The Research On The Role Of GRK3 To Neuroendocrine Prostate Cancer By The Induction Of Androgen Deprivation Therapy

Prostate cancer is one of the male reproductive system malignant tumor and it is very common, as well as the most common malignant tumor in the western countries, especially in the United States, the incidence of prostate cancer patients is still higher than lung cancer, ranked the first place. The incidence of men with prostate cancer is a growing trend in our country. Due to the hidden prostate cancer symptoms, at least 50% of the patients for locally advanced tumor diagnosis and treatment and prevention is particularly important to improve the survival rate. The leading causes of death in patients are metastasis and recurrence. Neuroendocrine cells have been acknowledged as the normal prostate pipeline and an integral part of acini. Nearly 50% of clinical type front splenic carcinoma with neuroendocrine cells mutation. Neuroendocrine prostate cancer, also known as small cell undifferentiated carcinoma, is a highly invasive prostate cancer subtypes. It can be primary, but its most advanced from prostatic adenocarcinoma(PCA) after hormone treatment. To clarify the molecular mechanism of NEPC generation and development of targeted therapy drugs NEPC is currently a hot research topic in the field of prostate cancer.In this study, we develop prostate gland cancer cells induced by hormone-free LNCa P transformation towards NE direction, make preparation of the ADT induction type of neuroendocrine prostate cancer cells, called t- NEPC(ADT- induced NE differentiated cell line),and studied role of protein kinase GRK3 in the process of thet- NEPC celltransformation.The aim of this study is to provide new therapeutic targets and experiment basis for the treatment of NEPC.Objective: By preparing the neuroendocrine prostate cancer cells of ADT-induction type, and study the role of protein kinase GRK3 in the process of prostate gland cancer cells to the neuroendocrine transformation, to provide new therapeutic targets and experimental basis for the treatment of NEPC.Methods:1 The cell cultureLNCa P human prostate gland cancer cells, and induced t-ADT NEPC cells, human prostate gland cancer cells, both in containing 5% fetal bovine serum glutamine, concentration of 1% and 1% of penicillin and streptomycin, to phenol red in RPMI 1640 added.2 Experimental methodsDirection transformation from human prostate gland cancer LNCa P cell to neuroendocrine by hormone-free RPMI1640 medium, successful preparation of prostate gland cancer cell source of neuroendocrine t-NEPC cells, using rear time PCR detection of two kinds of cells of PSA, AR and NE markers CHGA, CHGB and ENO2 expression changes, through the rear time PCR and western blot method GRK3 expression change detection of two kinds of cells.Virus infection method is adopted to establish the stable express sh GRK3 t-NEPC cells, and USES the rear time polymerase chain reaction(PCR) to detect low GRK3 striking NE t-NEPC cells markers CHGA, CHGB and ENO2 expression change.Results:1 The ADT can induce human prostatic adenocarcinoma LNCa P cell towards the direction of neuroendocrine differentiation First we pass on to phenol red RPMI1640, containing 5% fetal bovine serum, concentration of 1% of glutamine and 1% of penicillin and streptomycin medium long-term training to get a t-NEPC LNCa P cells. These cells are derived from long-term treatment with ADT LNCa P human prostate gland carcinoma cells.Results show that the LNCa P cells showed epithelial morphology and t-NEPC cells visible inclusions, it become a lot of neurons around cells called synapses, cell morphological changes.We again by real-time quantitative PCR in LNCa P and t-NEPC compare the expression of AR in the two cell lines and its target gene prostate specific antigen(PSA) expression of two NE markers.Results show that the expression of PSA in LNCa P cells is 10 times higher than in t-NEPC cells, AR in LNCa P cell than in t-NEPC cell 5 times higher than that of the expression, in LNCa P cell CHGA rose 6 times, CHGB rose by 4 times, ENO2 increased 20 times from earlier years.2 The expression changes of GRK3 in human prostatic adenocarcinoma in the direction of neuroendocrine differentiation We studied the GRK3 in LNCa P and t-NEPC expression in cells.Western blot-results show that compared with the LNCa P cell GRK3 ADT-induced t-NEPC level of protein in the cell obviously increased;By real-time quantitative PCR detection results show that the ADT-induced t-NEPC cells compared with LNCa P cell significantly increased by two times.3 Phenotypic effects on t-NEPC after knockout GRK3We use the GRK3 sh RNA infected t-NEPC cell, first observe the cell morphological changes, cell transfection GRK3 sh RNA, cell tentacles, morphological reverse change, then we again by real-time quantitative polymerase chain reaction(PCR) to detect t-NEPC shscramble and t-NEPC sh GRK3 to assess whether knockout GRK3 experiment is successful, the results showed: t-NEPC expression of sh GRK3 group was obviously lower than the control group t-NEPC shscramble 3 times, GRK3 sh RNA infected into t-NEPC cells reduced the endogenous GRK3, GRK3 knock out successfully.We further conducted a NE markers: CHGA CHGB and ENO2 express, through the real time quantitative PCR analysis t-NEPC sh GRK3 was t-NEPC shscramble NE marker expression decreased obviously.Conclusions:1 The ADT can induce human prostatic adenocarcinoma LNCa P cell in the direction of neuroendocrine differentiation.2 The GRK3 promoted the human prostatic adenocarcinoma in the direction of neuroendocrine differentiation.3 After knocking GRK3, t-NEPC phenotype changes reversely, NE markers CHGA, CHGB and ENO2 expression were significantly decreased.