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OncoTargets and Therapy Volume 11, 2018 - Issue
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Original Research

Hsa_circ_0003998 promotes cell proliferation and invasion by targeting miR-326 in non-small cell lung cancer

Wanjun Yu1 Department of Central Laboratory, Yinzhou People’s Hospital, Yinzhou Hospital Affiliated to Medical School of Ningbo University, Ningbo 315040, China, [email protected];2 Department of Respiratory and Critical Care Medicine, Yinzhou People’s Hospital, Yinzhou Hospital Affiliated to Medical School of Ningbo University, Ningbo 315040, China
,
Hao Jiang2 Department of Respiratory and Critical Care Medicine, Yinzhou People’s Hospital, Yinzhou Hospital Affiliated to Medical School of Ningbo University, Ningbo 315040, China
,
Huihui Zhang2 Department of Respiratory and Critical Care Medicine, Yinzhou People’s Hospital, Yinzhou Hospital Affiliated to Medical School of Ningbo University, Ningbo 315040, China
&
Jipeng Li1 Department of Central Laboratory, Yinzhou People’s Hospital, Yinzhou Hospital Affiliated to Medical School of Ningbo University, Ningbo 315040, China, [email protected]Correspondence[email protected]
Pages 5569-5577 | Published online: 07 Sep 2018

Figures & data

Figure 1 Hsa_circ_0003998 expression was upregulated in NSCLC cells and tissues.

Notes: Expression of hsa_circ_0003998 in four NSCLC cells and one cultured human lung epithelial cell line (A). Relative expression levels of hsa_circ_0003998 were determined in 60 paired NSCLC tissue samples and their corresponding normal samples (B). Upregulation of hsa_circ_0003998 in NSCLC was associated with tumor size (C) and lymph-node metastasis (D). NSCLC patients with higher expression of hsa_circ_0003998 had shorter overall survival time than those with lower expression of has-circ_0003998 (E). The values are the average of three independent experiments. **P<0.01.
Abbreviation: NSCLC, non-small cell lung cancer.
Figure 1 Hsa_circ_0003998 expression was upregulated in NSCLC cells and tissues.

Figure 2 Hsa_circ_0003998 knockdown suppressed NSCLC cell proliferation and invasion in vitro.

Notes: Specific siRNA targeting hsa_circ_0003998 was transfected into A549 and H1299 cells (A). CCK-8 proliferation assay showed the proliferation in the hsa_circ_0003998 knockdown group and the control group (B, C). Colony formation assays showed the clone number in cells transfected with siRNA or control (D). Invasion assays were used to measure invasion ability (E). The values are the average of three independent experiments. **P<0.01.
Abbreviations: NSCLC, non-small cell lung cancer; siRNA, small interfering RNA; NC, normal control.
Figure 2 Hsa_circ_0003998 knockdown suppressed NSCLC cell proliferation and invasion in vitro.

Figure 3 The interaction of hsa_circ_0003998 and miR-326.

Notes: The predicted miR-326/hsa_circ_0003998 binding site and its mutated version by site mutagenesis are as shown (A). The relative expression levels of miR-326 were detected in 30 paired NSCLC and their corresponding normal samples via qRT-PCR (B). Expression levels of miR-326 are negatively correlated with hsa_circ_0003998 among NSCLC samples, as indicated by two-tailed Pearson’s correlation analysis (r=−0.526, P<0.01) (C). Knockdown of hsa_circ_0003998 increased miR-326 expression (D). Luciferase assays were performed to determine the interaction between miR-326 and hsa_circ_0003998 in H1299 cells (E). The values are the average of three independent experiments. **P<0.01.
Abbreviations: NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction.
Figure 3 The interaction of hsa_circ_0003998 and miR-326.

Figure 4 miR-326 inhibitor rescued the function of hsa_circ_0003998 knockdown in H1299 cells.

Notes: miR-326 expression in four NSCLC cell lines detected by RT-PCR (A). miR-326 expression in H1299 cells transfected with si-hsa_circ_0003998 and/or miR-326 inhibitor (B). CCK-8 assay (C). Colony formation assay (D). Invasion assay (E). The values are the average of three independent experiments. *P<0.05, **P<0.01.
Abbreviations: NSCLC, non-small cell lung cancer; RT-PCR, real-time polymerase chain reaction; NC, normal control.
Figure 4 miR-326 inhibitor rescued the function of hsa_circ_0003998 knockdown in H1299 cells.

Figure 5 Hsa_circ_0003998 positively regulated the miR-326 target gene Notch1.

Notes: Notch1 mRNA (A) and protein (B) expressions in H1299 cells transfected with si-hsa_circ_0003998 and/or the miR-326 inhibitor. Notch1 mRNA levels were negatively correlated with miR-326 levels (C) and positively correlated with has_circ_0003998 levels (D) among NSCLC samples, as indicated by two-tailed Pearson’s correlation analysis. The values are the average of three independent experiments.
Abbreviation: NSCLC, non-small cell lung cancer; NC, normal control.
Figure 5 Hsa_circ_0003998 positively regulated the miR-326 target gene Notch1.
Figure 5 Hsa_circ_0003998 positively regulated the miR-326 target gene Notch1.

Figure S1 Relative expression levels of Notch1 mRNA were detected in four NSCLC cells and one cultured human lung epithelial cells via qRT-PCR.

Note: *P<0.05, **P<0.01.
Abbreviations: NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction.
Figure S1 Relative expression levels of Notch1 mRNA were detected in four NSCLC cells and one cultured human lung epithelial cells via qRT-PCR.

Table S1 Association between hsa_circ_0003998 expression and clinicopathological characteristics

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