长链非编码RNA RUSC1-AS1对肝细胞癌恶性生物学行的影响及其与微小RNA-326的关系

背景与目的:有研究表明长链非编码RNARUSC1-AS1(lnc RNARUSC1-AS1)与肿瘤的恶性生物学行为密切相关,但其对肝细胞癌(肝癌)的影响尚不清楚。笔者前期研究显示,lnc RNARUSC1-AS1与微小RNA-326(mi R-326)存在结合位点,因此本研究探讨lnc RNARUSC1-AS1在肝癌中的表达,以及是否通过靶向mi R-326调控肝癌细胞生物学行为。方法:用q RT-PCR检测41例肝癌组织和对应癌旁组织中lnc RNARUSC1-AS1与mi R-326的表达。以lnc RNARUSC1-AS1表达抑制质粒/阴性对照质粒、mi R-326模拟物/阴性对照序列、mi R-326抑制物/阴性对照序列为工具,采用MTT法、Transwell法、流式细胞术、Westernblot法观察接受不同转染处理的MHCC97-H细胞的增殖能力、迁移和侵袭能力、凋亡以及相关蛋白表达的变化。采用荧光素酶报告实验分析lnc RNARUSC1-AS1和mi R-326的靶向关系,并用q RT-PCR验证。结果:与癌旁组织比较,肝癌组织中lnc RNARUSC1-AS1表达水平明显升高,mi R-326表达水平明显降低(均P0.05)。转染lnc RNARUSC1-AS1表达抑制质粒或mi R-326模拟物后,肝癌MHCC97-H细胞的增殖能力以及迁移与侵袭能力明显降低,细胞凋亡率明显升高,cyclinD1、MMP-2、MMP-9、Bcl-2蛋白表达水平明显降低,P21、Bax蛋白表达水平明显升高(均P0.05)。MHCC97-H细胞转染lnc RNARUSC1-AS1表达抑制质粒的同时mi R-326抑制物,前者对MHCC97-H细胞以上作用被取消(均P0.05)。双荧光素酶报告实验及q RT-PCR验证结果显示,mi R-326为lnc RNARUSC1-AS1的靶分子。结论:lnc RNARUSC1-AS1在肝癌中表达上调,其可通过靶向调控mi R-326的表达促进肝癌细胞的恶性生物学行。

Influence of long non-coding RNA RUSC1-AS1 on malignant biological behaviors of hepatocellular carcinoma and its relationship with microRNA-326

Background and Aims: Studies have demonstrated that long non-coding RNA RUSC1-AS1 (lncRNA RUSC1-AS1) has a close relationship with the malignant biological behaviors of tumors. However, its role in hepatocellular carcinoma (HCC) is still unclear. Our previous study demonstrated that there are binding interactions between lncRNA RUSC1-AS1 and microRNA-326 (miR-326). Therefore, this study was conducted to investigate the expression of lncRNA RUSC1-AS1 in HCC, and whether it regulates the biological behaviors of HCC cells by targeting miR-326.  Methods: The expressions of lncRNA RUSC1-AS1 and miR-326 in 41 paired specimens of HCC tissue and tumor adjacent were detected by qRT-PCR. Using lncRNA RUSC1-AS1 expression inhibition plasmid/negative control plasmid, miR-326 mimics/negative control sequences and miR-326 inhibitors/negative control sequences as tools, the changes in proliferative ability, migration and invasion abilities, and apoptosis as well as the expressions of the related proteins in HCC MHCC97-H cells after different transfection treatments were determined by MTT assay, Transwell assay, flow cytometry and western blot analysis, respectively. The targeting relationship between lncRNA RUSC1-AS1 and miR-326 was analyzed by luciferase report assay, and then validated by qRT-PCR. Results: In HCC tissue compared with adjacent tissue, the expression level of lncRNA RUSC1-AS1 was significantly increased, and the expression level of miR-326 was significantly decreased (both P<0.05). In MHCC97-H cells after transfection with lncRNA RUSC1-AS1 expression inhibition plasmid or miR-326 mimics, the proliferative ability as well as the migration and invasion abilities were significantly reduced, while the apoptosis rate was significantly increased, and the protein expression levels of cyclin D1, MMP-2, MMP-9, Bcl-2 were significantly reduced, while the protein expression levels of P21, Bax were significantly increased (all P<0.05). In MHCC97-H cells transfected with lncRNA RUSC1-AS1 expression inhibition plasmid with simultaneous transfection of miR-326 inhibitors, the influences of the former on MHCC97-H cells were abolished (all P<0.05). The results of luciferase report assay and qRT-PCR validation showed that miR-326 was a target molecule for lncRNA RUSC1-AS1.  Conclusion: The expression of lncRNA RUSC1-AS1 is up-regulated in HCC, and it can promote the malignant biological behaviors of HCC cells by targeting miR-326.