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Construction of microRNA-199a Lentiviral Vector and It's Effects on the Biological Functions of Androgen-independent Prostate Cancer Cells

Author: ZhangDongXu
Tutor: XuDanFeng
School: Second Military Medical University
Course: Surgery
Keywords: miR-199a lentiviral vectors proatate cancer DU-145 oncobiology
CLC: R737.25
Type: Master's thesis
Year: 2013
Downloads: 21
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Abstract


[Background and Objective]Prostate cancer (PCa) is common in older men urinary malignancies. Themorbidity and mortality were ranked the global male cancer incidence in the secondand sixth place. In the United States, the incidence of prostate cancer was ranked inthe first place among male malignancy and second place of cancer death in men overthe age of40. In China and other Asian countries, prostate cancer incidence is muchlower than in Europe and America and other developed regions, but with the increaseddegree of aging of Chinese society, the improve tumor detection means and diet,living habits, living environment changes, the incidence was significant growth.Therefore, prostate cancer is more and more attention. Its occurrence, development,deterioration mechanisms and treatment research has become one of the hot researchof Urology filed.Early prostate cancer (PCa) patients often present no clinical symptoms, thoughthe widely application of serum prostate-specific antigen (PSA) screening means, itsdetection rate improved significantly, but there is still a considerable proportion ofdiagnosed prostate cancer patients in the disease progression period: high serum PSAvalues, high Gleason score, and the occurrence of local or distant bone metastases,thus losing the opportunity to select endocrine therapy instead of radicalprostatectomy. Castration treatment of androgen-dependent prostate cancer is efficacy,including surgical and medical castration. These two methods may promote tumor cellapoptosis, and lower serum PSA values, relieve pain and other symptoms, andeffectively delay tumor progression and metastasis.Endocrine therapy namelymaximum androgen blockade (MAB), its effitive time is short with only14-30months median time, and in the late stage, almost all patients will developandrogen-independent prostate cancer (AIPC).Micro-RNA was endogenous non-coding RNA molecules, which was composedof about18to25single-stranded nucleotides. Studies have shown that manyMicro-RNA not only play a role in the synthesis of a polypeptide chain, but also caneffectively regulate transcriptional gene expression, cell proliferation, differentiation,apoptosis, and metabolic processes. It also play an important regulatory role of tumorcell biologythe characteristics and the regulation mechanism is the most popular in recent years in areas such as medicine, biology, which prompted RNA-mediatedgenetic information regulatory networks were exited in vivo. A Micro-RNA canregulate a plurality of target genes, and a target gene can be simultaneously regulatedin a plurality of Micro-RNA, the different types of tumors as well as tumor formation,progression, deterioration in the different stages, and each has its own uniqueMicro-RNA abnormal expression of the spectrum. Generally believed that in animalcells, Micro-RNA mainly through the not completely complementary with theencoded protein mRNA base pairing binding, causing inactivation of the targetmRNA, degradation or translation hindered, resulting in the post-transcriptional levelgene regulation.Studies suggest that microRNA-199a (abbreviated as miR-199a) inhibit thegrowth of tumor cells in many human malignancies, such as breast cancer, colorectalcancer, osteosarcoma, and so on, but whether miR-199a paly regulatory role inprostate cancer cells and related regulatory mechanism has so far not been reported inthe literature. In view of the lentiviral vector system can be efficiently, stably importgene into mammalian primary or tumor cell lines and can be long-term and stableexpression in vivo. Besides, it has the advantages of high security for body’s immunesystem and the Internal Environment impact relative small. We intend to through genemicroarray verify that miR-199a is differentially expressed in prostate cancer tissueand surrounding normal prostate tissue, and build miR-199a lentiviral excessiveexpression vector, studying whether miR-199a expression levels change has impact tothe prostate cancer androgen-independent cell tumor biological characteristics, andlay the foundation for miR-199a molecular targeted therapy for the prostate cancerpatients.[Methods and Results]Part I: Construction of microRNA-199a overexpressionlentiviral vector and its efficiency validation.The lentiviral vector system can be stably and efficiently to import the desiredgene into mammalian primary or tumor cell lines. Besides, it has the advantages ofhigh security for body’s immune system and the Internal Environment impact relativesmall. Therefore, we chose a lentiviral vector system to build pCDH-miR-199a vector.First, according to the reported Genebank query gene sequence, synthetic specific primers (with restriction sites), the real-time PCR technology to amplify the targetgene fragment, RT-PCR product and vector was digested before connected to pCDHsystem, after the positive plasmid transfected293T cells, lentiviral got,concentrated,and titer determination, vector has high concentration titer lentiviral throughfluorescence microscopy imaging. The pCDH-miR-199a lentivirus vector infected theprostate cancer androgen-independent DU-145cells, detected by PCR, the resultsconfirmed that the pCDH-miR-199a lentivirus vector can significantly raised the levelof miRNA-199a expression in DU-145cells. The above results lay the foundation forthe subsequent study of how the changes in the miRNA-199a expression levels impactthe DU-145cell tumor biological characteristics.Part II: Significantly lower expression of microRNA-199a inclinical prostate cancer (PCa) tissues compare with adjacentnormal tissues (16pairs of tumor and adjacent tissues).Firstly, we use gene chip technology found that miR-199a in the presence ofsignificant expression differences between clinical prostate cancer specimens andadjacent normal prostate tissue.16pair of the surgical removal of the prostate cancerand cancer adjacent normal tissues were used for the experimental study, thepositioning of the tumor tissue generally based on the preoperative magneticresonance imaging (MRI) examination, preoperative puncture site of pathology reportpositive tumors, prostatectomy tissue hardness touch. Above freezing and fixedspecimens through RNA extraction, poly (A) tail, reverse transcription reaction andRT-PCR reaction, the results found that miR-199a expression level was significantdifferences in these16pair pathological defined diagnosis as prostate cancer and thesurrounding normal prostate tissue. The result show that compare with normalparacancerous tissues, The expression levels of miR-199a decreased significantly inprostate tumor tissues (P=0.07). The above results indicate that miR-199a is highlyinvolved in the regulation of pathological process of the prostate cancer formation anddevelop to the hormone independent PCa.Part III: High expression of miR-199a suppresses DU-145prostate cancer cell proliferation, migration, colony formation,apoptosis and induces cell cycle arrest at G0/G1phase.In the third part of this experiment, we use lentiviral-mediated miR-199a high expression vector to study the impact of miR-199a on the prostate cancerhormone-independent cell biological characteristics. DU-145cell lines were chosenfor the study, prostate cancer DU-145cells were effectively infected by lentiviralvector. In the fifth day after the viral infection we found lentiviral infection efficiencyof DU-145cells was high in an inverted fluorescence microscope. We use cellproliferation assay DU-145cell growth condition, and found that compared with theblank and negative control group, high expression of miRNA-199a can significantlyinhibited DU-145cell proliferation and growth, and OD value in the Reader595nmDetection was lower. We designed Transwell experiments to study the impact of highexpression of miRNA-199a on the migration ability of DU-145cells, found that highexpression of miRNA-199a significantly inhibited the migration of prostate cancerDU-145cells. Tumorigenic ability of tumor cells was measured by colony formingability of DU-145cells in the plate, we found that compared with the con, Lv-shcongroup, Lv-oxmiRNA-199a group significantly suppress the colony-forming ability ofDU-145cells, with the performance of small number and size of clones, and poorability of clone dispersed into the group. To further investigate the impact ofmiRNA-199a on DU-145cell cycle, we use PI staining flow cytometry technique todetect cell cycle distribution and apoptosis proportion. According to the various stagesof the cell cycle contained different DNA, we apply this theory to determine the cellcycle distribution and found that highly expressed miRNA-199a in DU-145cells caninduce G1/S phase arrest. In addition, highly expression of miRNA-199a canpromote DU-145cells apoptosis.[Conclusions]Based on the above three parts experimental results, we consider: from chiptesting and clinical tissue samples verify that miRNA-199a expressed significantlylower in prostate cancer. Lentiviral vectors can efficiently raised target moleculemiRNA-199a expression level in prostate cancer cells. MiRNA-199a can be used as a tumor suppressor to inhibit tumor biology functions and features of prostate cancercells. In view of prostate cancer from hormone-dependent to non-dependentmechanism is not yet clear, miRNA-199a may be designed to suppress and becomemolecular target for the treatment of androgen-independent prostate cancer.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Male genitalia tumors > Prostate cancer
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