Circular RNA SOX13 promotes malignant behavior and cisplatin resistance in non-small cell lung cancer through targeting microRNA-3194-3p/microtubule-associated protein RP/EB family member 1

Abstract

Circular RNA (circRNA) presents an essential regulatory role in affecting the occurrence and acquired resistance in non-small cell lung cancer (NSCLC), but how circSOX13 impacts NSCLC is unclear. In this work it was found that compared with adjacent normal tissues, circSOX13 and the microtubule-associated protein RP/EB family member 1 (MAPRE1) were signally up-regulated in NSCLC while miR-3194-3p was signally lowered. Pulmonary function tests (PETs) revealed that knocking down circSOX13 or overexpressing miR-3194-3p inhibited NSCLC proliferation, invasion and migration but promoted its apoptosis. The promoting effect of overexpressing circSOX13 on NSCLC was reversed via knocking down MAPRE1. Additionally, knocking down circSOX13 reduced cisplatin resistance in NSCLC. Furthermore, circSOX13 mediated MAPRE1 expression via competitively binding miR-3194-3p to exert its tumorigenic impact. To conclude, this work clarified the carcinogenic impact of circSOX13-miR-3194-3p-MAPRE1 axis on NSCLC and DDP resistance. CircSOX13 can be a potential diagnostic marker and therapeutic target for NSCLC, thus providing a new insight for clinically reversing its acquired resistance.

Keywords: CircSOX13; cisplatin resistance; marep1; mir-3194-3p; non-small cell lung cancer.

Figure 1.

CircSOX13 is highly expressed in NSCLC. A. RT-qPCR to detect circSOX13 expression in NSCLC tissues and normal tissues adjacent to cancer; B. RT-qPCR to detect circSOX13 expression in serum of normal subjects and patients with NSCLC; C. RT-qPCR to detectcircSOX13 expression in PC9, SPC-A1, H1975, H1299 and A549 and 16HBE; the values were expressed by mean ± SD (C, n = 3); versus the normal tissue group, * P < 0.05; versus the 16HBE group,^ P < 0.05.

Figure 2.

Knocking down circSOX13 inhibits NSCLC proliferation, migration and invasion but promotes apoptosis. After transfecting circSOX13-shRNA, A. RT-qPCR to detect circSOX13 expression in A549 and H1299; B. CCK-8 to detect the viability of A549 and H1299; C. Edu analysis to detect A549 and H1299 proliferation cell; D. Flow cytometry to detect A549 and H1299 apoptosis rate; E. Transwell to detect A549 and H1299 migration and invasion capabilities; the values were expressed by mean ± SD (n = 3); versus sh-NC group, * P < 0.05.

Figure 3.

CircSOX13 acts as a sponge for miR-3194-3p. A. Via bioinformatics website http://starbase.sysu.edu.cn/ to query the potential binding sites of miR-3194-3p and circSOX13; B. RT-qPCR to detect miR-3194-3p in NSCLC tissues and normal tissue adjacent to cancer; C. RT-qPCR to detect miR-3194-3p expression in PC9, SPC-A1, H1975, H1299 and A549 and 16HBE; D. RT-qPCR to detect miR-3194-3p expression in A549 and H1299 after transfecting circSOX13-shRNA; E. DLR experiment to detect the targeted binding relationship between miR-3194-3p and circSOX13; the values were mean ± SD (n = 3); versus the normal tissue group, * P < 0.05; versus the 16HBE group, ^ P < 0.05; versus the sh-NC group, ^# P < 0.05; versus the mimic NC group, ^& P < 0.05.

Figure 4.

CircSOX13 influences malignant behavior of NSCLC by regulating miR-3194-3p. After transfecting miR-3194-3p mimic, A. RT-qPCR to detect miR-3194-3p expression in A549 and H1299 cells; B. CCK-8 to detect A549 and H1299 cell viability; C. Edu analysis to detect A549 and H1299 proliferation; D. Flow cytometry to detect A549 and H1299 apoptosis rate; E. Transwell to detect A549 and H1299 invasion and migration ability; the values were expressed as mean ± SD (n = 3); versus the mimic NC group, * P < 0.05.

Figure 5.

MAPRE1 is the target gene of miR-3194-3p. A. RT-qPCR to detect MAPRI1 expression in NSCLC tissues and normal tissues adjacent to cancer; B. Western blot to detect MAPRI1 expression in PC9, SPC-A1, H1975, H1299 and A549 and 16HBE C. Western blot to detect MAPRE1 expression in A549 and H1299 cells after transfecting miR-3194-3p mimic; D. Via bioinformatics website http://starbase.sysu.edu.cn/ to query the potential binding sites of miR-3194-3p and MAPRE1; E. DLR experiment to check the targeting relationship between miR-3194-3p and MAPRE1; the values were expressed as mean ± SD (n = 3); versus the normal tissue group, * P < 0.05; versus the 16HBE group, ^ P < 0.05; versus the mimic NC group, ^# P < 0.05.

Figure 6.

CircSOX13 promotes the malignant behavior of NSCLC via miR-3194-3p/MAPRE1 axis. After transfecting oe-SOX13 and si-MAPRE1, A. RT-qPCR or western blot to detect circSOX13, miR-3194-3p and MAPRI1 expression; B. CCK-8 to detect A549 and H1299 viability; C. Edu analysis to detect A549 and H1299 proliferation; D. Flow cytometry to detect A549 and H1299 apoptosis rate; E. Transwell to detect A549 and H1299 invasion and migration abilities; the values were expressed as mean ± SD (n = 3); versus the oe-NC group, * P < 0.05; versus the oe-SOX13 + si-NC group, ^ P < 0.05.

Figure 7.

CircSOX13accelerates NSCLC cisplatin resistance. A. CCK-8 to detect the IC50 value of A549 and H1299 as well as A549/DDP and H1299/DDP; B. RT-qPCR or western blot to detect circSOX13, MAPRE1 and miR-3194-3p expression in A549 and H1299 and A549/DDP and H1299/DDP; After transfecting circSOX13-shRNA, C. RT-qPCR to detect circSOX13 expression in A549/DDP and H1299/DDP; D. CCK-8 to detect IC₅₀ value in A549/DDP and H1299/DDP; E. DNA replication of A549/DDP and H1299/DDP cells detected by Edu analysis; F. Flow cytometry to detect the apoptosis of A549/DDP and H1299/DDP; G. Transwell to detect A549/DDP and H1299/DDP invasion and migration abilities; the values were expressed by mean ± SD (n = 3); versus the A549 group or the sh-NC group, * P < 0.05.

Figure 8.

CircSOX13 promotes cisplatin resistance in NSCLC via miR-3194-3p /MAPRE1 axis. A. The expression of MAPRE1 and miR-3194-3p in A549/DDP and H1299/DDP cells after co-transfection oe-SOX13 and miR-3194-3p mimic detected by RT-qPCR or Western blot; B. IC₅₀ values of A549/DDP and H1299/DDP cells after co-transfection oe-circSOX13 and miR-3194-3p mimic detected by CCK-8; C. DNA replication in A549/DDP and H1299/DDP cells after co-transfection with oe-circSOX13 and miR-3194-3p mimic detected by Edu; D. Apoptosis of A549/DDP and H1299/DDP cells after co-transfection with oe-circSOX13 and miR-3194-3p mimic detected by flow cytometry; E. Transwell to detect the invasion and migration abilities of A549/DDP and H1299/DDP cells after co-transfection with oe-circSOX13 and miR-3194-3p mimic. The values were expressed as mean ± SD (n = 3); versus the oe-NC group, * P < 0.05; versus the oe-SOX13 + mimic NC group, ^ P < 0.05.

Figure 9.

CircSOX13 promotes NSCLC tumor growth and cisplatin resistance. A. Representative image of tumor; B. Tumor volume; C. Tumor weight; D. Tumor Ki-67 expression detected by immunohistochemistry; E. MAPRE1 protein expression detected by Western blot. The values were expressed as mean ± SD (n = 5); Versus the sh-NC group, * P < 0.05; versus the PBS group, ^ P < 0.05; versus the DDP + sh-NC group, ^# P < 0.05.