Objective: To investigate the expression of long non-coding RNA LOC101927476 (LncRNA LOC101927476) in ovarian cancer and its effect on the biological characteristics of ovarian cancer. Methods: Patients with ovarian cancer who underwent surgery in Cancer Hospital of Chinese Academy of Medical Sciences from 2018 to 2019 were selected. The expressions of LOC101927476 in ovarian cancer cells 3AO, OVCA429, TOV21G, A2780, SKOV3, as well as 22 primary tumor tissues and their matched metastatic tumor tissues were detected by real-time quantitative polymerase chain reaction (RT-PCR). Ovarian cancer transcriptome sequencing data from the TCGA database was used to verify the expressions of LOC101927476 and GATA4. 3AO and OVCA429 cells were infected with lentivirus plasmid containing OE-LOC101927476 and single guide RNA (sg-RNA) targeting LOC101927476, respectively. The effects of LOC101927476 on migration and invasion were detected by Transwell and wound healing assay. The effect of LOC101927476 on cell proliferation was detected by cell counting kit-8 (CCK-8) assay. Results: RT-PCR assay showed that 20 out of 22 patients had significantly lower expression of LOC101927476 in their metastatic tumors compared with primary tumors. Transwell assay showed that overexpression of LOC101927476 significantly inhibited the invasion and migration capacities of 3AO cells. The numbers of invading and migrating 3AO cells infected with OE-LOC101927476 lentivirus were (357±63) and (699±65), respectively, lower than (661±95) and (1 024±76) in OE-EV group (P<0.050). In contrast, the numbers of invading and migrating OVCA429 cells with LOC101927476 knockdown were (512±72) and (472±40), respectively, higher than (309±13) and (363±27) in sg-Control group (P<0.050). Wound healing assay results showed that after 48 hours, the percentage of scratch healing of 3AO cells in OE-LOC101927476 group was (10.86±0.63)%, significantly lower than (57.38±4.42)% of OE-EV group (P=0.009). After 24 hours, the percentage of scratch healing of OCVA429 cells in sg-LOC101927476 group was (59.98±1.34)%, significantly higher than (23.15±2.03)% of sg-Control group (P=0.004). CCK-8 assays showed that the OD value of 3AO cells in OE-LOC101927476 group was (2.07±0.08), significantly lower than (2.29±0.04) of OE-EV group (P=0.009). The OD value of OVCA429 cells in sg-LOC101927476 group was (2.13±0.03), significantly higher than (1.93±0.03) of sg-Control group (P=0.001). The relative expression of GATA4 in OE-LOC101927476 group was (1.86±0.25), significantly higher than 1.00 of OE-EV group (P=0.001). In patients with high expression of LncRNA LOC101927476, the expression level of GATA4 was (2.93±0.35), which was higher than (0.29±0.06) of LOC101927476 low expression group (P=0.001). Conclusion: LncRNA LOC101927476 can inhibit the invasion, migration and proliferation of ovarian cancer cells.
目的： 探讨长链非编码RNA(LncRNA)LOC101927476在卵巢癌细胞中的表达及其对卵巢癌生物学特征的影响。 方法： 选取2018—2019年于中国医学科学院肿瘤医院手术治疗的卵巢癌患者。采用实时荧光定量PCR(real-time PCR)检测卵巢癌细胞系3AO、OVCA429、TOV21G、A2780、SKOV3以及22例卵巢癌原发瘤和转移瘤组织中LncRNA LOC101927476的表达水平，采用TCGA数据库中卵巢癌转录组测序数据验证LncRNA LOC101927476和GATA4的表达，采用慢病毒包装体系构建过表达LOC101927476(OE-LOC101927476组)和空载(OE-EV组)慢病毒并转染至3AO细胞。设计单链向导RNA(sgRNA)，利用CRISPR/Cas9构建LncRNA LOC101927476敲除细胞系。采用Transwell法和划痕实验检测LncRNA LOC101927476对卵巢癌细胞侵袭和转移能力的影响。采用细胞计数盒8(CCK-8)法检测细胞的增殖能力。 结果： real-time PCR显示，22例卵巢癌患者中，20例转移灶中LncRNA LOC101927476的表达水平低于原发灶。Transwell迁移实验显示，OE-EV组3AO细胞的穿膜细胞数为(1 024±76)个，高于OE-LOC101927476组[(699±65)个，P＝0.003]；sg-Control组OVCA429细胞的穿膜细胞数为(363±27)个，低于sg-LOC101927476组[(472±40)个] ，差异有统计学意义(P＝0.011)。Transwell侵袭实验显示，OE-EV组3AO细胞的穿膜细胞数为(661±95)个，高于OE-LOC101927476组[(357±63)个]，差异有统计学意义(P＝0.006)；sg-Control组OVCA429细胞的穿膜细胞数为(309±13)个，低于sg-LOC101927476组[(512±72)个] ，差异有统计学意义(P＝0.005)。划痕实验显示，培养48 h时，OE-LOC101927476组3AO细胞的划痕愈合比例为(10.86±0.63)%，低于OE-EV组[(57.38±4.42)%]，差异有统计学意义(P＝0.009)；培养24 h时，sg-LOC101927476组OVCA429细胞的划痕愈合比例为(59.98±1.34)%，高于sg-Control组[(23.15±2.03)%]，差异有统计学意义(P＝0.004)。CCK-8结果显示，OE-LOC101927476组3AO细胞的增殖能力明显减弱，在过表达LncRNA LOC101927476后第4天时，OE-LOC101927476组3AO细胞的吸光度(A)值为2.07±0.08，与OE-EV组(2.29±0.04)比较，差异有统计学意义(P＝0.009)。sg-LOC101927476组OVCA429细胞的增殖能力提高，在敲降LncRNA LOC101927476的表达后第4天时，sg-LOC101927476组OVCA429细胞的A值为(2.13±0.03)，与sg-Control组(1.93±0.03)比较，差异有统计学意义(P＝0.001)。OE-LOC101927476组3AO细胞中GATA4的相对表达水平为1.86±0.25，与OE-EV组(1.00±0.00)比较，差异有统计学意义(P＝0.001)。在LncRNA LOC101927476高表达的患者中，GATA4的表达水平为(2.93±0.35)，高于LncRNA LOC101927476低表达的患者(0.29±0.06)，差异有统计学意义(P＝0.001)。 结论： LncRNA LOC101927476能够抑制卵巢癌的侵袭、迁移和增殖。.
Keywords: Invasion; Long non-coding RNA; Migration; Ovarian neoplasms; Proliferation.