Experimental Study Of MicroRNA-198Down Regulates Livin Gene Expression And Interferes The Apoptosis And Invasion In Prostate Cancer Cells

Prostate cancer is a malignancy and it can hazard the health of old men, theincidence was increasing in our country. Patients normally died of tumor cellsmetastasis. Livin gene is one member of IAPs family, and it can inhibit the endogenousand exogenous apoptotic pathway to increase the anti-apoptotic ability. Livin encodestwo isoforms, which called Livin-α and Livin-β, and previous studies suggested that itstissue expression and anti-apoptotic mechanism wasn’t consistent. Livin was specificexpression in tumor tissues and no expression or only a small amount in normal tissues.However, recent studies have shown that the regulation of tumor cell invasion andmetastasis may be the general properties of multiple IAPs family members, and Livingene may also regulate tumor cell invasion and metastasis directly.MircorRNAs（miRNAs）are non-coding RNAs, which can degrade the mRNA orinhibit the mRNA translation to decrease the expression of target protein, thus it canplay the role of modification in regulating the protein post-transcription. The previousstudy in our group found that miRNA-198could down regulate the Livin expression inprostate cancer cells.Objective1. Detect the expression of Livin gene in prostate cancer tissues, and study therelationship between the Livin gene expression and clinical stage, pathological grade,prognosis.2. Investigate the mechanism of Livin gene in regulating the prostate cancer cellsinvasion, and investigate the different mechanism in regulating prostate cancer cellsinvasion of two isoforms for further research.3. Investigate the effect of miRNA-198on cell proliferation, apoptosis, thesensitivity to chemotherapy, invasion and cell cycle in prostate cancer cells. Methods1. Prostate cancer tissues were used immunohistochemistry firstly, and43caseswere grouped by pathological grade and clinical stage. Pathological grade groupedaccording to Gleason score:2-4was divide into highly differentiated carcinoma（6cases）,5-7was divide into moderately differentiated carcinoma（21cases）,8-10wasdivide into poorly differentiated carcinoma（16cases）; Clinical stage grouped accordingto TNM staging:27cases were non-metastatic tumors（T1-4N0M0） and16cases weremetastatic tumors（T1-4N1-2M0or T1-4N0-2M1）. Immunohistochemical score wasaccording with the number of positive cells on immunohistochemical staining and dyingdegree, and all patients were follow-up.2. Transfected siRNA and plasmid firstly, and we used western blot assay to detectthe expression of Livin in LNCaP, PC-3, DU-145cell lines, then used the transwellassay to detect the cells invasion, followed by using the MTT assay to detect the cellsviability. In order to study the mechanism of regulating tumor cell invasion by Livin,then we used the immunofluorescence assay to detect the NF-κB activity and westernblot assay to search the downstream molecular.3. Transfected miRNA-198mimic firstly, and we used RT-PCR and western blotassay to detect the Livin expression and screen the density for next experiments, thenused MTT assay, transwell assay, etc. to detect the prostate cancer cells proliferation,apoptosis, sensitivity to chemotherapy, invasion and cycle change.Results1. Livin gene expresses in prostate cancer tissues and its clinical significanceThere were35cases expression of Livin gene and mainly expressed in thecytoplasm, the positive expression rate was81.4%. Then we found that Livin expressionwas higher in poorly differentiated group than highly differentiated group, and themoderately differentiated group was also higher than highly differentiated group.However, compared poorly differentiated group with moderately differentiated group,the statistic analysis showed no difference in Livin expression; in addition, Livinexpression was higher in the metastatic group than non-metastatic group. There was7cases recurrence by follow-up, and the recurrence rate was16.3%：the recurrence ratewas20%in Livin expression positive group and the recurrence rate was0in Livinexpression negative group, and the statistic analysis showed no difference.2. Experimental study of the Livin gene regulates prostate cancer cell invasionWe found that PC-3cells with high invasion ability expressed Livin-α and Livin-β, and LNCaP cells with low invasion ability only expressed Livin-α. Next,downregulating/upregulating the Livin expression could inhibit PC-3/increase LNCaPcells invasion ability. Then the same result was also showed in DU-145cells with highinvasion ability. In addition, downregulating/upregulating the Livin-α and Livin-βexpression in PC-3/LNCaP cells respectively, then we found that both isoforms couldregulate prostate cancer cells invasion and no difference in regulating capacity. In theend, downregulating/upregulating the Livin expression could inhibit/active the NF-κBsignaling pathway and then inhibit/increase the expression of Fibronectin and CXCR4.3. Experimental study of miRNA-198mimic inhibits the Livin gene expressionand interferes the apoptosis and invasion in prostate cancer cellsTransfected miRNA-198mimic into DU-145cell line, then used western blot todetect the Livin expression in DU-145cells, screened50nM as the minimum effectdensity and150nM as the maximum effect density for the next experiments. Then wefound that it could inhibit DU-145cells proliferation, induce apoptosis, increase theapoptotic sensitivity and inhibit invasion. In addition, transfected miRNA-198mimicinto PC-3cell line could degradate Livin-α mRNA and Livin-β mRNA, then made PC-3cells G₀/G₁phase increased and S phase decreased, which promoted prostate cancercells apoptosis.Conclusions1. Livin mainly expressed in the prostate cancer cells cytoplasm, and there werethe relationships between the Livin gene expression and clinical stage, pathologicalgrade; in addition, Livin expression group was more like to relapse, which there mayexist the relevance between Livin expression and tumor recurrence.2. Livin regulates prostate cancer cells invasion by impacting the NF-κB signalingpathway and the expression of Fibronectin and CXCR4. In addition, the two isoformscould regulate prostate cancer cells invasion respectively.3. miRNA-198mimic could inhibit prostate cancer cells proliferation, induceapoptosis, increase the apoptotic sensitivity and inhibit invasion. Then it could causeprostate cancer cells in the G₁/S cell cycle control point to regulate cell proliferation,which miRNA-198showed tumor suppressor in prostate cancer cells.