Biological Effects And Specific Identification Of The Peptide NYZL1 Binding To Human Bladder Carcinoma Cell

Objective:1. To study the effects of NYZL1 on proliferation, migration, apoptosis and other characteristics of bladder cancers cells in vitro, to provide the reference information for looking for NYZL1 receptor.2. The molecular probe of FITC-NYZL1 specifically bound bladder cancers were identified by immunofluorescence detection, in order to provide the ideal vehicle for bladder cancer targeting therapy.3. On the basis of the above experiment, collected fresh urine of clinical patients, analyzed the probe of FITC-NYZL1 targeting urine exfoliated cells and its practicality in diagnosing and monitoring bladder tumors. Methods:1. The peptide NYZL1 and its control peptide svNYZL1 were synthesized by solid-phase peptide synthesis. Purified and marked green fluorescent dye fluorescein isothiocyanate)(FITC).Using MTT and Wound-Healing experiment in vitro to study the NYZL1 effects of proliferation and migration on BIU-87 cell; Using flow cytometry technology to analysis the cell cycle distribution and cell apoptosis induced by NYZL1 treatment, Exploring the effect of NYZL1 peptide on biology behavior of BIU-87 cell.2. By laser scanning confocal microscope(LSCM) to detect the specificity of FITC-NYZL1 combined with bladder cancer BIU- 87, T24, E-J and renal cancer cells.3. Collected urine of bladder cancer patients in clinical, fresh urine was centrifuged, dropped pieces, added FITC-NYZL1 probe to incubate. The LSCM was used to observe the combination of probe after the cells were incubated with probe. At the same time, fresh urine was collected for Wright-Giemsa examination. Through cell chemical dyeing method, detected the positive rate of bladder cancer cells in urine exfoliated to verify the combination of the target peptide NYZL1 on bladder cancer cell specificity. Results:1. The peptide was successfully synthesized by NYZL1(CSSPIGRHC) peptides and sv-NYZL1(CRIGSPHSC) peptide(control peptide that is the amino acid sequence and structure NYZL1 the same, but different combinations of sequence) by Biochemical Co., Ltd. in Hangzhou. FITC-labeled, polypeptide FITC NYZL1 and randomized polypeptide FITC-svNYZL1 by HPLC and MS purification and identification of the synthesis of cyclic peptide amino acid sequence synthetic peptide purity of 98% or higher.2. MTT assay results showed that, compared with the control peptide, NYZL1 has no obvious inhibitory effect cell proliferation of bladder cancer BIU-87(P>0.05), the cell growth curve showed that the bladder cancer cell with NYZL1 has no significant difference with the other two groups(P>0.05). There was no statistically significant difference, suggesting NYZL1 does not directly affect the growth of tumor cells. We used wound healing test to study NYZL1 role in bladder cancer cell migration aspects. The results showed that with the extension of the time, scratch the NYZL1 group cell coverage area and the area of the control group and blank control group showed no significant difference, suggesting NYZL1 on migration BIU-87 cells had no effect.3. We used flow cytometry to analysis the distribution of BIU-87 cell cycle which was binding to NYZL1 probe. The results showed that: Compared with the control peptide, NYZL1 had no significant effect(P>0.05) on bladder cancer BIU-87 cell cycle distribution, there was no statistically significant difference. We then examined the apoptosis of BIU-87 cells, compared with the control group, with the increase of NYZL1 concentration, cell apoptosis had no significant effect(P>0.05), suggesting that NYZL1 distribution of cell apoptosis and cycle had no significant effect.4. Immunofluorescence identification NYZL1 specific shows that compared with the control peptide, FITC-NYZL1 addition to binding to BIU-87 cells, can also be combined with other bladder cancer, and there is no significant difference among groups binding(P > 0.05), confirming NYZL1 specifically bind to bladder cancer cells.5. FITC-NYZL1 can mark all patients with bladder cancer in urine exfoliated cancer cells, and in different stages of bladder cancer, the probe cell binding rate is also different, the higher of the tumor staging, FITC- NYZL1 combined with cell specificity rate is higher. Combined with the low rate of the control group, the difference of the experimental group and control group was statistically significant(P <0.001). Conclusions:1. Targeting peptide NYZL1 has no inhibitory effect on the proliferation, migration, apoptosis, and other biology behavior of bladder cancer cell.2. Peptide NYZL1 has significant targeting effect in bladder transitional cell carcinoma. It has specific binding sites on the surface of cancer cells. The results provides reference information for clinical application of NYZL1, screening and identification of NYZL1 receptors.We may choose NYZL1 as a candidate molecule for bladder cancer diagnosis and targeted therapy used in clinical studies.3. FITC-CSSPIGRHC can specifically bind to bladder cancer cells. The detection rate of bladder cancer cells had improved in urine exfoliated cells. This suggests that targeting peptide may serve as a new technique for the noninvasive diagnosis and clinical monitoring of bladder tumor, and lay the theoretical basis for early diagnosis of bladder cancer and targeted drug research in the future.