miR-30 Family Reduction Maintains Self-Renewal and Promotes Tumorigenesis in NSCLC-Initiating Cells by Targeting Oncogene TM4SF1

Abstract

Increasing evidence indicates that tumor-initiating cells (TICs) are responsible for the occurrence, development, recurrence, and development of the drug resistance of cancer. MicroRNA (miRNA) plays a significant functional role by directly regulating targets of TIC-triggered non-small-cell lung cancer (NSCLC), but little is known about the function of the miR-30 family in TICs. In this study, we found the miR-30 family to be downregulated during the spheroid formation of NSCLC cells, and patients with lower miR-30a/c expression had shorter overall survival (OS) and progression-free survival (PFS). Moreover, transmembrane 4 super family member 1 (TM4SF1) was confirmed to be a direct target of miR-30a/c. Concomitant low expression of miR-30a/c and high expression of TM4SF1 correlated with a shorter median OS and PFS in NSCLC patients. miR-30a/c significantly inhibited stem-like characteristics in vitro and in vivo via suppression of its target gene TM4SF1, and then it inhibited the activity of the mTOR/AKT-signaling pathway. Thus, our data provide the first evidence that TM4SF1 is a direct target of miR-30a/c and miR-30a/c inhibits the stemness and proliferation of NSCLC cells by targeting TM4SF1, suggesting that miR-30a/c and TM4SF1 may be useful as tumor biomarkers for the diagnosis and treatment of NSCLC patients.

Keywords: NSCLC; TICs; TM4SF1; biomarker; miR-30 family.

Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Isolation and Identification of Lung Cancer Stem Cells (A and B) CD133 and CD326 mRNA expression during spheroid formation of SPC-A1 (A) and NCI-H1650 (B) cell lines by qRT-PCR. (C) CD133 and CD326 protein expression during spheroid formation of SPC-A1 and NCI-H1650 cell lines by western blot. (D and E) Embryonic stem cell markers (OCT 4 and Bmi-1) expression during spheroid formation of SPC-A1 and NCI-H1650 cell lines by qRT-PCR (D) and western blot (E). (F) Lung stem cell markers (CCSP and TTF-1) expression during spheroid formation of SPC-A1 and NCI-H1650 cell lines by qRT-PCR. (G) Unsupervised hierarchical clustering of the expression data during spheroid formation of SPC-A1 and NCI-H1650 cell lines. (H) Expression of miR-30c/a during spheroid formation of SPC-A1 and NCI-H1650 cell lines by qRT-PCR. Data are shown as the means ± SDs of three independent experiments. Statistical analyses were performed with one-way ANOVA (**p 

Figure 2

Cancer Stem Cell Marker Expression of Spheroid Cell and Differentiated Cells (A) qRT-PCR to evaluate CD133 expression of tumor sphere cell and differentiated cell in SPC-A1 and NCI-H1650 cell lines. (B) qRT-PCR to evaluate CD326 expression of tumor sphere cell and differentiated cell in SPC-A1 and NCI-H1650 cell lines. (C) Western blot analysis to evaluate CD133 and CD326 expression of tumor sphere cell and differentiated cell in SPC-A1 and NCI-H1650 cell lines. (D–F) qRT-PCR (D and E) and western blot (F) to evaluate the expression of embryonic stem cell markers OCT 4 (D and F) and Bmi-1 (E and F). (G) qRT-PCR to evaluate the expression of lung stem cell markers CCSP and TTF-1. (H) qRT-PCR to evaluate the expression of miR-30c, -30a, -30e, and -30d in tumor spheres and differentiated cells. Data are shown as the means ± SDs of three independent experiments. Statistical analyses were performed with one-way ANOVA (*p 

Figure 3

Knockdown and Overexpression Experiments, Spheroid Formation Assay, Cell Growth Assays, and Apoptosis Assay (A) Overexpression and knockdown of miR-30c/a verified by qRT-PCR in the SPC-A1 cell line. (B) Overexpression and knockdown of miR-30c/a verified by qRT-PCR in the NCI-H1650 cell line. (C) Spheroid formation assay of the control group and knockdown groups of miR-30c/a at day 7. (D) Cell growth assays of the control group and knockdown groups of miR-30c/a. (E) Apoptosis assay of the control group and knockdown groups of miR-30c/a. (F) Spheroid formation assay of the control group and overexpression groups of miR-30c/a. (G) Cell growth assays of the control group and overexpression groups of miR-30c/a. (H) Apoptosis assay of the control group and overexpression groups of miR-30c/a. Data are shown as the means ± SDs of three independent experiments. Statistical analyses were performed with one-way ANOVA (*p 

Figure 4

Prediction of Target Genes of hsa-miR-30c and hsa-miR-30a (A) Prediction of target genes of hsa-miR-30c and hsa-miR-30a using 4 target gene prediction software packages: miRanda, miRDB, DIANA-MICROT, and StarBase. 301 common target genes were found. (B) GO analysis of the 301 common target genes. (C) Prediction of potential target miRNA of TM4SF1 using 4 target gene prediction software. (D) TM4SF1 expression examined by qRT-PCR when hsa-miR-30c/a were knocked down. (E) TM4SF1 expression examined by western blot when hsa-miR-30c/a were knocked down. (F) TM4SF1 expression examined by qRT-PCR when hsa-miR-30c/a were overexpressed. (G) TM4SF1 expression examined by western blot when hsa-miR-30c/a were overexpressed. (H) The 3′ UTR-binding site and mutation site of miR-30 family of the TM4SF1 gene. (I) Luciferase reporter assay to verify whether hsa-miR-30c/a directly targeted TM4SF1. (J) RIP-IP assays were performed to co-immunoprecipitate the Ago2 complexes from SPC-A1 cells transfected with either hsa-miR-30c/a mimic or negative control mimic. Real-time PCR assays were performed on RNA samples isolated from the Ago2 coIP fractions to measure the relative enrichment of the TM4SF1 mRNA. Data are shown as the means ± SDs of three independent experiments. Statistical analyses were performed with one-way ANOVA (**p  0.05 vs. control).

Figure 5

TM4SF1 and miR-30c/a Expression in Different Differentiated Cells (A) qRT-PCR to investigate the expression of TM4SF1 in 0-, 3-, and 7-day SPC-A1 and NCI-H1650 cells of spheroid culture. (B) Western blot to investigate the expression of TM4SF1 in 0-, 3-, and 7-day SPC-A1 and NCI-H1650 cells of spheroid culture. (C) Correction analysis of has-miR-30c and TM4SF1 expression. (D) Correction analysis of has-miR-30a and TM4SF1 expression. (E) qRT-PCR analysis of spheroid cells (day 7) and differentiated cells. (F) Blot analysis of spheroid cells (day 7) and differentiated cells. (G) Correction analysis of has-miR-30c and TM4SF1 expression. (H) Correction analysis of has-miR-30a and TM4SF1 expression. (I) Overexpression of TM4SF1 verified by qRT-PCR and western blot. (J) Spheroid formation assay of the control group and the TM4SF1 overexpression group at day 7. (K) Cell growth assays of the control group and the TM4SF1 overexpression group. (L) Apoptosis assay of the control group and the TM4SF1 overexpression group. Data are shown as the means ± SDs of three independent experiments. Statistical analyses were performed with one-way ANOVA (*p 

Figure 6

miR-30a/c Regulate TM4SF1 Expression (A) qRT-PCR and western blot analysis of TM4SF1 expression in 3 groups of cells: the control group, miR-30c group, and miR-30c + TM4SF1 group. (B) qRT-PCR and western blot analysis of TM4SF1 expression in 3 groups of cells: the control group, miR-30a group, and miR-30a + TM4SF1 group. (C) Spheroid formation assay of the control group, miR-30c group, and miR-30c + TM4SF1 group. (D) Cell growth assays of the control group, miR-30c group, and miR-30c + TM4SF1 group. (E) Apoptosis assay of the control group, miR-30c group, and miR-30c + TM4SF1 group. (F) Spheroid formation assay of the control group, miR-30a group, and miR-30a + TM4SF1 group. (G) Cell growth assays of the control group, miR-30a group, and miR-30a + TM4SF1 group. (H) Apoptosis assay of the control group, miR-30a group, and miR-30a + TM4SF1 group. Data are shown as the means ± SDs of three independent experiments. Statistical analyses were performed with one-way ANOVA (*p 

Figure 7

miR-30c/a Inhibit Tumor Growth In Vivo by Targeting TM4SF1 (A) Tumor volume curves of the control group, miR-30c group, TM4SF1 group, and miR-30c + TM4SF1 group. (B) Tumor volume curves of the control group, miR-30a group, TM4SF1 group, and miR-30a + TM4SF1 group. (C) Staining of cancer stem cell surface markers CD326 and CD133. (D) Quantitative analysis of relative fluorescence intensity of the para group, control group, miR-30c group, TM4SF1 group, and miR-30c + TM4SF1 group in SPC-A1 cells. Green, CD326; red, CD133. (E) Quantitative analysis of relative fluorescence intensity of the para group, control group, miR-30c group, TM4SF1 group, and miR-30c + TM4SF1 group in HCI-H1650 cells. Green, CD326; red, CD133. (F) Apoptosis assay of control group, miR-30c group, TM4SF1 group, and miR-30c + TM4SF1 group in HCI-H1650 cells. (G) Influence of miR-30c and TM4SF1 expression on apoptotic signal molecules cleaved-caspase-3 expression by western blot. (H) Influence of miR-30c and TM4SF1 expression on AKT/mTOR pathway-associated protein expressions by western blot. Data are shown as the means ± SDs of three independent experiments. Statistical analyses were performed with one-way ANOVA (*P

Figure 8

miR-30c/a and TM4SF1 Expression in NSCLC Tissue (A) miR-30c expression by qRT-PCR in 36 paired NSCLC tissues and 124 normal lung tissues. (B) miR-30a expression by qRT-PCR in 36 paired NSCLC tissues and 124 normal lung tissues. (C) IHC staining of TM4SF1 expression in normal and NSCLC tissues. (D) qRT-PCR analysis of TM4SF1 expression in normal and NSCLC tissues. (E) Correlation analysis. (F) Correlation analysis of miR-30c and TM4SF1 expression. (G) Correlation analysis of miR-30a and TM4SF1 expression.