Panoply™ Human CNR1 Knockdown Stable Cell Line

Endocrine therapy is a promising treatment for fertility preservation in endometrial cancer (EC), but progesterone resistance remains a major challenge. Analysis of the Cancer Genome Atlas (TCGA) database revealed that CNR1 is strongly negatively correlated with overall survival (OS) and recurrence-free survival (RFS) in endometrial cancer. To investigate the role of CNR1 in progesterone resistance in endometrial cancer and its potential molecular regulatory mechanisms, researchers established a stable progesterone-resistant cell line (IshikawaPR) by rendering a common cancer cell line (Ishikawa) progesterone-tolerant. CNR1 expression differences between the two cell lines were assessed using MTT, RT-PCR, Western blot, and immunofluorescence. Subsequently, a CNR1 knockdown lentiviral construct was constructed using GV248 as a tool vector and transfected into IshikawaPR cells. Cell biological behavior and progesterone sensitivity were assessed using plate cloning assays, EdU assays, flow cytometry cycling analysis, Transwell assays, and Scratch test. Results showed that CNR1 knockdown reduced the proliferation and migration abilities of IshikawaPR cells and enhanced their progesterone sensitivity. Additionally, knockdown of CNR1 can also downregulate the expression and activation of ERK and NF-κB.

Immunofluorescence analysis showed that both CNR1 and CyclinD1 were expressed in both control and CNR1-knockdown cells (Figure 1A). A comparison of the changes in proliferation activity and clonogenic capacity of control and CNR1-knockdown cell lines after treatment with 20 μM progesterone revealed that CNR1 knockdown significantly reduced cell proliferation activity and clonogenic capacity (Figures 1B and 1C). The number of actively dividing cells in sh-NC cells increased significantly, and CNR1 knockdown shifted cells from G1 to S phase (Figure 1D). Conversely, CNR1 deficiency slowed wound healing in drug-resistant cells. When both cell lines were treated with 20 μM progesterone, the migration rate of sh-NC cells was less affected by progesterone (Figure 1E). After CNR1 knockdown, the mRNA levels of CNR1, PGR, and total ERK were measured, revealing a decrease in the relative amount of ERK and an increase in the relative amount of PGR (Figure 1F).

Figure 1. Knockdown of CNR1 can decrease the proliferative activity and migration ability in resistant cells. (Ding F, et al., 2021)