| Background and objectives:Renal cell carcinoma (RCC) is one of the most common seen tumors in urinarysystem and the mechanism of RCC remains unclear, a highly aggressive RCC, early detection is not easy, the time of diagnosis about 35% of patients have been transferred, 2-year mortality rate is as high as 95%, about 40% of patients with metastasis or recurrence, 3-year survival rate is less than 5%. Once lymph node metastasis, even if the radical lymph node dissection, the patient survival time is less than five years. The general conservative treatment including radiotherapy, chemiotherapy and immunotherapy has unsatisfactory results, radiotherapy and chemotherapy is not used as adjuvant therapy. In recent years, biological and targeted therapy of advanced renal cell carcinoma has made some achievements, but the effect is still very limited. With the diagnostic and surgical techniques improved, the prognosis of renal cell carcinoma has a certain degree of improvement. However, recurrence and metastasis is still a threat to the patients. Therefore, prevention and treatment of metastatic renal cell carcinoma is the focus of research. As we all know metastasis with malignant tumors are the main causes of death and are one of the characteristics, enhance of tumor cell motility lead to invasive growth and migration, any molecular involved in tumor cell motility are likely to become a key elements of tumor metastasis, actin protein expression and spatial arrangement lead to the changes of cell motility, many factors both inside and outside cells involved with the change of actin protein expression and distribution.Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family , the linker between membrane protein and cytoskeleton, plays an important role in the cellular morphology, cytoskeleton reorganization, adhesion, invasion and metastasis. Recently, many studies have indicated that ezrin proteins are up-regulated in various kinds of tumors .However; such studies have not yet been done on human RCC.Objective:1. To study the association of ezrin protein and ezrin mRNA expression in RCC and its significance.2. To construct a eukaryotic expression vector encoding a shRNA targeting ezrin, using RNA interference inhibited ezrin expression in human RCC cell line 786-0, and observed the changes in biological characteristics of 786-0 cell. On the one hand, to clarify the role of ezrin in the occurrence and development of RCC, On the other hand to explore the value of RNA interference technology in gene therapy of RCC.Material and methodsThe expression of ezrin in renal cell carcinoma and its clinical significance.1. Collection of 80 cases of renal cell carcinoma and 40 cases of corresponding adjacent normal renal tissue, the expression of ezrin were detected by using of tissue microarray and immunohistochemical staining; Ezrin immunoreactivity was scored 0 to 12 (absent, weak, moderate or strong staining). One independent observer was calculated.2. The renal cell carcinoma tissues were divided into the single renal cell carcinoma tissue (neither lymph node metastasis nor tumor thrombus of the inferior vena cava), the renal cell carcinoma tissue with lymph node metastasis,the renal cell carcinoma tissue with tumor thrombus of the inferior vena cava .the expression of ezrin mRNA and protein were compared using reverse transcriptase-polymerase chain reaction (RT-PCR),and Western blot. Ezrin mRNA and protein expression level in tumors and adjacent normal tissues were calculated from the signal intensity. Gene expression levels were normalized by the corresponding gene expression levels of at b-actin.To construct a eukaryotic expression vector encoding a shRNA targeting ezrin.According to the ezrin mRNA sequence in GeneBank,2 pair oligo nucleotides were designed and synthesized . After primer annealing, they were inserted into plasmid pGeneSil-1 to construct the shRNA eukaryotic expression vector. The recombinant plasmid were transformed into DH5a,and the positive strain were identified by enzyme digestion and sequence analysis.The recombinant plasmid were transfected into 786-0 cells with Lipofectamine 2000, transfected cells were grown for 48 hours and then examined by fluorescence microscopy, the transfection efficiency were evaluate by fluorescence microscope.Effects of inhibition of ezrin by RNA interference on proliferation,apoptosis and invasion of human renal carcinoma cell line 786-0 cell.1. To evaluate the inhibition efficiency:①Cells in exponential growth phase were plated in 24-well plates containing antibiotic-free medium at 30% confluence and incubated overnight, then transfected with shRNA (shRNA-ezrin or shRNA-hk) using lipofectamine 2000 and Opti-MEM according to the manufacturer's protocol. Silencing was examined 48h after transfection.total RNA was extracted from cells using TRIzol according to the manufacturer's protocol.Real-time quantitative reverse transcription PCR (qRT-PCR) was performed with SYBR PrimeScript, b-actin was used as an internal control,relative mRNA level of ezrin was calculated according to the equation: △△Ct={Ct(ezrin)-Ct(β-actin)}transfected cells-{Ct (ezrin)-Ct (β-actin)}untransfectedcells. Normalized by b-actin level.Relative mRNA level of ezrin = 2-average△△CTinhibition efficiency = (1 -2-average△△CT)×100%.All experiments were performed in triplicate.②786-0 cells at 80-90% confluence were transfected with shRNA (shRNA-ezrin orshRNA-hk) using lipofectamine 2000 after 48h; total proteins were extracted fromcells for Western blot.2. MTT 786-0 cells were plated into 96-well plates at 4×106 per well in the presence or absence of chitosan nanopar-ticles for 1-7 days. During the last 4 hours of each treatment, cells were plused with MTT fter 30 minutes, OD570 was determined using a microplate reader.3. Cell proliferation and apoptosis Flow cytometry was used to further assess apoptosis in cells,cells were collected at 48h after transfection, and then cells were washed with 1×PBS and fixed with70% ethanol,finally 1×106 cells were analyzed by flow cytometry, the percentage of apoptotic cells in sub-Gl phase was calculated using Multicycle software. The changes of ultrastructure in 786-0 line cell were oberseved by Projection electron microscopy.4. Cell adhesion ananlysis To observe cell adhesion changes of 786-0 cells after inhibiting expression of ezrin by RNAi.5. Chemotactic motion assay The altered chemotactic motility of 786-0 cells mediated by RNAi was observed by chemotactic motion assay.6. Soft agar colony-formation assay Soft agar colony-formation assay was employed by RNAi was observed the altered anchor independent growth ability of 786-0 cells mediated by RNAi.Results 1. Ezrin protein expression and localization in the cell cytoplasm and membrane ,the expression of ezrin protein showed a significant difference between the renal cell carcinoma (95% ,76/80)and the adjacent normal renal tissue ( 75% , 30 /40 ) (X2=8.501 P=0.004);the mean weighted ezrin score of ezrin immunostaining in the group of lymph node metastasis were higher than those in no lymph node metastasis group (9.04±1.37 vs 7.45±1.75, t=4.385,P<0.001), the group of of tumor thrombus of the inferior vena cava (IVC) were higher than those in no tumor thrombus of the inferior vena cava group(9.00±1.79 vs 7.63±2.03, t=2.480, P=0.015);there was no correlation between the expression of ezrin protein and age,gender,stage,tumor size,histological grade and histological subtype of renal cell carcinoma (P > 0.05).2. A significant difference of ezrin mRNA and protein was observed in adjacent normal renal tissues and renal carcinoma tissue by RT-PCR and Western blot.the expression level of ezrin mRNA and protein were up-regulated in renal carcinoma tissues as compared with adjacent normal renal tissues (P< 0.01, P< 0.05) ; the expression level of ezrin mRNA in the group of lymph node metastasis and the group of tumor thrombus of the inferior vena cava were higher than those in the single renal cell carcinoma tissue (P< 0.01, P< 0.05) ; There was no significant difference of ezrin mRNA and protein between the group of lymph node metastasis and the group of tumor thrombus of the inferior vena cava (P = 0.318, P = 0.462).3. To construct an eukaryotic expression vector encoding an shRNA targeting ezrin. CD The DNA fragments were insert into vector pGeneSil-1, which acted as a template for the synthesis of shRNAs that corresponded to ezrin (shRNA- ezrin and shRNA-hk), under the control of the U6 promoter that directs the synthesis of a polymeraseⅢ-specific RNA transcript. The shRNAs were composed of 2 identical 19-n sequence motifs in an inverted orientation that were separated by a 9-nt spacer.Recombinant ezrin eukaryotic expression vector pGenSil-1-ezrin was constructed successfully.②786-0 cells transfected with pGeneSil-1-ezrin were cultured successfully. After 24hour incubation,GFP(green fluorescent protein) expression cells was assessed withafluorescence microscopy , number of GFP positive cells per field of view atmagnification of 200 was counted ,the GFP-positive cells showed no differencebetween the lipofectamine-shRNA-treated cells( P > 0. 05). in each group the numberof GFP-positive cells accounted for 80-83% ,this result demonstrated thatlipofectamine was able to delivery of shRNA into cells.4. Effects of inhibition of ezrin by RNA interference on proliferation,apoptosisand invasion of renal cell line 786-0①Fluorescence quantitative PCR results showed that: transfected cells were grownfor 48 hours, all groups have some degrees of ezrin gene amplification,the relativeexpression levels of ezrin mRNA were 100%, 94.61% , 33.45%, 47.63% respectivelyin the group of p-shRNA-0,p-shRNA-hk,p-shRNA-ezrinl and p-shRNA-ezrin2,inhibition efficiency of shRNA-ezrin1 group is up to 66.55%.②Western blot showed that :there was no significant difference of ezrin among ofthe groups after transfected 24h (F=0.793, P=0.512) ,the expression of ezrin proteincould be markedly inhibited by shRNA-ezrinl and shRNA-ezrin2 after transfected72h (F= 11.549,P< 0.001) .③MTT assay: the proliferation rate of 786-0 cells expressing shRNA-ezrin wasreduced as demonstrated by a decrease OD values in a time-dependent manner,significant differences in viability were observed after 3-7 days, compared with theuntransfected 786-0 cells.④Projection electron microscopy: 786-0 cells transfected were found somephenomenon of nuclear margination, and fragmentation.⑤Apoptosis of 786-Ocells after 48h treatment of shRNA-ezrin detected by flow cytometry.Apoptotic rate of cells treated with shRNA-ezrinl for 48 hour was significantly higher than those of untransfected cells and 786-0cells transfected with shRNA-hk (p<0.01) ; Compared with the cells treated with shRNA-ezrin,the percentage of G0/G1 cell was significantly higher than those of untransfected cells and 786-0cells transfected with shRNA-hk (p<0.01) and PI(proliferation index) was decreased in the cells treated with shRNA-ezrin compared with those of untransfected cells and 786-0cells transfected with shRNA-hk (p<0.01) .⑥Heterotypic cell adhesion ananlysis showed the numbers of adhesive cell in 786-0 cells transfected with shRNA-ezrinl were less than the 786-0 cells untransfected and transfected with shRNA-hk(p<0.01).⑦Chemotactic motion decreased in 786-0 cells transfected with shRNA-ezrinl,and the transwell 786-0 cells transfected with shRNA-ezrinl, 786-0 cells untransfected and 786-0 cells transfected shRNA-hk were 8.000±1.789,11.500±1.871,12.667±1.367 under×200 visual field respectively (p<0.001) .⑧Soft agar colony-forming assay showed less 786-0 cells transfected with shRNA-ezrinl colonies than 786-0 cells untransfected and 786-0 cells transfected by shRNA-hk,with 17.500±2.258, 29.333±2.066, 30.500±2.074 respectively (p<0.001) .Conclusions1. Ezrin was expressed in the renal cell carcinoma tissue and adjacent normal renal tissues and correlated with adverse prognostic factors.2.The ezrin expressed in lymph node metastasis and tumor thrombus of the inferior vena cava renal carcinoma were higher than those in no lymph node metastasis and no tumor thrombus of the inferior vena cava renal carcinoma; there were no correlation between the expression of ezrin protein and age,gender,stage,tumor size,histological grade and histological subtype of renal cell carcinoma. 3. Recombinant ezrin eukaryotic expression vector pGeneSil-1-ezrin was constructed successfully.4. 786-0 cells transfected with p-GeneSil-1-ezrin were cultured successfully, fluorescence quantitative PCR results showed that inhibition efficiency of shRNA-ezrinl is up to 66.55%.5. After transfected with p-GeneSil-1-ezrin, the capablity of 786-0 cells of proliferation, invasion and adhersion were decreased dramatically; the percentage of G0/G1 cell and apoptotic rate was significantly higher than those of untransfected cells.Summarily, membranous translocation of ezrin might play a role during malignant transformation and disease progression of RCC, ezrin expression represents a prognostic factor for RCC.
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