Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells

Abstract

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

ZNF671 DNA methylation as a molecular predictor for the early recurrence of serous ovarian cancer

“…Next, we performed a Matrigel invasion assay. JHOS‐2 was less invasive compared with SKOV3 and NIH‐OVCAR3, which showed intrinsic invasive ability . Cell invasion was significantly increased by ZNF671 depletion in the SKOV3 and NIH‐OVCAR3 cell line, whereas ZNF671 depletion had a minimal effect on cell invasion by JHOS‐2 (Figure and Figure ).…”

Silencing miR-16 Expression Promotes Angiotensin II Stimulated Vascular Smooth Muscle Cell Growth

“…Cell proliferation was calculated using the ratio of BrdU positive cells to cell nuclei stained with propidium-iodide (PI) or DAPI. Cell proliferation was also examined with MTT assay as we published previously [30]. …”

“…A scratch was created using a sterile 20 μl pipette-tip and washed 3 times with PBS. Fresh growth medium was added for additional 24 h. Migration rate was calculated using to (area of the wound area at 0 h− the wound area at 24 h)/the wound area at 0 h. Cell migration was also examined using trans well plates as we published previously [30]. …”

CRISPR/Cas9-mediated noncoding RNA editing in human cancers

“…Using this method, they successfully disrupted the expression of miR-21 and found that disruption of pre-miR-21 sequences results in decreased cell proliferation, migration and invasion in ovarian cancer cells. 45 Except for editing/regulating these miRNAs discussed above, CRISPR/Cas9 has also shown vast applications toward other miRNAs, like, miR-137, miR-93, miR-309, miR-126a/b etc., in various cancer cells or organisms. [46][47][48][49][50][51][52] CRISPR/Cas9 for lncRNA editing in cancer In addition to miRNAs, lncRNAs have been reported to be successfully edited/regulated by CRISPR/Cas9 system.…”