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. 2023 Nov 1;24(11):3715-3727.
doi: 10.31557/APJCP.2023.24.11.3715.

Metformin Caused Radiosensitivity of Breast Cancer Cells through the Expression Modulation of miR-21-5p/SESN1axis

Fatemeh Saffari  1 , Abdoalmabood Momeni  2 , Mehrzad Ramezani  2 , Yasamin Ansari  3 , Sahar Moghbelinejad  4
Affiliations

Metformin Caused Radiosensitivity of Breast Cancer Cells through the Expression Modulation of miR-21-5p/SESN1axis

Fatemeh Saffari et al. Asian Pac J Cancer Prev. .

Abstract

Objective: In this research we evaluated molecular mechanism of effect of metformin in radio sensitivity of breast cancer cells.

Methods: This research was done in cellular and molecular research center of Qazvin university of Medical science in 1399 to 1401. Studied samples were two breast cancer cell lines (MCF-7 and MDA-MB-231) they are derived from primary and secondary tumors resected from a single patient. We exposed them to cumulative 50 Gy radiation and constructed radio resistant cell lines. Then resistant cell lines were treated with 50µm of metformin. Our studied groups were resistant cells treated and un treated with metformin. Then, the expression rate of miR-21-5p and SESN1 gene in resistant and control cells was checked by Quantitative Real-time PCR(qRTPCR). After the cell lines were treated with different concentrations of metformin at different intervals, the rate of cell proliferation and cell death was checked by CCK-8 assay and flow cytometry. The Western blot method was also used to confirm the expression of some genes.

Results: Our results showed that the expression of miR-21-5p was upregulated in radiation-resistant cancer cells (1.8±0.65) (P<0.0001) MCF-7 cell line and (1.6±0.42)(P<0.001) MBA-MD-231 cell line, while the expression of SESN1 was down regulated (0.46±0.12) (P<0.0001) MCF-7 cell line and (0.42±0.22) (P<0.001) MBA-MD-231 cell line. Metformin enhanced the radio sensitivity of breast cancer cells in a dose and time-dependent manner. Also, metformin treatment decreased the expression of miR-21-5p (0.47±0.32) (P<0.0001) MCF-7 Cell line and (0.45±0.21)(P<0.001) MBA-MD-231 cell line and increased the expression of SESN1 (1.65±0.72)(P<0.0001)MCF-7 cell line and (1.73±0.52)(P<0.0001) MBA-MD-231 cell line. The function of metformin was reversed by miR-21-5p inhibitors or the transfection of SESN1 overexpressing plasmids.

Conclusion: In conclusion, based on this research results, metformin enhanced the radio sensitivity of breast cancer cells via modulating the expression of miR-21-5p and SESN1.

Keywords: Metformin; Radio resistant; SESN1 gene; breast cancer; miR-21-5p.

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Conflict of interest statement

The authors declare no conflicts of interest for this work.

Figures

Figure 1
Figure 1
After Radiosensitive Cell Lines were Constructed (a-a'): CCK-8 assay was used to confirm the successful construction of radio-resistant cell lines exposed to different doses of radiation (b-b'): Shows significant differential expression of miR-21-5p and SESN1 gene between resistant and sensitive cell lines. *p<0.05 **p < 0.01 and ***p < 0.001
Figure 2
Figure 2
MiR-21-5p Directly Targeted SESN1. (a) Shows potential binding site for miR-21-5p and SESN1 (Target Scan Release 7.2) . (b) Luciferase activity was analyzed in 293T cells co transfected with SESN1-WT or SESN1-MUT in conjunction with miR-21-5p or miR-NC. **p < 0.01, *** p < 0.001
Figure 3
Figure 3
miR-21-5p was Involved in Regulating Radioresistance miR-21-5p Mimics and Inhibitorswere Transfected into MCF-7/R and MDA-MB-231/R Cells, Successful Transfection was Confirmed by qRT-PCR (a-a' and b-b'). CCK-8 assay and flow cytometry were used to detect the proliferation and apoptosis of breast cancer cells, respectively(c-c', d-d' , e-e', f-f'). *p<0.05 **p < 0.01 and ***p < 0.001
Figure 3
Figure 3
miR-21-5p was Involved in Regulating Radioresistance miR-21-5p Mimics and Inhibitorswere Transfected into MCF-7/R and MDA-MB-231/R Cells, Successful Transfection was Confirmed by qRT-PCR (a-a' and b-b'). CCK-8 assay and flow cytometry were used to detect the proliferation and apoptosis of breast cancer cells, respectively(c-c', d-d' , e-e', f-f'). *p<0.05 **p < 0.01 and ***p < 0.001
Figure 4
Figure 4
miR-21-5p/SESN1 are Involved in Radio Sensitivity of Breast Cancer Cells .miR-21-5p mimics and pcDNA-SESN1 were co-transfected into MCF-7/R cells, while miR-21-5p inhibitors and si-SESN1 was transfected into MDA-MB-231/R cells, and successful transfection was confirmed by qRT-PCR. (a-a', b-b') CCK-8 assay and flow cytometry were used to detect the proliferation and apoptosis of breast cancer cells, respectively(c-c', d-d' , e-e', f-f'). *p<0.05, **p < 0.01 and ***p < 0.001
Figure 4
Figure 4
miR-21-5p/SESN1 are Involved in Radio Sensitivity of Breast Cancer Cells .miR-21-5p mimics and pcDNA-SESN1 were co-transfected into MCF-7/R cells, while miR-21-5p inhibitors and si-SESN1 was transfected into MDA-MB-231/R cells, and successful transfection was confirmed by qRT-PCR. (a-a', b-b') CCK-8 assay and flow cytometry were used to detect the proliferation and apoptosis of breast cancer cells, respectively(c-c', d-d' , e-e', f-f'). *p<0.05, **p < 0.01 and ***p < 0.001
Figure 5.
Figure 5.
Metformin Radio Sensitized, Radioresistant Breast Cancer Cells (a-a') After treatment with different concentrations of metformin, CCK-8 assay was used to detect breast cells viability after 48 hr. (b-b') CCK-8 assay was used to detect the cells viability of MCF-7/R and MDA-MB-231/R cells in different rate of miR-21-5p in MCF-7/R and MDA-MB-231/R cells were detected by qRT-PCR after treatment times after treatment with 50 μm metformin. (c-c') Apoptosis rate was detected 48 h after treatment with 50 µm metformin. *p<0.05, **p < 0.01 and ***p < 0.001
Figure 6
Figure 6
Metformin Decreased the Expression of miR-21-5p and Increased the Expression of SESN1 in a Dose and Time-Dependent Manner. (a-a´) The expression with different concentrations of metformin for 48 h. (b-b') Shows the expression rate of miR-21-5p after MCF-7/R and MDA-MB-231/R cells were treated with 50 μm of metformin at different times (c-c') which shows the expression rate of SESN1 in MCF-7/R and MDA-MB-231/R cells after treatment with different concentrations of metformin for 48 hr. (d-d') Shows expression rate of SESN1 after MCF-7/R and MDA-MB-231/R cells were treated with 50 μm of metformin for different times. *p<0.05, **p < 0.01 and ***p < 0.001
Figure 7
Figure 7
Inhibition of miR-21-5p Reversed the Function of Metformin. (a-a') shows the results of qRT-PCR after transfection of miR-21-5p inhibitor in studied cell lines (b-b') shows cells viability after transfection of miR-21-5p inhibitor in studied cell lines. (c-c') Shows apoptosis rate after transfection of miR-21-5p inhibitor in studied cell lines. **p < 0.01 and ****p < 0.001
Figure 8
Figure 8
SESN1 Expression Reversed the Function of Metformin (a-a') Shows results of successful transfection in MCF-7 cells by western blot method. (b-b') Shows results of successful transfection in MCF-7 and MDA-MB-231 cells by QRT PCR method (c-c') Shows results of cell viability after transfection (d-d') Results of apoptosis rate after transfection **p < 0.01 and ****p < 0.001
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