Aberrant microRNAs expression in CD133⁺/CD326⁺ human lung adenocarcinoma initiating cells from A549

Abstract

Increasing evidence demonstrates that miRNAs are involved in the dysregulation of tumor initiating cells (TICs) in various tumors. Due to a lack of definitive markers, cell sorting is not an ideal separation method for lung adenocarcinoma initiating cells. In this study, we combined paclitaxel with serum-free medium cultivation (inverse-induction) to enrich TICs from A549 cells, marked by CD133/CD326, defined features of stemness. We next investigated aberrant microRNAs in this subpopulation compared to normal cells with miRNA microarray and found that 50 miRNAs exhibited a greater than 2-fold change in expression. As further validation, 10 miRNAs were chosen to perform quantitative RT-PCR on the A549 cell line and primary samples. The results suggest that aberrant expression of miRNAs such as miR-29ab, miR-183, miR-17-5p and miR-127-3P may play an important role in regulating the bio-behavior of TICs.

Fig. 1.

Separation of the CD133⁺/CD326⁺ subpopulation by inverse-induction combined with paclitaxel treatment from A549 cells. (A) Cytometric CD133/CD326 expression in normal A549 cells, (B) secondary passage spheroids induced by single inverse-induction, (C) the progression of regrowing into spheres after treatment with paclitaxel (from left to right, showed the morphology on the 4^th day, 10^th day and 14^th day, 200×) (D) Cytometric CD133/CD326 expression in secondary passage spheroids after paclitaxel treatment.

Fig. 2.

Identification of the CD133⁺/CD326⁺ subpopulation as CSCs of A549. (A) Immunofluorescence of CD133 (green) and CD326 (red) expression on spheroids after paclitaxel treatment. (B) Differentiation of spheroids into progeny upon conditioning with complete medium. From left to right: morphology change on the second day, sixth day and expression of CK8/CK18 on progeny (200×). (C) Subcutaneous xenografts on nude mice (left armpit of forelimb, 1 × 10⁴ CD133⁺/CD326⁺ cells; right armpit of forelimb, 1 × 10⁴ normal A549 cells), all mice were sacrificed after injection for 3 weeks and observed by histology (200×). (D) Detection of stem cell-associated genes between the CD133⁺/CD326⁺ cells and normal A549 cells (defined as 1) by quantitative RT-PCR.

Fig. 3.

CD133⁺/CD326⁺ subpopulation co-located in primary tumor samples and sorted by MACS. (A) Immunofluorescence of CD133 (green) and CD326 (red) on fresh sections. (B) Postoperative lung cancer samples were confirmed to be adenocarcinoma by histology. (C) Confirmation of expression by flow cytometry. Positivity for CD133/CD326 of cells before sorting was 1.2% (left) and of cells after sorting was 93.2% (right). (D) Cell morphology: before sorting, cells were adherent (left), and after sorting, cells grew into tight spheres. (right, 200×).

Fig. 4.

Aberrant expression of miRNAs in the CD133⁺/CD326⁺ subpopulation derived from A549 cells and validated by qRT-PCR. (A) Partial relative miRNAs exhibited a greater than 2-fold expression change in the CD133⁺/CD326⁺ subpopulation compared to normal A549 cells (defined as 1) by microarray. (B) Validation of expression of 10 selected miRNAs expression in CD133⁺/CD326⁺ cells at the cell line level and (C) further investigation on primary samples by qRT-PCR. (D) Venn analysis shows the up-regulation and down-regulation of the miRNAs in the A549 cell line (left) and primary samples (right). Trends consistent with array data were underlined, and the overlapping area shows validation of miRNA expression in primary samples that showed trends consistent with those in the A549 cell line.