Bone Marrow-mesenchymal Stem Cells Repress The Prostate Cancer Cells By Mirna-29c

BackgroundProstate cancer (Pca) is one of the common malignant tumor in men. In the United Statesthe new cases of prostate cancer is241740in2012, and Pca is the second diagnosedmalignancy and the first cause of death. In our country, as a result of population aging,changes in diet and the improvement of diagnostic technology, the morbidity and mortality ofprostate cancer has gradually risen. Although the incidence of prostate cancer in China is farlower than the western countries, there is no denying that it has been increasing dramatically.Pca as an important disease endangering health of older men is worthy of attention. In ourcountry, most patients had lost the opportunity of surgical therapy when Pca was detected atadvanced stage. For those patients with advanced Pca, standard treatment is to adopt theendocrine and chemical medication therapy, to achieve the goal of castration (that is, castrationtherapy). For the advanced patients with metastasis occurred, the effective rate of castrationtherapy is satisfied, but most of these patients will relapses in3years or so. And at present thelack of effective treatment for advanced Pca eventually lead to death of the patients.Stem cells have the ability of self-renewal, high proliferation and multi-lineagedifferentiation potential, who as a new treatment has broad application prospects. Autologousstem cell is easy to obtain, but also avoid the problem of the histocompatibility. In addition, itwas found that the relationship between mesenchymal stem cell (MSCs) and tumor cells are soclose. MSCs can be recruited to tumor tissue to participate in the reshaping of the tumormicroenvironment, and crosstalk between the two produce significant influence on themechanics biological behavior, including the tumor cell growth, proliferation, invasion andmetastasis, but the action mechanism of crosstalk remains unclear.miRNA was recovered in recent years as a kind of endogenous non-coding small RNAmolecules. Recent studies have shown that miRNA play an important role in cancer initiationand development by negatively control the expression of tumor associated genes. IndividualmiRNA can regulate the expression of multiple target genes at the same time, so targetedmiRNA interference in theory can more effectively inhibit the biological behavior of the tumor.So far, only a handful of miRNA proved to involved in the progress of prostate cancer. In the usual study, we first found that androgen-independent prostate cancer and androgen-dependentprostate cancer have multiple differently expressed miRNA molecules by the miRNAexpression profile chip technology,and preliminarily confirmed that miR-663can be used asthe independent prognostic molecule for advanced prostate cancer. Alex Yuan et al found thatMSCs can carry and release of micrornas molecules to play a corresponding role. Combinedwith the interaction of MSCs to tumors, we have reason to believe that the miRNA can serveas important clues to reveal biological effects of MSCs on prostate cancer.ObjectiveFor MSCs can significantly inhibit proliferation and invasion of prostate cancer LNCaPcell, we can get the differently expressed the miRNA expression spectrum between Pca cellssuppressed by MSCs and wild Pca cells. To clear the miRNA of most obvious differentexpression and validate it in cells and cell culture level, we use multiple bioinformaticssoftware to predict the potential target genes of miR-29c. The predicted target genes will begiven GO function clustering and pathway analysis, and we preliminarily judge and verifypotential target genes of miR-29c(such as C7ORF24、COL6A2and PIK4CA). The functionand possible mechanism of miR-29c/C7ORF24, COL6A2, PIK4CA pathways were furtherexplored in the process of MSCs suppress prostate cancer cells, which offers a new strategyand thought for clinical treatment of prostate cancer.Methods1. Extracting, separating, purifying and cultivatingBM-MSCsfrom healthy adult bonemarrow tissue, and identifying MSCs through morphology and flow cytometry technology tolay the right foundation for subsequent work.2. MSCs was cultured with prostate cancer LNCaP cell by Transwell technology.Observing proliferation and invasion of prostate cancer LNCaP cells, and clearing thebiological effect of MSCs to prostate cancer LNCaP cells.3. Screening miRNA from prostate cancer LNCaP cell cultured with MSCs and wild typeprostate cancer LNCaP cells, and selecting the miRNA of obvious different expression(miR-29c)and validating it in cells and cell culture level. Clearing miR-29c which secreted byMSCs are passed to prostate cancer LNCaP cells, and examining the biological effect ofmiR-29c to prostate cancer LNCaP cells.4. Through the miRNA database and bioinformatics analysis, screen the target protein genes of miR-29c and clear target genes exact binding site. Through the real time-PCR andWestern blot test, we can clear the relationship of target genes controlled by miR-29c and thedisease.Results1. Separating and purifying MSCs from healthy adult bone marrow tissue Successfully,which present morphologic stability, better refraction and growth liking fish samples. Flowcytometry test showed that MSCs express marker molecules on the surface of mesenchymalstem cell which include CD29, CD90, CD73and CD105, without expressing CD14, CD34and CD45.2. It was found in our study that MSCs can significantly inhibit proliferation and invasionof prostate cancer cells by Transwell technology, which proved that MSCs can inhibit thebiological function of prostate cancer cells.3. Using the miRNA expression profile chip technology, we select the differentexpression of miRNA between Pca suppressed by MSCs and parent Pca,and miR-29c havemost obvious different expression. real-time PCR was performed to verify that miR-29cexpression in prostate cancer cells cultured with MSCs is obviously higher than that inparental cells.And it is found that miR-29c expression in prostate cancer cells culture solutioncultured with MSCs is obviously higher than that in parental cells culture solution, whichfurther proving miR-29c are secreted by MSCs and are passed to prostate cancer cells.4. CCK-8method to detect that prostate cancer LNCaP cell proliferation was significantlysuppressed which was transfected miR-29c. Flow cytology was performed to detect that Sphase cells of prostate cancer LNCaP cell proliferation obviously decrease which wastransfected miR-29c. It is proved that miR-29c can inhibit the growth and division of prostatecancer cells.5. Transwell invasion experiment found that the number of prostate cancer LNCaP cellstransfected miR-29c through the matrigel was decreased obviously than that of control groups,which was prompted that miR-29c inhibits invasive ability of LNCaP cell.6. Multiple bioinformatics software was performed to predict that potential target genesof miR-29c,then the predicted target genes be given GO function clustering and pathwayanalysis, and we preliminarily judge potential target genes of miR-29c are C7ORF24、COL6A2and PIK4CA. After prostate cancer LNCaP cells transfected miR-29c,the real time-PCR and Western blot test were performed to confirme that C7orf24, COL6A2, PIK4CAin the mRNA and protein levels decreased significantly.It is showed that C7orf24, COL6A2,PIK4CA may be target genes of miR-29c.Conclusions1. This study by invitro function test of MSCs act on prostate cancer LNCaP cell isshowed that, MSCs can promote LNCaP cell proliferation by increasing the number of S phasecells of LNCaP cell. At the same time we also confirmed that MSCs increases LNCaP cellinvasive ability by increasing the cells number of prostate cancer LNCaP cells through theTranswel matrigel. So it is showed that MSCs play an important role in the growth andprogression of prostate cancer.2. Using the miRNA expression profile chip technology, we select the most obviousdifferent expression of miR-29c between Pca suppressed by MSCs and parent Pca, and verifyin cells and cell culture level miR-29c differentially expressed. It is showed that miR-29cwhich secreted by MSCs are passed to prostate cancer LNCaP cell, and miR-29c may be thekey molecules of MSCs inhibit prostate cancer cells.3. Through LNCaP cell transfected miR-29c of simulation test, We found that miR-29ccan obviously inhibits the proliferation and invasive ability of LNCaP cell. To some extent,this also proves that miR-29c can inhibit the biological behaviour of prostate cancer.4. And by searching for its target genes it is find C7orf24, COL6A2, PIK4CA may beimportant target proteins which change the proliferation and invasive ability of LNCaPcell.However,miR-29c often acts on multiple pathways and protein,and the combination ofmiR-29c and protein is still need to improve by reporter gene assay.