miR‑21‑5p confers doxorubicin resistance in gastric cancer cells by targeting PTEN and TIMP3

Abstract

Drug resistance and disease recurrence are major obstacles to the effective treatment of cancer, including gastric cancer (GC). However, the mechanisms of drug resistance remain to be fully elucidated. The present study investigated the roles of microRNA (miR)‑21‑5p in the doxorubicin (DOX) resistance of GC cells and the underlying mechanisms. miR‑21‑5p expression levels were identified to be inversely correlated with two well‑known tumor suppressor genes, phosphatase and tensin homologue and tissue inhibitor of matrix metalloproteinases 3, and were upregulated in GC cell lines in proportion to their degree of resistance. Suppressing miR‑21‑5p expression partially sensitized SGC7901/DOX cells to DOX, suggesting that knockdown of miR‑21‑5p expression may be used as a therapeutic strategy to improve GC cell resistance. Importantly, increased miR‑21‑5p expression levels at diagnosis were correlated with clinicopathological characteristics including advanced stage and poor prognosis, further implying that a relapse of GC may be a consequence of miR‑21‑5p upregulation, thus providing evidence for the potential utility of miR‑21‑5p antagonism to sensitize GC cells to DOX chemotherapy.

Figure 1

Differential expression of miR-21-5p in GC patients. (A) Relative expression of miR-21-5p (normalized to U6) in GC and matched adjacent NC tissues was measured by reverse transcription quantitative-polymerase chain reaction analysis. (B) Primary GC samples (n=30) were divided according to clinical outcomes. Patients who developed distant recurrence or succumbed had significantly higher miR-21-5p expression than those who had CR after treatment. CR, complete remission; miR, microRNA; NC, normal control; GC, gastric cancer.

Figure 2

Expression levels of miR-21-5p in SGC7901 and SGC7901/DOX cells. (A) Native SGC7901 cells those with acquired resistance to three different concentrations of DOX were incubated with various doses of DOX for 48 h, and the cell viability was determined using a cell counting kit-8 assay. (B) The IC₅₀ values of SGC7901 and three DOX-resistant cell lines were calculated. (C) Reverse transcription quantitative-polymerase chain reaction analysis indicated that miR-21-5p was significantly upregulated in SGC7901/DOX cells compared with those in SGC7901 cells. ^**P<0.01 and ^***P<0.001. DOX, doxorubicin; SGC7901/DOX, DOX-resistant SGC7901 cell line; IC₅₀, concentration causing 50% growth inhibition; miR, microRNA.

Figure 3

Effect of miR-21-5p antagomir on DOX sensitivity of SGC7901/DOX-2 μg/ml cells. (A) Transfection of anti-miR-21 decreased miR-21-5p expression levels in SGC7901/DOX-2 μg/ml cells to 44.8% of those in the anti-miR-NC group. (B) After transfection of SGC7901/DOX cells with anti-miR-21 or anti-miR-NC for 24 h and treatment with various doses of DOX for 48 h, the cell viability was assessed. (C) Flow cytometric analysis determined the influence of miR-21-5p on the apoptosis of SGC7901/DOX-2 μg/ml cells following treatment with DOX (1 μg/ml) for 48 h. The proportions of dead cells (Q1: Annexin V-FITC⁻/PI⁺), late apoptotic or necrotic cells (Q2: Annexin V-FITC⁺/PI⁺), early apoptotic cells (Q3: Annexin V-FITC⁺/PI⁻) and live cells (Q4: Annexin V-FITC⁻/PI⁻) are displayed. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. anti-miR-NC. miR, microRNA; FITC, fluorescein isothiocyanate; PI, propidium iodide; DOX, doxorubicin; SGC7901/DOX, DOX-resistant SGC7901 cell line; NC, negative control; Q, quadrant.

Figure 4

Overexpression of miR-21 increases DOX resistance of SGC7901 cells. (A) A reverse transcription quantitative-polymerase chain reaction assay was performed to examine miR-21-5p expression levels after transfection of its mimics (or miR-NC). (B) A cell counting kit-8 assay was used to determine the viability of SGC7901 cells after transfection with miR-21 mimics (or miR-NC) following exposure to various doses of DOX for 48 h. (C) Apoptotic rates of SGC7901 cells were analyzed to determine the role of miR-21-5p on the effects of DOX treatment (1 μg/ml) for 48 h. Late apoptotic or necrotic cells and early apoptotic cells are displayed in Q2 and Q3, respectively. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. miR-NC. miR, microRNA; FITC, fluorescein isothiocyanate; DOX, doxorubicin; NC, negative control; Q, quadrant.

Figure 5

PTEN and TIMP3 are targets of miR-21-5p. (A) The 3′-UTRs of PTEN and TIMP3 were identified as putative targets of miR-21-5p with 7-mer or 8-mer complementarity regions. Seven nucleotides in the miR-21-5p seed region (gray font) were mutated using polymerase chain reaction site-directed mutagenesis. (B) A dual-luciferase reporter assay was performed to assess the influence of miR-21-5p on luciferase intensity controlled by the 3′-UTR of potential targets. (C and D) Western blot analysis indicated a decrease or increase in PTEN and TIMP3 protein levels following transfection of miR-21 mimics or anti-miR-21, respectively. Western blot images are representative of at least three independent experiments. The value under each sample indicates the fold change of PTEN or TIMP3 protein levels relative to the control. ^*P<0.05 vs. miR-NC. PTEN, phosphatase and tensin homologue; TIMP3, tissue inhibitor of matrix metalloproteinases 3; UTR, untranslated region; miR, microRNA; NC, negative control; Hsa, Homo sapiens; MUT, mutant; SGC7901/DOX, doxorubicin-resistant SGC7901 cell line.

Figure 6

PTEN and TIMP3 have a key role in the chemoresistance of SGC7901 cells. (A and B) mRNA levels of PTEN and TIMP3 in SGC7901 cells were reduced after transfection with specific siRNA. (C and D) The protein expression of PTEN and TIMP3 in SGC7901 cells was reduced after transfection with specific siRNA. (E) A cell counting kit-8 assay was performed to detect the effect of siRNA-mediated PTEN and TIMP3 knockdown on the viability of SGC7901 cells. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. miR-NC. PTEN, phosphatase and tensin homologue; TIMP3, tissue inhibitor of matrix metalloprotein-ases 3; siRNA, small interfering RNA; miR, microRNA; NC, negative control; DOX, doxorubicin.

Figure 7

PTEN and TIMP3 are downregulated in GC tumor tissues, which negatively correlates with miR-21-5p levels. (A and B) A reverse transcription quantitative-polymerase chain reaction assay was used to measure the expression levels of PTEN and TIMP3 in GC samples and matched adjacent normal tissues. (C) A statistically significant negative correlation between the expression levels of miR-21-5p and PTEN was observed in GC samples (r=−0.4175, P=0.0039). (D) A statistically significant negative correlation between the expression levels of miR-21-5p and TIMP3 was observed in GC samples (r=−0.3263, P=0.004). GC, gastric cancer; PTEN, phosphatase and tensin homologue; TIMP3, tissue inhibitor of matrix metalloproteinases 3; miR, microRNA.