Knockdown Cell Clones
166 Material and Methods
4.2
All methods for analysis of AQP gene expression, transfection of AQP3 shRNA into MDA-MB-231 cells, Western blotting and immunocytochemical analysis of cells were performed as described in Chapter 2.
To investigate the gene expression of all 13 AQPs in MDA-MB-231 cell line, SYBR® green real-time PCR reaction was performed. The expression of housekeeping genes was also analysed under the same conditions in order to use these as internal controls. The reaction conditions and amount of cDNA used for real time PCR were mentioned in detail in chapter 2. AQP3 expression was investigated by immunocytochemical assay and was performed on MDA-MB-231 cells as described in section 2.13.
Once enough cells were available, cell pellets were collected for each clone of the shRNA constructs for RNA and protein extraction. Total RNA from all clones of each shRNA construct was extracted and reverse transcribed as mentioned in chapter 2. The AQP3 gene expression in all clones was investigated by performing qPCR. The reaction conditions were same as described before in Chapter 2. Western Blotting (as described in Chapter 2) was also performed on selected clones to further verify the AQP3 protein expression.
Statistical analysis of data was performed using GraphPad Prism 6 software. One-way ANOVA with test post-hoc test (Dunnett's multiple comparisons) for comparisons of > 3 groups or Student’s t-test for comparisons of <3 groups were performed for the statistical analysis between experimental conditions and p values < 0.05 were considered significant.
167 Results
4.3
mRNA expression profile of AQPs in MDA-MB-231 cell line 4.3.1
SYBR® Green real-time PCR (qPCR) was performed to investigate the gene expression of all AQPs and housekeeping genes (beta actin and YWHAZ) in the MB-231 cell line. MDA-MB-231 cells exhibited expression of several AQP family members but expressed AQP3 at a significantly higher level compared to all other AQPs. The expression of AQPs was normalised to beta actin and YWHAZ housekeeping genes. The amplification and dissociation curves for all 13 AQPs plus the normalising genes beta actin and YWHAZ are displayed in Figures 4.1 to 4.15.
Relative expression of all AQPs was compared to AQP0 using the 2-∆∆Ct method. The averaged Ct values generated in qPCR reaction were shown in Table 4.1. The mRNA expression of AQP3 (see Figure 4.16) was significantly (relative mean fold value 17.24, p <0.0001) higher than all other AQPs demonstrated by One way ANOVA with Dunnett’s post-hoc test.
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Figure 4.1 SYBR® Green real-time PCR analysis of beta actin gene expression.
Gene expression of beta actin was assessed in diluted cDNA samples of MDA-MB-231 cells. (A) Amplification curves showed that the mean Ct value for beta actin in these cells was 20.61. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 89°C suggesting excellent primer specificity. n=3.
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Figure 4.2 SYBR® Green real-time PCR analysis of YWHAZ gene expression.
Gene expression of YWHAZ was assessed in diluted cDNA samples of MDA-MB-231 cells. (A) Amplification curves showed that the mean Ct value for YWHAZ in these cells was 21.28. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 81°C suggesting excellent primer specificity; n=3.
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Figure 4.3 SYBR® Green real-time PCR analysis of AQP0 gene expression.
Gene expression of AQP0 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP0 in these cells was 30.02. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 84°C suggesting excellent primer specificity; n=3.
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Figure 4.4 SYBR® Green real-time PCR analysis of AQP1 gene expression.
Gene expression of AQP1 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP1 in these cells was 32.81. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 84°C suggesting excellent primer specificity. n=3.
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Figure 4.5 SYBR® Green real-time PCR analysis of AQP2 gene expression.
Gene expression of AQP2 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP2 in these cells was 36.05. (B) Dissociation curve analysis showed that the amplicon from the PCR reaction dissociated at range of temperatures suggesting nonspecific primer binding; n=3.
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Figure 4.6 SYBR® Green real-time PCR analysis of AQP3 gene expression.
Gene expression of AQP3 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP3 in these cells was 26.00. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 85°C suggesting excellent primer specificity; n=3.
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Figure 4.7 SYBR® Green real-time PCR analysis of AQP4 gene expression.
Gene expression of AQP4 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP4 in these cells was 31.43. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 79°C showing adequate primer specificity. n=3.
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Figure 4.8 SYBR® Green real-time PCR analysis of AQP5 gene expression.
Gene expression of AQP5 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP5 in these cells was 31.34. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 85°C suggesting excellent primer specificity; n=3.
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Figure 4.9 SYBR® Green real-time PCR analysis of AQP6 gene expression.
Gene expression of AQP6 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP6 in these cells was 30.57. (B) Dissociation curve analysis showed that the amplicon from the PCR reaction dissociated at approximately 89°C suggesting adequate primer specificity; n=3.
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Figure 4.10 SYBR® Green real-time PCR analysis of AQP7 gene expression.
Gene expression of AQP7 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP7 in these cells was 30.30. (B) Dissociation curve analysis showed that the amplicon from the PCR reaction dissociated at approximately 89°C suggesting excellent primer specificity; n=3.
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Figure 4.11 SYBR® Green real-time PCR analysis of AQP8 gene expression.
Gene expression of AQP8 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP8 in these cells was 31.31. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 82°C suggesting excellent primer specificity; n=3.
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Figure 4.12 SYBR® Green real-time PCR analysis of AQP9 gene expression.
Gene expression of AQP9 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP9 in these cells was 32.89. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 82°C suggesting excellent primer specificity; n=3.
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Figure 4.13 SYBR® Green real-time PCR analysis of AQP10 gene expression.
Gene expression of AQP10 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP10 in these cells was 35. (B) Dissociation curve analysis showed that the amplicon from the PCR reaction dissociated at approximately 78°C showing adequate primer specificity; n=3.
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Figure 4.14 SYBR® Green real-time PCR analysis of AQP11 gene expression.
Gene expression of AQP11 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP11 in these cells was 27.52. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 89°C suggesting excellent primer specificity; n=3.
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Figure 4.15 SYBR® Green real-time PCR analysis of AQP12 gene expression.
Gene expression of AQP12 was assessed in diluted cDNA samples of MDA-MB-231 cells using pre-validated primers. (A) Amplification curves showed that the mean Ct value for AQP12 in these cells was 28.89. (B) Dissociation curve analysis showed one dissociation peak that dissociated at approximately 88°C suggesting excellent primer specificity; n=3.
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Table 4.1 Mean Ct values from real-time PCR reactions for housekeeping genes and all AQPs in MDA-MB-231 cells.
Figure 4.16 Relative AQP mRNA Expression in MDA-MB-231 cells.
The expression of AQPs was normalised to beta actin and YWHAZ housekeeping genes.
Relative expression of all AQPs was compared to AQP0, calculated by 2-∆∆Ct by using Ct values shown in Table 4.1. The mRNA expression of AQP3 was significantly higher than all other AQPs. Data are shown as mean ± SEM; n = 3, ****p <0.0001.
184
Relative AQP3 mRNA expression in MCF-7 and MDA-MB-231 cells 4.3.2
Real-time PCR reaction was performed to further investigate the AQP3 gene expression in MCF-7 (ER and PR positive cell line) and MDA-MB-231 (TNBC cell line) cells. The expression of AQP3 was normalised to beta actin and YWHAZ housekeeping genes. Relative expression of AQP3 in MCF-7 and MDA-MB-231 cell lines was compared using the 2-∆∆Ct method. The mRNA expression of AQP3 was significantly reduced in MCF-7 cells (mean fold value 1.40, p <0.05) than MDA-MB-231 cells. Statistical significance was assessed by Student’s t test (see Figure 4.17).
Figure 4.17 Expression of AQP3 mRNA in MCF-7 and MDA-MB-231 cells.
qPCR analysis of AQP3 mRNA expression in MCF-7 and MDA-MB-231cells showed that AQP3 expression level in MDA-MB-231 cells was significantly higher than in MCF-7 cells.
Bar graph displaying the relative fold difference of AQP3 in both cell lines internally normalisedtohousekeepinggenes.Datarepresentmean±SEM;*p = 0.043, n=3.
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Expression of AQP3 by Immunocytochemistry in MDA-MB-231 cells 4.3.3
To examine the AQP3 protein expression in MDA-MB-231 cells, immunofluorescence staining of AQP3 peptide was performed. The cells were stained with rabbit anti-AQP3 antibody and viewed on a Leica DMI4000B inverted microscope by using GFP (for DyLight488) and A4 (for DAPI) filter cubes. The results demonstrated the AQP3 expression in cytoplasm as well as on the cell membrane of MDA-MB-231 cells (see Figure 4.18). The result further confirmed the expression of AQP3 in MDA-MB-231 cells. This finding also suggested that MDA-MB-231 cells mostly express AQP3 in cytoplasm which might be translocated to the cell membrane like most AQPs.
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Figure 4.18 Fluorescence Immunocytochemistry for AQP3 in MDA-MB-231 cells Immunocytochemical characterisation of AQP3 protein expression in MDA-MB-231 cells stained with (A) DyLight488 dye-conjugated AQP3 antibody for AQP3 protein and (B) DAPI for nucleus. (C) Overlaid image of A and B. (D) Negative control showing DAPI staining only. Bar =30 μm. Image is representative of n=5 experiments.
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