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Cancer stem cells (CSCs) are a subpopulation of neoplastic cells with self-renewal capacity and limitless proliferative potential as well as high invasion and migration capacity. These cells are commonly associated with epithelial-mesenchymal transition (EMT), which is also critical for tumor metastasis. Recent studies illustrate a direct link between EMT and stemness of cancer cells. Long non-coding RNAs (lncRNAs) have emerged as important new players in the regulation of multiple cellular processes in various diseases. To date, the role of lncRNAs in EMT-associated CSC stemness acquisition and maintenance remains unclear. In this study, we discovered that a set of lncRNAs were dysregulated in Twist-positive mammosphere cells using lncRNA microarray analysis. Multiple lncRNAs-associated canonical signaling pathways were identified via bioinformatics analysis. Especially, the Shh-GLI1 pathway associated lncRNA-Hh, transcriptionally regulated by Twist, directly targets GAS1 to stimulate the activation of hedgehog signaling (Hh). The activated Hh increases GLI1 expression, and enhances the expression of SOX2 and OCT4 to play a regulatory role in CSC maintenance. Thus, the mammosphere-formation efficiency (MFE) and the self-renewal capacity in vitro, and oncogenicity in vivo in Twist-positive breast cancer cells are elevated. lncRNA-Hh silence in Twist-positive breast cells attenuates the activated Shh-GLI1 signaling and decreases the CSC-associated SOX and OCT4 levels, thus reduces the MFE and tumorigenesis of transplanted tumor. Our results reveal that lncRNAs function as an important regulator endowing Twist-induced EMT cells to gain the CSC-like stemness properties. S tem C ells 2016;34:55-66 [ABSTRACT FROM AUTHOR]

Background: Gastric cancer (GC) is a primary cause of cancer death around the world. Previous studies have found that Drosha plays a significant role in the development of tumor cells. Soon after, we unexpectedly found that the expression of microRNA6778-5p (miR6778-5p) is unconventionally high in the gastric cancer cells low-expressing Drosha. So, we designed the Drosha interference sequence and recombined it into a lentiviral vector to construct Drosha knockdown lentivirus and transfected the Drosha knockdown lentivirus into gastric cancer cells to establish Drosha knockdown gastric cancer cell lines. We aimed to explore the effect of microRNA6778-5p on the proliferation of gastric cancer cells with Drosha knockdown and its intrinsic mechanism., Methods: We designed the Drosha interference sequence and recombined it into a lentiviral vector to construct Drosha knockdown lentivirus and transfected the Drosha knockdown lentivirus into gastric cancer cells to establish Drosha knockdown gastric cancer cell lines. After transfecting miR6778-5p mimics and inhibitor into gastric cancer cell lines with Drosha knockdown, the expression levels of miR6778-5p mimics in Drosha low-expressing gastric cancer cells increased, while miR6778-5p inhibitor decreased the expression levels of miR6778-5p. The Cell Counting Kit-8 (CCK-8) experiment was used to detect the proliferation ability of gastric cancer cells after overexpression or knockdown of miR6778-5p and bioinformatics predicted the relationship between miR6778-5p and glycogen synthase kinase-3 β (GSK3 β )., Results: After infection with the Drosha knockdown lentivirus, Drosha's mRNA and protein levels were significantly downregulated in gastric cancer cells. The expression levels of miR6778-5p mimics in Drosha low-expressing gastric cancer cells increased, while miR6778-5p inhibitor decreased the expression levels of miR6778-5p. Overexpression of miR6778-5p significantly enhanced the proliferation ability of Drosha low-expression gastric cancer cells; on the contrary, knocking down miR6778-5p weakened the proliferation ability of Drosha low-expression gastric cancer cells. Bioinformatics predicted that miR6778-5p targeted glycogen synthase kinase-3 β (GSK3 β ) and the mRNA and protein levels of GSK3 β decreased significantly after overexpression of miR6778-5p., Conclusion: miR6778-5p promotes the proliferation of Drosha low-expressing gastric cancer cells by targeting GSK3 β ., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 Mingjun Ren et al.)

Objective: To investigate the effect of Twist gene deficiency on the enrichment and migration of breast cancer stem-like cells (CSCs)., Methods: Twist gene in the BT-549 cell line was knocked down by shRNA interference technology, and the cell line BT-549-shVec was used as the negative control group. Interference efficiency was determined by real-time quantitative RT-PCR (qRT-PCR) and Western blotting. Mammospheres with the characteristics of stem-like cells were collected by long-term serum-free suspension cultivation. The migration ability of mammospheres was analyzed by Transwell(TM) assay., Results: The BT-549-shTwist breast cancer cells were successfully established by shRNA interference technology. The enrichment and migration abilities of CSCs from the BT-549-shTwist cell line decreased significantly., Conclusion: Twist gene promotes the enrichment and migration of CSCs from the BT-549 cell line.

Objective: To study the application of different metabolic activation systems in benzo(a)pyrene [B(a)P]-induced human bronchial epithelium cell HBETR transformation., Methods: In vitro metabolic activations of B(a)P were compared with rat liver S9 fraction mix, overexpression of a key enzyme (P450 CYP1A1), and prior low dose B(a)P (1 micromol/L) induction. Using soft agar assay and tumorigenicity assay, the different metabolic activation systems were compared to the influence on transformation of human bronchial epithelium cell HBETR., Results: Both immunoblotting and enzyme activity showed that cells overexpressing CYP1A1 (HBETR-1A1) and 48 h after low dose B(a)P induction (HBETR-IN) had high-level expression of CYP1A1. There were no obvious changes in the biology characteristic of these cells. The latencies of cell transformation in HBETR-1A1 and HBETR-IN cells were 11 weeks when cells were treated with B(a)P at concentration of 20 micromol/L, while it took 14 weeks to achieve cell transformation in their control cells. The latencies of malignant transformation in HBETR cells in presence or absence of S9-mix were 14 weeks and 20 weeks, respectively. The efficiencies of cell transformation were in consonance with the protein level of endogenous CYP1A1 enzyme and its enzyme activity., Conclusion: The three metabolic conditions of the addition of rat liver S9 fraction mix, overexpression of a key enzyme (CYP1A1), and low dose B(a) P induction could enhance the B(a)P metabolic activation and shorten the latency of malignant transformation. In terms of the feasibility, difficulty of manipulation, stability, and reliability, low dose B(a)P induction could seem to be a prospective system used in metabolic activation in comparison with rat liver S9 fraction mix addition.