| Prostate cancer is a common malignant tumor in male. The morbidity and mortality of prostate cancer is keeping in high level in developed countries. In our country, the incidence of this desease is on the rise in recent years.Now, more and more evidence shows that mi RNAs are involved in the development of tumor by playing oncogenes or tumor suppressor genes function. mi RNA is a non-coding single stranded RNA of about22 nucleotides in length in cells. Through binding to the 3’UTR of target m RNA, mi RNA regulates the expression of target gene by inhibiting the translation of target m RNA or degrading the target m RNA, mi R-141 is a member of the mi R-200 family, which is located on chromosome 12. The pre-mi RNA precursor of mi R-141 in the cytoplasm can be processed to produce mi R-141-3p and mi R-141-5p two kinds of mature mi RNA.Research shows that, compared with the normal prostate tissue, mi R-141(mi R-141-5p)expression in prostate cancer tissues is up-regulated. And also, the expression of mi R-141 is able to be detected in the peripheral blood of prostate cancer patients. A small samples study, aming to early phase or metastatic prostate cancer patients, showed that mi R-141 and mi R-375 are the molecular biomarkers for diagnosis and prognosis assessment of prognosis. Analysis of mi RNA expression profile in serum of prostate cancer patients showed that compared with normal control, 15 kinds of mi RNAs are up-regulated. Of which, mi R-141 was correlated with locally advanced prostate cancer. The mi R-141 used in the relevant literature are mi R-141-5p(5’-UAACACUGUCUGGUAAAGAUGG-3’).Whether the expression of mi R-141-3p play a role in prostate carcinogenesis and development or not, needs to be deciphered. It was reported that AR signaling pathway is closely related with the development of prostate cancer. By binding to AR, androgen delivers the extracellular signal to the intra-cell and influences the expression of down-stream target genes. For early stage prostate cancer cells, the growth of cells is androgen dependent and the therapy is effective by blocking androgen pathway. And recent studies have shown that, for those of androgen independent prostate cancer cells,endocrine therapy can not obtain effective treatment. At the same time, the expression of AR in tumor cells and the amount of AR positive cells did not decrease significantly in the whole tumor tissues. Other study show that mi R-141 is able to promote the transcriptional activiety of AR by targeting degrading the expression of SHP, indicating that there is an interaction between mi R-141 and AR. Our preliminary study showed that mi RNA-141-3p is lower expressed in prostate cancer cells compared to normal cells. By using Targetscan and Micro Cosm, we found that this is possible binding site in 3’UTR of AR gene. Further to explore the function of lower expression of mi R-141-3p on the growth and proliferation of LNCa P cells and its mechanism is conducive to understanding the mechanism of prostate cancer from a new perspective. This study will proved new theoretical and experiment basis for prostate cancer therapy.Method:(1) Real-time PCR was used to detect the expression of mi R-141-3p in LNCa P and PC-3 prostate cancer cells, Sao S-2 osteosarcoma cells, Si Ha cervical cancer cells,BEAS-2B normal lung epithelial cells and HUVEC vein endothelial cells;(2) MTT assay,colony formation assay and flow cytometry apoptosis Detection technology were used to detect the effects of mi R-141-3p on LNCa P cell proliferation and apoptosis;(3) Targetscan and Micro Cosm software were used to analysis the target gene of mi R-141-3p;(4) By transfecting mi R-141-3p mimics into LNCa P cells, RT-PCR and western blot were used to detect the expression of AR;(5) p MIR-AR-3’UTR luciferase reporter vector, internal control plasmid p RL-luciferase, negative control mimics, mi R-141-3p mimics or negative control inhibitor, mi R-141-3p LNCa P cells were transfected into LNCa P cells, luciferase reporter detection system was used to detect the activity of luciferase;(6)p MIR-AR-3’UTR-Mut vectors were constructed. p MIR-AR-3’UTR-mut vectors,p RL-luciferase, negative control mimics, mi R-141-3p mimics, negative control inhibitor,mi R-141-3p inhibitor were transfected into LNCa P cells, luciferase reporter detection system was used to detect the activity of luciferase;(7) Chemical synthesis ARE sequence was ligated into p GL3-Promoter vector and p GL3- ARE vector was constructed, luciferase reporter detected system was used to detect the dual luciferase activity;(8) RT-PCR was used to dectect the expression of CDC6 and MKX.Results:(1) RT-PCR results showed that compared with normal cells, BEAS-2B and HUVEC, expression of mi R-141-3p in LNCa P cells was lower, while the expression of mi R-141-3p in Sao S-2 Si Ha were higher.(2) The results of MTT detection, plate clone formation assay and flow cytometry showed mi R-141-3p can inhibit the proliferation of LNCa P cells and promote apoptosis of LNCa P cells.(3) Analysis by Targetscan and Micro Cosm showed that mi R-141-3p is highly complementary to 3’UTR of AR m RNA.(4) Compared with the control group(control transfected with Negative mimics), the expression levels of AR m RNA and AR protein in experimental group decreased significantly.(5) Luciferase reporter assay system results showed that mi R-141-3p mimics decreased the luciferase activity of p MIR-AR-3’UTR comparing to the control group.While, mi R-141-3p inhibitor increased the luciferase activity of p MIR-AR-3’UTR compared to the control group.(6) Dual luciferase reporter assay system for p MIR-AR-Mut results showed that co-transfection of p MIR-AR-Mut, p RL-luciferase,mi R-141-3p mimics/mi R-141-3p inhibitor did not change the activity of luciferase compared to the control group, which was transfected with p MIR-AR-Mut-NC mimics or p MIR-AR-Mut-NC inhibitor.(7) The results showed that mi R-141-3p was able to decrease the luciferase activity of p GL3-ARE compared to the control group.(8)Real-time PCR results showed that mi R-141-3p mimics transfection decreased the m RNA expression levels of CDC6 and MKX compared to the control group; while mi R-141-3p inhibitor transfection increased the m RNA expression levels of CDC6 and MKX compared to the control group.Conclusion: mi R-141-3p was lower expressed in LNCa P cells; mi R-141-3p can inhibit the proliferation of LNCa P cells and promote the apoptosis of LNCa P cells;This study confirmed that AR is the target gene of mi R-141-3p;ARE dual luciferase reporter detection showed that mi R-141-3p expression can decrease the expression of CDC6 and MKX,which are AR specific down-stream genes. |
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