Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jan 31;13(1):9.
doi: 10.1186/s13045-020-0844-0.

MiR-509-3 augments the synthetic lethality of PARPi by regulating HR repair in PDX model of HGSOC

Chenggong Sun  1   2 , Wenyu Cao  1   2 , Chunping Qiu  1   2 , Chengcheng Li  1   2 , Samina Dongol  1   2 , Zhiwei Zhang  1   2 , Ruifen Dong  1   2 , Kun Song  1   2 , Xingsheng Yang  1   2 , Qing Zhang  3   4 , Beihua Kong  5   6
Affiliations

MiR-509-3 augments the synthetic lethality of PARPi by regulating HR repair in PDX model of HGSOC

Chenggong Sun et al. J Hematol Oncol. .

Abstract

Background: PARP inhibitors have been the most promising target drugs with widely proven benefits among ovarian cancer patients. Although platinum-response, HR-related genes, or HRD genomic scar detection are acceptably used in assessment of Olaparib response, there are still evident limitations in the present approaches. Therefore, we aim to investigate more accurate approaches to predict Olaparib sensitivity and effective synergistic treatment strategies.

Methods: We probed two databases (TCGA and Qilu Hospital) in order to quest novel miRNAs associated with platinum-sensitivity or HR-related genes. Cellular experiments in vitro or in vivo and PDX models were utilized to validate their role in tumor suppression and Olaparib sensitizing. Furthermore, HR gene mutation was analyzed through WES to explore the relation between HR gene mutation and Olaparib response.

Results: High miR-509-3 expression indicated better response to platinum and longer progression-free and overall survival in two independent ovarian cancer patient cohorts (high vs. low miR-509-3 expression; PFS: TCGA P < 0.05, Qilu P < 0.05; OS: TCGA P < 0.05, Qilu P < 0.01). MiR-509-3 could impair the proliferation, migration, and invasion ability but enhance the sensitivity to Olaparib of ovarian cancer cell in vitro and in vivo by directly targeting HMGA2 and RAD51. In two PDX cases (PDX1 and PDX9), miR-509-3 could significantly increase the sensitivity to Olaparib along with the decrease of RAD51 positive rate (mean tumor weight NC + Olaparib vs. miR-509 + Olaparib; PDX1 P < 0.05, PDX9 P < 0.05). Additionally, in PDX8, miR-509-3 treatment dramatically reversed the Olaparib insensitivity (P < 0.05) by downregulating RAD51 expression. RAD51 functional detection revealed that all Olaparib sensitive cases exhibited low RAD51 positive rate (lesser than 50%) in treated groups. Furthermore, among the four HR gene mutation patients, three harbored HR core gene mutation and were sensitive to Olaparib while the remaining one with non-HR core gene mutation did not respond well to Olaparib.

Conclusions: MiR-509-3 can sensitize ovarian cancer cells to Olaparib by impeding HR, which makes it a potential target in PARPi synergistic treatment. HR core gene analysis and RAD51 functional detection are prospectively feasible in prediction of PARPi response.

Keywords: PARPi; PDX; RAD51; Synthetic lethality; miR-509-3.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
MiR-509-3 is significantly increased in P-sen group and indicated better prognosis. a The TCGA cohort was divided into two groups according to the clinical dataset: platinum-sensitive group (n = 159) and platinum-resistant group (n = 140). Heat map was drawn by DEGseq R package and illustrated the six top increased miRNAs and 6 top decreased miRNAs between two groups. b MiR-509-3 expression is elevated in platinum-sensitive group compared with platinum-resistant group in TCGA cohort (P < 0.05). c Overall survival (OS) and d progression-free survival (PFS) in survival curves for TCGA cohort with miR-509-3 low expression or high expression (high vs low miR-509-3 expression: OS, hazard ratio = 0.644, 95% confidence interval (CI) = 0.48 to 0.99 vs. hazard ratio = 1.551, 95% CI = 1.008 to 2.083, P < 0.04; PFS, hazard ratio = 0.708, 95% CI = 0.546 to 0.956 vs. hazard ratio = 1.412, 95% CI = 1.045 to 1.829, P < 0.02). e Relative miR-509-3 expression level was detected by RT-qPCR in 126 HGSOC patients from Qilu hospital. The platinum-sensitive group exhibited higher miR-509-3 expression level than platinum-resistant group (P < 0.01). f OS and g PFS in Qilu hospital cohort which was stratified into miR-509-3 low and high expression group whose cutoff value was defined by the medium (high vs. low miR-509-3 expression: OS, hazard ratio = 0.550, 95% CI = 0.334 to 0.830 vs. hazard ratio = 1.817, 95% CI = 1.205 to 2.987, P = 0.007; PFS, hazard ratio = 0.657, 95% CI = 0.432 to 0.929 vs. hazard ratio = 1.522, 95% CI = 1.076 to 2.314, P = 0.024)
Fig. 2
Fig. 2
MiR-509-3 inhibits ovarian cancer cells proliferation and invasion. a Cell cycle analysis of A2780, HEY, and UWB1.289 when transfected with miR-509-3 mimics. miR-509-3 increased the percentage of G0/G1 phase significantly in A2780, HEY, and UWB1.289 cell lines. b MTT and plate clonogenic assay of three ovarian cancer cells. MiR-509-3 decreased relative cell viability at different time points (day 2 to day 5) and cell clonal number in 6-well plates. c Transwell assay was performed to determinate the effects of miR-509-3 overexpression on migration and invasion of A2780, HEY, and UWB1.289 cells. MiR-509-3 decreased the number of migrated cells. d Intraperitoneal tumor formation assay of HEY or UWB 1.289 in nude mice. MiR-509-3 impaired the number of abdominal metastasis (HEY, 15.33 ± 2.40 vs. 7.33 ± 1.20, P < 0.05; UWB1.289 27 ± 4.35 vs. 9 ± 1.55, P < 0.05) and tumor burden (HEY, 0.52 ± 0.07 vs. 0.25 ± 0.04, P < 0.05; UWB1.289 0.39 ± 0.07 vs. 0.15 ± 0.02, P < 0.05) in vivo. The red arrows indicate the tumor metastatic nodules. e Western blot of the EMT and cell cycle associated markers in miR-509-3 overexpressing cells. MiR-509-3 significantly downregulated ZEB1, N-cad, β-Catenin, Vimentin, Snail, and Slug but upregulated E-cad expression. G0/G1 arrest related markers CDK4, CDK6, CCND1, and p21 were also downregulated
Fig. 3
Fig. 3
MiR-509-3 augments ovarian cancer cell sensitivity to cisplatin and Olaparib in vivo and vitro. a Relative cell viability of HEY and UWB1.289 which were exposed to different dose of cisplatin for at least 36 h and then measured by MTT method. MiR-509-3 decreased relative cell viability at various drug concentrations (HEY: 2, 4, 8 μg/mL, P < 0.05; UWB1.289: 2, 4, 8 μg/mL, P < 0.05) when compared with NC cells. b Relative cell viability of HEY and UWB1.289 which were treated with different dose of Olaparib (HEY: 10, 20, 30, 40, 80 μM; UWB1.289: 20, 40, 80, 120, 160 μM) for over 48 h. MiR-509-3 increased the response to Olaparib in HEY (10, 20, 30, 40 μM, P < 0.05) and UWB1.289 (20, 40, 80 μM, P < 0.05) cell lines. c Experimental design of tumor formation test in BALB/c nude mice. Four-week-old mice were subcutaneously injected with HEY or UWB1.289 cells (8 × 106 cells per site) overexpressing miR-509-3 or corresponding NC. At the sixth week, tumor-bearing mice received intraperitoneal injection of Olaparib (50 mg/kg, once a day) for 2 weeks and then were sacrificed to harbor tumor tissues which were weighted and photographed. d, e At the eighth week of mice life, tumors harbored from each group were showed and the tumor weight was compared among four groups (data are mean ± SD, n = 5) in HEY and UWB1.289. Olaparib and miR-509-3 combined treatment indicated the lowest tumor weight (g) among four subgroups (HEY, 0.76 ± 0.05, 0.39 ± 0.14, 0.33 ± 0.14, 0.12 ± 0.06; UWB1.289, 1.02 ± 0.17, 0.48 ± 0.26, 0.41 ± 0.14, 0.15 ± 0.008)
Fig. 4
Fig. 4
HMGA2 and RAD51 are direct targets of miR-509-3. a Putative binding sites in the HMGA2 3’UTR. There were three putative miR-509-3 binding sites and binding sequence was deleted as mutated type, which were cloned into pmirGLO vector respectively. b HEK293T cell was used in the Dual-Glo luciferase assay system. The luciferase activity of pmirGLO plasmid with wild-type (WT) HMGA2 3′UTR is weaker than that with mutant-type (MT) in HEK293T cell (site2, P < 0.001; site3, P < 0.0001). c Correlation between miR-509-3 and HMGA2. Relative HMGA2 mRNA level was negatively correlated with miR-509-3 level in RT-qPCR assay of tumor samples (Pearson r = − 0.3844, P = 0.015). d–f Exhibited the similar research toward RAD51 and concluded that miR-509-3 also directly targeted RAD51. g, h HMGA2 and RAD51 mRNA level in miR-509-3 overexpressing cells. Relative mRNA expressions were downregulated when cells were transfected with miR-509-3 mimics in A2780, HEY and UWB1.289. i Western blot assay demonstrated that miR-509-3 remarkably reduced protein expression level of HMGA2 and RAD51 in HEY and UWB1.289. h IHC representative image of subcutaneous tumors. The tumor tissues were fixed by formalin and cut into 4-μm-thick sections to be incubated by HMGA2 or RAD51 primary antibody. The expression of HMGA2 and RAD51 were both reduced by miR-509-3 in IHC images of HEY and UWB1.289
Fig. 5
Fig. 5
MiR-509-3 increases sensitivity to Olaparib by downregulating RAD51 and impairing HMGA2-ATM axis in ovarian cancer cell. a, b Relative cell viability of HEY and UWB1.289 which were transfected with two HMGA2 siRNAs and then treated with Olaparib for 48 h in accordance with the previous concentration gradient. The viability curve illustrated that HMGA2 downregulation enhanced the cell response to Olaparib significantly. c Western blot assay confirmed the interference efficiency of two HMGA2 siRNAs. And the total ATM and activated ATM protein level were both decreased along with the down-regulation of HMGA2. d Co-immunoprecipitation of HMGA2 and ATM protein. HEY cells were lysed and mixed with rabbit anti-human HMGA2 polyclonal antibody or normal rabbit IgG. Anti-HMGA2 antibody could co-immunoprecipitate ATM and HMGA2 while immunoglobin G (IgG) had no effect. e, f Rescue impact of HMGA2 on the response to Olaparib. Transfection of HMGA2 cDNA (in pEnter vector) could abrogate the miR-509-3-induced Olaparib sensitivity to Olaparib in HEY and UWB1.289 cell lines. The cell viability curves of different groups were drawn by four colors. g Protein level of four rescue groups. For the four treatment groups, transfection of HMGA2 cDNA could indeed upregulate the HMGA2 protein level and total ATM expression down-regulation was rescued by up-regulation of HMGA2 in miR-509-3 overexpressing cells. h HEY and UWB1.289 cells with miR-509-3 or NC were exposed to irradiation at different dose (0, 2, 4, 6 Gy) to induce DNA damage and repair system. MiR-509-3 overexpressing group significantly exhibited a lower protein level of ATM-CHK1/2 pathway (phospho-ATM, phospho-CHK1, phospho-CHK2 and phospho-H2AX.) and RAD51. i IF images of RAD51 foci in HEY and UWB1.289. Cells were treated with irradiation of 4Gy and 4 h later, RAD51 functional nuclear foci formation was detected by IF photographed and was found to be remarkably reduced by miR-509-3. Nuclei were stained with DAPI (blue)
Fig. 6
Fig. 6
Genomic characterizations of the established PDX cases and Olaparib or miR-509-3 treatment responses in PDX models. a Ovarian cancer-driven gene mutation landscape of cases. Tumor and paired normal tissues of nine enrolled patients were sequenced by WES and 157 genes associated with ovarian and/or breast cancer susceptibility or tumorigenesis were annotated. The landscape of 9 PDX cases revealed the chief mutated genes in these HGSOC patients. The TP53 mutation rate is 88.89% (8/9). b HR pathway gene mutation landscape of cases. The landscape demonstrated that 44.44% (4/9) cases harbored HR gene mutation in which BRCA1/2 accounted for 75% (3/4). c The procedure of PDX model establishment and application in this study. P0 tumor tissues were obtained from gynecological surgery and implanted subcutaneously as P1. In the treatment groups (P3), AAV-miR-509-3 or AAV-NC were injected intraperitoneally twice within one week and then treated with Olaparib (50 mg/kg) once a day for 2 weeks. d Typical cases of miR-509-3 sensitization. PDX1 and PDX9 were representative cases in which miR-509-3 could enhance the sensitivity to Olaparib (mean tumor weights of NC + DMSO, NC + Olaparib, or miR-509-3 + Olaparib group respectively: PDX1, 1.073 ± 0.244, 0.553 ± 0.135, 0.143 ± 0.042; PDX9, 0.973 ± 0.195, 0.570 ± 0.087, 0.183 ± 0.110). Dotted red lines circled the tumor site in the abdomen of NCG mice. e The summarized list of detailed characteristics of each PDX cases including treatment status, sample origins, HR gene mutation status, endogenic miR-509-3 expression, HMGA2-positive rate, RAD51-positive rate, and mean tumor weight of three treatment groups (showed as means ± SD). In this figure, label “*” means this case is sensitive to Olaparib. Label “#” means miR-509-3 has sensitizing effect
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69(1):7–34. doi: 10.3322/caac.21551. - DOI - PubMed
    1. Bookman MA. Optimal primary therapy of ovarian cancer. Ann Oncol. 2016;27(Suppl 1):i58–i62. doi: 10.1093/annonc/mdw088. - DOI - PubMed
    1. Vaughan S, Coward JI, Bast RC, Jr, Berchuck A, Berek JS, Brenton JD, et al. Rethinking ovarian cancer: recommendations for improving outcomes. Nat Rev Cancer. 2011;11(10):719–725. doi: 10.1038/nrc3144. - DOI - PMC - PubMed
    1. Roos WP, Thomas AD, Kaina B. DNA damage and the balance between survival and death in cancer biology. Nat Rev Cancer. 2016;16(1):20–33. doi: 10.1038/nrc.2015.2. - DOI - PubMed
    1. Pilie PG, Tang C, Mills GB, Yap TA. State-of-the-art strategies for targeting the DNA damage response in cancer. Nat Rev Clin Oncol. 2019;16(2):81–104. doi: 10.1038/s41571-018-0114-z. - DOI - PMC - PubMed

Publication types

MeSH terms

-