Spinal G9a May Contribute To Bone Cancer Pain In Rats

Researches showed that patients with advanced cancer are often suffering severe pains,which seriously affect the quality of life of patients,and bone cancer pain(BCP)is a common one.Under the current clinical treatment,45%of patients are still suffering pain without effectively controlled.The mechanism of bone cancer pain is still not completely clear,which is the reason for the insufficient pain relief,so it is very important to explore the mechanism of bone cancer pain.G9a is a histone methyltransferase that can regulate the expression of various genes and participate in various pathophysiological regulatory processes.A variety of histone methyltransferases play important roles in bone cancer pain in rats.However,the role of spinal G9a in bone cancer pain in rats is seldom reported.Therefore,this study intends to explore the role of spinal cord G9a in bone cancer pain in rats.Part one The role of spinal G9a in bone cancer pain ratsObjective:To establish a model of tibia cancer pain in rats and observe the changes of spinal G9a mRNA and protein expression of BCP rats.Methods:40 female rats were randomly divided into normal group,n=8;sham group,n=8;BCP group,n=24.The normal group was left untreated;10μl of heat-killed Walker 256 tumor cells(1×10~5)were injected into the left tibia bone marrow cavity of the sham group rats,and the rest of the treatment was the same as rats in the BCP group.10μl of Walker 256 cells were inoculated into the left tibial bone marrow cavity of rats in BCP group.The changes in ambulatory score(AS)and von Frey-induced paw withdrawal threshold(PWT)of rats before modeling and 1,3,6,9,12,15,18 days after modeling were measured(n=8);X-rays of the left tibia of each group of rats were taken on 6,12,18 d,and the tibial bone destructions of the rats were observed to further verify whether the rat bone cancer pain model build successfully(n=4).The spinal cord lumbar enlargement of normal group,sham group,BCP6d,BCP12d and BCP18d rats were taken.The changes of G9a mRNA and protein expression in the spinal cord were measured by real-time quantitative PCR(RT-qPCR)and western blot(WB).Results:During the 18 days observation period,there was no statistically difference in AS and PWT between the normal group and the sham group(P>0.05).Compared with the normal group and sham group,the AS of rats in the BCP group increased progressively and the PWT decreased progressively from 6th day to 18th day after modeling,and the difference was statistically significant(P<0.01).X-rays of rats in each group were taken.The radiographs showed that in BCP group,bone marrow damage and trabecular bone defects were seen on the tibial epiphysis on the 6th day after modeling.On the 12th day after modeling,bone destruction of the tibial epiphysis was significantly increased,and trabecular bone damage was further aggravated.On the 18th day,severe damage to the tibial bone marrow even cortical bone damage,and swelling of the surrounding soft tissues ware seen.The left tibia of the sham group and the normal group was normal,and no bone structure was seen.There was no significant difference in G9a mRNA and protein expression in the spinal cord between the normal group and the sham group(P>0.05).Compared with the normal group,the G9a mRNA in the spinal cord of BCP6d,BCP12d,and BCP18d rats was 4.42±0.43,2.09±0.24,1.65±0.18 times that of the normal group,and the G9a protein was 2.88±0.49,1.81±0.28,1.76±0.2 times that of the normal group(P<0.01).Conclusion:G9a mRNA and protein in the spinal cord of BCP rats increased significantly.Part two Intrathecal BIX-01294 could attenuate bone cancer pain in BCP ratsObjective: To observe the effect of intrathecal injection of BIX-01294 on BCP rats.Methods: 56 female SD rats were randomly divided into normal group,sham group,BCP group,BCP + Dimethyl sulfoxide(DMSO)group,BCP + BIX-01294 group,normal + DMSO group,normal + BIX-01294 Group,n = 8.BCP + DMSO group,BCP + BIX-01294 group,normal + DMSO group,normal + BIX-01294 group were intrathecal injection of DMSO or BIX-01294 5 μg on the 3rd day after modeling.The volume of intrathecal injection of drugs in each group was 10 μl,once a day,for three consecutive days.The changes of AS score and PWT after intrathecal injection were observed in each group.Results: Compared with the BCP group,intrathecal injection of BIX-01294 could reduce AS scores of BCP rats and increase PWT of BCP rats(P < 0.05 or 0.01).Intrathecal injection of BIX-01294 had no effect on AS scores and PWT of normal rats(P > 0.05).Conclusion: Intrathecal injection BIX-01294 can significantly alleviate bone cancer pain in rats.