microRNA-877 contributes to decreased non-small cell lung cancer cell growth via the PI3K/AKT pathway by targeting tartrate resistant acid phosphatase 5 activity

Abstract

Non-small cell lung cancer (NSCLC) is a leading cause of cancer death in both men and women. microRNAs (miRs) can exert important functions in cancer development. However, the role of miR-877 in NSCLC as it relates to tartrate resistant acid phosphatase 5 (ACP5) is unknown. For this study, the gain-and-loss-of-function experiments were performed to explore the effects of miR-877 and ACP5 on NSCLC. miR-877 expression in LC and paracancerous tissues, lung epithelial cell line and NSCLC cell lines was detected, and the association between miR-877 expression and clinical features of LC patients was analyzed. The levels of ACP5, epithelial-mesenchymal transition (EMT) markers and apoptosis-related proteins were measured. In vivo experiments were conducted for further validation. Consequently, we found that miR-877 expression was lowered in LC tissues and cell lines, and correlated with clinical stage, differentiation, lymph node metastasis and prognosis of NSCLC patients. Additionally, miR-877 was determined to inhibit ACP5 activity, and miR-877 downregulated the PI3K/AKT pathway by silencing ACP5. Furthermore, overexpression of miR-877 inhibited the viability, migration, invasion and EMT of NSCLC cells, but promoted cell apoptosis. In conclusion, miR-877 overexpression inhibited malignant biological behaviors of NSCLC cells by downregulating ACP5 and inactivating the PI3K/AKT pathway.

Keywords: Non-small cell lung cancer; PI3K/AKT pathway; microRNA-877; tartrate resistant acid phosphatase 5.

Figure 1.

miR-877 is poorly expressed in LC. (a). ACP5 could bind to multiple miRs at the 3ʹUTR region; (b). miR-877 is downregulated in lung adenocarcinoma tissues, * p < 0.05, ** p < 0.01; (c). miR-877 binds to ACP5 mRNA and inhibits its activity; (d). miR-877 is downregulated in LC tissues, n = 80; (e). The miR-877 level is negatively correlated to the ACP5 level in LC patients, n = 80; (f). Relative miR-877 expression in L9981, 95 C, H1299, A549, SPC-A-1 and BEAS-2B cell lines, n = 3; All * p < 0.05. miR-877, microRNA-877; LC, lung cancer; ACP5, tartrate resistant acid phosphatase 5

Figure 2.

miR-877 expression is related to prognosis of LC patients. miR-877, microRNA-877; LC, lung cancer

Figure 3.

miR-877 overexpression decreases NSCLC cell viability. In the SPC-A-1 cells miR-877 was overexpressed and in the 95 C cells the miR-877 was inhibited. (a). Relative OD value detected using MTT assay; (b). Representative images and statistical chart of relative colony counts detected by colony formation assay; (c). Representative images and statistical chart of EdU-positive cells detected by EdU assay. The experiment was performed three times independently. The results are presented as the mean ± standard deviation. Compared to the miR-NC group, * p < 0.05, ** p < 0.01. miR-877, microRNA-877; LC, lung cancer; OD, optical density; EdU, 5-ethynyl-2ʹ-deoxyuridine; NC, negative control

Figure 4.

Overexpression of miR-877 promotes NSCLC cell apoptosis and cell cycle arrest. In the SPC-A-1 cells miR-877 was overexpressed and in the 95 C cells the miR-877 was inhibited. (a). Cell cycle distribution of SPC-A-1 cells and 95 C cells by flow cytometry; (b). Apoptosis rate of SPC-A-1 cells and 95 C cells by flow cytometry; (c). Representative images of morphological changes of apoptotic cells by Hoechst 33,258 staining; (d). Relative levels of apoptosis-related markers detected by western blot analysis. The experiment was performed three times independently. The results are presented as the mean ± standard deviation. Compared to the miR-NC group, * p < 0.05, ** p < 0.01. miR-877, microRNA-877; LC, lung cancer; NC, negative control

Figure 5.

Overexpression of miR-877 inhibits the EMT, cell migration and invasion of LC cells. In the SPC-A-1 cells miR-877 was overexpressed and in the 95 C cells the miR-877 was inhibited. (a). Relative mRNA expression of EMT markers; (b). Relative protein levels of EMT markers; (c). Relative E-cadherin positive cells and decreased vimentin in SPC-A-1 cells and 95 C cells by immunofluorescence assay; (d). Relative migration ability of SPC-A-1 cells and 95 C cells in vitro; (e). Relative invasive ability of SPC-A-1 cells and 95 C cells in vitro. The experiment was performed three times independently. The results are presented as the mean ± standard deviation. Compared to the miR-NC group, * p < 0.05, ** p < 0.01. miR-877, microRNA-877; LC, lung cancer; EMT, Epithelial-mesenchymal transition; NC, negative control

Figure 6.

miR-877 suppresses the growth, invasion and migration of NSCLC cells by decreasing ACP5. (a). Relative mRNA expression of ACP5 in SPC-A-1 cells and 95 C cells measured by qRT-PCR; (b). Relative protein level of ACP5 in SPC-A-1 cells and 95 C cells detected by western blot analysis; (c). Relative OD value detected by MTT assay; (d). Relative EdU positive ratio detected by EdU assay; (e). Relative colony formation ability of cells determined by colony formation assay; (f). Relative cell cycle distribution detected by flow cytometry; (g). Relative apoptosis rate detected using flow cytometry; H. Representative images of apoptotic cells measured by Hoechst 33,258 staining; (i). Relative mRNA expression of E-cadherin and N-cadherin measured by qRT-PCR; (j). Relative protein levels of E-cadherin and N-cadherin detected by western blot analysis; (k). Representative images of E-cadherin positive cells decreased and vimentin-positive cells obtained by immunofluorescent staining; (l). Relative migration ability of cells determined by scratch test; (m). relative invasion ability of cells determined by Transwell assay. The experiment was performed three times independently. The results are presented as the mean ± standard deviation. Compared to the miR-NC group, * p < 0.05, ** p < 0.01. miR-877, microRNA-877; LC, lung cancer; ACP5, tartrate resistant acid phosphatase 5; OD, optical density; EdU, 5-ethynyl-2ʹ-deoxyuridine; NC, negative control

Figure 7.

miR-877 inhibits NSCLC cell growth by targeting ACP5 and inactivating the PI3K/AKT pathway. The experiment was performed three times independently. The results are presented as the mean ± standard deviation. Compared to the miR-NC group, * p < 0.05, ** p < 0.01; compared to the miR-877 group, # p < 0.05. miR-877, microRNA-877; LC, lung cancer; ACP5, tartrate resistant acid phosphatase 5; PI3K, phosphatidylinositol-3 kinase; AKT, protein kinase B; NC, negative control

Figure 8.

Overexpression of miR-877 inhibits LC cell growth in vivo. (a). Relative tumor volume in each group; (b). Relative tumor weight in each group; (c). Relative positive rate of Ki67 in tumor cells by immunohistochemistry; (d). Representative images of TUNEL-positive rate in mouse tumors. Compared to the miR-NC group, * p < 0.05, ** p < 0.01, n = 5. miR-877, microRNA-877; LC, lung cancer; NC, negative control