Overexpression of hsa_circ_0008274 inhibited the progression of lung adenocarcinoma by regulating HMGA2 via sponging miR-578

Abstract

Background: Circular RNAs (circRNAs) had been identified as a non-coding RNA associated with many types of cancer in recent years. However, the involvement of hsa_circ_0008274 in lung adenocarcinoma (LUAD) has not been explored. The aim of our research is to explore the biological mechanism and function of hsa_circ_0008274 in LUAD.

Methods: The hsa_circ_0008274, miR-578, and high mobility group AT-Hook 2 (HMGA2) mRNA expression levels were detected via qRT-PCR. Cell Counting Kit-8 (CCK-8) Transwell assay and wound healing assay were performed to measure the cell proliferation, invasion, and migration ability. Luciferase reporter and Western blotting experiments were performed to characterize the competing endogenous RNA (ceRNA) mechanism of hsa_circ_0008274.

Results: Our findings determined that the expression of hsa_circ_0008274 in LUAD was significantly decreased. Cell experiments showed that overexpressed hsa_circ_0008274 could reduce the proliferation and invasion ability of LUAD cells. Moreover, miRNA-578 could identify as a miRNA sponge of hsa_circ_0008274. Overexpressed hsa_circ_0008274 reduced the proliferation and invasion of LUAD cells caused by miR-578 mimics. Increasing the expression of miR-578 can aggravate the proliferation and invasion of LUAD cells and block the inhibition of proliferation and invasion of LUAD cells mediated by overexpressed hsa_circ_0008274. Subsequent data indicate that HMGA2 of the tumor-promoting gene is the target gene of miR-578. The upregulation of HMGA2 partially reversed the tumor inhibitory effect of LUAD cells induced by overexpressed hsa_circ_0008274 or miR-578 mimics.

Conclusions: In summary, our data show that the overexpression of hsa_circ_0008274 repressed the proliferation and invasion of LUAD through downregulating miR-578 and activating HMGA2.

Keywords: HMGA2; hsa_circ_0008274; lung adenocarcinoma; miR-578.

FIGURE 1

Hsa_circ_0008274 expression analysis in LUAD tissues and cell lines. (a) The expression levels of circular and linear UGGT2 in A549 and SPC‐A‐1 cells treated with RNase R. (b) qRT‐PCR analysis of hsa_circ_0008274 expression in A549, CALU3, SPC‐A‐1, GLC‐82, and human bronchial epithelial cells (HBE). (c) Hsa_circ_0008274 expression analysis of LUAD and paired normal lung tissues. (d) The OS rates of LUAD patients with high and low expression of mRNA UGGT2 was evaluated by Kaplan–Meier method. **p < 0.01, ***p < 0.001

FIGURE 2

Overexpressed hsa_circ_0008274 inhibited the proliferation and invasion of LUAD cells. (a) After overexpressing, relative hsa_circ_0008274 expression was determined by RT‐qPCR. (b) CCK‐8 assay was assessed to detect cell proliferation. (c) Colony‐forming ability was applied by colony formation assay. (d),(e) Evaluation of invasion and metastasis potential were measured by Transwell invasion assay and wounding assay. **p < 0.01 and ***p < 0.001

FIGURE 3

miR‐578 restrained the proliferation and invasion of LUAD cells. (a) MiR‐578 and miR‐633 are the intersection of circBank and circinteractome databases. (b) The expression of miR‐578 and miR‐633 in lung adenocarcinoma cell lines compared to normal lung cell, only miR‐578 expression was increased. After miR‐578 mimics and inhibitor, (c,e) cell proliferation was measured by the CCK‐8 assay, and (d) Transwell invasion assay was applied to examine invasive potential. (f),(g) After overexpressed circRNA with or without miR‐578, the invasion of cell was detected. *p < 0.05, **p < 0.01

FIGURE 4

HMGA2 overexpression reversed hsa_circ_0008274 upregulated‐ or miR‐578 mimics‐mediated antitumor function. (a) The binding sequences between miR‐578 and HMGA2‐3′UTR were shown. The red sequences represent its mutant. (b),(c) The luciferase activity of HMGA2‐wt and HMGA2‐mut in A549 and SPC‐A‐1 cells was detected by dual‐luciferase reporter assay after transfection of miR‐578 or miR‐NC. (d) HMGA2 expression was determined by Western blot with or without miR‐578 mimics. (e),(f) Cell invasion was evaluated by the Transwell invasion. (g) The HMGA2 protein expression was measured after overexpressed circRNA cotransfected with or without miR‐578 mimics. (h),(i) Cell invasion was evaluated by the Transwell invasion. (j) The schematic diagram revealed that the downregulated hsa_circ_0008274 released miR‐578 and silences HMGA2 expression, which, in turn, affected the proliferation and invasion of LUAD