摘要
研究表明多种microRNA（miRNA）可能在肝癌的发生发展中发挥重要作用，其作用机制仍值得进一步研究和探讨。因此，本研究从已报道的肝癌差异表达miRNA中进一步筛选关键miRNA，并验证和探讨其作用机制。
从已发表的研究中筛选出肝癌组织及肝癌患者血清/血浆中与正常肝组织及正常血清/血浆中共同的差异表达miRNA；用qRT-PCR在正常肝细胞与肝癌细胞中对筛选出的目标miRNA表达情况进行验证；用过表达和抑制的方法观察目标miRNA对肝癌细胞侵袭能力（Transwell实验）与增殖能力（MTT实验）的影响，以及在30例临床标本中检测目标miRNA的表达并通过KM plotter网站分析其对肝癌患者生存的影响；通过miRDB和GEPIA数据库预测和分析目标miRNA的靶基因，并用逆转实验和双荧光素酶报告实验进一步验证。
在肝癌组织（vs.正常肝组织）及肝癌患者血清/血浆（vs.正常人血清/血浆）中共同高表达的miRNA有4个（miR-18a-3p、miR-221-3p、miR-222-3p、miR-224-3p），共同低表达的miRNA有2个（miR-26a-3p、miR-125b-3p）。qRT-PCR实验证实，与正常肝细胞比较，miR-18a在肝癌细胞中高表达，miR-26a在肝癌细胞中低表达（均P<0.05）。过表达/抑制miR-18a-3p表达能促进/降低肝癌细胞的侵袭及生长能力（均P<0.05），而过表达/抑制miR-26a-3p对肝癌细胞的侵袭及生长能力影响无不法确定。分析结果显示，ADCY1是miR-18a-3p的靶基因，过表达ADCY1能部分逆转miR-18a-3p对肝癌细胞的上述作用，同时，表达上调的miR-18a-3p能通过结合到ADCY1 mRNA 3'UTR抑制ADCY1的表达。
肝癌是世界上第2位最常见恶性肿
microRNA（miRNA）是大小为17~25个核苷酸的短RNA分子，可通过转录后调控来抑制靶基因的表达。单个miRNA可以靶向数百个mRNA，并影响通常参与功能相互作用途径的许多基因的表达。miRNA已被证明与许多疾病的发病机制有关，并且参与众多肿瘤的发生、发展、转移及耐药等过
腺苷酸环化酶1（adenylate cyclase 1，ADCY1）是ADCY超家族的成员，其位于7p12.3，包含22个外显子；蛋白质产物的分子量为130 kD。已有相关研究报道了ADCY1在结肠
本研究通过筛选并分析相关测序数据，证实了miR-18a-3p在肝癌进展中发挥重要作用，并能通过转录后调控抑制下游ADCY1基因的表达来增加肝癌细胞的侵袭及生长能力。
本研究中所使用正常肝组织细胞L-02、肝癌细胞SK-HEP-1、HA22T均来自中科院上海细胞库，由本实验室保存。选取中南大学湘雅医院普通外科30例肝癌患者的癌组织及癌旁组织作为临床标本，患者入院时均签署知情同意告知书。
正常肝细胞L-02使用1640培养基进行培养、肝癌细胞SK-HEP-1、HA22T使用DMEM培养基进行培养（加入10%胎牛血清、1%谷氨酰胺、1%青霉素-链霉素双抗），并置于含5%CO2的37 ℃细胞培养箱中进行培养。
将基底胶按1∶20比例稀释后，每个transwell小室加入100 μL，并置于37 ℃细胞培养箱中使其凝固。2 h后，于上室加入150 μL含100 000个细胞的无血清培养基，下室加入含血清培养基，再放入37 ℃细胞培养箱中培养12~24 h。取出上室，进行清洗、固定、染色、显微镜下观察、拍照等。每组实验设置3个副孔。
TRIzol试剂提取肝癌细胞总RNA，取2 μg总RNA进行逆转录。 使用Bio-Rad CFX96系统进行实验，计算RNA（mRNA和miRNA）的表达。GAPDH（用于mRNA）或U6（用于miRNA）标准化数据，并通过ΔΔCt值评估相对表达。所有引物均购自Integrated DNA Technologies公司（美国）。
用含10%胎小牛血清的培养液配成单个细胞悬液，以每孔1 000~10 000个细胞接种到96孔板，每孔体积200 μL。培养3~5 d后，每孔MTT溶液（5 mg/mL用PBS配）20 μL继续孵育4 h，终止培养，小心吸弃孔内培养上清液，对于悬浮细胞需要离心后再吸弃孔内培养上清液。每孔加150 μL DMSO，振荡10 min，使结晶物充分溶解。选择490 nm波长，在酶联免疫监测仪上测定各孔光吸收值，记录结果，以时间为横坐标，吸光值为纵坐标绘制细胞生长曲线。
过表达miR-520F-3p采用pLV慢病毒质粒作为载体，序列（上游：CGC GTA CTG CCC TAA GTG CTC CTT CTG GGT CGA CCC AGA AGG AGC ACT TAG GGC AGT TTT TTG，下游：CGC AAA AAA CTG CCC TAA GTG CTC CTT CTG GGT CGA CCC AGA AGG AGC ACT TAG GGC AGT A）由 Integrated DNA Technologies公司（美国）提供。新构建质粒经小提、酶切验证后再经大提扩增，然后经慢病毒包装、转染，形成稳定转染细胞后行下一步功能实验。
miR-18a-3p 抑制剂质粒购买于Integrated DNA Technologies公司（美国），具体序列为rCrCrA rGrArA rGrGrA rGrCrA rCrUrU rArGrG rGrCrA rGrU。瞬转具体步骤：⑴ 当肝癌细胞长至60%~80%汇合度时转染，6孔板：贴壁细胞0.25~1×1
根据Targetscan网站（https://www.targetscan.org/vert_71）对ADCY1 3'UTR序列分析结果，发现ADCY1 3'UTR共9 121个碱基序列，为分析miR-18a-3p对ADCY1 3'UTR的调控功能，选取ADCY1 3'UTR前2 200个碱基序列构建野生型及突变型质粒。ADCY1的3'UTR采用psiCHECK-2质粒为载体。野生型引物序列（上游：CAG TAA TTC TAG GCG ATC GCA GGA GCC CAC GTG GGC CTCT，下游：AGA TAT TTT ATT GCG GCC AGC CCC AGT AGC AGC GAG AGG CC）；突变型引物序列（上游：CAG TAA TTC TAG GCG ATC GCA GGA GCC CAC GTG GGC CTCT，下游：AGA TAT TTT ATT GCG GCC AGC CAA AGT CTC CAG TGG GTC CA）由Integrated DNA Technologies公司（美国）提供。新构建质粒经小提、酶切验证后再经大提扩增，然后经慢病毒包装、转染，形成稳定转染细胞后行下一步功能实验。
首先，从已发
图1 差异表达miRNA筛选及验证 A：从已发表文章中筛选出肝癌中差异表达的6个miRNA；B：6个miRNA在正常肝细胞L-02及肝癌细胞HA22T中的表达情况；C：6个miRNA在正常肝细胞L-02及肝癌细胞SK-HEP-1中的表达情况
Figure 1 Screening and validation of the differentially-expressed miRNAs A: Six miRNAs screened out from the published studies; B: Expression of 6 miRNAs in normal hepatic L-02 cells and liver cancer HA22T cells; C: Expression of 6 miRNAs in normal hepatic L-02 cells and liver cancer SK-HEP-1 cells
过表达miR-18a-3p/miR-18a-3p抑制剂能明显促进/抑制肝癌细胞的侵袭及增殖能力（均P<0.05）（
图2 miR-18a-3p、miR-26a-3p对肝癌细胞侵袭及增殖能力的影响 A-B：过表达miR-18a-3p/抑制miR-18a-3p后肝癌细胞侵袭能力的变化；C-D：过表达miR-18a-3p/抑制miR-18a-3p后肝癌细胞增殖能力的变化；E-F：过表达miR-26a-3p/miR-26a-3p 抑制剂后肝癌细胞侵袭能力的变化；G-H：过表达miR-26a-3p/miR-26a-3p 抑制剂后肝癌细胞增殖能力的变化
Figure 2 Influences of miR-18a-3p and miR-26a-3p on invasion and proliferation abilities of liver cancer cells A-B: Changes in invasion ability of liver cancer cells after overexpression miR-18a-3p/miR-18a-3p inhibitor; C-D: Changes in proliferation ability of liver cancer cells after overexpression miR-18a-3p/miR-18a-3p inhibitor; E-F: Changes in invasion ability of liver cancer cells after miR-26a-3p overexpression/miR-26a-3p inhibiton; G-H: Changes in proliferation ability of liver cancer cells after miR-26a-3p overexpression/miR-26a-3p inhibiton
在30例肝癌患者的癌和癌旁组织标本中检验了miR-18a-3p的表达情况，结果显示肝癌组织中miR-18a-3p的表达明显高于癌旁组织（P<0.01）（
图3 组织标本验证miR-18a-3p的表达及miR-18a的表达与肝癌患者的生存关系 A：30对临床标本中miR-18a-3p的表达情况；B：KM plotter网站分析miR-18a的表达与肝癌的生存关系
Figure 3 Validation of miR-18a-3p expression in tissue samples and relationship between miR-18a expression and survival of liver cancer patients A: The miR-18a-3p expressions in 30 paired clinical specimens; B: Analysis of the relationship between miR-18a expression and survival of liver cancer patients using KM plotter website
为了进一步研究miR-18a调控肝癌进展的具体机制，通过miRDB网站（http://mirdb.org/）预测能被miR-18a-3p靶向调控的下游基因，并选取其中得分>95分的基因（SNX8、FNDC5、EFNA1、ZBBX、ADCY1、PDP1）（
图4 miR-18a-3p与ADCY1的关系分析 A：miRDB网站预测能被miR-18a-3p靶向调控的6个下游基因；B：GEPIA数据库分析以上6个基因在肝癌组织及癌旁组织中的表达情况；C-D：同时过表达miR-18a-3p及ADCY1后检测肝癌细胞的侵袭及增殖能力
Figure 4 Analysis of relationship between miR-18a-3p and ADCY1 A: The 6 downstream genes potentially regulated by miR-18a-3p predicted by miRDB website; B: Analysis the expressions of the 6 genes in liver cancer and adjacent tissues using GEPIA database; C-D: Invasion and proliferation abilities of liver cancer cells after co-overexpression of miR-18a-3p and ADCY1
为研究miR-18a-3p调控ADCY1的机制，预测ADCY1 mRNA 3'UTR前2 200 bp区域能与miR-18a-3p结合的靶点，并构建相应的野生型和突变型质粒（
图5 双荧光素酶报告实验 A：miR-18a-3p与ADCY1结合位点的模式图及野生型、突变型质粒构建原理；B：转染野生型或突变型质粒后肝癌细胞的荧光素酶活性
Figure 5 Dual-luciferase reporter assay A: Pattern of the binding site between miR-18a-3p and ADCY1, and construction principle of the wild-type and mutant-type plasmids; B: Luciferase activities of the liver cancer cells after transfection with wild-type and mutant-type plasmids
全世界每年有超过850 000例诊断为肝癌的患
非编码RNA已经被证实在多种疾病发生、发展、转移及耐药过程中发挥重要作
ADCY1是ADCY超家族的成员，它位于7p12.3，包含22个外显子；其蛋白质产物的分子量为130 kD。ADCY1主要表达于脑、睾丸、甲状腺、肝脏、前列腺、子宫内膜、心脏等组织器官。已有相关研究报道了ADCY1在许多疾病中发挥重要作用。研
miR-18a-3p在肝癌组织及细胞中高表达，并可通过转录后调控抑制下游ADCY1基因的表达，以此促进肝癌细胞的侵袭及增殖能力。本研究结果可为寻找肝癌治疗的新靶点提供实验基础。
利益冲突
所有作者均声明不存在利益冲突。
参考文献
Siegel RL, Miller KD, Fuchs HE, et al. Cancer statistics, 2022[J]. CA Cancer J Clin, 2022, 72(1):7-33. doi: 10.3322/caac.21708. [百度学术]
Siegel RL, Miller KD, Fuchs HE, et al. Cancer statistics, 2021[J]. CA Cancer J Clin, 2021, 71(1):7-33. doi: 10.3322/caac.21654. [百度学术]
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020[J]. CA Cancer J Clin, 2020, 70(1):7-30. doi: 10.3322/caac.21590. [百度学术]
中华人民共和国国家卫生健康委员会医政医管局. 原发性肝癌诊疗规范(2019年版)[J]. 肿瘤综合治疗电子杂志, 2020, 6(2):55-85. doi: 10.12151/JMCM.2020.02-10. [百度学术]
National Health Commission of the People's Republic of China. Primary hepatic carcinoma diagnosis and treatment specification(2019 edition)[J]. Journal of Multidisciplinary Cancer Management: Electronic Version, 2020, 6(2):55-85. doi: 10.12151/JMCM.2020.02-10. [百度学术]
Erstad DJ, Tanabe KK. Hepatocellular carcinoma: early-stage management challenges[J]. J Hepatocell Carcinoma, 2017, 4:81-92. doi: 10.2147/JHC.S107370. [百度学术]
Jiang HB, Ge RX, Chen SW, et al. miRNA-204-5p acts as tumor suppressor to influence the invasion and migration of astrocytoma by targeting ezrin and is downregulated by DNA methylation[J]. Bioengineered, 2021, 12(2):9301-9312. doi: 10.1080/21655979.2021.2000244. [百度学术]
Safi A, Delgir S, Ilkhani K, et al. The expression of miRNA-152-3p and miRNA-185 in tumor tissues versus margin tissues of patients with chemo-treated breast cancer[J]. BMC Res Notes, 2021, 14(1):234. doi: 10.1186/s13104-021-05647-z. [百度学术]
Zhang JZ, Zhong Y, Sang YM, et al. miRNA-144-5p/ITGA3 suppressed the tumor-promoting behaviors of thyroid cancer cells by downregulating ITGA3[J]. Comput Math Methods Med, 2021, 2021:9181941. doi: 10.1155/2021/9181941. [百度学术]
Li YF, Guan B, Liu JT, et al. microRNA-200b is downregulated and suppresses metastasis by targeting LAMA4 in renal cell carcinoma[J]. EBioMedicine, 2019, 44:439-451. doi: 10.1016/j.ebiom.2019.05.041. [百度学术]
Fang C, Dai CY, Mei Z, et al. microRNA-193a stimulates pancreatic cancer cell repopulation and metastasis through modulating TGF-β2/TGF-βRIII signalings[J]. J Exp Clin Cancer Res, 2018, 37(1):25. doi: 10.1186/s13046-018-0697-3. [百度学术]
Xu WG, Sun DP, Wang YQ, et al. Inhibitory effect of microRNA-608 on lung cancer cell proliferation, migration, and invasion by targeting BRD4 through the JAK2/STAT3 pathway[J]. Bosn J Basic Med Sci, 2020, 20(3):347-356. doi: 10.17305/bjbms.2019.4216. [百度学术]
李乾, 郭清皓. 肝细胞癌中miR-574-5p的表达与作用及其与预后的关系[J]. 中国普通外科杂志, 2021, 30(8):926-933. doi: 10.7659/j.issn.1005-6947.2021.08.007. [百度学术]
Li Q, Guo QH. Expression and action of miR-574-5p in hepatocellular carcinoma and its association with prognosis[J]. Chinese Journal of General Surgery, 2021, 30(8):926-933. doi: 10.7659/j.issn.1005-6947.2021.08.007. [百度学术]
Liu G, Zhao H, Song Q, et al. Long non-coding RNA DPP10-AS1 exerts anti-tumor effects on colon cancer via the upregulation of ADCY1 by regulating microRNA-127-3p[J]. Aging (Albany NY), 2021, 13(7):9748-9765. doi: 10.18632/aging.202729. [百度学术]
Ma M, Dai J, Tang H, et al. microRNA-23a-3p inhibits mucosal melanoma growth and progression through targeting adenylate cyclase 1 and attenuating cAMP and MAPK pathways[J]. Theranostics, 2019, 9(4):945-960. doi: 10.7150/thno.30516. [百度学术]
Zou T, Liu JY, She L, et al. A perspective profile of ADCY1 in cAMP signaling with drug-resistance in lung cancer[J]. J Cancer, 2019, 10(27):6848-6857. doi: 10.7150/jca.36614. [百度学术]
Pang WJ, Yao WY, Dai X, et al. Pancreatic cancer-derived exosomal microRNA-19a induces β-cell dysfunction by targeting ADCY1 and EPAC2[J]. Int J Biol Sci, 2021, 17(13):3622-3633. doi: 10.7150/ijbs.56271. [百度学术]
Zhang YF, Yang JR, Wang XY, et al. GNG7 and ADCY1 as diagnostic and prognostic biomarkers for pancreatic adenocarcinoma through bioinformatic-based analyses[J]. Sci Rep, 2021, 11(1):20441. doi: 10.1038/s41598-021-99544-x. [百度学术]
Xu JH, An P, Winkler CA, et al. Dysregulated microRNAs in hepatitis B virus-related hepatocellular carcinoma: potential as biomarkers and therapeutic targets[J]. Front Oncol, 2020, 10:1271. doi: 10.3389/fonc.2020.01271. [百度学术]
Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012[J]. CA Cancer J Clin, 2015, 65(2):87-108. doi: 10.3322/caac.21262. [百度学术]
GBD Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013[J]. Lancet, 2015, 385(9963):117-171. doi: 10.1016/S0140-6736(14)61682-2. [百度学术]
European Association For The Study Of The Liver, European Organisation For Research And Treatment Of Cancer. EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma[J]. J Hepatol, 2012, 56(4):908-943. doi: 10.1016/j.jhep.2011.12.001. [百度学术]
Schultheiß M, Bengsch B, Thimme R. Hepatocellular Carcinoma[J]. Dtsch Med Wochenschr, 2021,146(21):1411-1420. doi: 10.1055/a-1226-3047. [百度学术]
国家卫生健康委办公厅. 原发性肝癌诊疗指南(2022年版)[J]. 临床肝胆病杂志, 2022, 38(2):288-303. doi:10.3969/j.issn.1001-5256.2022.02.009. [百度学术]
General Office of National Health Commission. Standard for diagnosis and treatment of primary liver cancer (2022 edition)[J]. Journal of Clinical Hepatology, 2022, 38(2):288-303. doi:10.3969/j.issn.1001-5256.2022.02.009. [百度学术]
郝芬林, 李涛, 姚宏, 等. 环状RNA在肝细胞癌发生发展中的作用研究进展[J]. 中国普通外科杂志, 2022, 31(1):107-115. doi: 10.7659/j.issn.1005-6947.2022.01.012. [百度学术]
Hao FL, Li T, Yao H, et al. Association of circRNAs with the occurrence and development of hepatocellular carcinoma: recent progress[J]. Chinese Journal of General Surgery, 2022, 31(1):107-115. doi: 10.7659/j.issn.1005-6947.2022.01.012. [百度学术]
Ambros V, Bartel B, Bartel DP, et al. A uniform system for microRNA annotation[J]. RNA, 2003, 9(3):277-279. doi: 10.1261/rna.2183803. [百度学术]
Wei YT, Guo DW, Hou XZ, et al. miRNA-223 suppresses FOXO1 and functions as a potential tumor marker in breast cancer[J]. Cell Mol Biol (Noisy-le-grand), 2017, 63(5):113-118. doi: 10.14715/cmb/2017.63.5.21. [百度学术]
Liu GD, Ouyang XW, Sun Y, et al. The miR-92a-2-5p in exosomes from macrophages increases liver cancer cells invasion via altering the AR/PHLPP/p-Akt/β-catenin signaling[J]. Cell Death Differ, 2020, 27(12):3258-3272. doi: 10.1038/s41418-020-0575-3. [百度学术]
Chen XF, Guo JF, Xu JF, et al. MiRNA-206 inhibits proliferation of renal clear cell carcinoma by targeting ZEB2[J]. Eur Rev Med Pharmacol Sci, 2019, 23(18):7826-7834. doi: 10.26355/eurrev_201909_18992. [百度学术]
Chen J, Wu F, Shi Y, et al. Identification of key candidate genes involved in melanoma metastasis[J]. Mol Med Rep, 2019, 20(2):903-914. doi: 10.3892/mmr.2019.10314. [百度学术]
He RQ, Li XJ, Liang L, et al. The suppressive role of miR-542-5p in NSCLC: the evidence from clinical data and in vivo validation using a chick chorioallantoic membrane model[J]. BMC Cancer, 2017, 17(1):655. doi: 10.1186/s12885-017-3646-1. [百度学术]
Ma JG, Hou X, Li MX, et al. Genome-wide methylation profiling reveals new biomarkers for prognosis prediction of glioblastoma[J]. J Cancer Res Ther, 2015, 11(Suppl 2):C212-215. doi: 10.4103/0973-1482.168188. [百度学术]