POPDC1 Suppression in Cancer Progression

Introduction

This study will be demonstrating the loss and suppression of POPDC1 cell membrane localization. Secondly, POPDC1 suppression promotes proliferation and cell migration in breast cancer cells, which are inhibited significantly by POPDC1 over expression. Thirdly, the interaction of the cAMP with and regulation pertaining to POPDC1 expression takes place in the breast cancer cells. Lastly, inhibition mediated by cAMP of the breast cancer cell proliferation and migration is mediated potentially via POPDC1 signaling. For students needing support in related research topics, seeking healthcare dissertation help can provide valuable guidance in navigating complex concepts.

The research question:

Does dysregulation of POPDC1 promoting malignant phenotypes in breast cancer and POPDC1 restoration have the potential of inhibiting cell proliferation and migration and reverting cells to a less malignant phenotype?

For answering this research question, POPDC1 expression level is first determined in breast cancer cells comparing with the non-malignant breast cells. Secondly, gain and loss of POPDC1 functions and its effect is assessed on breast cancer cell proliferation and migration. Thirdly, whether cAMP does interaction with and regulation of POPDC1 levels in breast cancer cells is determined. Finally, whether cell proliferation and migration’s inhibition that is mediated by cAMP is facilitated potentially via POPDC1 signaling is assessed.

Method and materials

Cell culture

ATCC or MCF10A cells have been subject to growth in the growth of mammary epithelial cell medium bullet kit (Lonza). SKBR3 cells (ATCC), MDA231 cells (ATCC) and MCF7 cells (ATCC) have been subject to growth in Dulbecco’s Modified Eagle’s medium (DMEM) (Sigma) in supplementation with 10 percent FCS (foetal calf serum), 1 percent streptomycin/penicillin (streptomycin 10 mg/ml; penicillin 10,000 units/ml).

Western blot

The lysed sells in RIPA (radioimmunoprecipitation assay) with containing buffer of 50 mM Tris/HCl at 0.5% Nonidet P-40, 1% protease inhibitor (Sigma), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 0.1% Triton-X 100, 150 mM sodium chloride, pH 8, and 0.1% phosphatase inhibitor (Sigma).

Immunocytochemistry

Coverslips of sterile glass have been pre-coated with 10 μg/ml PLL (poly-l-lysine) and under sterile conditions have dried. There will be seeding of a total of 2 × 104 cells on PLL coated glass coverslips and incubation of 37°C, 5% CO2 in a overnight humidified incubator.

siRNA with protein suppression

A growth of a total of 2 × 104 cells per/ml in complete medium 24 h before the transient transfection with scrambled siRNA (Qiagen) or POPDC1 siRNA (Qiagen).

Stable cell lines generation

mPOPDC1 cDNA contains G418 resistance cassette that Professor Thomas Brand (Imperial College London) has gifted. The transfection of plasmid DNA to SKBR3, MDA231, and MCF7 cells with the use of Amaxa Nucleofector™ II (Lonza) in generating stable cell line that over-expresses POPDC1; MCF7 POP1++, MDA POP1++ and SKBR3 POP1++.

Boyden migration assay

The migration assay of the Boyden chamber will be performed in assessing the cell migration with the use of 8 μm pore polycarbonate membranes. The experiments having the POPDC1 effects knocking down and over expression of POPDC1 have been assessed on cell migration, and the Boyden chamber’s lower cells have been loaded with the medium of serum free that lacks any migration solution in ensuring that the differences observed in migration was because of the effects of POPDC1 difference in expression level.

Expected Results

The POPDC1 expression is subject to suppression in the lines of breast cancer comparing with non-malignant cells

POPDC1 localization of cell membrane is subject to reduction in the lines of breast cancer

The POPDC suppression promotes cell proliferation and migration in the breast cancer cells

The POPDC1 over expression does suppression cell proliferation and migration in breast cancer cell lines

Interaction of cAMP does interaction and up-regulates POPDC1 in breast cancer cells

cAMP does inhibition of cell proliferation and migration in breast cancer cells

POPDC1 suppression is overcome by SP-8-Br-cAMPS and this does rescuing of inhibited cell proliferation and migration.

Discussion and Conclusion

This study will be showing that in breast cells, there is expression of POPDC1, although the expression is suppressed significantly in aggressive SKBR3 and MDA231 breast cancer cells comparing with the breast cells of non-malignant MCF10A. This has consistency with POPDC1 protein and RNA suppression observable in a number of cancers that includes hepatocellular carcinoma, colorectal cancer and gastric cancer.

This study will show further that POPDC1 localization’s differential in breast cancer cells is compared with non-malignant breast cells. The localization of the cell membrane of POPDC1 has been subject to reduction in SKBR3 and MDA231 compared with POPDC1 localization to the cell membrane in normal MCF10A breast cells.

To conclude, POPDC1 is the representative of a druggable target having the potential of being subject to manipulation in inhibiting breast cancer cell proliferation and migration.