Cisplatin Regulates SH-SY5Y Cell Growth Through Downregulation Of BDNF Via MiR-16

Neuroblastoma is one of the most common childhood tumors, usually diagnosed at a median age of17months. The tumor originates from neural-crest tissues, deeply in the adrenal medulla and paraspinal ganglia, with no specific clinical presentations. About50%to60%patients are diagnosed with neuroblastoma in advanced stages. In spite of multidisciplinary care, even the introduction of dose intensive chemotherapy, outcome of high risk neuroblastoma has no significant improvement, with5-year survival rate of<50%.MicroRNAs (miRNAs) are small non-coding RNA, involved in post-transcriptional regulation of gene expression. Through binding to the imperfect sequence of the target mRNAs, miRNAs result in translational inhibition or destabilization of target mRNAs. MiRNAs can act as tumor suppressors or as oncogenes. MiR-106a was showed to be upregulated in gastric cancer, having pro-tumorigenic effects. MiR-16functions as a tumor suppressor and was downregulated in mantle cell lymphoma SP cells by regulating Bmil, leading to reduction in tumor size following lymphoma xenografts.Brain-derived neurotropic factor (BDNF), a member of neurotropin family, plays a critical role in neuron survival, differentiation and axon wiring through binding to its preferred receptor TrkB (tyrosine kinase receptor B). Researches have demonstrated the BDNF-TrkB signals can not only regulate the growth of nerve neutron, but also affect many tumors development, invasion and outcome, including lung cancer, bladder, pancreatic, etc. A recent study showed that BDNF could enhance the proliferation and survival of transitional cell carcinoma. High expression levels of BDNF are associated with more aggressive malignant behavior in human cancers, including pancreatic cancer and breast cancer.Cisplatin is an effective drug for the treatment of neuroblastoma, which can inhibit the growth of neuroblastoma cells. However, the molecular mechanism of cisplatin in anti-tumor remain elusive. In previous studies, we found that the expression of miR-21significantly decreased following cisplatin treatment, and miR-21could down-regulate the expression level of MSH2in cells. The results suggest we should explore the changes of miRNA expression caused by cisplatin, and detest the changes of downstream molecules, so that we can further clarify the antitumor mechanism of cisplatin, and provide scientific basis for the development of (?) new anti-cancer drugs. Based on the currently accepted effect of miR-16and BDNF and the predictable correlation between them, in this study, we choose miR-16and BDNF to explore the molecular mechanism of cisplatin in antitumor.To study the expression of miR-16and BDNF in neuroblastoma cells, and the relationship among cisplatin, miR-16and BDNF, three experiment parts were designed. The first part is to study cisplatin inhibiting the growth of SH-SY5Y cells and the expression of related molecular, miR-16and BDNF. The second is to study the mechanism of miR-16suppressing SH-SY5Y cells growth. The third is to study cisplatin inhibiting growth of SH-SY5Y cell xenografts and its mechanism.Part1The study of cisplatin inhibiting growth of SH-SY5Y cells and the expression of related molecules, miR-16and BDNFObjective:To study growth of SH-SY5Y cells treated with cisplatin and then elucidate the expression of related molecular, miR-16and BDNF in SH-SY5Y cells. Methods:SH-SY5Y cells in good condition were planted into cell culture plates. On the following day, different concentrations of cisplatin(0,1.5,3,4.5,6and12μg/ml, separately) were dissolved in the culture medium. After48h, experiments were respectively carried out as follows:(1) The inverted phase contrast microscope was used to observe the state and quantity of the cells;(2) MTT was added into each well,4h later the culture medium was discarded,100μl DMSO was added in order to dissolve the crystals, the OD value was measured with an enzyme-linked immunosorbent assay reader at570nm and cell growth inhibition rate was calculated;(3) The collected cells were double-dyed by Annexin V-FITC and PI, the apoptosis rate under different cisplatin concentration was analyzed by flow cytometry instrument;(4) SH-SY5Y cells following cisplain treatment were collected to extract the total RNAs, the expression of miR-16was detected by real-time quantitative PCR;(5) Total protein was extracted for electrophoresis and transferred to PVDF membrane, hybrided with anti-BDNF polyclonal antibody, then enzymed-second antibody was used to make the substrate show color, finally the expression of BDNF in cells was observed after being took picture.Results:The density of living cells gradually reduced with the increasing concentration of cisplatin, and the number of floating dead cells increased. MTT results showed that the cell growth inhibition rate reached more than half with6μg/ml cisplatin, and the inhibition rate was positively correlated with cisplatin concentration; Flow cytometry instrument testing results showed that the apoptosis rate of0μg/ml group was obviously lower than those of other groups, cell apoptosis rate increased with the increase of cisplatin concentration; The result of real-time quantitative PCR demonstrated that the expression of miR-16was significantly rised in SH-SY5Y cells after cisplatin treatment; The result of western blotting showed that the expression of BDNF was significantly reduced with3μg/ml cisplatin.Conclusions: Cisplatin can inhibit the growth of SH-SY5Y cells and increase cells apoptosis; at the same time, it can also elevate the expression of miR-16; otherwise, the expression of BDNF decreased after cisplatin treatment.Part2Research on mechanism of miR-16inhibiting SH-SY5Y cell growthObjective:To clarify the influence of miR-16on the growth of SH-SY5Y cells and predict the correlation between miR-16and BDNF, and finally prove the influences of miR-16on the expression of BDNF.Methods:Chemically synthesized miR-16or negative control oligonucleotides were transferred into SH-SY5Y cells by liposome, when the cells density of SH-SY5Y in culture plates reached about60%. They were incubated for48h after replacing medium.48h later, subsequent experiments were carried out as follows:(1) The state and quantity of the cells were observed under the inverted phase contrast microscope;(2) The OD value of each group was measured by MTT assay, cell growth inhibition rate was calculated by formula;(3)After double-dyed by Annexin V-FITC and PI, the cell apoptosis rate induced by miR-16was analyzed by flow cytometry instrument;(4) To predict the correlation between miR-16and BDNF by bioinformatics analysis; to detect the expression of BDNF in SH-SY5Y cells transfected with miR-16by Western blotting.Results:The quantity and density of SH SY5Y cells transfected with miR-16decreased significantly than normal and NC group, cell morphology and the ticking to the wall changed; MTT assay showed cell proliferation inhibition rate was obviously higher in miR-16transfected group(figure7), there was statistically significant difference(p< 0.01) using the statistical t-test analysis. With V-FITC/PI double dye, cell apoptosis rate was33.7%in miR-16transfected group, which was significantly increased compared with the control group; In order to confirm the correlation between miR-16and BDNF, bioinformatics analysis was performed to find that BDNF-3’-UTR targed to miR-16; Further by Western blotting test, the result showed that BDNF expression decreased obviously after miR-16transfected.Conclusions:MiR-16can act as tumor suppressor gene, which can inhibit cell proliferation and promote cell apoptosis; There is a matching site between BDNF-3’-UTR and miR-16; MiR-16can down-regulate expression of BDNF to play a tumor suppressor role.Part3Inhibitory effect of cisplatin on SH-SY5Y cell xenografts and its mechanismObjective:At individual level, we explored the influence of cisplatin to the growth of nude mice xenografts; We further improved that cisplatin could influent the express of miR-16and BDNF in the xenografts cells, and increase the express of miR-16which could further downregulate the expression of BDNF.Methods:Plenty of SH-SY5Y cells were cultured to be injected subcutaneously into the nude mice back for making animal model; after being narcotized with pentobarbital sodium, nude mice were injected with1×107SH-SY5Y cells each point. When the tumor volume grew to100mm3, experimental group were intraperitoneally injected with3mg/kg cisplatin and the control group were injected with the same amount of saline. Cisplan was injected every four days, and4times all together; Four days after the last treatment, nude mice were sacrificed, then tumors were extracted from the back skin. We observed tumors from the same group, took pictures; weighed each tumor with electronic scales, recorded and calculated the average. RNA was extracted from the tumor cells, and then real-time quantitative PCR was performed to detect miR-16expression, protein was extracted for electrophoresis, transfer printed, antibody hybridization, finaly chemical spectral imager was applied to observe the protein bands, analysed BDNF expression in transplanted tumor.Results:After the nude mice were subcutaneously injecting SH-SY5Y cells, we observed the tumor every day, and estimated the size of the tumors; About5to7days later, when the tumor volume reached about100mm3, the mice were treated with ciplatin; After the last treatment, the xenografts tumors were took out, we foud that tumors volume with cisplatin treatment was obviously reduced, so cisplatin can inhibit the SH-SY5Y cell proliferation in nude mice; We weighed the tumors using precised electronic scales, the weight of saline group was0.135±0.015g, the weight of cisplatin group was0.057±0.018g; The result of real-time quantitative PCR showed that the expression of miR-16in the tumor treated with cisplatin was significantly increased compared with the control, this was consistant with experimental results of cellular level. Western blotting result showed that after being treated with cisplatin, the expression of BDNF in SH-SY5Y cell xenografts was obviously reduced.Conclusions:Cisplatin can restrain the growth of SH-SY5Y cell xenografts in the nude mice and increase the expression of miR-16; while the expression of BDNF was reduced.Overall Conclusions:Cisplatin can inhibit the proliferation of SH-SY5Y cells, promote cell apoptosis, as well as inhibit the growth of SH-SY5Y cell xenografts in nude mice; Cisplatin can play the role by increasing the expression of miR-16, and miR-16can inhibit the expression of BDNF in both the cells and the nude mice xenografts; At the level of cell and individual, cisplatin both plays the role of inhibiting the cell growth via up-regulating miR-16expression, and finally down-regulating BDNF expression.