据，有望改善EOC患者的临床预后。

Part One

Esophageal cancer (EC) is a malignant digestive tract cancer with a high incidence and poor prognosis in China. According to the data published by the National Cancer Center in 2022, esophageal cancer ranks seventh in incidence and fifth in mortality among all tumor types. Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) are the two main pathological subtypes of esophageal cancer, and ESCC is more prevalent in China, accounting for 85.79% of all EC subtypes. Although the incidence and mortality of ESCC have been declining in China due to the breakthroughs in screening, surgical procedures, and novel treatment modalities, most ESCC patients are diagnosed in advanced stages and have poor prognosis due to the feature of concealed pathogenesis, high malignancy and heterogeneity. Therefore, elucidating the underlying molecular mechanisms of the occurrence and progression of ESCC, and identifying early diagnostic biomarkers and effective therapeutic targets are of great clinical significance.

Lactate was once considered as a “metabolic by-product”, and the discovery of lysine lactylation (Kla) built a bridge between metabolism and gene expression regulation. Lactylation is a lactate (LA) or acetyl coenzyme A (acetyl-CoA)-derived post-translational modification (PTM), and the substrates of lactylation include histones and non-histone proteins. Alanyl-tRNA synthetases (AARSs) and histone acetyltransferases (HATs) mediate the covalent binding of lactyl group to lysine residues, while deacetylases (HDAC1-3 and SIRT1-3) catalyze reversible de-lactylation modification. The Warburg effect, also known as aerobic glycolysis, promotes lactate accumulation in tumor microenvironment, which drives high lactylation levels. Lactylation has been reported to play an important role in various tumor types, contributing to a range of malignant features including tumor occurrence and progression, resistance to radiotherapy and chemotherapy, immune evasion, and metabolic reprogramming. As a recently discovered PTM, lactylation is closely associated with tumor metabolic characteristics, while the reader, writer, eraser proteins and substrates involved require further research. Exploring the functions and molecular mechanisms of lactylation in different biological stages of tumor progression provides new insights for tumor diagnosis and treatment.

With the continuous development of proteomics and translatomics technologies in recent years, long noncoding RNAs (LncRNAs), once known as a non-coding RNA, have been reported to contain short open reading frames (sORFs), which have the ability to encode functional peptides. The peptides encoded by lncRNA participate in various stages of tumorigenesis and progression through DNA damage repair regulation, transcriptional regulation, post-transcriptional regulation, translational regulation and metabolic regulation. Studying the functional mechanisms of lncRNA-encoded peptides in tumors provides new potential strategies for tumor diagnosis and treatment.

LncRNA-J23 is a long non-coding RNA previously identified by our laboratory, which is closely associated with stemness and is of vital importance in ESCC. The research on the non-coding function of lncRNA-J23 was completed by Fan Xinyi, a graduated PhD student from our laboratory (for detailed information, refer to the doctoral thesis of Fan Xinyi). The first part of this thesis focuses on the functional peptide JF2 encoded by lncRNA-J23, which functions as an oncogenic peptide in ESCC, and further explores the function and molecular mechanism of peptide JF2 independent from lncRNA-J23 as a non-coding RNA in promoting ESCC occurrence and progression. Firstly, we predicted the encoding potential of lncRNA-J23 using ORFfinder, and identified two open reading frames, suggesting that lncRNA-J23 had the potential to encode functional peptides. We further constructed JF2-GFP and JF2-Flag fusion plasmids, transfected ESCC cells with the fusion plasmids, and confirmed the exogenous expression of peptide JF2 through immunofluorescence assay and western blot. Peptide JF2 was chosen as the research object for further study.

In order to explore the endogenous expression of peptides JF2, we customized polyclonal antibodies targeting peptide JF2. Antibody anti-JF2-R3 was screened from six polyclonal antibodies, and identified that anti-JF2-R3 could specifically recognize peptide JF2 by western blot. Using customized antibody anti-JF2-R3, we revealed that lncRNA-J23 could encode peptide JF2 in ESCC cells, and peptide JF2 was highly expressed in ESCC cell lines compared to immortalized esophageal epithelial cells. Nuclear and cytoplasmic protein extraction experiment showed that peptide JF2 was mainly localized in the cytoplasm. Functional experiments demonstrated that peptide JF2 promoted the proliferation, colony formation, invasion, and migration of ESCC cells. The expression level of peptide JF2 was detected by western blot using customized antibody anti-JF2-R3 in 6 pairs of ESCC and paired adjacent tissues. The results showed that peptide JF2 was significantly highly expressed in ESCC tissues than that in paired adjacent tissues.

To explore the function of peptide JF2 independent from lncRNA-J23 as a ncRNA, we constructed and screened monoclonal ESCC cell lines with stable peptide JF2 knock-down using CRISPR/Cas9 single-vector lentivirus, which enabled peptide JF2 knock down without affecting the expression level of lncRNA-J23. Transcriptome sequencing and mass spectrometry were used to identify biological processes and potential binding proteins associated with peptide JF2 in ESCC, suggesting that peptide JF2 was closely related to glycolysis and lactate synthesis and transport. It was hypothesized that small peptide JF2 might be involved in the regulation of lactylation of ESCC. To further identify downstream lactylated target proteins regulated by peptide JF2, we performed global lactylome analysis on monoclonal ESCC cell lines with stable peptide JF2 knock-down, and GO enrichment analysis of proteins with down-regulated lactylation in sg/nc revealed association with RNA splicing and mRNA processing. Among these proteins, splicing factors related to alternative splicing included RBMX, BRDX, and BRDY. We detected lactylation of canidate target proteins by co-immunoprecipitation using anti-L-Lactyl Lysine antibody, and results showed that peptide JF2 positively regulated the lactylation of the alternative splicing factor RBMX. We simulated the acidic microenvironment by adding sodium lactate (NaLc) exogenously, which promoted the expression of peptide JF2 encoded by lncRNA-J23 without affecting the expression level of lncRNA-J23.

In conclusion, the expression of peptide JF2 encoded by lncRNA-J23 is significantly up-regulated in ESCC, which can regulate alternative splicing by promoting the lactylation of alternative splicing factor, RBMX, and promote the occurrence and progression of ESCC. We still need to further explore the effect of peptide JF2 on the in-vivo function of ESCC cells. Exploring the molecular mechanism of peptide JF2 in promoting RBMX lactylation and further regulating downstream alternative splicing events provide a new diagnostic marker or therapeutic target for ESCC patients.

Part Two

Non-coding RNA (ncRNA) is defined as a class of RNA molecules that do not encode proteins. MicroRNA (miRNA) is a class of single-stranded non-coding small RNA molecules with the approximate length of 21 nucleotides. MicroRNAs are highly conserved, stable in tissues and blood, easy to detect, and participated in the regulation of tumorigenesis and progression, which suggest that microRNAs are promising candidates for tumor diagnostic biomarkers. In the first part of this thesis, we explored the non-classical functions of peptides encoded by lncRNA in ESCC. In the second part, we focused on microRNA, a type of non-coding RNA, to explore its classical non-coding functions in ovarian cancer chemoresistance.

Ovarian cancer (OC) is the most lethal cause of gynecologic cancer worldwide. Epithelial ovarian cancer (EOC) is the most common histological type in ovarian cancer, accounting for over 90% of all ovarian cancers. Due to the lack of effective methods for EOC early detection and screening, about 70% of EOC patients are diagnosed at stage III or IV. The upfront treatment mainly includes debulking surgery followed by platinum-based chemotherapy, and cisplatin (DDP) is the first-line platinum-based drug for advanced EOC. Chemotherapy resistance is a significant cause for poor prognosis of EOC. Even though the initial response rate reaches 70%-75%, almost all patients will relapse and develop platium resistance eventually. However, the molecular mechanism of chemo-resistance remains incomplete. Therefore, the second part of this thesis aims to elucidate the mechanisms of cisplatin resistance in EOC, and develop available therapies and predictive biomarkers for resistant EOC.

Stemness signaling has been reported to be closely related to chemo-resistance. Before the publication of the second part of the thesis, there have been studies on ncRNAs in regulating the mechanism of tumor chemotherapy resistance, with screening criteria mostly based on TCGA database analysis or sequencing of cisplatin-resistant and sensitive tumor cell lines. However, few research subjects have been originated from EOC tissues derived from chemotherapy-resistant and sensitive EOC patients. Considering the close association between EOC chemoresistance and stemness characteristics, we combined the EOC spheroid cells tumorigenicity model with transcriptome sequencing of EOC tissues from patients sensitive or resistant to chemotherapy, and screened for microRNAs related to stemness characteristics and highly expressed in tissues from chemo-resistant EOC patients. Compared with chemo-resistant ovarian cancer cell lines, sequencing of resistant EOC tissues can reflect the microRNAs profiling of pathological state of EOC patients more directly.

The expression level of miR-6836 was detected in 57 sensitive and 30 resistant EOC tissues, and the results confirmed that the expression of miR-6836 in resistant EOC tissues was significantly higher than in sensitive EOC tissues. The expression level of miR-6836 was further detected in serum samples from 65 EOC patients and 39 healthy individuals or patients with benign ovarian tumors, and the results indicated that the expression of miR-6836 in EOC serum samples was significantly higher than in healthy individuals or patients with benign tumors. The analysis results of KMplot online database revealed that the high expression level of miR-6836 was associated with poor prognosis in EOC patients. These findings suggested that miR-6836 had the potential as a molecular marker for the diagnosis and chemotherapy sensitivity prediction of EOC patients.

Functionally, miR-6836 promoted EOC cell cisplatin resistance by increasing stemness and suppressing apoptosis. Furthermore, miR-6836 could be packaged into secreted exosomes in cisplatin-resistant EOC cells and exosomal miR-6836 was able to be delivered into cisplatin-sensitive EOC cells and reverse their cisplatin response. Animal experiments further confirmed that miR-6836 enhanced the stemness, tumorigenicity, and peritoneal metastatic capabilities of EOC cells in vivo. Mechanistically, miR-6836 directly targeted DLG2 (Discs Large Homolog 2) to enhance Yap1 nuclear translocation, and was regulated by TEAD1 forming the positive feedback loop: miR-6836-DLG2-Yap1-TEAD1, which continuously drives the stemness, drug resistance and metastasis phenotypes of EOC.

In order to assess the potential of miR-6836 as an effective therapeutic target for EOC chemotherapy sensitization, we customized a chemically modified miR-6836 antagonist (antagomir). As an oligonucleotide drug, antagomir-miR-6836 had the potential to sensitize chemo-resistant EOC. We established a EOC subcutaneous transplantation mouse model and treated platinum-resistant EOC xenograft tumor with the combined treatment of antagomir-miR-6836 and cisplatin, which significantly inhibited the proliferation of subcutaneous EOC tumors. In vivo, the miR-6836 antagonist targeted DLG2 to inhibit the nuclear translocation of YAP1, thereby enhancing the sensitivity of EOC cells to cisplatin. We revealed a novel functional mechanism of EOC chemoresistance and suggested that miR-6836 could serve as a potential molecular marker and therapeutic target for EOC chemoresistance, providing a new perspective for the diagnosis and treatment of EOC chemoresistance. Our study revealed the molecular mechanisms of EOC chemotherapy resistance, and identified miR-6836 as the possible therapeutic target and effective biopsy marker for resistant EOC.

In conclusion, the second part of the thesis revealed the critical role of miR-6836 in EOC chemoresistance and proposed the molecular mechanisms of exosomal miR-6836 conferring the resistant phenotype in EOC tumor microenvironment. These findings provide experimental evidence for the development of possible therapeutic targets and effective biopsy markers for resistant EOC, and are expected to improve the prognosis of resistant EOC patients.