Fig 1.
MiR-582-5p is down-regulated in NSCLC tissues compared with para-carcinoma tissue.
(A) 30 paired NSCLC tissues and para-carcinoma tissues were collected and the expression level of miR-582-5p in both kinds of tissues was detected by using qPCR assay. (B) The expression of miR-582-5p is markedly lower in NSCLC tissues than that in para-carcinoma tissues. (C) qPCR assay was used to determine miR-582-5p expression in the BEAS-2B immortalized human bronchial epithelial cell line and NSCLC cell lines, including A549, NCI-H23, NCI-H358, NCI-H1975 and PC-9. Data are expressed as mean ± SD. *P<0.05, **P<0.01 and ***P < 0.001 versus the normal tissues’ group or BEAS-2B group.
Fig 2.
Overexpression of miR-582-5p inhibits proliferation and invasion of NCI-H358 cell.
(A) NCI-H358 cells were transfected with miR-582-5p mimics and then miR-582-5p expression was examined with qPCR assay. (B) After transfection with miR-582-5p mimics, NCI-H358 cell proliferation was measured by using CCK-8 assay at different times (0 h, 24 h, 48 h and 72 h). (C) Western blot assay was performed to determine the expression of E-cadherin and Vimentin. (D) Relative expression of E-cadherin and Vimentin was quantified by using Image J software and GraphPad Prism 5.0. (E) Transwell assay was conducted to evaluate the invasive potential of NCI-H358 cells. (F) Relative invasive cells were estimated with Image-Pro Plus tool and GraphPad Prism 5.0. Data are showed as the mean ± SD (n = 3). *P<0.05, **P<0.01 and **P<0.001 versus the scramble group.
Fig 3.
Knockdown of miR-582-5p promotes proliferation and invasion of PC-9 cell.
(A) MiR-582-5p inhibitors were transfected into PC-9 cells and the silencing effect was assessed by using qPCR assay. (B) Cell growth rate was measured with CCK-8 assay. (C) The protein level of E-cadherin and Vimentin was detected by Western blot assay after PC-9 cells were transfected with miR-582-5p inhibitors. (D) Relative expression of E-cadherin and Vimentin was analyzed by using Image J software and GraphPad Prism 5.0. (E) The effect on PC-9 cell invasion mediated by miR-582-5p inhibitors was evaluated by Transwell assay. (F) The number of invasive cells was quantified with Image-Pro Plus and GraphPad Prism 5.0. Data are presented as the mean ± SD (n = 3). *P<0.05, **P<0.01 and **P<0.001 versus the scramble group.
Fig 4.
MiR-582-5p directly binds to NOTH1 3’UTR in NCI-H358 cell.
(A) Illustration showed the predicted binding site between miR-582-5p and WT-3'UTR-NOTCH1 or Mut-3'UTR- NOTCH1. (B) Dual luciferase reporter assay was conducted to verify the presumptive binding sites. (C) The mRNA level of NOTCH1 was determined with qPCR assay after NCI-H358 cells were transfected with miR-582-5p mimics. (D) The protein level of NOTCH1, NICD1 and Hes-1 was detected by Western blot assay after NCI-H358 cells were transfected with miR-582-5p mimics. (E) Relative expression of NOTCH1 was quantified with Image J and GraphPad Prism 5.0. Data are expressed as the mean ± SD (n = 3). **P<0.01 and ***P<0.001 versus the scramble group.
Fig 5.
NOTCH1 is up-regulated in NSCLC tissues compared with para-carcinoma tissues.
(A) The mRNA level of NOTCH1 in 30 paired NSCLC tissues and para-carcinoma tissues was detected with qPCR assay. The mRNA level of NOTCH1 is significantly higher in NSCLC tissues than that in para-carcinoma tissues. (B) The correlation between miR-582-5p and NOTCH1 is shown. (C) The mRNA expression of NOTCH1 in in the BEAS-2B immortalized human bronchial epithelial cell line and NSCLC cell lines (A549, NCI-H23, NCI-H358, NCI-H1975 and PC-9) was measured by using qPCR assay. Data are expressed as mean ± SD. *P<0.05 and ***P < 0.001 versus the normal tissues’ group or the BEAS-2B cell.
Fig 6.
Overexpression of NOTCH1 promotes cell proliferation and invasion.
(A) NCI-H358 cells were transfected with NOTCH1 plasmids and the mRNA expression of NOTCH1 was determined with qPCR assay. (B) Western blot assay was performed to detect the expression of NOTCH1 after NCI-H358 cells were transfected with NOTCH1 plasmids. (C) Relative protein expression of NOTCH1 is shown. (D) Cell proliferation was detected by CCK-8 assay after NCI-H358 cells were transfected with NOTCH1 plasmids or pCMV6 vectors. (E) The protein level of E-cadherin and Vimentin was assessed by Western blot assay. (F) Relative protein expression of E-cadherin and Vimentin is shown. (G) Transwell assay was used to determine cell invasive ability. (H) Relative number of invaded cells is shown. Data are shown as mean ± SD (n = 3). **P<0.01 and ***P<0.001 versus the pCMV6 group.
Fig 7.
Overexpression of NOTCH1 rescues the miR-582-5p-mediated inhibition in NCI-H358 cell proliferation and invasion.
(A) NCI-H358 cells were co-transfected with miR-582-5p mimics and NOTCH1 plasmids or vectors. And cell proliferation was assessed by CCK-8 assay. (B) Western blot assay was applied to examine EMT markers’ expression. (C) Relative protein expression of E-cadherin and Vimentin is shown. (D) Cell invasion was evaluated with Transwell assay. (E) Relative number of invaded cells is shown. Data are presented as mean ± SD (n = 3). *P<0.05, **P<0.01 and ***P<0.001 versus the scramble group. #P<0.05, ##P<0.01 versus the miR-582-5p mimic group.