关键词: 肉瘤, 卡波西, 人脐静脉内皮细胞, 卡波西肉瘤相关疱疹病毒, 环状RNA, RELL1, MAPK信号通路

Abstract: 【Abstract】 Objective To explore the role and mechanisms of action of human circular RNA 0001400 （hsa_circ_0001400） and its linear transcript RELL1 in the development of Kaposi's sarcoma （KS）. Methods KS-associated herpesvirus （KSHV） was induced and extracted from BCBL-1 cells by phorbol ester. Human umbilical vein endothelial cells （HUVECs） were then infected with KSHV, inverted microscopy and real-time fluorescence-based quantitative PCR (qRT-PCR) were performed to observe cellular morphology and detect the expression of KSHV lytic gene ORF50 and latent gene LANA, respectively, so as to verify whether the infection was successful. HUVECs in the logarithmic growth phase were divided into 3 groups: HUVEC group （uninfected HUVECs）, KSHV + HUVEC group （HUVECs infected with KSHV at a multiplicity of infection ［MOI］ of 0.5）, and mitogen-activated protein kinase （MAPK） inhibitor group （HUVECs infected with KSHV at a MOI of 0.5 for 6 hours, followed by the treatment with 1 μmol/L MAPK inhibitor）. Cell counting kit （CCK8） assay and flow cytometry were performed to assess the cellular proliferative ability and detect apoptosis in the above 3 groups, respectively. qRT-PCR and Western blot analysis were conducted to determine the transcription and protein expression levels of hsa_circ_0001400, its linear transcript RELL1, and rat sarcoma viral oncogene homolog （RAS）/MAPK signaling pathway-related genes in the HUVEC group and KSHV + HUVEC group. Five pairs of KS tissues and paraneoplastic tissues were collected, and mRNA expression of the above genes was verified by qRT-PCR in the KS tissue samples. Statistical analyses were performed using two-way analysis of variance, one-way analysis of variance, and t test. Results Compared with the uninfected HUVECs, the infected HUVECs became rounder and grew more densely at 48 hours after the infection with KSHV. The mRNA expression of ORF50 and LANA genes could be detected in the KSHV-infected HUVECs, and their mRNA expression levels were significantly higher than those in the uninfected HUVECs （both P < 0.001）, indicating successful infection of HUVEC by KSHV. The cellular proliferative rate in the KSHV + HUVEC group gradually increased over time during 24 to 72 hours after the infection, while that in the MAPK inhibitor group was markedly inhibited. The total apoptosis rate at 48 hours significantly differed among the 3 groups （F = 673.98, P < 0.001）, and was significantly higher in the MAPK inhibitor group than in the KSHV + HUVEC group and the HUVEC group （both P < 0.001）, while there was no significant difference between the KSHV + HUVEC group and the HUVEC group （P > 0.05）. qRT-PCR showed that the mRNA expression of hsa_circ_0001400, RELL1, Kirsten rat sarcoma viral oncogene homologue （KRAS）, MAPK11, and extracellular signal-regulated kinase （ERK）-2 was significantly higher in the KSHV + HUVEC group than in the HUVEC group （all P < 0.05）, while ERK1 mRNA expression did not significantly differ between the 2 groups （t = 0.92, P = 0.410）. Western blot assay showed that the protein expression of RELL1, KRAS, MAPK11, ERK1, and ERK2 was significantly higher in the KSHV + HUVEC group than in the HUVEC group （all P < 0.01）. The mRNA expression of RELL1, ERK1, and ERK2 was significantly higher in the KS tissues than in the paraneoplastic tissues （all P < 0.05）. Conclusion KSHV infection may regulate the occurrence and development of KS by inducing the expression of hsa_circ_0001400 and its linear transcript RELL1, as well as activating the MAPK signaling pathway.

Key words: Sarcoma, Kaposi, Human umbilical vein endothelial cells, Kaposi's sarcoma-associated herpesvirus, Circular RNA, RELL1, MAPK signaling pathway