| Purpose:(1) McroRNAs are widely expressed in various tumor tissues, nevertheless serum microRNA is seldom reported, especially in prostate cancer. According to the existing expression profile of microRNA, miR-20a, miR-21, miR-125b, and miR-141 have been documented to overexpress in prostate cancer. We designed to detect these four microRNAs in the serum of patients with prostate cancer, and to compare different expression pattern in different stage of prostate cancer, to achieve an overview of the expression profile of these microRNAs and validate their role in predicting treatment efficacy and prognosis of patients.(2) According to the findings in the first part of our study, the levels of serum miR-21 were significantly higher in hormone refractory prostate cancer patients than those in hormone-sensitive prostate cancer patients, and also correlating to serum PSA levels, which indicated that miR-21 might play an important role in the proliferation of prostate cancer cells. Inhibiting the expression of miR-21 might slow down the growth of tumor cells, even reversing the androgen-independent status. In order to investigate the intrinsic molecular mechanism, we further studied the effect of specific miR-21 antisense oligonucleotides on the growth of LNCap prostate cancer cells. MiR-21 is a potentially novel target of gene therapy.Materials and methods:(1) Specific TaqMan probes for real-time quantitative PCR were employed to detect serum miR-20a, miR-21, miR-125b, and miR-141 in 56 patients (including 20 cases of localized prostate cancer,20 cases of hormone sensitive prostate cancer with bone metastasis,10 cases of hormone refractory prostate cancer with bone metastasis, and 6 patients of benign prostatic hyperplasia). U6 snRNA was used as an inner standard. One-Way ANOVA test was applied to compare the differences of serum miR-20a, miR-21, miR-125b, miR-141 in the above 4 groups of patients, to find out the novel predictors for the diagnosis, treatment and prognosis of patients with prostate cancer.(2) The specific miR-21 antisense oligonucleotides were transfected to LNCaP prostate cancer cells through Lipofectamine 2000. Growth curves were drawn to observe effect of miR-21 antisense oligonucleotides on the proliferation of tumor cells. Forty-eight hours after transfection, proteins were extracted from both the cytoplasm and nucleus of tumor cells. Protein levels of PTEN, mTOR, AKT, AR, and PSA were detected by western-blotting method in the cytoplasm and nuclear proteins, seperately.Results:(1) The levels of serum miR-21 were significantly higher in hormone refractory prostate cancer patients than in hormone-sensitive prostate cancer patients, p =0.016. Stratified analysis in patients with hormone sensitive prostate cancer showed that levels of serum miR-21 were lower in patients with PSA levels of less than 4ng/ml than in those with PSA levels of more than 4ng/ml. Furthermore, Serum miR-21 levels were higher in patients who were resistant to docetaxel-based chemotherapy when compared to those sensitive to chemotherapy, p=0.032. The expressions of serum miR-20a and miR-125b showed no statistical differences between the 4 groups of patients. Serum miR-141 expressed higher in patients with bone metastases than in those with localized prostate cancer and those with benign prostatic hyperplasia, p=0.006, and its level even correlated to the number of bone lesions, p<0.001. What’s more, serum miR-21 and miR-141 were also found to be positively related to Gleason Score, and patients with higher Gleason Score had higher serum miR-21 and miR-141 levels, p=0.001and p<0.001, respectively.(2) The cell growth curve showed that the specific miR-21 antisense oligonucleotides could inhibit the growth of LNCaP prostate cancer cells. After transfection, the AR in nucleus was down regulated, which induced the reduction of cytoplasm PSA expression. Furthermore, the nuclear AKT level also decreased after the transfection of specific miR-21 antisense oligonucleotides, which might cause the mTOR protein traslocating from nucleus to cytoplasm.Conclusion:(1) It is feasible to detect MicroRNAs in the serum of prostate cancer patients. Serum miR-21 was highly expressed in hormone refractory prostate cancer patients with bone metastases, and it might have predictive value for the efficacy of docetaxel-based chemotherapy. Serum miR-141 overexpressed in patients with bone metastasis. Thus, serum miR-21 can be a potential predictor for hormone-refractory prostate cancer, and serum miR-141 be used as a biomarker for bone metastasis.(2) The proliferation of LNCaP prostate cancer cells can be significantly inhibited by down regulating the expression of miR-21 in tumor cells. MiR-21 may have the function of up-regulating AR and AKT in prostate cancer cells, and the expression of mTOR protein might traslocate from the cytoplasm to the nucleus. Therefore, after inhibition of miR-21, expressions AR, mTOR and AKT proteins in the nucleus decreased, leading to the restriction of cell proliferation. |
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