Long noncoding RNA MALAT1 knockdown reverses chemoresistance to temozolomide via promoting microRNA-101 in glioblastoma

Abstract

Glioblastoma (GBM) is the most common and lethal tumor of the central nervous system with highly infiltrative and resistant to chemotherapy. Temozolomide (TMZ) is widely used as the first-line treatment for the therapy of GBM. However, a considerable percentage inherent or acquired resistance in GBM accounts for many treatment failures of the TMZ chemotherapy. Therefore, a deeper understanding of the molecular characteristics underlying TMZ resistance and the identification of novel therapeutic target is urgent. Here, we show that MALAT1 was significantly upregulated in TMZ-resistant GBM cells. On the other hand, MALAT1 knockdown reduces TMZ resistance of GBM cells both in vitro and in vivo by inhibiting cell proliferation and promoting apoptosis. We also show that miR-101 overexpression reduced TMZ resistance of GBM cells and played an antagonistic role compared with MALAT1. Importantly, we demonstrate that MALAT1 promoted the chemoresistance through suppressing miR-101 signaling pathway via directly binding it in GBM cells. In conclusion, our study indicates that knockdown of MALAT1 reverses chemoresistance to TMZ via promoting miR-101 regulatory network in GBM and thus offers a novel prognostic marker and potential target for GBM TMZ-based chemotherapy.

Keywords: Chemoresistance; LncRNA MALAT1; glioblastoma; miR-101; temozolomide.

Figure 1

High expression of MALAT1 was associated with TMZ resistance in glioma cells. (A) Cell survival rates of GBM cell lines U251 and U251/TMZ were assessed by MTT assay. (B) The IC50 values of TMZ in U251 and U251/TMZ. (C) The expressions of MRP1, MGMT, and P‐gp were evaluated by Western blotting, and β‐actin was used as control. (D) The mRNA expressions of MALAT1 in cell lines U251 and U251/TMZ were assessed by qRT‐PCR analysis, and β‐actin was used as control. (E) Representative images of ISH for MALAT1 in U251 and U251/TMZ cells. All data represent the means ± SD, and all experiments were performed in triplicate. **P < 0.01.

Figure 2

Knockdown of MALAT1 reduces chemoresistance in TMZ‐resistant GBM cells in vitro and in vivo. (A) The relative expressions of MALAT1 in the U251/TMZ cells transduced with shMALAT1 or shNC were determined by qRT‐PCR, and β‐actin was used as control. (B) The cell survival rates of transgenic cell lines with TMZ (50–400 μmol/L) treatments were determined by MTT assay. (C) Colony formation assay showed the numbers of colonies of U251/TMZ cells transduced with shMALAT1 or shNC in the presence or absence of TMZ. (D) Annexin V/PI staining and flow cytometry analysis was used to assess apoptosis in GBM cell lines. (E) Representative DAPI‐stained nuclei and terminal deoxynucleotide transferase dUTP nick end labeling‐stained apoptotic nuclei of each group. (F) Tumor growth curve was based on the tumor volumes which were calculated every 5 days, and the TMZ treatment (5 mg/kg/day) began at 25th day after injection. (G) Tumors were harvested and measured at the 35th day after injection. All data represent the means ± SD of three replications. *P < 0.05, **P < 0.01.

Figure 3

MALAT1 is a direct target of miR‐101. (A) Representation of the miR‐101 binding site in MALAT1 based on the online database microRNA.org. (B) Luciferase activity of the reporter construct containing the wild‐type or mutant miR‐101 binding site was measured after cotransfection with 50 nmol/L microRNA. (C and D) The expression of miR‐101 and MALAT1 was determined by qRT‐PCR. All experiments were performed in triplicate, *P < 0.05.

Figure 4

Overexpression of miR‐101 reduces TMZ resistance of GBM cells. (A) The relative expressions of miR‐101 in the U251/TMZ cells were determined by qRT‐PCR, and β‐actin was used as control. (B) The expressions of miR‐101 in the U251/TMZ cells with overexpression of miR‐101. (C) MTT assay was used to determine the cell survival rates of transgenic cell lines with TMZ (50–400 μmol/L) treatments. (D) Colony formation assay of U251/TMZ cells transduced with miR‐101 mimics or miR‐NC in the presence or absence of TMZ. (E) Flow cytometry analysis indicated the apoptosis rates in GBM cell lines. (F) Representative DAPI‐stained nuclei and terminal deoxynucleotide transferase dUTP nick end labeling‐stained apoptotic nuclei of each group. All data represent the means ± SD of three replications. *P < 0.05, **P < 0.01.

Figure 5

MALAT1 inhibition reverses TMZ resistance by upregulating miR‐101. (A) MTT assay was used to determine the cell survival rates of transgenic cell lines with TMZ (50–400 μmol/L) treatments. (B) Colony formation assay of transgenic U251/TMZ in the presence or absence of TMZ (300 μmol/L). (C) The apoptosis rate was determined by flow cytometry analysis. (D) The protein level of cleaved‐PARP and cleaved‐Caspase9 was assessed by Western blotting, and β‐actin was used as control. All experiments were performed in triplicate. *P < 0.05, **P < 0.01.

Figure 6

MALAT1 knockdown suppresses the expression of GSK3β and MGMT by upregulating miR‐101. (A) The protein levels of GSK3β and MGMT were determined by Western blotting, and β‐actin was used as control. (B) The promoter methylation of MGMT was evaluated by BSP assay. All data represent the means ± SD of three replications. *P < 0.05, **P < 0.01.