Loss of miR‐6844 alters stemness/self‐renewal and cancer hallmark(s) markers through CD44‐JAK2‐STAT3 signaling axis in breast cancer stem‐like cells

Abstract

MicroRNAs regulate breast stemness and self‐renewal properties in breast cancer cells at the molecular level. Recently we reported the clinical relevance and in vitro expression profile of novel miR‐6844 in breast cancer and ‐derived stem‐like cells (mammosphere). In the present study, we first time explore the functional role of loss of miR‐6844 in breast cancer cells derived mammosphere. Down expression of miR‐6844 significantly decreased cell proliferation in MCF‐7 and T47D cells derived mammosphere in a time‐dependent manner. MiR‐6844 down expression reduced the sphere formation in terms of size and number in test cells. Loss of miR‐6844 significantly altered stemness and self‐renewal markers (Bmi‐1, Nanog, c‐Myc, Sox2, and CD44) in mammosphere compared to negative control spheres. Moreover, loss of miR‐6844 inhibits the JAK2‐STAT3 signaling pathway by decreasing p‐JAK2 and p‐STAT3 levels in breast cancer cells derived mammosphere. Loss of miR‐6844 expression significantly decreased CCND1 and CDK4 mRNA/protein levels and arrested breast cancer stem‐like cells in G2/M phase. Reduced expression of miR‐6844 increased Bax/Bcl‐2 ratio, late apoptotic cell population, and Caspase 9 and 3/7 activity in the mammosphere. Low expression of miR‐6844 decreased migratory and invasive cells by altering the expression of Snail, E‐cad, and Vimentin at mRNA/protein levels. In conclusion, loss of miR‐6844 decreases stemness/self‐renewal and other cancer hallmark in breast cancer stem‐like cells through CD44‐JAK2‐STAT3 axis. Thus, downregulation of miR‐6844 by therapeutic agents might be a novel strategy to target breast cancer stemness and self‐renewal.

Kurarinone targets JAK2‐STAT3 signaling in colon cancer‐stem‐like cells

“…The cell cycle analysis was performed on flow cytometer (BD Accuri C6). 5 2.8 | Annexin V-FITC/PI staining Annexin V and PI staining is mostly used for measuring cellular death through apoptosis. Annexin V detects early apoptotic cells and PI binds with DNA and it can enter into necrotic or late apoptotic cells.…”

“…After incubation (15 min) 400 μL of annexin binding buffer was added and the samples were analyzed by flow cytometer (BD Accuri C6) as per method discussed elsewhere. 5 2.9 | RNA isolation, cDNA synthesis, and quantitative RT-PCR…”

“…The mRNA expression was quantified by using QuantStudio (R) 3 Real-Time PCR machine (Applied Biosystems) as per standard protocol described elsewhere. 5 The list and sequence of primers are given in Table 1. β-actin was used as an internal control for normalization.…”

“…The JAK/STAT proteins are receptor tyrosine kinases and forms key component of the JAK/STAT pathway. 5 The intracellular signaling pathway regulates several key cellular events such as cell division, apoptosis, and cell death. 6 Constitutive activation of JAK/ STAT pathway has been associated with tumor initiation and progression by regulating the expression of genes involved in cell division and apoptosis.…”

“…6 Constitutive activation of JAK/ STAT pathway has been associated with tumor initiation and progression by regulating the expression of genes involved in cell division and apoptosis. 5,6 CCSCs have been found to overexpress JAK2 which phosphorylates STAT proteins (especially STAT3). The phosphorylated STAT3 directly increase cyclin D1 (CCND1) expression requires for active growth of the cancer stem cells.…”