TY - JOUR
T1 - Functional Screen for microRNAs Suppressing Anchorage-Independent Growth in Human Cervical Cancer Cells
AU - Huseinovic, Angelina
AU - Jaspers, Annelieke
AU - van Splunter, Annina P.
AU - Sørgård, Hanne
AU - Wilting, Saskia M.
AU - Swarts, Dorian R.A.
AU - van der Meulen, Ida H.
AU - van Beusechem, Victor W.
AU - de Menezes, Renée X.
AU - Steenbergen, Renske D.M.
N1 - Funding Information: Funding: The project is funded by Dutch Cancer Society, grant number KWF-11312/2017-2 and Stichting Cancer Center Amsterdam, grant number CCA2014-5-15.
Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2022/5/1
Y1 - 2022/5/1
N2 - The progression of anchorage-dependent epithelial cells to anchorage-independent growth represents a critical hallmark of malignant transformation. Using an in vitro model of human papillomavirus (HPV)-induced transformation, we previously showed that acquisition of anchorage-independent growth is associated with marked (epi)genetic changes, including altered expression of microRNAs. However, the laborious nature of the conventional growth method in soft agar to measure this phenotype hampers a high-throughput analysis. We developed alternative functional screening methods using 96-and 384-well ultra-low attachment plates to systematically investigate microRNAs regulating anchorage-independent growth. SiHa cervical cancer cells were transfected with a microRNA mimic library (n = 2019) and evaluated for cell viability. We identified 84 microRNAs that consistently suppressed growth in three independent experiments. Further validation in three cell lines and comparison of growth in adherent and ultra-low attachment plates yielded 40 microRNAs that specifically reduced anchorage-independent growth. In conclusion, ultra-low attachment plates are a promising alternative for soft-agar assays to study anchorage-independent growth and are suitable for high-throughput functional screening. Anchorage independence suppressing microRNAs identified through our screen were successfully validated in three cell lines. These microRNAs may provide specific biomarkers for detecting and treating HPV-induced precancerous lesions progressing to invasive cancer, the most critical stage during cervical cancer development.
AB - The progression of anchorage-dependent epithelial cells to anchorage-independent growth represents a critical hallmark of malignant transformation. Using an in vitro model of human papillomavirus (HPV)-induced transformation, we previously showed that acquisition of anchorage-independent growth is associated with marked (epi)genetic changes, including altered expression of microRNAs. However, the laborious nature of the conventional growth method in soft agar to measure this phenotype hampers a high-throughput analysis. We developed alternative functional screening methods using 96-and 384-well ultra-low attachment plates to systematically investigate microRNAs regulating anchorage-independent growth. SiHa cervical cancer cells were transfected with a microRNA mimic library (n = 2019) and evaluated for cell viability. We identified 84 microRNAs that consistently suppressed growth in three independent experiments. Further validation in three cell lines and comparison of growth in adherent and ultra-low attachment plates yielded 40 microRNAs that specifically reduced anchorage-independent growth. In conclusion, ultra-low attachment plates are a promising alternative for soft-agar assays to study anchorage-independent growth and are suitable for high-throughput functional screening. Anchorage independence suppressing microRNAs identified through our screen were successfully validated in three cell lines. These microRNAs may provide specific biomarkers for detecting and treating HPV-induced precancerous lesions progressing to invasive cancer, the most critical stage during cervical cancer development.
UR - http://www.scopus.com/inward/record.url?scp=85128783622&partnerID=8YFLogxK
U2 - 10.3390/ijms23094791
DO - 10.3390/ijms23094791
M3 - Article
C2 - 35563182
AN - SCOPUS:85128783622
SN - 1661-6596
VL - 23
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 9
M1 - 4791
ER -