The Effects Of FHF2Mediated By Sodium Channels On Pain And Sodium Channels Expression Profiles In Human Prostatic Cancer

Fibroblast growth factor homologous factors (FHFs) belong to aspecial subfamily of fibroblast growth factor (FGF) because of theirlacking of secretion property. FHFs include four members: FHF1-FHF4,which can interact intracellularly with voltage gated sodium (Nav)channel and affect channel expression and function as well as neuronalexcitability. FHF4deficient mice develop ataxia and severe mentalretardation was found in FHF2knockout mice. DRG neurons act aspimary neurons of pain conduction, and a wide range of Nav channels areexpressed in DRG neurons. Among those, Nav1.7is preferentiallyexpressed in unmyelinated small DRG neurons and amplifiessubthreshold of depolarizations, and set up threshold of Nav1.8andNav1.9opening, and regulates neurons excitability and pain conduction.The function of FHFs, one of the major modulators of Nav channels, onsodium channel in DRG neurons and pain are largely unknown. Thus thepresent study will first investigate the expression property of FHFs inDRG neurons, and then determine the functional effect of FHFs onNav1.7channel in heterlogous expression system and in rat DRG neuronsby using adenovirus mediated shRNA to knockdown endogenous FHF2.Finally using FHF2conditional knockout mice study effects of FHF2onpain behavior responding to thermal and mechanical stimulation.Nav channels are expressed not only in excitable cells but also inmany cancer cells, including prostate cancer. Expressions of VGSCs insome cancers are related to cancer migration, invasion and metastasis invivo. It has been reported that the expression of some kinds of Navchannel α subtypes were up-regulated in prostate cancer cells. However, the detailed expression profiles of Nav channel α subunits in normalhuman prostate, in benign prostatic hyperplasia (BPH) and prostaticcancer (PC) are still controversial. Here, we used real-time quantitivePCR technique systematically detected all subtypes of Nav channel αsubunits in normal human prostate, benign prostatic hyperplasia (BPH)and prostatic cancer cells. Those studies will provide basis for thecorrelation of Nav channels with prostate cancers and will provide cluesfor clinical diagnosis and drug targets for treatment of prostate cancer.Part1The effects of FHF2mediated by sodium channels on painObjective:(1) To determine the expression property of FHFs in ratDRG neurons.(2) To study the effects of main FHFs isoforms on Nav1.7channels in CHO cell.(3) To study the effects of knockout FHF2onsodium channels in small DRG neurons.(4) Using FHF2conditionalknockout mice study FHF2effects on pain behavior.Methods:(1) Using whole cell patch clamp technique to study theeffects of FHF2on sodium currents.(2) Using quantitative RT-PCR(qPCR) and immunostaining assay and confocal microscopy to define theexpression property.(3) Using Von Frey filaments and radiate heat teststo test mechanical allodynia and thermal hyperalgesia in FHF2conditional knockout miceResults:(1) FHF2is the major isoform of FHFs in rat DRG neurons.FHF2preferentially expresses in small DRG neurons rather than mediumand large DRG neurons.(2) Coexpression of FHF2U with Nav1.7,doubled the current density and shifted the voltage dependent inactivationto depolarization direction and slow inactivation in CHO cells.(3)Knockout FHF2in rat DRG neurons by shRNA revealed a loss offunction of sodium channel in small DRG neurons. Immunostaininganalysis shows sodium channel protein is reduced on cell membranewhen knockout FHF2in DRG neurons.(4) Withdrawal latency to thermalstimulus in FHF2knockout mice is prolonged compared to wildtype mice(from10.08±0.59s to11.96±0.64s respectively)(P<0.05). The painful threshold to the mechanical stimulation in FHF2knockout mice isincreased compared to wildtype mice (from3.27±0.24g to4.14±0.23grespectively)(P <0.05).Conclusions:(1) FHF2is the major isoform of FHFs in rat DRGneurons（.2）Overexpression of FHF2U can significantally double Nav1.7current density, depolarize channel inactivation curve and slow channelinactivation.(3) Sodium channel display a loss of function phenotype inrat small DRG neurons after knockout FHF2by shRNA.(4) The latencyto thermal and mechanical stimulation was significantly increased inFHF2knockout mice, possibly due to FHF2related sodium channel loseof function.Part2Expression profiles of sodium channels in human prostaticcancer cellsObjective: To detect all subtypes of VGSCs α subunits expressionproperty in normal human prostate, human benign prostatic hyperplasia(BPH) and human prostatic cancer cells (PC-3and LnCap cells).Methods:(1) QPCR was used to detect all subtypes of VGSCs αsubunits in NP, BPH, PC-3and LnCap cells.(2) Whole cell patch clamptechnique was used to record the sodium channels.Results:(1) Nav1.5was the major isoform expressed in normalhuman prostate tissue. Both Nav1.5and Nav1.2were predominantisoforms in BPH tissue.(2) Both Nav1.6and Nav1.7were abundantlyexpressed in PC-3and LnCap cells.(3) By comparing the relativeexpression levels of Nav1.5, Nav1.6and Nav1.7in those tissues/cells,mRNA levels of Nav1.6and Nav1.7were6to27-fold higher in PC-3andLnCap cells than in either normal or BPH samples.(4) Tetrodotoxinsensitive sodium currents were successfully recorded in PC-3cells, butnot in LnCaP cells.Conclusions: We concluded that although all types of VGSCsexhibited low expression level in human normal prostate and BPH. BothNav1.6and Nav1.7were dramatically up-regulated in prostatic cancers, especially in highly metastatic PC-3, suggesting that both Nav1.6andNav1.7might be potential diagnostic markers for some kind of prostatecancer in human.