TINCR靶向microR-544a/FBXW7对人乳腺癌细胞增殖、侵袭的影响

Effects of lncRNA TINCR targeting miR-544a/FBXW7 on proliferation and invasion of human breast cancer cells

摘要:

目的 探讨长链非编码RNA（lncRNA）组织分化诱导非蛋白编码RNA（TINCR）靶向microRNA-544a（miR-544a）/FBXW7对人乳腺癌细胞增殖、侵袭的影响。方法 体外培养人乳腺癌MCF7细胞株，设置空白对照组（不转入任何载体）、TINCR NC组（pcDNA3.1空载体）和TINCR上调组（pcDNA3.1-TINCR表达载体）。采用实时荧光定量聚合酶链反应（qRT-PCR）检测MCF7细胞lncRNA TINCR、miR-544a相对表达量；CCK-8法检测MCF7细胞增殖情况；流式细胞术检测MCF7细胞凋亡情况；Transwell法检测MCF7细胞侵袭情况；Western blotting检测MCF7细胞FBXW7、增殖细胞核抗原（PCNA）、Bcl-2相关X蛋白（Bax）、基质金属蛋白酶-2（MMP-2）蛋白相对表达量。取TINCR上调组MCF7细胞，分为miR-544a NC组（转染miR-544a NC）和miR-544a上调组（转染miR-544a mimic），验证转染效率。结果 空白对照组、TINCR NC组MCF7细胞lncRNA TINCR、miR-544a相对表达量、OD值、细胞凋亡率、穿膜细胞数及FBXW7、PCNA、Bax、MMP-2蛋白相对表达量比较，差异无统计学意义（P >0.05）。与空白对照组、TINCR NC组比较，TINCR上调组MCF7细胞lncRNA TINCR相对表达量、细胞凋亡率及FBXW7、Bax蛋白相对表达量升高（P <0.05），miR-544a相对表达量、OD值、穿膜细胞数及PCNA、MMP-2蛋白相对表达量降低（P <0.05）。TINCR上调组与miR-544a NC组MCF7细胞OD值、侵袭细胞数及miR-544a、FBXW7蛋白相对表达量比较，差异无统计学意义（P >0.05）。与TINCR上调组和miR-544a NC组比较，miR-544a上调组MCF7细胞OD值、侵袭细胞数及miR-544a相对表达量升高（P <0.05），FBXW7蛋白相对表达量降低（P <0.05）。结论 lncRNA TINCR可能通过靶向miR-544a/FBXW7，抑制人乳腺癌细胞增殖、侵袭，并促进其凋亡。

Abstract:

Objective To investigate the effects of long non-coding RNA (lncRNA) tissue differentiation-inducing non-protein coding RNA (TINCR) targeting microRNA-544a (miR-544a)/F-box and WD-40 domain protein 7 (FBXW7) on the proliferation and invasion of human breast cancer cells.Methods Human breast cancer MCF7 cell line was cultured in vitro, and the blank control group (without any vector), TINCR NC group (pcDNA3.1 empty vector) and TINCR upregulated group (pcDNA3.1-TINCR expression vector) were set up. The expressions of lncRNA TINCR and miR-544a in MCF7 cells were detected by real-time quantitative polymerase chain reaction (qPCR). The proliferation of MCF7 cells was detected by cell counting kit-8 (CCK-8) method. The apoptosis of MCF7 cells was detected by flow cytometry. The invasion of MCF7 cells was detected by transwell assay. The expressions of FBXW7, proliferating cell nuclear antigen (PCNA), Bcl-2-associated X protein (Bax) and matrix metalloproteinase-2 (MMP-2) in MCF7 cells were detected by Western blotting. The MCF7 cells from TINCR upregulated group were further divided into miR-544a NC group (transfected with miR-544a negative control) and miR-544a upregulated group (transfected with miR-544a mimic), and the transfection efficiency was determined.Results There was no significant difference in the relative expression levels of lncRNA TINCR and miR-544a, optical density (OD) value, apoptosis rate, number of migrated cells, or levels of FBXW7, PCNA, Bax, MMP-2 proteins between the blank control group and TINCR NC group (P > 0.05). Compared with the blank control group and TINCR NC group, the expression level of lncRNA TINCR, apoptosis rate, and the expression levels of FBXW7 and Bax proteins in MCF7 cells in the TINCR upregulated group were higher (P < 0.05), while the expression level of miR-544a, OD value, number of migrated cells, and the expression levels of PCNA and MMP-2 proteins were lower (P < 0.05). There was no significant difference in OD value, number of invasive cells, or the expression levels of miR-544a and FBXW7 proteins in MCF7 cells between the TINCR upregulated group and the miR-544a NC group (P > 0.05). Compared with the TINCR upregulated group and the miR-544a NC group, OD value, number of invasive cells and expression level of miR-544a in the miR-544a upregulated group were higher (P < 0.05), while FBXW7 protein expression level was lower (P < 0.05).Conclusions LncRNA TINCR may target miR-544a/FBXW7 to inhibit the proliferation and invasion but to promote the apoptosis of human breast cancer cells.