miR‑448 promotes progression of non‑small‑cell lung cancer via targeting SIRT1

Abstract

Deregulation of microRNAs (miRs) has been demonstrated to be involved in both the initiation and the development of non-small-cell lung cancer (NSCLC). miR-448 has been identified as a tumor suppressor in several cancer types. The aim of the present study was to explore the role of miR-448 in NSCLC. Tumor tissues and paired normal tissues were obtained from patients with NSCLC. The viability and migration of A549 cells were determined by the Cell Counting kit-8 and wound-healing assays, respectively. Gene and protein levels were detected by reverse transcription-quantitative polymerase chain reaction analysis and western blotting, respectively. The interaction between the 3' untranslated region of sirtuin1 (SIRT1) and miR-448 was predicted by TargetScan and verified by dual luciferase reporter assay. miR-448 levels were revealed to be decreased whereas SIRT1 levels were increased in NSCLC tissues compared with normal tissues. Pearson's correlation analysis demonstrated that there was a negative correlation between miR-448 and SIRT1 mRNA levels. Overexpression of miR-448 led to reduced growth and migration ability of A549 cells. In addition, overexpression of miR-448 decreased SIRT1 mRNA and protein levels, thereby inhibiting epithelial-mesenchymal transition (EMT) and affecting EMT-associated molecules, including vimentin and E-cadherin. Dual luciferase reporter assay confirmed that SIRT1 was a direct target of miR-448. Notably, activation of SIRT1 by resveratrol treatment partially reversed the cell growth inhibition induced by miR-448 mimics. These findings suggested that the progression of NSCLC may be controlled by miR-448, which appears to hold promise as a therapeutic target for patients with NSCLC.

Down-Regulation of TMPO-AS1 Induces Apoptosis in Lung Carcinoma Cells by Regulating miR-143-3p/CDK1 Axis

“…The specific primers were as follows: TMPO-AS1: 5’-AGCCAGACCTCTACAATCGG-3’ (forward) and 5’-TTAGGATTCTTGCGGGTGGT-3’ (reverse); hsa-miR-143-3p: 5’-TGAGATGAAGCACTGTAGCTC-3’ (forward) and 5’-GAGCTACAGTGCTTCATCTCA-3’ (reverse); CDK1: 5’-TGGGGTCAGCTCGTTACTCA-3’ (forward) and 5’-CACTTCTGGCCACACTTCATTTA-3’ (reverse); U6: 5’-CTCGCTTCGGCAGCACA-3’ (forward) and 5’-AACGCTTCACGAATTTGCGT-3’ (reverse), GAPDH: 5’-CCTGCACCACCAACTGCTTA-3’ (forward) and 5’-GGCCATCCACAGTCTTCTGAG-3’ (reverse), 23 among which GAPDH and U6 were performed as internal reference control.…”

“…Then qRT-PCR was operated with BlazeTaq™ SYBR Green qRT-PCR Mix (BioCat, Heidelberg, Germany) in PCR machine CFX96 Touch (Bio-Rad, California, US) under following conditions: 2 minutes at 95 C, 45 cycles for 5 seconds at 94 C and 40 seconds at 60 C. Then the comparative cycle threshold (CT) method (2 ÀDDCT) was used to calculate the relative expression of each mRNA. 22 The specific primers were as follows: TMPO-AS1: 5'-AGC CAGACCTCTACAATCGG-3' (forward) and 5'-TTAG GATTCTTGCGGGTGGT-3' (reverse); hsa-miR-143-3p: 5'-TGAGATGAAGCACTGTAGCTC-3' (forward) and 5'-GAGCTACAGTGCTTCATCTCA-3' (reverse); CDK1: 5'-TGGGGTCAGCTCGTTACTCA-3' (forward) and 5'-CA CTTCTGGCCACACTTCATTTA-3' (reverse); U6: 5'-CTCG CTTCGGCAGCACA-3' (forward) and 5'-AACGCTTCACG AATTTGCGT-3' (reverse), GAPDH: 5'-CCTGCACCACC AACTGCTTA-3' (forward) and 5'-GGCCATCCACAGTC TTCTGAG-3' (reverse), 23 among which GAPDH and U6 were performed as internal reference control.…”

Construction of SIRT1 gene shRNA lentivirus vector and its effect on the proliferation of breast cancer cells

“…Qi et al confirmed that SIRT1 is the direct target of miR-448 through double luciferase reporter gene detection. Activation of SIRT1 would reverse the EMT growth inhibition ability of non-small cell lung cancer cells induced by miR-448 mimetics and play a destructive role (29). The above results showed that after lentivirus infected sh-SIRT1 interferes with SIRT1 and down-regulates expression, the expression level of adhesion molecules which aggregate among cells such as α-catenin, PTEN, and Ecadherin was up-regulated, and finally the proliferation and EMT of breast cancer cells were inhibited.…”

Long noncoding RNA HULC accelerates the growth of human liver cancer stem cells by upregulating CyclinD1 through miR675-PKM2 pathway via autophagy

“…In particular, Conditional Sirt1 deletion in the hematopoietic stem and progenitor system promotes hematopoietic stem and progenitor cell (HSPC) expansion under stress conditions [23]. Moreover, SIRT1 enhances progression and epithelial-mesenchymal transition in several cancer [24,25]. Furthermore, CyclinD1 promotes the cancer cell growth dependent on autophagy [26].…”