Functional Screen for microRNAs Suppressing Anchorage-Independent Growth in Human Cervical Cancer Cells

Abstract

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.

Downregulation of miR‐193a/b‐3p during HPV‐induced cervical carcinogenesis contributes to anchorage‐independent growth through PI3K–AKT pathway regulators

“…In earlier research, we showed that both miR‐193a‐3p and miR‐193b‐3p reduce anchorage‐independent growth in SiHa cervical cancer cells 11 . To further understand their role in the regulation of anchorage‐independent growth, we investigated the effect of overexpression of both miRNAs in late passages of FK16A and FK18B cells cultured using adherent plates and ULA plates, respectively.…”

“…Anchorage-independent growth was assessed using conventional soft agar assays and on ultra-low attachment (ULA) plates as described before. 11 For colony formation assay, 2500 cells were transfected and seeded in a medium containing 0.35% agarose (Sea-plague agarose; Lonza Group Ltd.) on a surface of 0.6% bottom agarose in a 48-well plate in duplicate. After 3 weeks, colonies exceeding ∼50 cells were counted.…”

“…In earlier research, we showed that both miR-193a-3p and miR-193b-3p reduce anchorage-independent growth in SiHa cervical cancer cells. 11 To further understand their role in the regulation of anchorage-independent growth, we investigated the effect of overexpression of both miRNAs in late passages of FK16A and FK18B share the same seed sequence, gene target prediction resulted in the same list of genes from both online tools. Therefore, we proceeded with the target prediction procedure for miR-193a-3p.…”

“…[8][9][10] Identification of biologically relevant miRNAs by a functional screen on SiHa cervical cancer cells, showed that miR-193a-3p and miR-193b-3p were involved in anchorage-independent growth. 11 To further investigate the role of miR-193a-3p and miR-193b-3p in HPV-induced carcinogenesis, the present study aimed to examine their functional involvement in HPV-transformed keratinocytes and to identify and validate their target genes in relation to anchorage-independent growth.…”

miR‐129‐5p inhibits anchorage‐independent growth through silencing of ACTN1 and the ELK4/c‐FOS axis in HPV‐transformed keratinocytes

“…For cell viability measurements, cells were transfected with 2 nM of miRNA mimics and the C2 in 384-well plates using DharmaFECT 4 as the transfection reagent, as described previously. 10 Cell viability was measured using the CellTiter-Glo 3D assay (Promega) following the manufacturer's instructions. Each measurement was performed in duplicate, and cell viability upon miR-129-5p transfection was normalized to the negative control C2.…”