CN112089840B - Application of inhibitors of KTN1-AS1 in the preparation of drugs for the treatment of bladder cancer - Google Patents
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Abstract
本发明公开了KTN1‑AS1的抑制剂在制备治疗膀胱癌药物中的应用。本发明研究表明，KTN1‑AS1在BUC细胞及组织中明显上调并与患者预后呈负相关，敲除和上调KTN1‑AS1的表达分别能增强或抑制体外和体内BUC细胞的增殖，并分别抑制或促进体外和体内细胞凋亡；此外，我们证明上调的KTN1‑AS1能募集EP300到KTN1启动子，激活KTN1表达，并通过激活KTN1/Rho GTPase途径从而加速膀胱癌的增殖和发展。因此，KTN1‑AS1的抑制剂在制备靶向负性调控KTN1表达制剂中可应用。同时，KTN1的抑制剂也可在制备治疗膀胱癌药物中应用。The invention discloses the application of a KTN1-AS1 inhibitor in preparing a medicine for treating bladder cancer. The research of the present invention shows that KTN1-AS1 is significantly up-regulated in BUC cells and tissues and is negatively correlated with the prognosis of patients, and knocking out and up-regulating the expression of KTN1-AS1 can respectively enhance or inhibit the proliferation of BUC cells in vitro and in vivo, and inhibit or inhibit the proliferation of BUC cells in vitro and in vivo, respectively. Promotes apoptosis in vitro and in vivo; furthermore, we demonstrate that up-regulated KTN1‑AS1 recruits EP300 to the KTN1 promoter, activates KTN1 expression, and accelerates bladder cancer proliferation and progression by activating the KTN1/Rho GTPase pathway. Therefore, inhibitors of KTN1-AS1 can be used in the preparation of preparations that target and negatively regulate KTN1 expression. At the same time, the inhibitor of KTN1 can also be used in the preparation of drugs for treating bladder cancer.
Description
技术领域technical field
本发明属于生物医药技术领域，具体涉及KTN1-AS1的抑制剂在制备治疗膀胱癌药物中的应用。The invention belongs to the technical field of biomedicine, and particularly relates to the application of an inhibitor of KTN1-AS1 in the preparation of a medicine for treating bladder cancer.
背景技术Background technique
膀胱癌是全球第十大最常见的癌症，其发病率和死亡率较高。根据《2018年全球癌症统计》显示，每年约诊断出549,000例新发膀胱癌病例，每年有多达200,000例的膀胱癌患者死亡。值得注意的是，虽然诊断出的膀胱癌中有70％至80％是非肌肉浸润性肿瘤，但20％至30％是肌肉浸润性肿瘤。然而，大约1/3的非肌肉浸润性肿瘤最终将发展为肌肉浸润性肿瘤或发生转移，这将导致肿瘤的进展。即使是接受最佳治疗（手术和化学疗法）的膀胱癌患者，远处转移的发生率也很高，临床结局和预后也很差。因此，我们迫切需要对驱动膀胱癌发生和发展的分子机制有更好的了解。Bladder cancer is the tenth most common cancer worldwide, with high morbidity and mortality. According to Global Cancer Statistics 2018, approximately 549,000 new cases of bladder cancer are diagnosed each year, and as many as 200,000 bladder cancer patients die each year. Notably, while 70% to 80% of diagnosed bladder cancers are non-muscle-invasive tumors, 20% to 30% are muscle-invasive tumors. However, approximately one-third of non-muscle-invasive tumors will eventually develop into muscle-invasive tumors or metastasize, which will lead to tumor progression. Even patients with bladder cancer who receive optimal treatment (surgery and chemotherapy) have a high incidence of distant metastases and poor clinical outcomes and prognosis. Therefore, we urgently need a better understanding of the molecular mechanisms driving bladder cancer initiation and progression.
越来越多的研究表明，长非编码RNA（lncRNA）参与多种生理和病理状况，例如胚胎发育，心血管疾病和癌症。LncRNA是长度大于200个核苷酸的特殊RNA转录物，没有（或具有有限的）蛋白质编码潜能。在功能上，lncRNA异常表达可能导致多种生物学效应，例如细胞增殖、分化和凋亡。从机制上来讲，lncRNA是通用分子，具有与DNA、RNA或蛋白质相互作用的潜能，可作为信号、诱饵、向导和支架的原型。LncRNA KTN 1反义RNA 1（KTN1-AS1）位于染色体14q22.3上，并映射在KTN1上游区域的反义DNA链。该lncRNA首先被报道在大肠癌细胞和组织中高表达，并且与肿瘤发生和不良的临床结果呈正相关。然而，在膀胱癌中KTN1-AS1是否上调及其相关潜在机制尚不清楚。因此，需要广泛研究KTN1-AS1在BUC中的功能及作用机制。A growing number of studies have shown that long noncoding RNAs (lncRNAs) are involved in a variety of physiological and pathological conditions, such as embryonic development, cardiovascular disease, and cancer. LncRNAs are specialized RNA transcripts greater than 200 nucleotides in length with no (or limited) protein-coding potential. Functionally, abnormal expression of lncRNAs may lead to various biological effects, such as cell proliferation, differentiation and apoptosis. Mechanistically, lncRNAs are general-purpose molecules with the potential to interact with DNA, RNA, or proteins, serving as prototypes for signals, decoys, guides, and scaffolds.
发明内容SUMMARY OF THE INVENTION
本发明旨在提供一种可以用于制备治疗膀胱癌药物的新靶点。The present invention aims to provide a new target that can be used to prepare a drug for treating bladder cancer.
本发明提供了KTN1-AS1的抑制剂在制备治疗膀胱癌药物中的应用。所述膀胱癌是指BUC。The invention provides the application of the inhibitor of KTN1-AS1 in the preparation of a medicine for treating bladder cancer. The bladder cancer refers to BUC.
本发明提供了KTN1-AS1的预测试剂在制备预测膀胱癌复发标记物中的应用。The invention provides the application of the KTN1-AS1 prediction reagent in the preparation of a marker for predicting the recurrence of bladder cancer.
本发明提供了KTN1-AS1的靶位点检测试剂在制备治疗膀胱癌分子标志物中的应用。The invention provides the application of the target site detection reagent of KTN1-AS1 in the preparation of molecular markers for the treatment of bladder cancer.
本发明提供了KTN1-AS1在制备靶向负性调控KTN1及Rho GTPase制剂中的应用。The present invention provides the application of KTN1-AS1 in the preparation of preparations that target and negatively regulate KTN1 and Rho GTPase.
本发明提供了KTN1作为靶位点在制备治疗膀胱癌药物中的应用。所述膀胱癌是指BUC。The invention provides the application of KTN1 as a target site in preparing a medicine for treating bladder cancer. The bladder cancer refers to BUC.
本发明提供了KTN1-AS1/KTN1/Rho GTPase调控制剂在制备治疗膀胱癌药物中的应用。所述膀胱癌是指BUC。The invention provides the application of a KTN1-AS1/KTN1/Rho GTPase regulating preparation in preparing a medicine for treating bladder cancer. The bladder cancer refers to BUC.
本发明研究表明，KTN1-AS1在膀胱癌细胞及组织中表达上调，并在II至IV期膀胱癌中观察到KTN1-AS1的表达较I期膀胱癌患者升高，且KTN1-AS1表达较高的患者预后更差。敲除或过表达KTN1-AS1可分别增强或抑制体外和体内BUC细胞的增殖和侵袭。此外，KTN1-AS1的上调促进了KTN1的表达水平。机制研究表明，KTN1-AS1能通过募集EP300到KTN1启动子区域来顺式激活KTN1的表达。因此，以上研究结果表明，KTN1-AS1/KTN1是BUC增殖及侵袭的重要参与者，并且KTN1-AS1/KTN1可作为BUC患者的新型生物标志物和治疗靶标。The research of the present invention shows that the expression of KTN1-AS1 is up-regulated in bladder cancer cells and tissues, and the expression of KTN1-AS1 in stage II to IV bladder cancer is observed to be higher than that in stage I bladder cancer patients, and the expression of KTN1-AS1 is higher patients have a worse prognosis. Knockdown or overexpression of KTN1-AS1 enhanced or inhibited the proliferation and invasion of BUC cells in vitro and in vivo, respectively. In addition, the upregulation of KTN1-AS1 promoted the expression level of KTN1. Mechanistic studies have shown that KTN1-AS1 can activate KTN1 expression in cis by recruiting EP300 to the KTN1 promoter region. Therefore, the above findings suggest that KTN1-AS1/KTN1 is an important player in BUC proliferation and invasion, and KTN1-AS1/KTN1 can be used as a novel biomarker and therapeutic target for BUC patients.
本发明还首次揭示在膀胱癌中，KTN1的表达在癌组织中较正常组织中显着升高，且GEO数据库中两个数据集（GSE3167和GSE138118）中也观察到KTN1的表达上调。敲除或过表达KTN1-AS1能影响KTN1的mRNA和蛋白质的表达，提示KTN1-AS1在顺式中起作用。通过分析不同数据库中KTN1-AS1和KTN1之间的表达相关性，我们发现KTN1-AS1与KTN1表达呈显著正相关。由于亚细胞分级分析显示KTN1-AS1在细胞核中的表达高于胞质溶胶，因此我们假设KTN1-AS1可能与核蛋白相互作用，在转录水平上调节KTN1的表达。The present invention also revealed for the first time that in bladder cancer, the expression of KTN1 was significantly increased in cancer tissues compared with normal tissues, and the up-regulation of KTN1 expression was also observed in two datasets (GSE3167 and GSE138118) in the GEO database. Knockdown or overexpression of KTN1-AS1 can affect the expression of KTN1 mRNA and protein, suggesting that KTN1-AS1 functions in cis. By analyzing the expression correlation between KTN1-AS1 and KTN1 in different databases, we found that KTN1-AS1 was significantly positively correlated with KTN1 expression. Since subcellular fractionation analysis showed that KTN1-AS1 was more expressed in the nucleus than in the cytosol, we hypothesized that KTN1-AS1 might interact with nuclear proteins to regulate KTN1 expression at the transcriptional level.
本研究探索了KTN1-AS1在膀胱癌中的作用机制，为了更好地了解KTN1-AS1和KTN1之间的关系，我们使用了UCSC在线数据库（http://genome.ucsc. edu/）分析发现，KTN1-AS1和KTN1启动子区域之间高度重叠，该区域高度富集H3K27Ac峰，而H3K27Ac是活性增强剂的标记物。因此，我们使用预测性RNAct算法寻找具有组蛋白乙酰转移酶（HAT）活性的KTN1-AS1结合蛋白。我们预测到EP300（HAT）可与KTN1-AS1结合。我们在RT4和T24细胞（空载体转染或过表达KTN1-AS1）中进行了RIP实验分析。结果表明，KTN1-AS1可通过EP300募集间接负责H3K27Ac的修饰。为了证明我们的假设，我们进行了ChIP-qPCR分析。我们在RT4和T24细胞中敲除KTN1-AS1后，KTN1启动子区域中的H3K27Ac缺失。相反，H3K27Ac在过表达KTN1-AS1的RT4和T24细胞中高度富集，这种表型在EP300敲除的情况下消失。以上结果表明，KTN1-AS1能通过募集EP300和在KTN1启动子区域富集H3K27Ac来促进KTN1的表达水平。This study explored the mechanism of action of KTN1-AS1 in bladder cancer. To better understand the relationship between KTN1-AS1 and KTN1, we used the UCSC online database (http://genome.ucsc.edu/) to analyze the findings. , a high overlap between the KTN1-AS1 and KTN1 promoter regions, which are highly enriched for the H3K27Ac peak, which is a marker for activity enhancers. Therefore, we used the predictive RNAct algorithm to search for KTN1-AS1-binding proteins with histone acetyltransferase (HAT) activity. We predicted that EP300 (HAT) could bind to KTN1-AS1. We performed RIP assay analysis in RT4 and T24 cells (transfected with empty vector or overexpressed KTN1-AS1). The results suggest that KTN1-AS1 may be indirectly responsible for the modification of H3K27Ac through EP300 recruitment. To prove our hypothesis, we performed ChIP-qPCR analysis. After we knocked down KTN1-AS1 in RT4 and T24 cells, H3K27Ac was deleted in the KTN1 promoter region. Conversely, H3K27Ac was highly enriched in RT4 and T24 cells overexpressing KTN1-AS1, a phenotype that disappeared in the case of EP300 knockdown. The above results indicated that KTN1-AS1 could promote the expression level of KTN1 by recruiting EP300 and enriching H3K27Ac in the KTN1 promoter region.
本发明进一步探讨了KTN1-AS1是否以KTN1依赖的方式促进膀胱癌的进展，我们进行了一系列功能获得/丧失的实验。与KTN1-AS1敲除后获得的结果一致，用sh-KTN1转染的膀胱癌细胞显示出明显更低的增殖、侵袭和迁移水平。同样，KTN1-AS1的过表达促进了膀胱癌细胞的迁移和侵袭。然而，该表型在过表达KTN1-AS1的细胞中同时沉默KTN1时部分丢失。这些结果表明KTN1在膀胱癌发生发展发挥着重要作用。从STRING和inBioMap数据库中检索到的数据的生物信息学分析显示，KTN1可能与几种蛋白质（RHOA，RAC1，RHOG，CDC42，KLC1和EEF1D）相互作用，其中，四种蛋白质（RAC1，RHOA，RHHOG和CDC42）是Rho GTPase家族的成员。以往研究报道，Rho GTPases参与细胞迁移、细胞极性、细胞周期调节和细胞骨架重构等生理病理活动。因此，我们探索了KTN1-AS1是否通过调节KTN1表达对Rho GTPase信号通路产生影响。Western印迹结果表明，沉默了KTN1的细胞中RAC1，RHOA和CDC42的蛋白水平显着降低；相反，在KTN1-AS1过表达的情况下，KTN1、RAC1、RHOA和CDC42蛋白的水平增加。在过表达KTN1-AS1的细胞中，同时敲除KTN1后，KTN1、RAC1、RHOA和CDC4的表达被部分下降。因此，以上研究结果表明KTN1-AS1可以通过调节KTN1 / Rho GTPase信号通路来促进膀胱癌的进展。The present invention further explored whether KTN1-AS1 promotes bladder cancer progression in a KTN1-dependent manner, and we performed a series of gain/loss experiments. Consistent with the results obtained after KTN1-AS1 knockout, bladder cancer cells transfected with sh-KTN1 showed significantly lower levels of proliferation, invasion and migration. Likewise, overexpression of KTN1-AS1 promoted the migration and invasion of bladder cancer cells. However, this phenotype was partially lost upon simultaneous silencing of KTN1 in cells overexpressing KTN1-AS1. These results suggest that KTN1 plays an important role in the development of bladder cancer. Bioinformatics analysis of data retrieved from STRING and inBioMap databases revealed that KTN1 may interact with several proteins (RHOA, RAC1, RHOG, CDC42, KLC1 and EEF1D), among which, four proteins (RAC1, RHOA, RHHOG) and CDC42) are members of the Rho GTPase family. Previous studies have reported that Rho GTPases are involved in physiological and pathological activities such as cell migration, cell polarity, cell cycle regulation, and cytoskeleton remodeling. Therefore, we explored whether KTN1-AS1 has an effect on the Rho GTPase signaling pathway by regulating KTN1 expression. Western blotting results showed that the protein levels of RAC1, RHOA and CDC42 were significantly decreased in KTN1-silenced cells; on the contrary, the protein levels of KTN1, RAC1, RHOA and CDC42 were increased in the case of KTN1-AS1 overexpression. In cells overexpressing KTN1-AS1, the expression of KTN1, RAC1, RHOA, and CDC4 was partially decreased after simultaneous KTN1 knockdown. Therefore, the above findings suggest that KTN1-AS1 can promote bladder cancer progression by regulating the KTN1/Rho GTPase signaling pathway.
本发明为了进一步研究KTN1-AS1是否在体内调节膀胱癌的生长，我们建立了皮下异种移植小鼠模型。T24细胞用对照载体KTN1-AS1或KTN1-AS1 + sh-KTN1稳定转染，然后注入裸鼠皮下。与对照组相比，膀胱癌细胞中KTN1-AS1的过表达能显着提高膀胱癌的生长速度（根据肿瘤体积测量），而在T24细胞中KTN1-AS1的过表达的情况下同时敲除KTN1的能消除这种效应。本发明通过使用IHC测定法观察增殖标志物Ki-67，进一步证实了KTN1-AS1的致瘤潜力。结果显示， KTN1-AS1过表达的动物组中的Ki-67水平显着增加，其增加程度远大于KTN1敲除组小鼠的Ki-67水平，且KTN1和RAC1的表达（也通过IHC评估）与上述体外结果一致。这些结果表明，KTN1-AS1能通过KTN1/Rho GTPase信号轴参与膀胱癌的发生发展。In the present invention, in order to further study whether KTN1-AS1 regulates the growth of bladder cancer in vivo, we established a subcutaneous xenograft mouse model. T24 cells were stably transfected with the control vector KTN1-AS1 or KTN1-AS1 + sh-KTN1 and then injected subcutaneously into nude mice. Overexpression of KTN1-AS1 in bladder cancer cells significantly increased the rate of bladder cancer growth (measured by tumor volume) compared to controls, while KTN1 knockdown in T24 cells was accompanied by overexpression of KTN1-AS1 can eliminate this effect. The present invention further confirmed the tumorigenic potential of KTN1-AS1 by observing the proliferation marker Ki-67 using IHC assay. The results showed that Ki-67 levels were significantly increased in the KTN1-AS1 overexpressing animal group to a much greater extent than that of KTN1 knockout mice, and the expression of KTN1 and RAC1 (also assessed by IHC) Consistent with the above in vitro results. These results suggest that KTN1-AS1 can participate in the occurrence and development of bladder cancer through the KTN1/Rho GTPase signaling axis.
总之，本研究详细描述了BUC中KTN1-AS1上调的表达模型，证实了KTN1-AS1在膀胱癌发生发展中的作用及其分子机制。高表达的KTN1-AS1能够通过顺式激活KTN1及其介导的Rho GTPase的信号传导级联反应，进而促进膀胱癌的增殖和侵袭，表明KTN1-AS1和KTN1在膀胱癌的进展中发挥着重要的作用。因此，KTN1-AS1的抑制剂可作为靶位点在制备治疗膀胱癌药物中应用。同时，KTN1的抑制剂也可作为靶位点在制备治疗膀胱癌药物中应用。In conclusion, this study describes in detail the expression model of KTN1-AS1 upregulation in BUC, confirming the role of KTN1-AS1 in the development and progression of bladder cancer and its molecular mechanism. Highly expressed KTN1-AS1 can promote the proliferation and invasion of bladder cancer by cis-activating KTN1 and its mediated signaling cascade of Rho GTPase, indicating that KTN1-AS1 and KTN1 play an important role in the progression of bladder cancer. effect. Therefore, the inhibitor of KTN1-AS1 can be used as a target site in the preparation of drugs for treating bladder cancer. At the same time, the inhibitor of KTN1 can also be used as a target site in the preparation of drugs for treating bladder cancer.
附图说明Description of drawings
图1. KTN1-AS1在膀胱癌组织中高表达。（A）KTN1-AS1在范癌中的表达。 （B，C）通过starBase和TANRIC数据库分析了差异表达的KTN1-AS1。（D）利用TANRIC数据库评估KTN1-AS1在不同膀胱癌肿瘤分级中的表达情况。（E）利用RT-qPCR在6对膀胱癌和邻近的非肿瘤组织中检测了KTN1-AS1表达。 （F）使用RT-qPCR在RT4和T24细胞中确定KTN1-AS1的细胞定位。*P <0.05，** P <0.01，*** P <0.001，**** P <0.0001。Figure 1. KTN1-AS1 is highly expressed in bladder cancer tissues. (A) Expression of KTN1-AS1 in paracarcinoma. (B,C) Differentially expressed KTN1-AS1 was analyzed by starBase and TANRIC databases. (D) The expression of KTN1-AS1 in different bladder cancer tumor grades was assessed using the TANRIC database. (E) KTN1-AS1 expression was detected in 6 pairs of bladder cancer and adjacent non-tumor tissues by RT-qPCR. (F) The cellular localization of KTN1-AS1 was determined in RT4 and T24 cells using RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
图2.敲除KTN1-AS1可抑制膀胱癌细胞的增殖、迁移和侵袭。在RT4和T24细胞中，通过sh-RNA使KTN1-AS1沉默。（A）使用RT-qPCR确认沉默效率。 （B）进行MTT法评估在沉默KTN1-AS1的RT4和T24细胞中的细胞增殖。 （C）在sh-KTN1-AS1转染后，用RT4和T24细胞进行克隆形成测定。（D）进行划痕实验来评估敲除KTN1-AS1后RT4和T24细胞的迁移。（E）利用Transwell实验研究敲除KTN1-AS1后RT4和T24细胞的侵袭能力。* P <0.05，** P <0.01，*** P <0.001。Figure 2. Knockdown of KTN1-AS1 inhibits proliferation, migration and invasion of bladder cancer cells. KTN1-AS1 was silenced by sh-RNA in RT4 and T24 cells. (A) Confirmation of silencing efficiency using RT-qPCR. (B) MTT assay was performed to assess cell proliferation in RT4 and T24 cells silenced KTN1-AS1. (C) Clonogenic assays were performed with RT4 and T24 cells after sh-KTN1-AS1 transfection. (D) Scratch experiments were performed to assess the migration of RT4 and T24 cells after knockdown of KTN1-AS1. (E) Transwell assay was used to study the invasive ability of RT4 and T24 cells after KTN1-AS1 knockout. *P < 0.05, **P < 0.01, ***P < 0.001.
图3.过表达KTN1-AS1能促进膀胱癌细胞的增殖、迁移和侵袭。（A）利用RT-qPCR确认过表达效率。（B）MTT法检测过表达KTN1-AS1的RT4和T24细胞中的细胞增殖情况。（C）在KTN1-AS1过表达后，用RT4和T24细胞进行克隆形成实验。（D）利用划痕实验评估过表达KTN1-AS1后的RT4和T24细胞迁移情况。（E）利用Transwell实验研究过表达KTN1-AS1后RT4和T24细胞的侵袭能力。* P <0.05，** P <0.01，*** P <0.001。Figure 3. Overexpression of KTN1-AS1 can promote the proliferation, migration and invasion of bladder cancer cells. (A) Confirmation of overexpression efficiency by RT-qPCR. (B) Cell proliferation in RT4 and T24 cells overexpressing KTN1-AS1 was detected by MTT assay. (C) Clonogenic experiments were performed with RT4 and T24 cells after KTN1-AS1 overexpression. (D) The migration of RT4 and T24 cells after KTN1-AS1 overexpression was assessed by scratch assay. (E) Transwell assay was used to study the invasive ability of RT4 and T24 cells after KTN1-AS1 overexpression. *P < 0.05, **P < 0.01, ***P < 0.001.
图4. KTN1在膀胱癌中高表达，并与KTN1-AS1正相关。（A，B）使用RT-qPCR和WB法检测6对膀胱癌和相邻的非肿瘤组织中KTN1的mRNA和蛋白质表达水平。（C，D）分析GEO数据库中的KTN1表达情况。（E-H）使用RT-qPCR和WB法在KTN1-AS1下调或上调后确定RT4和T24细胞中的KTN1的 mRNA和蛋白表达情况。（I-L）检索GEPIA和GEO数据库分析KTN1-AS1和KTN1水平之间的相关性情况。* P <0.05，** P <0.01，*** P <0.001。Figure 4. KTN1 is highly expressed in bladder cancer and positively correlates with KTN1-AS1. (A, B) The mRNA and protein expression levels of KTN1 in 6 pairs of bladder cancer and adjacent non-tumor tissues were detected by RT-qPCR and WB. (C, D) Analysis of KTN1 expression in the GEO database. (E-H) The mRNA and protein expression of KTN1 in RT4 and T24 cells were determined after KTN1-AS1 down-regulation or up-regulation using RT-qPCR and WB methods. (I-L) GEPIA and GEO databases were searched to analyze the correlation between KTN1-AS1 and KTN1 levels. *P < 0.05, **P < 0.01, ***P < 0.001.
图5. KTN1-AS1募集EP300到KTN1启动子，并与KTN1表达正相关。 （A）使用UCSC数据库观察KTN1-AS1和KTN1之间的位置关系。（B）利用RNAct算法预测潜在的KTN1-AS1结合蛋白，特别是组蛋白乙酰转移酶。（C）进行RIP实验鉴定KTN1-AS1与EP300蛋白的相互作用情况。（D，E）检索GEPIA和GEO数据库分析EP300和KTN1之间的相关性情况。（F）敲除EP300后，在RT4和T24细胞中检测到EP300和KTN1的蛋白表达情况。（G）行ChIP-qPCR法检测H3K27Ac，尤其是在KTN1启动子区域。* P <0.05，** P <0.01，*** P <0.001。Figure 5. KTN1-AS1 recruits EP300 to the KTN1 promoter and positively correlates with KTN1 expression. (A) The positional relationship between KTN1-AS1 and KTN1 was observed using the UCSC database. (B) Prediction of potential KTN1-AS1 binding proteins, particularly histone acetyltransferases, using the RNAct algorithm. (C) RIP experiments were performed to identify the interaction between KTN1-AS1 and EP300 protein. (D, E) Search GEPIA and GEO databases to analyze the correlation between EP300 and KTN1. (F) After knockdown of EP300, the protein expression of EP300 and KTN1 was detected in RT4 and T24 cells. (G) ChIP-qPCR was performed to detect H3K27Ac, especially in the KTN1 promoter region. *P < 0.05, **P < 0.01, ***P < 0.001.
图6. KTN1-AS1以KTN1依赖的方式促进膀胱癌细胞的增殖、迁移和侵袭。（A）使用RT-qPCR确认靶基因的下调和/或上调（转染效率）。（B）MTT法评估转染sh-KTN1，KTN1-AS1和KTN1-AS1 + sh-KTN1的膀胱癌细胞的增殖情况。（C）使用不同构建体转染的RT4和T24细胞进行克隆形成实验检测。 （D）利用划痕实验检测用不同构建体转染的RT4和T24细胞的迁移情况。（E）Transwell实验探讨用不同构建体转染的RT4和T24细胞的侵袭能力变化。* P <0.05，** P <0.01，*** P <0.001。Figure 6. KTN1-AS1 promotes proliferation, migration and invasion of bladder cancer cells in a KTN1-dependent manner. (A) Confirmation of down-regulation and/or up-regulation of target genes (transfection efficiency) using RT-qPCR. (B) The proliferation of bladder cancer cells transfected with sh-KTN1, KTN1-AS1 and KTN1-AS1 + sh-KTN1 was assessed by MTT assay. (C) Colony formation assays using RT4 and T24 cells transfected with different constructs. (D) Migration of RT4 and T24 cells transfected with different constructs was examined by scratch assay. (E) Transwell experiments to explore changes in the invasive ability of RT4 and T24 cells transfected with different constructs. *P < 0.05, **P < 0.01, ***P < 0.001.
图7. KTN1-AS1调节KTN1/Rho GTPase轴。（A-C）使用STRING和inBio-Map数据库预测到的KTN1潜在相互作用蛋白。（D）通过WB法检测在转染了sh-KTN1，KTN1-AS1和KTN1-AS1+sh-KTN1的RT4和T24细胞中KTN1，RAC1、RHOA、CDC42、RHOG和β-actin的表达情况。* P <0.05，** P <0.01，*** P <0.001。Figure 7. KTN1-AS1 regulates the KTN1/Rho GTPase axis. (A-C) Potential KTN1 interacting proteins predicted using STRING and inBio-Map databases. (D) The expression of KTN1, RAC1, RHOA, CDC42, RHOG and β-actin in RT4 and T24 cells transfected with sh-KTN1, KTN1-AS1 and KTN1-AS1+sh-KTN1 was detected by WB method. *P < 0.05, **P < 0.01, ***P < 0.001.
图8. KTN1-AS1促进KTN1表达来介导的体内膀胱癌的发生发展。（A）裸鼠皮下注射T24对照细胞（用空载体转染），用KTN1-AS1转染的T24细胞或用KTN1-AS1和sh-KTN1共转染的T24细胞。（B）从注射细胞后的第7天到第25天，每3天测量一次肿瘤体积情况。（C）通过免疫组织化学法检测肿瘤中的Ki-67、KTN1和RAC1表达情况。* P <0.05，** P <0.01，*** P <0.001。Figure 8. KTN1-AS1 promotes KTN1 expression to mediate the development of bladder cancer in vivo. (A) Nude mice were subcutaneously injected with T24 control cells (transfected with empty vector), T24 cells transfected with KTN1-AS1 or T24 cells co-transfected with KTN1-AS1 and sh-KTN1. (B) Tumor volume was measured every 3 days from
图9. KTN1-AS1通过顺式激活KTN1及其介导的Rho GTPase的信号传导级联反应来促进膀胱癌的肿瘤发生：示意图。Figure 9. KTN1-AS1 promotes bladder cancer tumorigenesis by cis-activating KTN1 and its mediated signaling cascade of Rho GTPase: schematic.
图10.（A-C）基于Cancer RNA-Seq Nexus数据库，分析KTN1-AS1在不同膀胱癌肿瘤分期（与相邻正常组织相比）的差异表达情况。Figure 10. (A-C) Differential expression of KTN1-AS1 in different bladder cancer tumor stages (compared to adjacent normal tissues) was analyzed based on Cancer RNA-Seq Nexus database.
图11.在RT4细胞中确定KTN1-AS1的敲低效率。Figure 11. Determination of KTN1-AS1 knockdown efficiency in RT4 cells.
图12.（A，B）从GEO数据库检索的数据集中KTN1-AS1和KTN1水平之间的相关性情况。Figure 12. (A,B) Correlation between KTN1-AS1 and KTN1 levels in datasets retrieved from the GEO database.
图13. KTN1与EP300正相关。（A）使用catRAPID数据库预测KTN1-AS1和EP300之间的相互作用情况。（B-O）GEO数据库检索的不同数据集中KTN1和EP300之间的相关性情况。Figure 13. KTN1 is positively correlated with EP300. (A) Prediction of the interaction between KTN1-AS1 and EP300 using the catRAPID database. (B-O) Correlation between KTN1 and EP300 in different datasets retrieved from the GEO database.
图14.（A，B）KTN1和KTN1-AS1在RT4和T24细胞中的干扰效率。Figure 14. (A,B) Interference efficiency of KTN1 and KTN1-AS1 in RT4 and T24 cells.
下面结合附图和实验数据对本发明做进一步的解释和说明：Below in conjunction with accompanying drawing and experimental data, the present invention is further explained and illustrated:
1、材料及方法1. Materials and methods
细胞培养及转染，qRT-PCR分析，蛋白质免疫印迹分析，免疫组织化学测定，MTT测定，划痕实验，Transwell实验，RNA pulldown，RIP，核质分离实验，染色质免疫共沉淀，双荧光素酶实验，以上方法均是现有方法，在此不再累述。Cell culture and transfection, qRT-PCR analysis, Western blot analysis, immunohistochemical assay, MTT assay, scratch assay, Transwell assay, RNA pulldown, RIP, nucleocytoplasmic separation assay, chromatin immunoprecipitation, dual fluorescein Enzyme experiments, the above methods are all existing methods, and will not be repeated here.
2、结果2. Results
2.1 LncRNA KTN1-AS1在膀胱癌中上调2.1 LncRNA KTN1-AS1 is upregulated in bladder cancer
为了揭示KTN1-AS1是否在膀胱癌中上调，我们首先从包括TCGA数据库在内的不同数据库中检索和分析了KTN1-AS1的表达数据。我们发现KTN1-AS1不仅在膀胱癌中显着升高，而且在其他几种癌症中也显着升高（图1A-C）。在Ⅱ至Ⅳ期膀胱癌中也发现了相似的结果（分别为图1D和图10A-C）。此外，与相邻的非肿瘤性膀胱组织相比，本研究中分析的膀胱癌组织还显示KTN1-AS1显着增加（图1E）。另外，我们发现KTN1-AS1主要定位于细胞核中（图1F），这表明KTN1-AS1功能与转录调控相关。总之，这些结果表明KTN1-AS1可能发挥致癌作用。To reveal whether KTN1-AS1 is upregulated in bladder cancer, we first retrieved and analyzed the expression data of KTN1-AS1 from different databases including the TCGA database. We found that KTN1-AS1 was significantly elevated not only in bladder cancer, but also in several other cancers (Fig. 1A–C). Similar results were found in stage II to IV bladder cancer (Fig. 1D and Fig. 10A-C, respectively). In addition, the bladder cancer tissue analyzed in this study also showed a significant increase in KTN1-AS1 compared with adjacent non-neoplastic bladder tissue (Fig. 1E). Additionally, we found that KTN1-AS1 was mainly localized in the nucleus (Fig. 1F), suggesting that KTN1-AS1 function is related to transcriptional regulation. Taken together, these results suggest that KTN1-AS1 may play an oncogenic role.
2.2敲除KTN1-AS1能抑制膀胱癌细胞的增殖、迁移和侵袭2.2 Knockout of KTN1-AS1 can inhibit the proliferation, migration and invasion of bladder cancer cells
为了评估KTN1-AS1在膀胱癌中的潜在作用，我们使用两种不同的膀胱癌细胞系进行了一系列体外沉默实验。值得注意的是，在RT4和T24细胞中均证实了KTN1-AS1沉默（分别为图2A和图11）。首先，我们评估了敲除KTN1-AS1对膀胱癌细胞生长的影响。MTT实验表明，沉默KTN1-AS1能显着降低膀胱癌细胞的增殖（图2B）。同样，克隆形成试验表明，敲除KTN1-AS1能显着抑制膀胱癌细胞的克隆形成能力（图2C）。另外，为探讨KTN1-AS1在癌症转移中的作用，我们进行了划痕实验和Transwell实验，结果表明敲除KTN1-AS1可显著抑制伤口愈合能力和膀胱癌细胞的侵袭潜能（分别见图2D、2E）。这些结果表明，敲除KTN1-AS1能抑制膀胱癌细胞的增殖和转移。To assess the potential role of KTN1-AS1 in bladder cancer, we performed a series of in vitro silencing experiments using two different bladder cancer cell lines. Notably, KTN1-AS1 silencing was confirmed in both RT4 and T24 cells (Fig. 2A and Fig. 11, respectively). First, we evaluated the effect of KTN1-AS1 knockdown on bladder cancer cell growth. MTT experiments showed that silencing KTN1-AS1 significantly reduced the proliferation of bladder cancer cells (Fig. 2B). Likewise, clonogenic assays showed that knockdown of KTN1-AS1 significantly inhibited the clonogenic ability of bladder cancer cells (Fig. 2C). In addition, to explore the role of KTN1-AS1 in cancer metastasis, we performed scratch assay and Transwell assay, and the results showed that knockout of KTN1-AS1 could significantly inhibit the wound healing ability and the invasive potential of bladder cancer cells (see Figure 2D, 2E). These results suggest that knockdown of KTN1-AS1 inhibits the proliferation and metastasis of bladder cancer cells.
2.3过表达促进膀胱癌细胞的迁移和侵袭2.3 Overexpression promotes the migration and invasion of bladder cancer cells
为了进一步探讨KTN1-AS1的致癌作用，我们建立了（稳定地）过表达KTN1-AS1的细胞系。我们使用RT-qPCR在RT4和T24细胞中均证实了KTN1-AS1过表达效率（图3A）。与沉默KTN1-AS1观察到的实验结果相反，过表达KTN1-AS1能明显促进细胞活力并增加细胞集落形成能力（分别为图3B、3C）。划痕实验表明，在KTN1-AS1过表达后，膀胱癌细胞的迁移率显着增加（图3D）。此外，根据transwell实验的结果，与对照组相比，过表达KTN1-AS1能显着增强膀胱癌细胞的侵袭能力（图3E）。这些发现表明KTN1-AS1的过表达能显著促进膀胱癌细胞的增殖和侵袭。To further explore the oncogenic role of KTN1-AS1, we established a cell line (stably) overexpressing KTN1-AS1. We confirmed KTN1-AS1 overexpression efficiency in both RT4 and T24 cells using RT-qPCR (Fig. 3A). Contrary to the experimental results observed by silencing KTN1-AS1, overexpression of KTN1-AS1 significantly promoted cell viability and increased cell colony-forming ability (Fig. 3B, 3C, respectively). Scratch experiments showed that the migration rate of bladder cancer cells was significantly increased after KTN1-AS1 overexpression (Fig. 3D). In addition, according to the results of transwell experiments, overexpression of KTN1-AS1 significantly enhanced the invasive ability of bladder cancer cells compared with the control group (Fig. 3E). These findings suggest that overexpression of KTN1-AS1 can significantly promote the proliferation and invasion of bladder cancer cells.
2.4在膀胱癌中上调，并与KTN1-AS1的表达呈正相关2.4 was upregulated in bladder cancer and positively correlated with the expression of KTN1-AS1
考虑到KTN1-AS1在膀胱癌中的潜在作用，我们猜想lncRNA是否可以顺式或反式作用，特别是影响其邻近基因KTN1的表达（KTN1-AS1映射到上游启动子区域的反义DNA链KTN1）。我们在收集的膀胱癌样品检测KTN1的表达，与正常组织相比，KTN1在癌组织中的mRNA和蛋白质水平的表达均显着增加（分别为图4A和B）。此外，GEO数据库中的两个数据集（GSE3167和GSE138118）也显示KTN1在膀胱癌中显着上调（分别为图4C和D）。值得注意的是，我们观察到KTNI-AS1的敲除或过表达能影响KTN1的mRNA和蛋白水平（图4E-H），这表明lncRNA可能起顺式作用。我们进一步分析了KTN1-AS1和KTN1表达之间的相关性。从不同数据库中检索到的数据的分析表明，确认KTN1-AS1与KTN1表达呈显著正相关（图4I-L），其与GEO数据库中的分析结果一致（图12A、12B）。由于细胞定位分析显示KTN1-AS1在细胞核中的表达高于细胞质（图1F），因此我们假设KTN1-AS1可能与核蛋白相互作用，在转录水平上调节KTN1的表达。Considering the potential role of KTN1-AS1 in bladder cancer, we wondered whether the lncRNA could act in cis or trans, especially affecting the expression of its neighboring gene KTN1 (KTN1-AS1 maps to the antisense DNA strand in the upstream promoter region of KTN1) ). We detected the expression of KTN1 in the collected bladder cancer samples, and the expression of KTN1 at both mRNA and protein levels was significantly increased in cancer tissues compared with normal tissues (Fig. 4A and B, respectively). In addition, two datasets in the GEO database (GSE3167 and GSE138118) also showed that KTN1 was significantly upregulated in bladder cancer (Fig. 4C and D, respectively). Notably, we observed that knockdown or overexpression of KTNI-AS1 could affect the mRNA and protein levels of KTN1 (Fig. 4E–H), suggesting that lncRNAs may act in cis. We further analyzed the correlation between KTN1-AS1 and KTN1 expression. Analysis of data retrieved from different databases showed that KTN1-AS1 was confirmed to be significantly positively correlated with KTN1 expression (Fig. 4I-L), which was consistent with the analysis results in the GEO database (Fig. 12A, 12B). Since cellular localization analysis showed that KTN1-AS1 was more expressed in the nucleus than in the cytoplasm (Fig. 1F), we hypothesized that KTN1-AS1 might interact with nuclear proteins to regulate KTN1 expression at the transcriptional level.
2.5通过募集EP300蛋白和下游表观遗传修饰来调节KTN1表达2.5 Regulation of KTN1 expression by recruiting EP300 protein and downstream epigenetic modifications
为了更好地了解KTN1-AS1和KTN1之间的关系，我们使用了UCSC数据库分析。结果显示，KTN1-AS1和KTN1启动子区域之间高度重叠，该区域高度富集H3K27Ac峰（图5A）；值得注意的是，H3K27Ac是活性增强剂的标记物。该观察结果使我们使用预测性RNAct算法寻找具有组蛋白乙酰转移酶（HAT）活性的KTN1-AS1结合蛋白。我们预测到EP300（HAT）能与KTN1-AS1结合（图5B）。为了验证该猜想，我们在RT4和T24细胞（空载体转染或过表达KTN1-AS1）的情况下进行了RIP分析。结果显示，KTN1-AS1能与EP300一起被下拉，与阴性对照细胞相比，过表达KTN1-AS1的丰度更大（图5C）。然后，我们使用检索到的TCGA和GEO数据库研究了其他相关关系，膀胱癌样品中KTN1的表达与EP300呈显著正相关（分别为图5D和E）。值得注意的是，我们还观察到敲除EP300后KTN1蛋白水平表达下降，这进一步证实了EP300和KTN1之间的关系（图5F）。因此，我们推测KTN1-AS1通过募集EP300间接负责H3K27Ac的修饰。为了证明我们的假设，我们进行了ChIP-qPCR分析，结果示在RT4和T24细胞中敲除KTN1-AS1的情况下，KTN1启动子区域中的H3K27Ac缺失（图5G）。相反，H3K27Ac在过表达KTN1-AS1的RT4和T24细胞中呈现高度富集，这种现象在敲除EP300的情况下消失（图5G）。总之，这些数据表明，KTN1-AS1通过募集EP300至KTN1启动子区域富集H3K27Ac来促进KTN1的表达水平。To better understand the relationship between KTN1-AS1 and KTN1, we used UCSC database analysis. The results showed high overlap between the KTN1-AS1 and KTN1 promoter regions, which are highly enriched for the H3K27Ac peak (Fig. 5A); notably, H3K27Ac is a marker of activity enhancers. This observation led us to search for KTN1-AS1-binding proteins with histone acetyltransferase (HAT) activity using a predictive RNAct algorithm. We predicted that EP300 (HAT) could bind to KTN1-AS1 (Fig. 5B). To test this conjecture, we performed RIP analysis in RT4 and T24 cells (either transfected with empty vector or overexpressing KTN1-AS1). The results showed that KTN1-AS1 could be pulled down together with EP300, and the abundance of overexpressing KTN1-AS1 was greater than that of negative control cells (Fig. 5C). We then investigated other correlations using the retrieved TCGA and GEO databases, and the expression of KTN1 in bladder cancer samples was significantly positively correlated with EP300 (Fig. 5D and E, respectively). Notably, we also observed a decrease in KTN1 protein level expression after knockdown of EP300, which further confirmed the relationship between EP300 and KTN1 (Fig. 5F). Therefore, we speculate that KTN1-AS1 is indirectly responsible for the modification of H3K27Ac by recruiting EP300. To prove our hypothesis, we performed ChIP-qPCR analysis, which showed deletion of H3K27Ac in the KTN1 promoter region in the presence of KTN1-AS1 knockdown in RT4 and T24 cells (Fig. 5G). In contrast, H3K27Ac was highly enriched in RT4 and T24 cells overexpressing KTN1-AS1, a phenomenon that was abolished in the knockdown of EP300 (Fig. 5G). Taken together, these data suggest that KTN1-AS1 promotes the expression level of KTN1 by recruiting EP300 to the KTN1 promoter region to enrich for H3K27Ac.
2.6通过KTN1/Rho GTPase信号通路促进膀胱癌进展2.6 Promote bladder cancer progression through KTN1/Rho GTPase signaling pathway
为了进一步探讨KTN1-AS1是否以KTN1依赖的方式促进膀胱癌的进展，我们进行了一系列功能获得/丧失的实验。与KTN1-AS1敲除后获得的结果一致，用sh-KTN1转染的膀胱癌细胞显示更低的增殖、侵袭和迁移水平（图6B-E）。同样，KTN1-AS1的过表达能促进膀胱癌细胞的迁移和侵袭。然而，该表型在过表达KTN1-AS1的细胞中在敲除KTN1的情况下部分丢失（图6B-E）。这些结果表明KTN1也是膀胱癌中的致癌基因。对从STRING和inBioMap数据库中检索到的数据的生物信息学分析显示，KTN1可能与几种蛋白质（RHOA，RAC1，RHOG，CDC42，KLC1和EEF1D）相互作用（图7A-C）。其中，有四种蛋白质（RAC1，RHOA，RHHOG和CDC42）是RhoGTPase家族的成员。越来越多的证据表明，Rho GTPases参与细胞迁移、细胞极性、细胞周期调节和细胞骨架重构等生理病理过程。因此，我们检查了KTN1-AS1是否通过调节KTN1表达对Rho GTPase信号通路产生影响。WB实验结果表明，敲除KTN1的细胞中RAC1、RHOA和CDC42的蛋白水平显着降低；然而，对于RHOG蛋白水平却并非如此（图7D）。相反，在KTN1-AS1过表达的情况下，KTN1、RAC1、RHOA和CDC42蛋白的水平增加。与我们之前的推测一致，在过表达KTN1-AS1的细胞中，sh-KTN1转染后，KTN1、RAC1、RHOA和CDC4的表达被部分恢复（图7D）。两者合计，KTN1-AS1可以通过调节KTN1/Rho GTPase信号通路来促进膀胱癌的进展。To further explore whether KTN1-AS1 promotes bladder cancer progression in a KTN1-dependent manner, we performed a series of gain/loss experiments. Consistent with the results obtained after KTN1-AS1 knockdown, bladder cancer cells transfected with sh-KTN1 showed lower levels of proliferation, invasion and migration (Fig. 6B–E). Similarly, overexpression of KTN1-AS1 can promote the migration and invasion of bladder cancer cells. However, this phenotype was partially lost with KTN1 knockdown in KTN1-AS1-overexpressing cells (Fig. 6B–E). These results suggest that KTN1 is also an oncogene in bladder cancer. Bioinformatic analysis of data retrieved from STRING and inBioMap databases revealed that KTN1 may interact with several proteins (RHOA, RAC1, RHOG, CDC42, KLC1 and EEF1D) (Fig. 7A–C). Among them, four proteins (RAC1, RHOA, RHHOG and CDC42) are members of the RhoGTPase family. Accumulating evidence suggests that Rho GTPases are involved in physiopathological processes such as cell migration, cell polarity, cell cycle regulation, and cytoskeleton remodeling. Therefore, we examined whether KTN1-AS1 has an effect on the Rho GTPase signaling pathway by regulating KTN1 expression. WB experiments showed that the protein levels of RAC1, RHOA and CDC42 were significantly reduced in KTN1 knockout cells; however, this was not the case for RHOG protein levels (Fig. 7D). In contrast, the levels of KTN1, RAC1, RHOA and CDC42 proteins increased in the presence of KTN1-AS1 overexpression. Consistent with our previous speculation, the expression of KTN1, RAC1, RHOA and CDC4 was partially restored after sh-KTN1 transfection in cells overexpressing KTN1-AS1 (Fig. 7D). Taken together, KTN1-AS1 can promote bladder cancer progression by regulating the KTN1/Rho GTPase signaling pathway.
2.7在体内促进KTN1介导的膀胱癌肿瘤发生2.7 Promotes KTN1-mediated bladder cancer tumorigenesis in vivo
为了进一步研究KTN1-AS1是否在体内调节膀胱癌的生长，我们建立了皮下异种移植小鼠模型。T24细胞用对照载体KTN1-AS1或KTN1-AS1 + sh-KTN1稳定转染，然后注入裸鼠皮下（图8A）。值得注意的是，与对照组相比，膀胱癌细胞中过表达KTN1-AS1能显着提高了癌症的生长速度（根据肿瘤体积测量），而在T24细胞中KTN1-AS1的过表达的情况下敲除KTN1能部分消除观察到的现象。癌症的促进作用（图8B）。通过使用IHC测定法观察增殖标志物Ki-67，进一步证实了致瘤潜力。与上述结果一致， KTN1-AS1过表达的动物组中的Ki-67水平显着增加，其程度远大于接受KTN1沉默，KTN1-AS1过表达的动物中的水平细胞（图8C）。另外，KTN1和RAC1的表达（也通过IHC评估）与上述体外结果一致（图8C）。总之，这些结果表明，KTN1-AS1可通过KTN1/Rho GTPase轴来参与膀胱癌的肿瘤发生。To further investigate whether KTN1-AS1 regulates bladder cancer growth in vivo, we established a subcutaneous xenograft mouse model. T24 cells were stably transfected with the control vector KTN1-AS1 or KTN1-AS1 + sh-KTN1 and then injected subcutaneously into nude mice (Fig. 8A). Notably, overexpression of KTN1-AS1 in bladder cancer cells significantly increased the rate of cancer growth (as measured by tumor volume) compared to controls, whereas overexpression of KTN1-AS1 in T24 cells Knockdown of KTN1 partially abolished the observed phenomenon. Cancer promotion (Fig. 8B). The tumorigenic potential was further confirmed by observing the proliferation marker Ki-67 using an IHC assay. Consistent with the above results, Ki-67 levels were significantly increased in the KTN1-AS1-overexpressing animal group to a much greater extent than in animals receiving KTN1-silenced, KTN1-AS1-overexpressing cells (Fig. 8C). Additionally, the expression of KTN1 and RAC1 (also assessed by IHC) was consistent with the above in vitro results (Fig. 8C). Taken together, these results suggest that KTN1-AS1 may be involved in bladder cancer tumorigenesis through the KTN1/Rho GTPase axis.
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