1- Morphological and molecular change of A549 cell line exposed to Cigarette smoke extract (CSE)
A549 cells (alveolar type II cells line) has been used in this study .A proximality 1×10⁶ cell/ml was seeded in which these cells distributed in three flask .The concentration of A549 cells line has been quantified before and after CSE exposed using haemocytometer chamber for different time period 24 ,48 and 72 hours at the same time compared the treated cells with untreated cells as it presented in table below before CSE exposed concentration of cells of both control and treated cells have quantified 47×10⁴ cell/ml and 56×10⁴ cell/ml respectively.
After exposed cells to CSE for 24 hours period the result shown a significant reduction of cell number compared before treated and the same on control cells. Subsequently at 48 incubation its shows that concentration of treated cells has been significantly increased compering to control. Whereas, at 72 hours more cell proliferated revealed in both treated and untreated cells which also shows higher in control cells
Morphological changed assessed after CSE has been exposed to A549 cell line which observed on treated cell and control cells for period of 24 and 27 hours by using inverted microscope. The cells line showed long fusiform shape, small in size and clear cell boundaries in both control and treated cells for period of 24 hours. Whereas at 72 hours incubation cells morphologies appeared less cells boundaries and more flatten.
Figure1
Table 1
| A549 cells line | Control/untreated | Treated with CSE |
| Before treatment | 47.5×10⁴cell/ml
|
56×10⁴cell/ml |
| After 24 hours | 27×10⁴ cell/ml | 21.5×10⁴cell/ml |
| After 48 hours | 41×10⁴ cell/ml | 49×10⁴cell/ml |
| After72houres | 57×10⁴cell/ml | 50×10⁴ cell/ml |
2- The concentration of miro RNA from exosome RNA shows in table below:
The concentration of miRNA in the exosome RNA has been measured using a qubit miRNA assays thus, the resultant shown in below table that concertation of miRNA in treated A549 cells line with CSE was more comparing to control A549 cell line which indicated more exposure of CSE to cells line formed more miRNA production.
| Untreated/Control A549 CELL | Treated A549 cell with CSE |
| 1.63ng/µl | 2.43ng/µl |
Cigarette smoke extract inhibit cells viability in A549 cell line.
In this research MTT assays has been used to determine the viability of A549 cell line exposed to cigarette smoke extract. two different media has been used PRMI and EDM (Exosome Depleted FBs medium) for cell growth and various concentration of CSE with different incubation timing 24,48&72 hours.
In figure 1 the viability of cell exposed to CSE and control has been studied. In treated cells the viability of cells was slightly increased to 120% at concentration 20% compering to control which was 100%. Substantial reduction of treated cell viability was shown from 120% to35% at concertation between 20-100% which also shown a significant reduction in cell viability comparing to control that can be shown in p- value.
Figure1: Different concertation of CSE has been treated to A549 cell line that grown on EDM and incubated for 24 hours, the MTT used to examine the cell viability. Thus, CSE concentration and cells viability were represented in % and C stated for control. The graph shows the Average ±SEM and ***p<0.005.vs control .
Similarly, in figure 2 the same plate at different column cells exposed to CSE with same concentration of CSE incubated for 24 hours but cells grown in RPMI media. The cells shown reduction of cell viability from 100% to 78% at concentration 10% that shown significate and followed by slightly increased at between 10%-20% concentration. Moreover, cell viability slightly reduced from 98%-78% at concentration 60%. afterward. Significant decrease of treated cells observed at concentration60%- 80% comparing to control cells remained at 100%growth.
Figure2 Different concertation of CSE has been treated to A549 cell line that grown on RPMI and incubated for 24 hours, the MTT used to examine the cell viability. Thus, CSE concentration and cells viability were represented in % and C stated for control. The graph shows the Average ±SEM and *p<0.05,**p<0.01***p<0. 005.vs control.
Moreover, figure 3 the cells grown in different medium as mentioned above but this time incubated for 48 hours. gradual reduction of cells viability grown in EDM has been seen at 10% concentration. Followed by gradual decreased of cells viability at concentration 10 to 20% and little stability at 40% concentration. Significant reduction observed at concertation of 40% -100% which shows the massive cells apoptosis on 20% cells viability
Figure3 Different concertation of CSE has been treated to A549 cell line that grown on RPMI and incubated for 24 hours, the MTT used to examine the cell viability. Thus, CSE concentration and cells viability were represented in % and C stated for control. The graph shows the Average ±SEM and ***p<0. 005.vs control.
Likewise the cell viability in RPMI medium showed a significate decreased of cell viability at 10%Concentration whereas the control was 100% viable cells followed by gradual reduction of cell viability from 10 %-60% .beside at 80% concentration onward shows significant different with control that can be indicated at p-value ***p<0.05.
Figure4 Different concertation of CSE has been treated to A549 cell line that grown on RPMI and incubated for 48 hours, the MTT used to examine the cell viability. Thus, CSE concentration and cells viability were represented in % and C stated for control. The graph shows the Average ±SEM and *p<0.05,**p<0.001 and***p<0. 005.vs control.
Moreover for 72 hours incubation the cell viability also assessed and found that double in increase of cell viability to 200% at concentration 10%. Also the cell viability has been reduced from 200% to10% with increasing of the CSE concentration between 10%to -80%. Thus, significant different in cell viability has been observed at 80%-100% compering to control which can be realised at p-valve ***p<0.001.
Figure5 Different concertation of CSE has been treated to A549 cell line that grown on EDM and incubated for 72 hours, the MTT used to examine the cell viability. Thus, CSE concentration and cells viability were represented in % and C stated for control. The graph shows the Average ±SEM and ***p<0. 005.vs control.
In the same way, at figure 6 after CSE exposed on A549 cells line and incubated for 72 house the viability of cells that grown under RPMI media were gradually raised up to 240% at concentration 10%-20%. Followed by significant reduction of cell viability at concentration of 80% which can show at p-value
Figure6 Different concertation of CSE has been treated to A549 cell line that grown on RPMI and incubated for 72 hours, the MTT used to examine the cell viability. Thus, CSE concentration and cells viability were represented in % and C stated for control. The graph shows the Average ±SEM and *p<0. 005.vs control.
4- Real time qPCR to evaluate gene expressions
Evaluation of CSE in gene expression of target mir17-5p and mir134-5p in A549 cell line has been performed by real time qPCR techniques and results shown miR17-5p was down regulated which was 0.2 compering to control 1 whereas, in miR 134-5p shown upregulated in 2.2 and significant different from control.
Figure 7 expression of miR 17-5p and miR134-5p in A549 cells line exposed to CSE and control .Real time quantitative PCR used to examine the miRNA expression . C stated for control. The graph shows the Average ±SEM and ***p<0. 005.vs control.
