miR-26 Induces Apoptosis and Inhibits Autophagy in Non-small Cell Lung Cancer Cells by Suppressing TGF-β1-JNK Signaling Pathway

Abstract

Non-small cell lung cancer (NSCLC) is one of the causes of cancer mortality worldwide. The role of miR-26 in the development and progression of NSCLC remains largely unknown. In this study we found an abnormal expression of miR-26 in human NSCLC tissues. It was found that miR-26 mimics induced cell apoptosis and promoted caspase-3, 9 activities in human NSCLC cells. The miR-26 inhibitor enhanced the expression of the light chain 3 (LC3) protein and the autophagy related genes in NSCLC cells. Moreover, miR-26 regulated apoptosis and autophagy by inhibiting TGF-β expression in a JNK dependent manner. In addition, miR-26 mimics induced cell apoptosis, was involved in the endoplasmic reticulum stress (ERS) signaling pathway. Down-regulation of the ERS, inhibited apoptosis which was induced by miR-26 mimics in NSCLC cells. In in vivo studies, TUNEL staining revealed that the number of TUNEL positive cells of the tumor tissue in the miR-26 treatment group, were significantly increased in comparison with the control group, while the number of TUNEL positive cells in the tumor tissue were remarkably decreased in the groups treated with miR-26, combined with the TGF-β1 inhibitor or JNK inhibitor. Additionally, the immunoreactivity of TGF-β1 in the cells treated with the miR-26 inhibitor, decreased in comparison to the control group. Our results indicated that miR-26 induced apoptosis and inhibited autophagy in human NSCLC cells through the TGF-β1-JNK signaling pathway, suggesting that miR-26 could be a potential novel target for the treatment of NSCLC.

Keywords: JNK; NSCLC; TGF-β; apoptosis; autophagy; miR-26.

FIGURE 1

The expression of miR-26 in non-small cell lung cancer. (A) The representative results of ISH staining in normal tissues and lung cancer tissues. (B) The miR-26 expression was detected in normal tissues and lung cancer tissues. Arrows represented the positive cells.

FIGURE 2

miR-26 induced cell apoptosis in non-small cell lung cancer cells. (A,B) Flow cytometry showed that miR-26 increased cell apoptosis in A549 cells. (C–E) Caspase activity assay showed increased Caspase-3 and caspase-9 activities after treatment with miR-26 in A549 cells. (F) Western blot showed a decreased expression of the Bcl protein and an increased expression of the Bax protein following miR-26 treatment in A549 cells. (G) miR-26 caused cell cycle arrest in A549 cells. ^∗P < 0.05 vs. Blank, ^∗∗P < 0.01 vs. Blank.

FIGURE 3

miR-26 inhibitor induced autophagy in non-small cell lung cancer cells. (A) Western blotting showed an increased expression of the LC3 protein following miR-26 inhibitor treatment in NSCLC cells. (B,C) Western blot and RT-PCR showed an increased expression of autophagy related protein molecules and mRNA of BECLIN1, ATG5, ATG7, and DAPK following miR-26 inhibitor treatment in A549 cells. ^∗P < 0.05 vs. Blank, ^∗∗P < 0.01 vs. Blank.

FIGURE 4

miR-26 inhibited TGF-β1 in non-small cell lung cancer cells. (A) Western blotting showed a decreased expression of the TGF-β1 protein following miR-26 treatment in A549 cells. (B) luciferase reporter assay revealed increased ASPP2 3’UTR luciferase activity in mutant A549 cells. ^∗∗P < 0.01 VS Con.

FIGURE 5

miR-26 inhibitor induced autophagy and apoptosis by targeting TGF-β1 in a JNK-dependent manner. (A) Western blot on the protein expression of JNK following treatment with the miR-26 inhibitor combined with the TGF-β1 or TGF-β1 inhibitor in A549 cells. (B) Flow cytometry on cell apoptosis in A549 cells treated with miR-26 followed by treatment with the TGF-β1 or TGF-β1 inhibitor. (C) Western blot on the protein expression of LC3 following treatment with the iR-26 inhibitor combined with the TGF-β1 or TGF-β1 inhibitor in A549 cells. (D) Flow cytometry on cell apoptosis in A549 cells treated with the miR-26 and the JNK or JNK inhibitor. (E) Western blot on the protein expression of LC3 following treatment with the miR-26 inhibitor combined with the JNK or JNK inhibitor in A549 cells. ^∗P < 0.05 VS miR-26 group, ^∗∗P < 0.01 VS miR-26 group.

FIGURE 6

miR-26 regulated autophagy and apoptosis were correlated with ERS signaling. (A) Western blot on the protein expression of Chop, ATF-4, Bip, and XBP-1 following miR-26 treatment in A549 cells. (B) RT-PCR on the mRNA expression of Chop, ATF-4, Bip, and XBP-1 following treatment with a siRNA of Chop, ATF-4, Bip, and XBP-1. (C) The mRNA expression of DR5 following a siRNA of Chop, ATF-4, Bip, and XBP-1 combined with the miR-26 treatment. (D) Western blot on the protein expression of Bcl and Bax following a siRNA of Chop, ATF-4, Bip, and XBP-1 combined with the miR-26 treatment. (E) Flow cytometry on cell apoptosis detected following a siRNA of Chop, ATF-4, Bip and XBP-1 combined with miR-26 treatment. (F) RT-PCR on the mRNA expression of BECLIN detected following a siRNA of Chop, ATF-4, Bip, and XBP-1 combined with miR-26 inhibitor treatment. (G) Western blot on the protein expression of LC3 detected following a siRNA of Chop, ATF-4, Bip, and XBP-1 combined with the miR-26 inhibitor treatment. ^∗P < 0.05 VS Con., ^∗∗P < 0.01 VS Con., ^∗∗∗P < 0.001 VS Con.

FIGURE 7

miR-26 inhibited NSCLC growth in vivo. (A) The tumor volume of mouse xenografts treated with the miR-26 and/or the TGF-β1 inhibitor or JNK inhibitor. (B) The protein expression of JNK and LC3 in tumors treated with the miR-26 inhibitor and/or the TGF-β1 inhibitor or JNK inhibitor. (C) Immunohistochemistry of TGF-β1 in the lung cancer tissues with different treatments. (D) TUNEL staining on cell apoptosis in the lung cancer xenograft tissues with different treatments. Arrows represented the positive cells.