MicroRNA-31 controls G protein alpha-13 (GNA13) expression and cell invasion in breast cancer cells
Suhail Ahmed Kabeer RasheedCui Rong TeoEmmanuel BeillardP. Mathijs VoorhoeveWei ZhouSujoy GhoshPatrick J. Casey
75
Citation
48
Reference
10
Related Paper
Citation Trend
Abstract:
Gα13 (GNA13) is the α subunit of a heterotrimeric G protein that mediates signaling through specific G protein-coupled receptors (GPCRs). Our recent study showed that control of GNA13 expression by specific microRNAs (miRNAs or miRs) is important for prostate cancer cell invasion. However, little is known about the control of GNA13 expression in breast cancers. This project was carried out to determine (i) whether enhanced GNA13 expression is important for breast cancer cell invasion, and (ii) if so, the mechanism of deregulation of GNA13 expression in breast cancers.To determine the probable miRNAs regulating GNA13, online miRNA target prediction tool Targetscan and Luciferase assays with GNA13-3'-UTR were used. Effect of miRNAs on GNA13 mRNA, protein and invasion was studied using RT-PCR, western blotting and in vitro Boyden chamber assay respectively. Cell proliferation was done using MTT assays.Overexpression of GNA13 in MCF-10a cells induced invasion, whereas knockdown of GNA13 expression in MDA-MB-231 cells inhibited invasion. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-31 exhibited an inverse correlation to GNA13 protein expression in breast cancer cells. Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3'-UTR-reporter activity. Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3'-UTR reporter activity. Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells. Examination of 48 human breast cancer tissues revealed that GNA13 mRNA levels were inversely correlated to miR-31 levels.These data provide strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms involving miR-31. Additionally our data shows that miR-31 regulates breast cancer cell invasion partially via targeting GNA13 expression in breast cancer cells. Loss of miR-31 expression and increased GNA13 expression could be used as biomarkers of breast cancer progression.Keywords:
Ectopic expression
HEK 293 cells
Various human host cell lines, which are more effective than the other original human cell lines, have been developed and used. Highly efficient human cell line can be obtained from the fusion between human embryonic kidney 293 (HEK293) and human Burkitt's lymphoma cells (Namalwa). Fused cell line has the advantages of both cell lines such as the high transfection efficacy of HEK293 cells and the constitutive expression of Epstein-Barr virus (EBV) genome which is related with high expression of target protein and anti-apoptotic growth of Namalwa cells. In this study, characterization of two original cell lines was performed by using design of experiment (DOE) considering cell maintenance, media development, optimization of culture condition, and scale-up. The formation of aggregates was apparent with high glutamine concentration at more than 6 mM. Supplementation of hydrolysates showed positive effects on the growth performances of HEK293 cells. On the contrary, Namalwa cells showed negative results. It was confirmed that Namalwa cells were more sensitive to lower temperature at $35^{\circ}C$ and hyperosmotic condition over 260 mOsm/kg. In addition, both cell lines showed limited growth in 3-L bioreactor due to shear stress.
HEK 293 cells
Growth inhibition
Cite
Citations (8)
Cite
Citations (22)
<p>Chemoresistance properties of MUC16 and effect of cisplatin on apoptosis of MUC16 knockdown cells. A & B, MUC16 knockdown (H1975-shMUC16 seq1 and 2) cells were highly sensitive to cisplatin (A) and gemcitabine (B). C, The percentage of apoptotic cells was significantly higher in MUC16 knockdown cells (H292-shMUC16) treated with 5μM cisplatin. In contrast, no significant change was observed in the untreated scramble (H292-SCR) and MUC16 knockdown cells. D, We performed stable knockdown of Muc16 in K1418, the result shows that Muc16 is significantly decreased as compared to scramble cells. E, The p53 target gene p21 was significantly increased in MUC16 knockdown (H292-shMUC16) cells. *P<0.05, **P<0.01, ***P<0.001, and NS non-significant.</p>
Cite
Citations (0)
RNA-mediated interference (RNAi) has become a promising biopesticide technology with which to direct sequence-specific gene knockdown of key targets in the potato psyllid (PoP) Bactericera cockerelli, resulting in significant mortality. In this study, three strategically selected target genes, ATF4, C7 and D24, essential for the biosynthesis and regulation of ecdysteroids, were evaluated for knockdown and mortality using oral delivery of individual, paired and all three double-stranded RNAs (dsRNAs), in five replicated experiments. Knockdown was determined as the fold-change in gene expression using a quantitative polymerase chain reaction.Knockdown of the D24 target, at 39%-45%, resulted in 51% PoP mortality by 10 days post-ingestion (dpi) of dsRNA. Knockdown of C7, at 38%-61%, resulted in 53% mortality by 10 dpi, whereas dsD24 ingestion resulted in 65% mortality by 10 dpi when dsD24 and dsC7 were co-delivered. Three phenotypes, INCOMEC, PREMEC and SWOLLEN, were observed at a frequency of 4%-12%, and are consistent with incomplete ecdysis in immature and/or adult PoP. Adult PoP exhibiting INCOMEC survived for several days but were unable to mate or fly, whereas SWOLLEN and PREMEC were lethal to the immature instars. Knockdown of ATF4 did not result in the mortality or malformations in immature and adult PoP.Compared with knockdown of individual D24 and C7 targets, significantly greater RNAi penetrance was achieved following delivery of combined dsRNAs. The highest knockdown that resulted in incomplete ecdysis and/or mortality was obtained for targets with predicted involvement in the same or interacting pathway(s). Knockdown of ATF4 was apparently "rescued" by uncharacterized compensatory gene(s) or effects. © 2022 Society of Chemical Industry.
Knockdown resistance
RNA Silencing
Cite
Citations (8)
<p>Chemoresistance properties of MUC16 and effect of cisplatin on apoptosis of MUC16 knockdown cells. A & B, MUC16 knockdown (H1975-shMUC16 seq1 and 2) cells were highly sensitive to cisplatin (A) and gemcitabine (B). C, The percentage of apoptotic cells was significantly higher in MUC16 knockdown cells (H292-shMUC16) treated with 5μM cisplatin. In contrast, no significant change was observed in the untreated scramble (H292-SCR) and MUC16 knockdown cells. D, We performed stable knockdown of Muc16 in K1418, the result shows that Muc16 is significantly decreased as compared to scramble cells. E, The p53 target gene p21 was significantly increased in MUC16 knockdown (H292-shMUC16) cells. *P<0.05, **P<0.01, ***P<0.001, and NS non-significant.</p>
Cite
Citations (0)
Animal cell-based expression platforms enable the production of complex biomolecules such as recombinant proteins and viral vectors. Although most biotherapeutics are produced in animal cell lines, production in human cell lines is expanding. One important advantage of using human cell lines is the increased potential that the resulting biotherapeutics would carry more “human-like” post-translational modifications. Among the human cell lines, HEK293 is widely utilized due to its high transfectivity, rapid growth rate, and ability to grow in a serum-free, suspension culture. In this review, we discuss the use of HEK293 cells and its subtypes in the production of biotherapeutics. We also compare their usage against other commonly used host cell lines in each category of biotherapeutics and summarise the factors influencing the choice of host cell lines used.
HEK 293 cells
Human cell
Cite
Citations (117)
Abstract We used the maternal-Gal4 shRNA system to knock down expression of dKDM5/lid in Drosophila melanogaster embryos, and analyzed the efficacy of the knockdown by qRT-PCR. Although average relative expression of lid was significantly lower in knockdown conditions compared to the driver-only control, we observed a wide and overlapping range of relative gene expression between individual control and knockdown embryos.
Cite
Citations (0)
Cite
Citations (0)
<p>Stable knockdown of TSPYL5 in lung cancer cells. A, MUC16 expression was significantly elevated in cisplatin resistant cell line. To determine the TSPYL5 role in lung cancer, we stably knocked down TSPYL5 in H292 cells. B, Upon TSPYL5 knockdown, the expression of p53 was increased as compared to scramble cells. C, Knockdown of TSPYL5 was also confirmed by QPCR analysis. β-actin was used as loading control. **P<0.01.</p>
Cite
Citations (0)
We recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense deoxyoligonucleotide (dO) into the somatic cells and reduces the cognate mRNA levels. In this paper, we tested whether we can piggyback more than one dO with one VMO. We designed various hybrids that had more than one dO that could be piggybacked with one VMO. We chose f7, f8, and αIIb genes and tested their knockdown by the appropriate assays. The knockdown with piggybacking either two or three dOs by one VMO yielded > 85% knockdown efficiency. We also performed knockdown of argonautes and rnaseh separately along with f7. We found the knockdown of f7 occurs when knockdown of argonautes happens and not when rnaseh knockdown was performed, suggesting that RNaseH is involved in mRNA degradation. In conclusion, we developed a method where we could knockdown three genes at one time, and by increasing the concentration of VMO by twofold, we could knockdown six genes simultaneously. These multiple gene knockdowns will not only increase the efficiency of the method in whole genome-wide knockdowns but will also be useful to study multifactorial disorders.
Cite
Citations (10)