The Effects And Molecular Mechanisms Of Neurotensin And Its Receptor NTSR1 On Proliferation Of Glioblastoma Cells

Malignant gliomas are highly invasive and lethal cancer which account for approximately 80% of adult primary malignant brain tumors. Although current therapy rang from surgery, chemotherapy to radiotherapy, the median length of survival with gliblastoma multiforme(GBM) is still less than 15 months. The rapid growth nature of glioblastoma is the result of a consequence of genomic instability and dysregulation of gene expression.Unlocking the rapid progression of glioblastoma requires an improved knowledge of the molecular mechanisms underlying glioblastoma cell proliferation and tumor growth.Neurotensin(NTS) is a 13 amino-acid peptide that was extensively studied as a neurotransmitter and hormone in the central nervous system as well as periphery(in particular digestive system). Additionally, NTS and its high affinity receptor(NTSR1) are involved in the progression of several malignant tumors and considered as a potential target for treatment. However, the biological function of NTS/NTSR1 in glioblastoma has been rarely studied. In our previous study, we found that NTS/NTSR1 signaling supports glioblastoma growth, yet, the detailed mechanisms, however, remain unknown.Micro RNAs(mi RNAs) are endogenous, short and highly conserved non-coding RNAs,which are incorporated into mi RNA-protein complexes. These complexes associate with the3’-untranslated regions(3’UTRs) of mRNAs and prevent translation or destabilize target m RNAs. Approximately 30-60% of the human genome, including cell cycle regulators, are regulated by mi RNAs.In this study, we aimed to evaluate the effect of NTS/NTSR1, mi RNAs and c-Myc on glioblastoma cell proliferation, G1/S phase transition and the growth of glioblastoma in vivo. miRNAs chip, RT-q PCR and Ch IP assay were employed to explore the effect of NTS/NTSR1 on miRNAs expression. Luciferase assay was used to evaluate the relationship between mi RNAs and CDKs. Western blot and RT-qPCR assay were employed to explore the effect of NTS/NTSR1 on CDK4 expression. This is the first report to demonstrate themolecular mechanisms underlying and favoring glioblastoma cell proliferation in response to NTS/NTSR1 signaling, which may help to develop new treatment method for slowing down the glioblastoma growth and improving the prognosis of patients.Results:1. NTS/NTSR1 promotes the glioblastoma cells proliferation, the transition of G1 phase into S phase and glioblastoma growthNTS is a facilitator in glioblastoma cell proliferation, which promotes glioblastoma cells from G1 phase into S phase in vitro, and this phenomenon is reversed by pharmacological antagonist SR48692 or si NTSR1. Silencing NTSR1 in glioblastoma cells could reduce the size of subcutaneous tumor. NTS/NTSR1 only regulates the G1-S phase transition, but does not seem correlated to glioblastoma cells apoptosis. NTS/NTSR1 promotes the expression of CDK4 at the level of transcription in glioblastoma cells,however, NTS/NTSR1 only modulates the expression of CDK6 at the post- transcriptional level.2. NTS/NTSR1 modulates the expression of CDK6 protein through c-Myc/ mi R-29b-1and mi R-129-3p in glioblastoma cellsNTS/NTSR1 represses the expression of mi R-29b-1 and mi R-129-3p in glioblastoma cells, these miRNAs interacts with the 3’UTR of CDK6 m RNA and repress the translation of CDK6 mRNA in glioblastoma cells; NTS/NTSR1 promotes the expression of c-Myc,which with the ability to interact with the promoter region of miR-29b-1 and repress the expression of mi R-29b-1.3. NTS/NTSR1 promotes the expression of CDK4 through transcription factor c-Myc at the level of transcription in glioblastoma cells.NTS/NTSR1 promotes the expression of c-Myc in glioblastoma cells; c-Myc binds to the promoter of CDK4 and promotes the expression of CDK4 m RNA and protein in glioblastoma cells.4. MiR-29b-1, mi R-129-3p and c-Myc regulates cell proliferation, G1/S phase transition, orthotopic implanted glioblastoma growth and survival duration.MiR-29b-1 and miR-129-3p represses the proliferation and G1/S phase transition of glioblastoma cells, however, miRNAs function disturbances can promote cells proliferation and G1/S phase transition; Transcription factor c-Myc and miRNAs function disturbancesalone could promotes glioblastoma cells proliferation and G1/S phase transition,combination of both synergistically enhance this effect; Intracranial transplant tumor assay confirm that silencing NTSR1 could inhibit tumor growth partial by increasing mi R-29b-1and mi R-129-3p level.Conclusions:In this study, we showed that NTS/NTSR1 stimulates glioblastoma cell proliferation and growth by promoting G1/S phase transition, which can be blocked by silencing NTSR1 in glioblastoma cells. On the top of that, we identified that the expression of mi R-29b-1 and mi R-129-3p is repressed by NTS/NTSR1 signaling, both of miRNAs with ability to inhibit the process of CDK6 translation. NTS/NTSR1 promotes the translation of CDK4 or represses the expression of mi R-29b-1 through transcription factor c-Myc in glioblastoma cells. In conclusion, the stimulation effect of NTS/NTSR1 on glioblastoma cell proliferation and growth in vitro and in vivo is dependent on mi RNAs/CDKs and c-Myc/CDKs axis.