第一部分

研究目的

结节性硬化症相关肾血管平滑肌脂肪瘤 （Tuberous sclerosis complex associated renal angiomyolipoma，TSC-RAML）是一种可累及双侧肾脏的罕见疾病。然而目前国内尚无系统的TSC-RAML相关数据。因此本部分研究旨在通过建立TSC-RAML临床数据库，了解国内TSC-RAML患者的临床信息。这项成果将加强对TSC-RAML患者的管理，并有助于后续的研究。

研究方法　

回顾性分析2010年1月至2023年1月就诊于北京协和医院泌尿外科门诊的TSC-RAML患者的资料。收集患者的病历资料：包括主诉、现病史、既往史、个人史、家族史等病史，收集年龄、性别、体格检查、影像学检查及其他TSC临床表现等信息。对于存在家族史的患者绘制家系系谱图并进行家系基因检测。

研究结果　

在该数据库中共包含186例TSC-RAML患者， 其中男性65例，女性121例， 男女比例为１：1.86。患者中位年龄为31岁。分级方面，117 例（62.9%）TSC-RAML为6级。22例（11.8%）存在肿瘤破裂出血病史。高分级与低分级肿瘤在破裂出血方面存在统计学差异（P = 0.0475）。合并TSC其他临床表现以血管纤维瘤／纤维斑块（155/186，83.3%）、室管膜下结节（103/146，70.5%）、肺淋巴管肌瘤病（102/157，65.0%）、色素脱失斑（114/186，61.3%）、 “鲨革” 斑（83/186，44.6%）最为常见。其中肺淋巴管肌瘤病（lymphangioleiomyomatosis，LAM）患者均为女性（P＜0.0001）。数据库中共包含12个TSC家系、共27例患者，其中男性12例，女性15例，年龄范围8 ~ 67岁。所有TSC家系患者均检出皮肤病变：面部血管纤维瘤25例，色素脱失斑18例，指/趾甲纤维瘤15例，“鲨革”13例。其他TSC合并临床表现包括：肾血管平滑肌脂肪瘤14例，室管膜下结节6例，肺淋巴管肌瘤病3例。对TSC家系患者进行二代基因检测。共报道TSC2突变家系8个，TSC1突变家系2个，无突变家系2个。在所有突变中，发现3个TSC2新致病突变（TSC2: c.208dup, c.1874C>G, c.1852del）。

研究结论　

在对数据库进行统计后，我们发现TSC-RAML患者以女性为主。皮肤病变和神经系统病变最为常见。大多数TSC-RAML分级为6级，LAM病仅累及女性。对数据库中TSC家系的基因检测结果进一步拓展了基因突变谱。TSC-RAML临床数据库的构建将有助于患者的管理、随访与治疗。

关键词: 结节性硬化症；肾血管平滑肌脂肪瘤；数据库；临床特征；性别分布；合并症；基因检测；家系

第二部分

研究目的

在前期研究中，我们发现miR-132-3p通过调节凋亡激活因子BCL2L11增加TSC细胞的增殖能力。然而该miRNA上游的调控机制尚不明确。基于circRNA能够对miRNA发挥调节作用这一理论，通过分析TSC-RAML及其配对正常肾组织的circRNA测序数据，探究表达异常的环状RNA在TSC-RAML中的作用。

研究方法

在TSC-RAML与瘤旁正常组织的circRNA差异表达谱中以截点值（cut-off）|差异倍数（fold change）| ＞2，P＜0.05为标准进行筛选，通过生信分析确定目标circRNA。利用CRISPR/Cas9系统敲除（Knock out, KO）NIH-3T3野生型细胞中的TSC2基因构建TSC模型细胞，并通过多聚酶链式反应 （PCR）、Western blot检测（WB）和mTOR抑制剂依维莫司（everolimus）对敲除结果进行验证。采用细胞计数试剂盒-8（Cell Counting Kit-8, CCK-8）、Transwell实验、流式细胞术检测该TSC模型细胞的增殖能力、侵袭能力以及凋亡情况。确认TSC模型细胞构建成功后，利用PCR、细胞增殖及侵袭实验、药敏实验等方法探究表达异常的目标circRNA对TSC模型细胞的调控作用。

研究结果

在对TSC-RAML及其配对瘤旁组织circRNA差异表达谱进行筛选后中，共有2308个差异表达的circRNA，其中hsa_circ_0022168在TSC-RAML中表达差异性下调。将hsa_circ_0022168作为研究目标后，我们开始构建TSC模型细胞系。我们利用CRISPR/Cas9系统成功敲除NIH-3T3细胞系的TSC2基因，通过PCR、WB、药敏、细胞增殖、侵袭等实验，证明TSC2 KO细胞系构建成功，该细胞系可作为TSC疾病的模型细胞。基于构建的细胞系，过表达hsa_circ_0022168后，miR-132-3p表达下降，并能够抑制TSC模型细胞的增殖和侵袭能力。

研究结论

通过对TSC-RAML组织中差异表达的circRNA进行筛选，我们发现hsa_circ_0022168差异性下调。利用构建的TSC模型细胞，我们进一步证明hsa_circ_0022168对miR-132-3p具有调控作用，并能够抑制TSC细胞系的增殖和侵袭，为探究TSC-RAML发病机制、寻找治疗新靶点提供了新的方向。

关键词: 结节性硬化症相关肾血管平滑肌脂肪瘤，成簇规律间隔回文短重复序列，基因敲除，TSC模型细胞，环状RNA，增殖，侵袭

Objective

Tuberous sclerosis complex associated renal angiomyolipoma (TSC-RAML) is a rare disease characterized by involving both kidneys. However, there is currently no systematic data of TSC-RAML in China. Therefore, this part aimed to clarify the clinical information of TSC-RAML patients in China by establishing a TSC-RAML clinical database. This accomplishment will enhance the management of TSC-RAML patients and contribute to future research.

Methods

The data of the TSC-RAML patients in Department of Urology, Peking Union Medical College Hospital, were retrospectively reviewed from January 2010 to January 2023. Medical records were retrieved, including chief complaint, present history, past history, personal history, family history, etc. Age, gender, physical examination, imagological examination and other TSC comorbidities were also added. Pedigree and next-generation sequencing were applied among patients in TSC families.

Results

A total of 186 TSC-RAML patients were included in the clinical database，65 of whom were males and 121 were females (male-to-female ratio 1:1.86). The median age of all patients was 31. Totally，117 cases (62.9%) of TSC-RAML were rated stage 6. Twenty-two cases (11.8%) had a history of tumor rupture and bleeding. Research showed statistical differences between high and low stage on TSC-RAML rupture bleeding (P=0.0475). Angiofibromas/fibrous cephalic plaque (155/186, 83.3%)，subependymal nodules (103/146, 70.5%), lymphangioleiomyomatosis (102/157, 65.0%), hypomelanotic macules(114/186，61.3%), shagreen patch (83/186，44.6%) were the most common clinical manifestations. All patients with lymphangioleiomyomatosis (LAM) were female (P＜0.0001). There were twenty-seven patients of 12 TSC families in this database, including 12 male and 15 female patients, aged 8-67 years. Skin lesions were detected among all TSC patients, including 25 cases of facial angiofibromas, 18 cases of hypomelanotic macules, 15 cases of ungual fibromas, and 13 cases of shagreen patch. Other clinical features were also revealed: 14 cases of renal angiomyolipoma, 6 cases of subependymal nodules, and 3 case of lymphangioleiomyomatosis. Next-generation sequencing was applied in TSC families. Totally, TSC2 mutations were reported in 8 families, TSC1 mutations were reported in 2 families and the last two families were genetically negative. The novel mutations (TSC2: c.208dup, c.1874C>G, c.1852del) were first reported in TSC.

Conclusion

After statistical analysis of the information in the database, we found that female patients predominated TSC-RAML patients. Dermatological and nervous lesions were the most common comorbidities in TSC-RAML patients. Most RAML were in stage 6. All patients with LAM in this study were female. The genetic sequencing results of TSC families in the database effectively expanded the mutation spectrum. The construction of TSC-RAML clinical database will be helpful for the management, follow-up, and treatment of TSC-RAML patients.

Keywords: tuberous sclerosis complex; renal angiomyolipoma; database; clinical features; gender distribution; comorbidities; next-generation sequencing; family.

Objective

Our previous study showed that miR-132-3p increased the proliferative ability of TSC cells through an apoptotic activator BCL2L11. However, the regulatory mechanism of this miRNA remains unclear. Based on the theory that circRNA can regulate miRNA, the role of abnormal expression of circular RNA in TSC-RAML was explored by analyzing the circRNA sequencing data of TSC-RAML and its paired normal kidney tissue.

Methods

The differently expressed profile of circRNAs between the TSC-RAML and adjacent non-neoplastic tissues was retrieved with the cut-off of |fold change| > 2 and P value < 0.05. The bioinformatics analysis was used for the target circRNA. TSC2 was knocked out by CRISPR/Cas9 system in the wide-type NIH-3T3 to construct TSC cellular model. The knockout results were verified by polymerase chain reaction sequencing (PCR), western blot assay (WB) and everolimus, a mammalian target of rapamycin (mTOR) inhibitor. Cell Counting Kit-8, Transwell assay, and flow cytometry were used to assess the proliferation, invasion, and apoptosis of the cell line. After confirming the successful construction of the TSC cellular model, we investigate the regulatory role of the target circRNA on TSC cellular model by PCR, cell proliferation and invasion assays, and drug sensitivity tests.

Results

After checking the circRNA differential expression profiles of TSC-RAML and its paired normal kidney tissue, a total of 2308 circRNAs were differentially screened out. The expression of hsa_circ_0022168 was downregulated in TSC-RAML. After selecting hsa_circ_0022168 as the research target, we proceeded to establish TSC cellular model. TSC2 was knocked out in NIH-3T3 cell lines by CRISPR/Cas9 system. PCR, WB, everolimus sensitivity test, proliferation and invasion assays showed that the TSC2 KO cell line was successfully constructed, which was suitable as a cellular model for TSC. Overexpression of hsa_circ_0022168 led to down-regulated expression of miR-132-3p, which could inhibit the proliferation and invasion ability in TSC cellular model.

Conclusion

After screening of differentially expressed circRNA in TSC-RAML, the downregulated hsa_circ_0022168 was identified as the target for our subsequent research. Based on the constructed TSC cellular model, further experiments showed that hsa_circ_0022168 had a regulatory effect on miR-132-3p and could inhibit the proliferation and invasion of the TSC cellular model, providing a new direction for exploring the pathogenesis of TSC-RAML and identifying new therapeutic targets.

Keywords TSC-RAML, CRISPR, knockout, TSC cellular model, circRNA, proliferation, invasion