MiR-32 Induces Radioresistance By Targeting DAB2IP And Regulating Autophagy In Prostate Cancer Cells

Objective:The aberrant expression of micro RNAs(mi RNAs) has been found in various types of cancer. mi R-32 is an oncomi R in prostate cancer, however, the mechanisms by which mi R-32 functions as a regulator of radiotherapy response and resistance in prostate cancer(PCa) are largely unknown.To study whether the carcinogenesis of mi R-21 upon prostatic cancer is related to radio-sensitivity via observing impact of the mi R-32 targeted DAB2 IP upon prostatic cancer cell lines PC3 and DU145 for the radiotherapy effect; to study the relationship between mi R-32 and radiotherapy induced autophagy and apoptosis; to study the direct target relationship of mi R-32 and DAB2 IP within prostatic cancer cells, and to explore possible relevant mechanisms and to offer reference basis for the radiotherapy of prostatic cancer.Methods: Section I: Endogenesis Detection 1. 4 cases of prostatic cancer tissues, 2 cases of control paracancer tissues and 2 cases of benign prostatic hyperplasia tissues were enrolled, and QPCR was adopted to determine mi R-32 levels;2. QPCR was adopted to determine mi R-32 levels of prostatic cancer cell lines PC3 and DU145 and normal human prostate epithelial cells RWPE-1. 3. PC3 and DU145 cell were treated with 0, 2, 4 and 6 Gy IR, and QPCR was adopted to determine mi R-32 levels. This would contribute to appropriate IR dose selection for following experiments; 4. Luciferase reporter gene is adopted to determine the direct targeting relationship between DAB2 IP and mi R-32 in prostatic cancer cells.Section II: Functional Modeling 1. To synthesize pre-mi R-32 and anti-mi R-32 respectively, and to transfect DU145 and PC3 cells. 2. QPCR was adopted to determine the expressions of transfected mi R-32 and DAB2 IP m RNA. 3. WB was adopted to detect the expression level changes of DAB2 IP within the functional model. Groups : Con, pre-con, pre-mi R-32, anti-con and anti-mi R-32 ones.Section III: Functional Experiment 1. The SF analysis experiment was adopted to detect the changes of radiosensitivity, whole IR was adopted to treat PC3 and DU145 functional model cells; Groups: Con, pre-con, pre-mi R-32, anti-con and anti-mi R-32IR concentration: 0, 2, 4 and 6 Gy. 2. IR-induced autophagy determination. IR of 2Gy was adopted to process cells, and WB was adopted to determine the LC3 B I/II and Beclin 1 protein expression levels after 24 hours to reflect the changes of autophagy capacity; Groups: Con, pre-con, pre-mi R-32, anti-con and anti-mi R-32 3. IR-induced flow apoptosis determination. Flow determination was adopted to detect apoptosis in 2 Gy IR treated cells; Groups I: Con, pre-con, pre-mi R-32, anti-con and anti-mi R-32 Groups II: con, pre-con, pre-mi R-32, pre-con+BFA(4n M) and pre-mi R-32+BFA(4n M); 4. Talen was adopted to knock off the DAB2 IP inside prostatic cancer cells and build stable lines; 5. IR of 2 Gy was adopted to process PC2-DAB(-) and DU145-DAB cells. WB was adopted to determine autophagy related factors LC3 B I/II and Beclin 1 after 24 hours. Groups : con, pre-con, pre-mi R-32, anti-con and anti-mi R-32; 6. IR-induced flow apoptosis determination. Flow determination was adopted to detect apoptosis in 2 Gy IR treated cells; Groups: con, pre-con, pre-mi R-32, pre-con+BFA(4n M),pre-mi R-32+BFA(4n M) 7.Section IV: Signal Path Study: to study whether the mediation of mi R-32 targeted DAB2 IP is via m TOR-p S6 K pathway. 1. WB was adopted to detect the expression level changes of DAB2 IP, pm TOR, m TOR, p S6 K, LC3 B and Beclin 1 within prostatic cancer cells. Groups: Con、pre-con、pre-mi R-32、anti-con、anti-mi R-32Results: In this study, we found that DAB2 IP, the mi R-32-dependent tumor-suppressor gene, was downregulated and induced autophagy and inhibited radiotherapy-induced apoptosis in the PCa cell. mi R-32 expression was upregulated or overexpressed in PCa, and mi R-32 inhibited DAB2 IP expression through a direct binding site within the DAB2 IP 3’ untranslated region. mi R-32 mimics enhanced tumor cell survival and decreased radiosensitivity in the PCa cells, which were reversed by mi R-32 inhibitor. Flowcytometric analysis revealed that overexpressed mi R-32, consistent with the DAB2 IP knockdown results, reduced IR-induced cell apoptosis, which was restored by 4n M BFA(Brefeldin A) treatment. More significantly, the overexpressed of mi R-32 and knockdown of DAB2 IP enhanced autophagy in IR-treated PCa. mi R-32 regulated the expression of autophagy-related proteins, such as DAB2 IP, Beclin 1, LC3 B I/II, as well as phosphorylation of S6 K and mammalian target of rapamycin(m TOR). In conclusion, these data provide new insights into the mechanisms governing the regulation ofDAB2IP expression by mi R-32 and their possible contribution to autophagy and radioresistance in PCa.Conclusion: this study demonstrated that mi R-32 targeted directly on DAB2 IP in PCa, and induced DAB2IP-deficient radioresistant human PCa cells. Moreover, our findings demonstrated the critical role for mi R-32 in inhibiting m TOR-S6 K pathway and suppressing autophagy by targeting DAB2 IP. On the basis of these results, mi R-32 appeared to be the novel tumor promoter and play an important role in radiotherapy resistance during the treatment of PCa.