microRNA-103 regulates the growth and invasion of endometrial cancer cells through the downregulation of tissue inhibitor of metalloproteinase 3

Abstract

Despite improvements in treatment over the past few decades, endometrial cancer remains one of the most common causes of mortality in women and there is an urgent need for the development of targeted therapies. The aim of this study was to confirm the target gene of miR-103 in human endometrial cancer and investigate the biological functions in which miR-103 is involved through the regulation of the expression of its target gene. This study may provide useful data to gain a better understanding of the effect of miR-103 in tumor formation. miR-103 expression levels were measured using real-time quantitative PCR. The effect of miR-103 on tissue inhibitor of metalloproteinase 3 (TIMP-3) expression was assessed in endometrial cancer cell lines with a miR-103 inhibitor to decrease the level of miR-103 expression. Furthermore, the roles of miR-103 in cell growth and invasion were analyzed using miR-103 inhibitor-transfected cells. The level of expression of miR-103 decreased following transfection with the miR-103 inhibitor. miR-103 inhibitor transfection increased the activity of the luciferase reporter assay containing the TIMP-3 3'-untranslated region (UTR) construct and increased the levels of the TIMP-3 protein but not its mRNA in endometrial cancer cell lines. Finally, miR-103 inhibitor-transfected cells exhibited reduced cell growth and invasive characteristics. Our data suggested that miR-103 post-transcriptionally downregulates the expression of the tumor suppressor TIMP-3 and stimulates growth and invasion in endometrial cancer cell lines. This provides a possible therapeutic target that may upregulate TIMP-3 in endometrial cancer.

Figure 1

TIMP-3 3’-UTR is a direct target for miR-103. (A) Suppression of miR-103 by anti-miR-103 in Ishikawa cells is shown. The cells were frst transfected with anti-miR-103 or negative control oligonucleotide. The cells were harvested 2 days later and total RNA was extracted using TRIzol reagent. Mature miR-103 was detected by SYBR qRT-PCR, using U6 RNA for normalization. (B) Suppression of miR-103 by anti-miR-103 in HEC-1B cells is shown. Following transfection with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control), mature miR-103 was detected as in (A). (C) An evolutionarily conserved target sequence for miR-103 is detected in the 3’-UTR of TIMP-3. (D and E) The luciferase assay was performed using Ishikawa and HEC-1B cells, respectively. The effect of miR-103 on the luciferase activity of pGL3-TIMP-3-wt and pGL3-TIMP-3-mut was measured as described in Materials and methods. A statistically significant upregulation in luciferase activity was found when the cells were transfected with pGL3-TIMP-3-wt together with anti-miR-103. pGL3-TIMP-3-wt, vector with wild-type TIMP-3 3’-UTR; pGL3-TIMP-3-mut, vector with mutated TIMP-3 3’-UTR. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05. qRT-PCR, quantitative reverse transcription PCR; UTR, untranslated region.

Figure 1

TIMP-3 3’-UTR is a direct target for miR-103. (A) Suppression of miR-103 by anti-miR-103 in Ishikawa cells is shown. The cells were frst transfected with anti-miR-103 or negative control oligonucleotide. The cells were harvested 2 days later and total RNA was extracted using TRIzol reagent. Mature miR-103 was detected by SYBR qRT-PCR, using U6 RNA for normalization. (B) Suppression of miR-103 by anti-miR-103 in HEC-1B cells is shown. Following transfection with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control), mature miR-103 was detected as in (A). (C) An evolutionarily conserved target sequence for miR-103 is detected in the 3’-UTR of TIMP-3. (D and E) The luciferase assay was performed using Ishikawa and HEC-1B cells, respectively. The effect of miR-103 on the luciferase activity of pGL3-TIMP-3-wt and pGL3-TIMP-3-mut was measured as described in Materials and methods. A statistically significant upregulation in luciferase activity was found when the cells were transfected with pGL3-TIMP-3-wt together with anti-miR-103. pGL3-TIMP-3-wt, vector with wild-type TIMP-3 3’-UTR; pGL3-TIMP-3-mut, vector with mutated TIMP-3 3’-UTR. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05. qRT-PCR, quantitative reverse transcription PCR; UTR, untranslated region.

Figure 1

TIMP-3 3’-UTR is a direct target for miR-103. (A) Suppression of miR-103 by anti-miR-103 in Ishikawa cells is shown. The cells were frst transfected with anti-miR-103 or negative control oligonucleotide. The cells were harvested 2 days later and total RNA was extracted using TRIzol reagent. Mature miR-103 was detected by SYBR qRT-PCR, using U6 RNA for normalization. (B) Suppression of miR-103 by anti-miR-103 in HEC-1B cells is shown. Following transfection with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control), mature miR-103 was detected as in (A). (C) An evolutionarily conserved target sequence for miR-103 is detected in the 3’-UTR of TIMP-3. (D and E) The luciferase assay was performed using Ishikawa and HEC-1B cells, respectively. The effect of miR-103 on the luciferase activity of pGL3-TIMP-3-wt and pGL3-TIMP-3-mut was measured as described in Materials and methods. A statistically significant upregulation in luciferase activity was found when the cells were transfected with pGL3-TIMP-3-wt together with anti-miR-103. pGL3-TIMP-3-wt, vector with wild-type TIMP-3 3’-UTR; pGL3-TIMP-3-mut, vector with mutated TIMP-3 3’-UTR. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05. qRT-PCR, quantitative reverse transcription PCR; UTR, untranslated region.

Figure 1

TIMP-3 3’-UTR is a direct target for miR-103. (A) Suppression of miR-103 by anti-miR-103 in Ishikawa cells is shown. The cells were frst transfected with anti-miR-103 or negative control oligonucleotide. The cells were harvested 2 days later and total RNA was extracted using TRIzol reagent. Mature miR-103 was detected by SYBR qRT-PCR, using U6 RNA for normalization. (B) Suppression of miR-103 by anti-miR-103 in HEC-1B cells is shown. Following transfection with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control), mature miR-103 was detected as in (A). (C) An evolutionarily conserved target sequence for miR-103 is detected in the 3’-UTR of TIMP-3. (D and E) The luciferase assay was performed using Ishikawa and HEC-1B cells, respectively. The effect of miR-103 on the luciferase activity of pGL3-TIMP-3-wt and pGL3-TIMP-3-mut was measured as described in Materials and methods. A statistically significant upregulation in luciferase activity was found when the cells were transfected with pGL3-TIMP-3-wt together with anti-miR-103. pGL3-TIMP-3-wt, vector with wild-type TIMP-3 3’-UTR; pGL3-TIMP-3-mut, vector with mutated TIMP-3 3’-UTR. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05. qRT-PCR, quantitative reverse transcription PCR; UTR, untranslated region.

Figure 1

TIMP-3 3’-UTR is a direct target for miR-103. (A) Suppression of miR-103 by anti-miR-103 in Ishikawa cells is shown. The cells were frst transfected with anti-miR-103 or negative control oligonucleotide. The cells were harvested 2 days later and total RNA was extracted using TRIzol reagent. Mature miR-103 was detected by SYBR qRT-PCR, using U6 RNA for normalization. (B) Suppression of miR-103 by anti-miR-103 in HEC-1B cells is shown. Following transfection with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control), mature miR-103 was detected as in (A). (C) An evolutionarily conserved target sequence for miR-103 is detected in the 3’-UTR of TIMP-3. (D and E) The luciferase assay was performed using Ishikawa and HEC-1B cells, respectively. The effect of miR-103 on the luciferase activity of pGL3-TIMP-3-wt and pGL3-TIMP-3-mut was measured as described in Materials and methods. A statistically significant upregulation in luciferase activity was found when the cells were transfected with pGL3-TIMP-3-wt together with anti-miR-103. pGL3-TIMP-3-wt, vector with wild-type TIMP-3 3’-UTR; pGL3-TIMP-3-mut, vector with mutated TIMP-3 3’-UTR. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05. qRT-PCR, quantitative reverse transcription PCR; UTR, untranslated region.

Figure 2

Inhibition of miR-103 upregulates TIMP-3 protein expression in endometrial cancer cells. Ishikawa and HEC-1B cells were transfected with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control) and the TIMP-3 protein level was detected by western blot analysis. The GAPDH protein was regarded as an endogenous normalizer. (A and C) Inhibition of miR-103 upregulates the endogenous TIMP-3 protein expression in Ishikawa and HEC-1B cells. (B and D) A significant increase in TIMP-3 activity was detected in Ishikawa and HEC-1B cells transfected with anti-miR-103, compared with the other two groups. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05.

Figure 2

Inhibition of miR-103 upregulates TIMP-3 protein expression in endometrial cancer cells. Ishikawa and HEC-1B cells were transfected with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control) and the TIMP-3 protein level was detected by western blot analysis. The GAPDH protein was regarded as an endogenous normalizer. (A and C) Inhibition of miR-103 upregulates the endogenous TIMP-3 protein expression in Ishikawa and HEC-1B cells. (B and D) A significant increase in TIMP-3 activity was detected in Ishikawa and HEC-1B cells transfected with anti-miR-103, compared with the other two groups. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05.

Figure 2

Inhibition of miR-103 upregulates TIMP-3 protein expression in endometrial cancer cells. Ishikawa and HEC-1B cells were transfected with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control) and the TIMP-3 protein level was detected by western blot analysis. The GAPDH protein was regarded as an endogenous normalizer. (A and C) Inhibition of miR-103 upregulates the endogenous TIMP-3 protein expression in Ishikawa and HEC-1B cells. (B and D) A significant increase in TIMP-3 activity was detected in Ishikawa and HEC-1B cells transfected with anti-miR-103, compared with the other two groups. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05.

Figure 2

Inhibition of miR-103 upregulates TIMP-3 protein expression in endometrial cancer cells. Ishikawa and HEC-1B cells were transfected with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control) and the TIMP-3 protein level was detected by western blot analysis. The GAPDH protein was regarded as an endogenous normalizer. (A and C) Inhibition of miR-103 upregulates the endogenous TIMP-3 protein expression in Ishikawa and HEC-1B cells. (B and D) A significant increase in TIMP-3 activity was detected in Ishikawa and HEC-1B cells transfected with anti-miR-103, compared with the other two groups. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05.

Figure 3

miR-103 affects endometrial cancer cell growth and invasion in vitro. (A) MTT assay was performed. The cells in the anti-miR-103 group showed slow proliferation compared with the untreated and negative control groups. The Ishikawa and HEC-1B cells were first transfected with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control) and then subjected to Matrigel chamber assays, as described in Materials and methods. After 48 h, the invasive cells were counted following staining with crystal violet. (B) Representative fields of invasive cells on the membrane. (C) The data are standardized against the negative control and presented as relative cell invasion numbers. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05.

Figure 3

miR-103 affects endometrial cancer cell growth and invasion in vitro. (A) MTT assay was performed. The cells in the anti-miR-103 group showed slow proliferation compared with the untreated and negative control groups. The Ishikawa and HEC-1B cells were first transfected with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control) and then subjected to Matrigel chamber assays, as described in Materials and methods. After 48 h, the invasive cells were counted following staining with crystal violet. (B) Representative fields of invasive cells on the membrane. (C) The data are standardized against the negative control and presented as relative cell invasion numbers. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05.

Figure 3

miR-103 affects endometrial cancer cell growth and invasion in vitro. (A) MTT assay was performed. The cells in the anti-miR-103 group showed slow proliferation compared with the untreated and negative control groups. The Ishikawa and HEC-1B cells were first transfected with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control) and then subjected to Matrigel chamber assays, as described in Materials and methods. After 48 h, the invasive cells were counted following staining with crystal violet. (B) Representative fields of invasive cells on the membrane. (C) The data are standardized against the negative control and presented as relative cell invasion numbers. Each bar is the mean ± SEM from three independent experiments. ^*P<0.05.