Circ-ZKSCAN1 regulates FAM83A expression and inactivates MAPK signaling by targeting miR-330-5p to promote non-small cell lung cancer progression

Abstract

Background: Circular RNAs (circRNAs) belong to a new type of endogenous non-coding RNA and plays a key role in carcinogenesis. Circ-ZKSCAN1 (hsa_circ_0001727) has been proven to be a tumor-dependent circRNA. However, its role in non-small cell lung cancer (NSCLC) has been underreported.

Methods: The expression patterns of circ-ZKSCAN1 were determined using qRT-PCR in NSCLC samples and cell lines. Cell proliferation was examined utilizing the CCK-8 assay. Cell migration and invasion were evaluated using the Transwell assay. The combination of circ-ZKSCAN1 and miR-330-5p in NSCLC cells was analyzed by RNA pull-down and luciferase reporter assay. We used the bioinformatics software circbank, CircInteractome, TargetScan and Miranda to predict circRNA-miRNA and miRNA-mRNA interactions.

Results: Our results showed that circ-ZKSCAN1 was significantly up-regulated in NSCLC, closely related to malignant characteristics and poor prognosis, and clinically related to tumor size and clinical stage. Subsequent experiments showed that circ-ZKSCAN1 could inhibit the growth of NSCLC cells in vitro and in vivo. Importantly, circ-ZKSCAN1 can act as a sponge of carcinogenic miR-330-5p to increase the expression of FAM83A, resulting in the inhibition of MAPK signal transduction pathway, thus promoting the progress of NSCLC. Interestingly, the increase in FAM83A expression caused by circ-ZKSCAN1 overexpression could in turn promote the expression of circ-ZKSCAN1.

Conclusions: Circ-ZKSCAN1 is a key positive regulator of NSCLC, and clarifies the potential molecular mechanism of the new circ-ZKSCAN1/miR-330-5p/FAM83A feedback loop in promoting the progress of NSCLC.

Keywords: FAM83A; Non-small cell lung cancer (NSCLC); circ-ZKSCAN1; miR-330-5p; prognosis.

Figure 1

Circ-ZKSCAN1 is highly up-expressed in NSCLC tissues and cells. (A) Relative circ-ZKSCAN1 expression in NSCLC tumor tissues compared with adjacent normal tissues (n=107) by qRT-PCR analysis. (B) Relative circ-ZKSCAN1 expression in NSCLC cell lines. (C) Relative circ-ZKSCAN1 expression in different clinical stages (I vs. II). (D) The overall survival (OS) probability of NSCLC patients with low- and high-circ-ZKSCAN1 expression. (**P<0.01, ***P<0.001). NSCLC, non-small cell lung cancer.

Figure 2

Circ-ZKSCAN1 inhibition retards NSCLC cells’ biological process. (A) Relative circ-ZKSCAN1 expression in A549 and H1299 cells after transfection with si-circ-ZKSCAN1 or control. CCK-8 assays (B) and colony formation assays (C,D,E) in A549 and H1299 cells treated with or without circ-ZKSCAN1 inhibition. (F,G,H) A549 and H1299 cells in transwell chambers were applied to cell migration assays (100×). (I,J,K) Cells treated with si-NC or si-circ-ZKSCAN1 to conduct wound healing assays. (L,M,N) Flow cytometry to determine cell apoptosis of A549 and H1299 after si-circ-ZKSCAN1 or si-NC. (**P<0.01, ***P<0.001). NSCLC, non-small cell lung cancer.

Figure 3

Circ-ZKSCAN1 can promote tumor formation in vivo. (A,B) After inoculating A549 cells for 40 days in nude mice. (C,D) The weight and volume of tumors in the control group and the OE-circ-ZKSCAN1 group. (E) Relative circ-ZKSCAN1 expression in tumors of nude mice. (F,G) Ki-67 proliferative marker compared controls with circ-ZKSCAN1-overexpressing xenografts, as determined by IHC staining. (**P<0.01) (200×). IHC, immunohistochemistry.

Figure 4

Circ-ZKSCAN1 can sponge and suppress miR-330-5p in NSCLC cells. (A,B) The relative indications of 4 miRNA expressions in NSCLC cells after transfection with the knock-down circ-ZKSCAN1 or control group. (C,D) Circ-ZKSCAN1-wild- or -mutant-type luciferase vector after transfection with miR-330-5p or control mimics in A549 and H1299 cells by luciferase activity. (E,F) Four indicated miRNA expressions after RNA pull-down assay with circ-ZKSCAN1 probe and control probe in A549 and H1299 cells. (G) MiR-330-5p expression in control or OE-circ-ZKSCAN1 A549 and H1299 cells. (H, I, J) Relative expression levels of miR-330-5p in xenografts of nude mice (H), NSCLC cells (I), and tissues (J). (K) The OS probability of NSCLC patients with low- and high-miR-330-5p expression. (L) The correlation between circ-ZKSCAN1 and miR-330-5p expression in NSCLC tissues. (*P<0.05, **P<0.01, ***P<0.001). NSCLC, non-small cell lung cancer.

Figure 5

A feedback loop in NSCLC cells comprising Circ-ZKSCAN1, miR-330-5p, and FAM83A. (A,B) The relative luciferase activity of FAM83A 3’-UTR-wild- or -mutant-type luciferase vector after transfection with miR-330-5p mimics or control in A549 and H1299 cells. (C) Relative expression of FAM83A in miR-330-5p or control mimics in A549 and H1299 cells. (D) Relative FAM83A expression in NSCLC and paired normal tissues. (E) The OS probability of low and high expression of FAM83A in NSCLC patients. (F) WB analysis for the protein expression of FAM83A in control or miR-330-5p mimics A549 and H1299 cells after transfection with OE-circ-ZKSCAN1 vector and control. β-actin as an internal reference. (G) Relative FAM83A expression in tumors of nude mice. (H) FAM83A in NSCLC tissues by IHC staining. (I) The correlation between FAM83A and circ-ZKSCAN1 expression in NSCLC tissues. (J) Colony formation assays for overexpressed-miR-330-5p or control in NSCLC cells after transfection with OE-circ-ZKSCAN1 or OE-FAM83A vector. (K) Relative expression of circ-ZKSCAN1 and miR-330-5p in control or OE-FAM83A of A549 and H1299 cells. (*P<0.05, **P<0.01, ***P<0.001). NSCLC, non-small cell lung cancer.

Figure 6

The overexpression of miR-330-5p or silencing of FAM83A could block the suppression of MAPK pathway caused by circ-ZKSCAN1 overexpression in NSCLC cells. (A,B) WB analysis for the expression of JNK, p38, and ERK1/2 in control or OE-circ-ZKSCAN1 A549 (A) and H1299 (B) cells with miR-330-5p mimics or si-FAM83A. (**P<0.01). NSCLC, non-small cell lung cancer.

Figure 7

Schematic diagram of the mechanism by which the circ-ZKSCAN1-miR-330-5p-FAM83A regulatory loop regulates lung cancer proliferation by inactivating MAPK signaling pathway.

Figure S1

Circ-ZKSCAN1 in HBE cells. (A) Expression levels of circ-ZKSCAN1 in NSCLC cells treated with siRNAs. (B) CCK8 analysis of HBE cells with silencing or stably overexpressing circ-ZKSCAN1. (C,D) Cells treated with control, OE-circ-ZKSCAN1, and si-circ-ZKSCAN1 to perform wound healing assays. (E,F) HBE cells in transwell chambers were applied to cell migration assays (100×). (*P<0.05, **P<0.01). HBE, human bronchial epithelial.

Figure S2

Circ-ZKSCAN1 targeting miRNA. (A) Intersection of miRNAs predicted by circBank and CircInteractome databases. (B) The binding sites of circ-ZKSCAN1 and miR-330-5p.