Department of Hepatobiliary Surgery, Affiliated Hospital of Hebei University, Baoding, Hebei 071000, China
背景与目的 肝细胞癌（HCC）是造成癌症相关性死亡的常见原因之一，研究表明长链非编码RNA（lncRNA）调控微小RNA（miRNA）的表达，进而通过抑制靶mRNA翻译或促进mRNA降解来参与肿瘤发生及进展过程。LINC00313作为一种具有致癌活性的lncRNA参与肿瘤发生及进展过程；膜联蛋白A2（ANXA2）在包括HCC的多种恶性肿瘤中表达上调，促进恶性表型的发生，并可能受上游miR-342-3p的调控。因此，本研究探讨LINC00313、miR-342-3p、ANXA2在HCC细胞中的表达及其相互关系。方法 用qRT-PCR与Western blot检测人肝实质细胞及HCC细胞系（Li-7、HuH-7、Hep3B2.1-7）中LINC00313、miR-342-3p及ANXA2表达。将体外培养的Li-7细胞分为空白对照组（无处理）、LINC00313 siRNA组（转染LINC00313 siRNA）、miR-342-3p模拟物组（转染miR-342-3p模拟物）、共转染阴性对照组（转染阴性siRNA序列与阴性miRNA序列）、共转染组（转染LINC00313 siRNA及miR-342-3p抑制物），用qRT-PCR与Western blot检测各组细胞LINC00313、miR-342-3p及ANXA2表达；MTT实验及平板集落形成实验检测各组细胞增殖；进行TUNEL染色检测各组细胞凋亡；Transwell侵袭及Western blot分别检测各组细胞侵袭数目及上皮-间充质转化（EMT）相关蛋白波形蛋白（vimentin）、E-钙黏蛋白（E-cadherin）表达；免疫荧光染色检测各组细胞Bcl-2关联X蛋白（Bax）/B淋巴细胞瘤-2（Bcl-2）；双荧光素酶报告实验分析Li-7细胞中LINC00313对miR-342-3p、miR-342-3p对ANXA2的靶向调控。建立皮下裸鼠异种移植瘤模型，验证LINC00313沉默对Li-7细胞体内生长的影响。结果 与人肝实质细胞比较，Li-7、HuH-7、Hep3B2.1-7细胞的LINC00313、ANXA2 mRNA及蛋白表达均明显升高，而miR-342-3p表达均明显降低（均P<0.05）。与对照组比较，LINC00313 siRNA组、miR-342-3p模拟物组细胞ANXA2 mRNA及蛋白表达、增殖率、集落生成率、侵袭细胞数目、vimentin蛋白表达均明显降低（P<0.05），miR-342-3p表达、凋亡率、E-cadherin蛋白表达、Bax/Bcl-2比值均明显升高（均P<0.05）；与LINC00313 siRNA组比较，共转染组细胞ANXA2 mRNA及蛋白表达、增殖率、集落生成率、侵袭细胞数目、vimentin蛋白表达均明显升高，而miR-342-3p表达、凋亡率、E-cadherin蛋白表达、Bax/Bcl-2比值均明显降低（均P<0.05）。Li-7细胞中，LINC00313可靶向下调miR-342-3p表达，miR-342-3p可靶向下调其ANXA2表达（均P<0.05）。体内实验结果显示，与无处理的Li-7细胞移植瘤比较，LINC00313敲低的Li-7细胞移植瘤的体积与质量均明显降低，肿瘤组织中LINC00313、ANXA2 mRNA及蛋白表达均明显降低，而miR-342-3p表达明显升高（均P<0.05）。结论 LINC00313在HCC细胞中的表达上调，LINC00313可能通过抑制miR-342-3p而增加后者靶基因ANXA2的表达，进而促进HCC细胞的恶性表型。
Background and Aims Hepatocellular carcinoma (HCC) is a common cause of cancer-related mortality. Studies have shown that long non-coding RNAs (lncRNAs) regulate the expression of microRNAs (miRNAs), which, in turn, participate in tumorigenesis and progression by inhibiting target mRNA translation or promoting mRNA degradation. LINC00313 is a cancer-promoting lncRNA that contributes to tumor development and progression. Annexin A2 (ANXA2) is upregulated in various malignant tumors, including HCC, promoting a malignant phenotype, and it may be regulated by upstream miR-342-3p. This study was conducted to explore the expression of LINC00313, miR-342-3p, and ANXA2 in HCC cells and their interrelationships.Methods The expressions of LINC00313, miR-342-3p, and ANXA2 were assessed in human liver tissue cells and HCC cell lines (Li-7, HuH-7, Hep3B2.1-7) using qRT-PCR and Western blot. Li-7 cells were cultured in vitro and divided into different groups: control (untreated), LINC00313 siRNA group (transfected with LINC00313 siRNA), miR-342-3p mimics group (transfected with miR-342-3p mimics), negative control co-transfection group (transfected with negative siRNA and negative miRNA), and co-transfection group (transfected with LINC00313 siRNA and miR-342-3p inhibitor). The expressions of LINC00313, miR-342-3p, and ANXA2 were analyzed in these groups using qRT-PCR and Western blot. Cell proliferation was assessed using MTT assay and colony formation assay. Apoptosis was detected by TUNEL staining. Transwell invasion assay and Western blot were performed to evaluate cell invasion and expressions of epithelial-mesenchymal transition-related proteins (vimentin and E-cadherin), respectively. Immunofluorescence staining was used to determine the Bax/Bcl-2 ratio in the cells. Dual-luciferase reporter assay was conducted to analyze the regulatory relationship between LINC00313 and miR-342-3p and between miR-342-3p and ANXA2 in Li-7 cells. Furthermore, a xenograft tumor model in nude mice was established to validate the impact of LINC00313 knockdown on Li-7 cell growth in vivo.Results Compared to human liver tissue cells, the expressions of LINC00313 as well as ANXA2 mRNA and protein was significantly upregulated in Li-7, HuH-7, and Hep3B2.1-7 cells, while miR-342-3p expression was significantly downregulated (all P<0.05). Compared to the control group, the LINC00313 siRNA group and miR-342-3p mimics group showed significant reductions in ANXA2 mRNA and protein expressions, cell proliferation, colony formation, and invasion, as well as decreased vimentin protein expression (all P<0.05). Furthermore, miR-342-3p expression, apoptosis rate, E-cadherin protein expression, and Bax/Bcl-2 ratio were significantly increased in these groups (all P<0.05). When compared to the LINC00313 siRNA group, the co-transfection group exhibited elevated ANXA2 mRNA and protein expressions, increased cell proliferation, colony formation, and invasion, as well as enhanced vimentin expression. However, it reduced miR-342-3p expression, apoptosis rate, E-cadherin protein expression, and Bax/Bcl-2 ratio (all P<0.05). In Li-7 cells, LINC00313 could negatively regulate miR-342-3p expression, while miR-342-3p could negatively regulate ANXA2 expression (both P<0.05). In vivo experiments demonstrated that compared to untreated Li-7 cell xenografts, LINC00313 knockdown in Li-7 cells significantly reduced tumor volume and mass. Additionally, LINC00313 and ANXA2 mRNA and protein expression in tumor tissues were markedly decreased, while miR-342-3p expression was significantly increased (all P<0.05).Conclusion LINC00313 is upregulated in HCC cells and may promote a malignant phenotype by inhibiting miR-342-3p, thereby increasing the expression of its target gene, ANXA2, in HCC cells.