Research paper 1 Flashcards
What is a summary of the paper?
- studied the expression pattern and role of miRNA-221 in non small cell lung carcinoma.
- studied using gain and loss of function analysis
- TIMP2 is a direct target of miRNA221
- miRNA221 promotes NSCLC growth and invasion through repressing the expression of TIMP2
- miRNA221 could be a potential target for the treatment of NSCLC
cell lines
- permanently established cell culture
- will proliferate indefinitely given appropriate fresh medium and space.
- Lines differ from cell strains in that they become immortalized.
what were the cell lines studied in the paper?
SPCA1
H1299
role of miRNAs?
- regulation of gene by regulating gene expression post transcriptionally
- proliferation, apoptosis, differentiation invasion
MiR-221
- belongs to Mir-221 and 22 cluster
- found to be upregulated in many cancers
- can be pro or anti oncogenic roles in different cell lines
Describe table 1
- correlation between clinicopathologic ( signs and symptoms+ lab results) characteristics of the patients and expression os miRNA 221 in NSCLC
- tissue samples were taken from cancer patients
- normal and cancer tissues
- this process was a way to collect tissue samples needed for the further experiments in order to study the role of miRNA221
Explain figures 1A and 1B
- Question asked: What are the differences in expression of miR221 within normal tissues, tumor tissues, and normal cell lines vs. tumor cell lines.
- qRT-PCR is a real-time PCR analysis, which is a tool for quick determination of the quantity of nucleic acids being produced. In this case, we are looking at the amount of miR-221 expression in NSCLC tissues as well as the cell lines.
- In figure 1A, we see that the level of miR221 expression in normal tissue samples was significantly lower than the tumor tissue samples. This makes sense as miRNA221 is found to be upregulated in many types of cancers.
- In figure 1B, we are given a few examples of cell lines taken from NSCLC. The normal cell line, known as MRC5 is shown to have significantly lower expression of miRNA221. Whereas all of the cell lines from NSCLC had significantly upregulated miR221 levels
- These figures suggest that miR-221 may play a significant role in the development of cancers such as NSCLC.
Explain figure 2:
Question: Now that we understand that miR221 plays a role in the development of NSCLC, what is that role?
- in order to understand the role or miR221, they looked at the effects of miR221 on cell proliferation and cell cycle in NSCLC
- On the left side are the results taken from the SPCA1 cell line and the right side shows the results taken from H1299 cell line.
- Figures A and B show the results from the proliferation assay, where cells may or may not be triggered to divide based on exposure to the miR221 mimic or the miR221 inhibitor. Figure A shows that upregulation of miR221 significantly promoted the proliferation of SPCA1 cells compared to the control. Figure B shows that downregulated miR221 inhibited the growth of H1299 compared to the controls
- Figures C and D are the results taken from a colony formation assay (whether or not a single cell can grow into a colony). On the left in figure C, the sample exposed to miR221 mimic had significantly higher colony formation. On the right in figure D, the sample exposed to miR221 inhibitor had significantly lower levels of colony formation compared to the control.
- figures E and F are the results of the cell cycle analysis. This process used flow cytometer to determine the impact of miR221 inhibitor on cell cycle progression. Flow cytometry can be used for cell cycle analysis to estimate the percentages of a cell population in the different phases of the cell cycle. On the x Axis, we see the different cell cycle stages (g0/g1 growth), S ( Dna synthesis) and g2/m ( preparing for mitosis/mitosis)
Figure E we see that miR221 mimic caused a significant increase in the growth phase compared to the control, with the opposite occuring in figure F. HOwever, there was a significant increase in the G2/M phase.
Overall, these results tell us that miR221 may play a significant role in the progression of the cell cycle.
Explain figure 3
Question: How does Mir221 effect migration and invasion in NSCLC.
A wound healing and transwell assay were performed in order to obtain these results. A wound healing, or scratch assay is the process of scratching a pipette tip in each well containing cells. The cells near the edge of the scratch will then migrate to the wound space. A transwell assay is a method of measuring migratory responses of cells .
- Figure A shows photos of the wound healing assay. H1299 cells seeded in wells were either transfected with miRNC or miR-221 inhibitor. After the scratch was applied, results were taken after 48 h. As shown here, there is a visible difference between the control and the inhibitor. We can conclude that the miR221 inhibitor blocked the migration of H1299 cells to the wound space.
- Figure B shows microscopic images of invasive cells under control vs inhibitor environments. This data can be shown quantitatively in figure C. The Y axis representing the number of cells and the X axis showing our two environments. As we can see here, there is a significantly lower number of cells per field when exposed to a miR221 inhibitor.
- Figure D shows the results from immunoblotting (laboratory technique used to detect a specific protein in a blood or tissue sample.) of Ecadherin, N cadherin and vimentin in H1299 cells. Figure E Y axis shows the relative expression of these proteins when they are transfected with two conditions. The miRNA221 inhibitor caused a significant increase in E cadherin and a significant decrase in both N-cadherin and Vimentin. I did some of my own searching to decipher what this means. Epithelial Mesenchymal transition is often promoted by decreased E-cadherin and overexpression of N-cadherin and Vimentin. This means miR221, when it is not inhibited, may play a role in promoting EMT.
- Figure F is the result of an immunoflorescent assay. (microscopic method that can detect and visualize the viral proteins expressed in cells via antigen—antibody reaction ).
Overall: these figures explain how miR-221 downregulation enhances E-cadherin while decreasing N-cadherin and Vimentin protein expression, which may indicate that miR221 induces EMT in H1299 cells.
E-Cadherin
- prevent dissociation of cells from original tumor mass
- loss of cell-cell adhesion and cell junctions allows cells to invade surrounding tissues and migrate to distant sites
N-Cadherin
- upregulation promotes metastasis
Vimentin
- When overexpressed in solid cancers, vimentin drives epithelial to mesenchymal transition (EMT) and ultimately, metastasis.
Explain Figure 4.
Question: What is the relationship between miR221 and TIMP2?
- Figure A: This figure is simply a display of the alignment between miR 221 and the 3’ UTR of TIMP1 mRNA.
- Figure B: A pearson’s correction analysis was run in order to find the relationship between expression of miR 221 and TIMP2 expression. This shows a significant inverse relationship.
- Figure C: Dual luciferase reporter assay (to study gene transcription and regulation.) was completed to further investigate if mir 221 binds to TIMP2. The results here show that there is a significant difference in the results of the wild type and the mutant samples. miR221 suppressed the luciferase activity of the wild type TIMP2 3’ UTR; however, not in the mutant of H1299. This shows that miR221 has an impact on the transcription of luciferase in wild type cells, but not necessarily in mutant cells.
- Figure D: To further investigate the relationship between TIMP2 and the cell lines, relative mRNA levels of TIMP2 was measured using a qRT-PCR analysis. The results show a significant difference between the mRNA expression in the control vs the samples transfected with miR221. This indicates that miR221 may have inhibiting control over TIMP2 mRNA expression.
- Figure E: To further investigate the relationship between TIMP2 and the cell lines, relative protein expression of TIMP2 was investigated using a western blot.
Overall, these figures can help us conclude that miR221 can directly target TIMP2 in NSCLC cells, more specifically miR221 negatively regulates TIMP2 by targeting the TIMP2 3’UTR
TIMP2
Tissue inhibitor of metallopeptidase 2
mir 221 binding site
Explain Figure 5..
Question: Does miR221 mediate NSCLC tumor growth?
These figures were the result of mice being infected with H1299 cells.
- Figure A shows the results of tumor volume in mice that were injected with H1299 cells transfected with either the control of the miR221 inhibitor. Shown on the X axis, growth measurements were taken every 2 days.
- Shifting to the right, figure B shows the tumor weight in mg of the control mice vs the mice injected with the miR221 inhibitor. These results show us that there was a significantly slower growth rate of the tumors in the mice injected with miR221 inhibitor. The overall volume and weight of the tumors were also significantly lower.
- Figure C, Question: what are the levels of EMT markers in vivo? once again shows a western blot to detect the proteins E-cadherin, N-cadherinm and vimentin protein levels in vivo, which are markers of EMT. The results of this Western blot are shown in figure D, which are similar results to what we saw in vitro.
- Figure E: question: Do cells grow slower from xenografted H1299 inhibitor transfected cell?
This first staining, the H and E staining, shows significantly less cell growth in the cells transfected with the inhibitor. On the bottom, we see the results of the , Immunohistochemistry (IHC) , which is a technique that exploits specific binding between an antigen and antibody to help detect specific antigens in cells. In this case, Ki67 is the antigen we are looking at. As seen in these images, there is less detection of Ki67 in the sample transfected with the miR221 inhibitor.
Overall, we can conclude from these figures that miR221 promotes the growth of tumor cells in vivo.
