Raphael et?al

Raphael et?al.48 found aberrations in the PI3K/AKT/mTOR pathway to be the most common genomic abnormalities in many kinds of breast cancer. target of miR-30a/c and miR-30a/c inhibits the stemness and proliferation of NSCLC cells by targeting TM4SF1, suggesting that miR-30a/c and TM4SF1 may be useful as tumor biomarkers for the diagnosis and treatment of NSCLC patients. by Targeting TM4SF1 To further investigate whether and how miR-30c/a affects lung cancer formation by targeting TM4SF1. Next, we observed staining of CSC surface markers CD326 and CD133 of 4 group tumor samples under a fluorescent microscope (Figure?7C). We quantitatively analyzed relative fluorescence intensity of the 4 samples. Figures 7D and 7E show that CD326 and CD133 expression was upregulated in NSCLC tissues compared with paracarcinoma tissues, TM4SF1 could promote CSC surface marker expression, and miR-30c could inhibit CSC surface marker expression by targeting TM4SF1. The apoptosis assay in Figure?7F showed that the rate of apoptosis was lower in NSCLC tissues compared with paracarcinoma tissues, TM4SF1 could inhibit cell apoptosis, and miR-30c could promote cell apoptosis by targeting TM4SF. We also investigated how TM4SF1 and miR-30c affect apoptotic signal molecules cleaved-caspase-3 by western blot. The results showed that miR-30c can promote cell apoptosis by targeting TM4SF (Figure?7G). Next, we performed western blot analysis to determine whether miR-30a/c and TM4SF1 affect the activity of the mTOR/AKT-signaling pathway. The results showed that miR-30a/c inhibited the activity of the mTOR/AKT-signaling pathway (Figure?7H). Open in a separate window Figure?7 miR-30c/a Inhibit Tumor Growth by Targeting TM4SF1 (A) Tumor volume curves of the control group, miR-30c group, TM4SF1 group, and miR-30c?+ TM4SF1 group. (B) Tumor volume curves of the control group, miR-30a group, TM4SF1 group, and miR-30a?+ TM4SF1 group. (C) Staining of cancer stem cell surface markers CD326 and CD133. (D) Quantitative analysis of relative fluorescence Rabbit Polyclonal to p50 Dynamitin intensity of the para group, control group, miR-30c group, TM4SF1 group, and miR-30c?+ TM4SF1 group in SPC-A1 cells. Green, CD326; red, CD133. CGP-42112 (E) Quantitative analysis of relative fluorescence intensity of the para group, control group, miR-30c group, TM4SF1 group, and miR-30c?+ TM4SF1 group in HCI-H1650 cells. Green, CD326; red, CD133. (F) Apoptosis assay of control group, miR-30c group, TM4SF1 group, and miR-30c?+ TM4SF1 group in HCI-H1650 cells. (G) Influence of miR-30c and TM4SF1 expression on apoptotic signal molecules cleaved-caspase-3 expression by western blot. (H) Influence of miR-30c and TM4SF1 expression on AKT/mTOR pathway-associated protein expressions by western blot. Data are shown as the means? SDs of three independent experiments. Statistical analyses were performed with one-way ANOVA (*P<0.05 and **P<0.01 vs. control). miR-30c/a and TM4SF1 Expression in NSCLC Tissue To further investigate miR-30c/a expression level in NSCLC tissue, we performed qRT-PCR in 36 paired NSCLC tissues and 124 normal lung tissues. The results showed that, when compared with normal tissues, miR-30c/a was significantly downregulated in NSCLC (Figures 8A and 8B). Immunohistochemistry (IHC) staining and qRT-PCR analysis showed that TM4SF1 was significantly upregulated in NSCLC (Figures 8CC8E). Next, correction analysis showed a significant CGP-42112 negative correlation between miR-30c/a and TM4SF1 expression (Figures 8F and 8G). Open in a separate window Figure?8 miR-30c/a and TM4SF1 Expression in NSCLC Tissue (A) miR-30c expression by qRT-PCR in 36 paired NSCLC tissues and 124 normal lung tissues. (B) miR-30a expression by qRT-PCR in 36 paired NSCLC tissues and 124 normal lung tissues. (C) IHC staining of TM4SF1 expression in normal and NSCLC tissues. (D) qRT-PCR analysis CGP-42112 of TM4SF1 expression in normal and CGP-42112 NSCLC tissues. (E)?Correlation analysis. (F) Correlation analysis of miR-30c and TM4SF1 expression. (G) Correlation analysis of miR-30a and TM4SF1 expression. Clinical Significance of miR-30c/a and TM4SF1 in NSCLC Kaplan-Meier survival curves were plotted and log rank analysis was? performed to evaluate the prognostic value of miR-30c/a and TM4SF1 in.