| Renal cell carcinoma accounts for third place of kidney cancer. Theincidence of RCC increased year by year, and there is prone in older,more common in men than women. Treatment is mainly dependent onrenal cell carcinoma and laparoscopic surgery etc, which often causedpostoperative complications and side effects of chemotherapy, greatlyreduce the quality of the life of patient. Therefore, finding noveltherapeutic targets and specific treatment is very important. Tumornecrosis factor-related apoptosis-inducing ligand (TRAIL) as a memberof the TNF family is capable of inducing apoptosis in renal cellcarcinoma. The purpose of this study is under the control of miRNAresponse element MRE, regulates the expression of TRAIL in cancertissue and normal tissue cells, thereby selectively killing tumor cells,without damage of normal tissues and organs. Specific studies are asfollows:1. Experimental Routes:1.1Expression of miR-138, miR-199within RCC cell line and tissuevs. normal cell line and tissue.1.1.1HK-2human normal kidney epithelium cell line, ACHN and786-O RCC cell lines and L-02human normal liver cells were cultured1.1.2Compare the differences of miR-138, miR-199expression level ofRCC cell line and normal via Real-time quantitative PCR.1.1.3Collected fresh tissue of patients with RCC and normal renaltissues. 1.1.4Compare the differences of miR-138, miR-199and expressionlevel of RCC tissue and normal renal tissue via Real-time quantitativePCR.1.2Expression of exogenous genes within RCC cells under thecontrol of MREs of miR-138, miR-199.1.2.1psiCheck2was reconstructed by inserting two copies of theseMREs into the downstream sites of luciferase coding region on thevectors, generating psiCheck2-3MREs.1.2.2Luciferase activity was determined in HK-2, L-02, ACHN and786-O cells,48h after the transfection of psiCheck2andpsiCheck2-3MREs, with Dual-Luciferase reporter system1.3Expression of TRAIL mediated by adenoviral vectors withinRCC cells under the regulation of MREs of miR-138, miR-199.1.3.1Ad-TRAIL-3MREs were generated with two copies of MREs ofmiR-138, miR-199immediately following the open reading frame.1.3.2Compare difference of TRAIL expression in L-02, and ACHNtransfected with Ad-TRAIL, Ad-TRAIL-3MREs and Ad-EGFP viawestern blot.1.3.3Compare difference of TRAIL expression in L-02, ACHN andliver cell transfected with Ad-TRAIL, Ad-TRAIL-3MREs and Ad-EGFPvia Real-time quantitative PCR.1.4Expression of TRAIL mediated by adenoviral vectors withinRCC cells under the regulation of abundance of miR-138, miR-199and miR-122.1.4.1Compare the differences TRAIL levels between L-02transfectedwith the inhibitor of miR-138, miR-199via western blot and Real-timequantitative PCR.1.4.2Compare the differences TRAIL levels between ACHN transfected with the mimics of miR-138, miR-199western blot and Real-timequantitative PCR.1.5Evaluate the apoptosis pathway (western bolt) and portion ofG0/G1cells of ACHN cells and L-02cells induced by Ad-TRAIL-3MREs.1.6MTT assay to examine the viability of RCC cell lines and normalcell lines, under the infection of recombinant adenoviruses.1.7Exam the growth of RCC tumors in mice by TRAIL-inducedapoptosis via the growth of ACTH, the expression of TRAIL and theapoptosis pathway under the injection of recombinant adenoviruses.1.8Investigate the hepatic tissue from TRAIL-induced cytotoxicity.1.8.1Harvest the blood and serum from the mice bear no tumoradministrated by PBS and recombinant adenoviruses.1.8.2Blood ALT level was evaluated by ELISA.1.8.3Exam the expression of TRAIL and the apoptotic pathway in livertissue administrated by recombinant adenoviruses via western blot.2. Result2.1evaluate the expression levels of miR-138, miR-199betweenRCC and normal cells.In primary RCC samples and RCC cell lines, miR-138, miR-199was found to be underexpressed, in comparison with normal control.miR-138, miR-199were overexpressed in normal kidney cells,HEK-293, and hepatic cells, L-02.2.2MREs of miR-138, miR-199and restrict the expression ofexogenous genes within RCC cells.The expression level of luciferase was greatly suppressed inHEK-293, and L-02transfected with psiCheck2-3MREs, compared with psiCheck2-transfected ones. However, there was no significantdifference in luciferase activity between RCC cell lines transfected withpsiCheck2and psiCheck2-3MREs.2.3Ad-TRAIL, Ad-TRAIL-3MREs and Ad-EGFP were added to theculture of L-02and ACHN cells, followed by the detection of TRAILprotein expression.The data indicated that Ad-TRAIL has no difference in its ability toexpress TRAIL between L-02and ACHN cells. In contrast, TRAILexpression was greatly suppressed in Ad-TRAIL-3MREs-infected L-02cells, but not in ACHN cells. qPCR assays further confirmed thatTRAIL mRNAs can be easily detected in Ad-TRAIL-andAd-TRAIL-3MREs-infected ACHN cells as well as Ad-TRAIL-infectedL-02cells. But there was no TRAIL mRNA in normal liver cellstransduced with Ad-TRAIL-3MREs.2.4The selective expression of TRAIL in Ad-TRAIL-3MREs-infected RCC cell lines is due to the regulation of miR-138, miR-199in cells.The immunoblot and qPCR assays indicated that TRAILexpression was partially restored in L-02cells transfected with theinhibitors of miR-138, miR-199. Accordingly, TRAIL expression wasmoderately reduced in ACHN RCC cells, which has increased levels ofmiR-138, miR-199due to the transfection of miRNA mimics.2.5The selective expression of TRAIL mediated by MREs-regulatedadenoviral vector can also lead to selective apoptosis in RCC cells.The results revealed that cleaved caspase3and PARP proteinswere highly expressed in Ad-TRAIL-infected L-02cells and ACHNcells treated with Ad-TRAIL or Ad-TRAIL-3MREs. In contrast, therewas no cleavage of caspase3and PARP in L-02cells transduced with Ad-TRAIL-3MREs.Increased portion of sub-G0/G1cells was observed in inAd-TRAIL-infected L-02cells and ACHN cells treated with Ad-TRAILor Ad-TRAIL-3MREs. However, the percentage of sub-G0/G1is quitelow in the L-02cell infected with Ad-TRAIL-3MREs.2.6Ad-TRAIL-3MREs was able to induce cytotoxicity to RCC cellswithout damage normal cells.The data indicated that Ad-TRAIL has no discrimination inreducing the survival of cancer cells and normal cells. In contrast,Ad-TRAIL-3MREs exerts a high selectivity to RCC cells in inducingcytotoxicity.2.7The in vivo growth of RCC can be inhibited by Ad TRAIL-3MREs treatmentThe growth of ACHN xenograft was greatly suppressed in the micetreated with Ad-TRAIL and Ad-TRAIL-3MREs. Furthermore,immunoblot analysis of TRAIL expression revealed that there was nosignificant difference in TRAIL expression between the tumors injectedwith Ad-TRAIL and Ad-TRAIL-3MREs. Also, the cleavage of caspase3and PARP was detected in the two groups of tumor.2.8Ad-TRAIL-3RMEs can protect liver from TRAIL-inducedapoptosisALT level was significantly heightened in the mice treated withAd-TRAIL. In contrast, serum ALT levels did not change in the micetreated with Ad-TRAIL-3MREs, in comparison with Ad-EGFP and PBS.Furthermore, immunoblot analysis of TRAIL and apoptosis-relatedproteins indicated that Ad-TRAIL activated the apoptotic pathway inliver tissue, whereas Ad-TRAIL-3MREs did not affect the apoptotic 3. Conclusions3.1The level of miR-138, miR-199was reduced in RCC cells and tissuecompared with the normal ones.3.2MREs of miR-138, miR-199can effectively suppress the expressionof exogenous genes in normal cells.3.3MREs of miR-138, miR-199can be used for restrictadenovirus-mediated TRAIL expression in RCC cells.3.4The selective expression of TRAIL mediated by adenoviral vectorresulted from the levels of miR-138, miR-199.3.5The selective TRAIL expressions were able to induce RCC-specificapoptotic event.3.6MREs-regulated adenovirus is a targeted anti-tumor agent withoutcytotoxicity to normal cells.3.7Ad-TRAIL-3MREs was able to suppress the growth of RCC tumorsin mice via TRAIL-induced apoptosis.3.8MREs-regulated TRAIL expression protected liver from TRAIL-induced toxicity. |
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