KLF10 inhibits cell growth by regulating PTTG1 in multiple myeloma under the regulation of microRNA-106b-5p

Abstract

Krüppel-like factor 10 (KLF10) has been identified as an important regulator in carcinogenesis and cancer progression. However, the role of KLF10 in multiply myeloma (MM) development and progression remains unknown. In present study, we found that KLF10 mRNA and protein were down-regulated in MM tissues and cell lines. Notably, KLF10 inhibited cell proliferation, cell cycle progression and promoted apoptosis in vitro and in vivo. Furthermore, we confirmed that KLF10 inhibited β-catenin nuclear translocation and inhibited PTTG1 transcription. PTTG1 knockdown could mimic the biological effects of KLF10. Moreover, we demonstrated that KLF10 expression was regulated by miR-106b-5p. In MM tissues, miR-106b-5p has an inverse correlation with KLF10 expression. Conclusively, our results demonstrated that KLF10 functions as a tumor suppressor in regulating tumor growth of MM under regulation of miR-106b-5p, supporting its potential therapeutic target for MM.

Keywords: KLF10; PTTG1; miR-106b-5p; multiply myeloma; proliferation.

Figure 1

KLF10 is significantly down-regulated in MM tissues and cell lines. (A) Relative KLF10 mRNA expression levels in MM and healthy donors were determined by qRT-PCR. (B) Representative Western blot analysis of KLF10 expression in the MM and healthy donors was shown. (C) KLF10 level was compared between MM tissues of different ISS stage. The expression of KLF10 mRNA (D) and protein (E) in three MM cell lines was significantly decreased compared to that in the nPCs cells. n = three repeats with similar results. *P < 0.05 by ANOVA. **P < 0.01.

Figure 2

KLF10 inhibits cell proliferation, cell-cycle progression and promotes apoptosis in MM cell. (A) RPMI8226 and U266 cells that were transduced with corresponding KLF10 overexpression vectors were subjected to WB for KLF10. (B) Flow cytometry checked the effects of KLF10 up-regulation on apoptosis. (C) KLF10 overexpression inhibited cell proliferation in RPMI8226 and U266 cells. (D) Effects of KLF10 overexpression on the cell cycle progression of MM cells were measured by flow cytometric analysis. (E) WB measured the cycle- and apoptosis-associated factors. n = six independent experiments. *P<0.05.

Figure 3

KLF10 inhibits tumor growth and promotes apoptosis in vivo. (A) Representative pictures of MM xenografts from RPMI8226-KLF10 and RPMI8226-control. (B) Tumor growth curve revealed that KLF10 overexpression significantly inhibited tumor growth in vivo. Tumor nodules were subjected to immunohistochemical staining for Ki-67 (C) and TUNEL (D) assays and quantitative analysis. Representative immunostaining and TUNEL assays revealed that KLF10 overexpression significantly decreased the number of Ki-67 positive cells and increased the number of apoptotic cells. *P<0.05.

Figure 4

KLF10 inhibits activation of Wnt signaling. (A) WB showed that KLF10 overexpression suppressed β-catenin nuclear accumulation in RPMI8226 and U266 cells. (B) WB revealed that KLF10 overexpression suppressed GSK3β phosphorylation in MM cells. n = six independent experiments. *P<0.05, **P<0.01.

Figure 5

KLF10 inhibited PTTG1 transcription in MM cells. (A) Relative PTTG1 mRNA expression levels in MM and healthy donors were determined by qRT-PCR. (B) Representative Western blot analysis of PTTG1 expression in the MM and healthy donors was shown. (C) A significant inverse correlation between the KLF10 and PTTG1 was observed in MM tissues. KLF10 overexpression inhibited PTTG1 mRNA (D) and protein (E) expression in MM cells. (F) Luciferase reporter assays confirmed that KLF10 inhibited PTTG1 transcription in HEK293 cells. n = three repeats with similar results. *P<0.05, n.s: no significance.

Figure 6

Inhibition of PTTG1 mimics KLF10-induced biological effects on MM. (A) WB showed the effects of PTTG1 siRNA to knockdown PTTG1. PTTG1 knockdown promoted apoptosis (B) and inhibited cell proliferation (C) and cell cycle progression (D). (E) WB measured the cycle- and apoptosis-associated factors. (F) WB showed that PTTG1 knockdown suppressed β-catenin nuclear accumulation in MM cells. (G) WB revealed that PTTG1 knockdown suppressed GSK3β phosphorylation in MM cells. n = six repeats with similar results. *P<0.05, n.s=no significance.

Figure 7

KLF10 is identified as a direct target of miR-106b-5p in MM. (A) miR-106b-5p and its putative binding sequence in the 3'-UTR of KLF10. The mutant binding site was generated in the complementary site for the seed region of miR-106b-5p. (B) miR-106b-5p was up-regulated in MM tissues compared to healthy donors. (C) miR-106-5p was knockdown by the miR-106b-5p inhibitors and measured by qRT-PCR. (D) qRT-PCR analysis of KLF10 mRNA expression in MM cells with anti-miR-106b-5p or anti-miR-NC vector transfection. (E) miR-106-5p knockdown increased the expression of KLF10 protein in MM cells. (F) miR-106b-5p significantly suppresses the luciferase activity that carried wild-type (wt) but not mutant (mt) 3'-UTR of KLF10. (G) A significant inverse correlation between the mRNA levels of KLF10 and miR-106b-5p was observed in MM tissues. n = six repeats with similar results. *P<0.05, n.s=no significance.