Differential transcriptome analysis in HPV-positive and HPV-negative cervical cancer cells through CRISPR knockout of miR-214

In this study we have investigated the effects of a tumour suppressor microRNA, miR-214, on gene expressionin HPV-positive (CaSki) and HPV-negative cervical cancer cells (C33A) by RNA sequencing using nextgeneration sequencing. The HPV-positive and HPV-negative cervical cancer cells were either miR-214-knocked-out or miR-214-overexpressed. Gene expression analysis showed that a total of 904 genes wereupregulated and 365 genes were downregulated between HPV-positive and HPV-negative cervical cancer cellswith a fold change of ±2. Furthermore, 11 differentially expressed and relevant genes (TNFAIP3, RAB25,MET, CYP1B1, NDRG1, CD24, LOXL2, CD44, PMS2, LATS1 and MDM1) which showed a fold change of ±5were selected to confirm by real-time PCR. This study represents the first report of miR-214 on global geneexpression in the context of HPV.

CRISPR-mediated knockdown of miR-214 modulates cell fate in response to anti-cancer drugs in HPV-negative and HPVpositive cervical cancer cells

Cervical cancer is the fourth most common cause of mortality in women worldwide. In this study weinvestigated the effect of a tumour suppressor microRNA miR-214 in modulating the cell death againstchemotherapeutic drugs like Doxorubicin, Cisplatin and Paclitaxel. CRISPR-facilitated knockdown andplasmid-based overexpression of miR-214 was performed in cervical cancer cell lines HeLa, C33A and CaSki.It was observed that knocking out miR-214 resulted in reduced apoptosis and cell migration upon drugtreatments; while overexpression of miR-214 resulted in marginal increase in apoptosis and cell migrationwhen treated with drugs. However, miR-214 had very little effect on production of reactive oxygen species.Our results also indicate that Doxorubicin was least effective and Paclitaxel most effective in inducing celldeath. A combination of miR-214 overexpression and Paclitaxel treatment was found to be most effective ininducing cell death in cervical cancer cells. Analysis of cell cycle phases followed by apoptotic markers alsoshowed that miR-214 overexpression along with Paclitaxel treatment caused an increase in PARP and declineof PI-3 kinase/Akt levels. Therefore, miR-214 levels determine the fate of the cancer cell duringchemotherapeutic treatment.