| Bone cancer pain(BCP)is a kind of chronic pain caused by cancer metastases to the bone.BCP is a major cause of moderate and severe pain in cancer patients,and it is a major risk factor to reduce the quality of life.However,the Three-step Pain-relieving Principles recommended by the WHO have been conducted,which did not provide an ideal easement of pain,which may due to the complex development mechanism of BCP.Therefore,probing the mechanism of BCP will of great assistance to clinical treatment.MicroRNAs(miRNAs)are approximately 22 nt long noncoding RNAs with post-transcriptional regulatory functions that are widely involved in the pathophysiological processes and are associated with pain as well.MiRNA-153 is widely expressed in various organs of the body,which can inhibit the invasion and migration of many kinds of carcinomas,participate in oxidative stress and control inflammation,but there is still a lack of research on pain.This study will construct models of BCP in the tibia of rats and explore the function of miR-153-5p in BCP.Chapter Ⅰ Construction of BCP modelObjective Construct and verify the BCP model of rat tibia.Methods 36 female SD rats with clean grade were divided into 3 groups randomly.The normal group(N group)had 12 rats;and other 12 rats were in the BCP group received 10μl cell suspensions with 1×10~5 walker256 breast cancer cells injection into the rat left tibial medullary cavity;the rest 12 rats were in the sham group injected with a 10μl heat-killed cells with the same method.The spontaneous pain behavior(ambulatory pain score,AS)and the mechanical pain sensitivity(paw withdrawal threshold,PWT)of rats were measured at 0,1,3,6,9,12,15,18 d after walker256 breast cancer cell injection.X-ray radiography was conducted to observe the tibial structure of the affected limb at 6,12,18 d.Results There was no significant difference in AS and PWT between sham group and N group(P>0.05).AS were gradually increased from 0±0 score to3.46±0.31 scores and PWT decreased from 30.25±5.87g to 3.33±1.01g in the BCP group from 0 to 18 days after modeling.And BCP group compared with the sham group,the AS and PWT were became statistical significance at the 6th day and last to the 18th day(P<0.05,0.01).After X-ray examination,there was no obvious abnormality in N group and Sham group,but the bone density of BCP rats decreased on the sixth day after modeling,and the range of bone density decreased on the 12th day,which could be accompanied by cortical defect.And on the 18th day,the swelling appeared,and the surrounding invasion was accompanied by morphological changes.Conclusion A model of BCP established by implanting walker 256 breast cancer cells into the rat tibial bone marrow cavity was demonstrated by imaging,pain behavior experiments to be stable and reliable,providing a fundamental platform for further mechanistic studies.Keywords BCP,Animal model,AS,PWTChapter Ⅱ The expression of miR-153-5p in spinal cord of BCP ratsObjective To investigate the expression of miR-153-5p in BCP rats.Methods 32 female SD rats with clean grade were divided into 2 groups randomly and every group had 16 rats.The construction methods of each group are identical to that of chapter I.Spinal lumbar enlargement were collected from each group on days 0,9,12 and 18.Then,the relative expression of miR-153-5p was determined by RT-qPCR.Results Compared with 0 d,the relative expression of miR-153-5p in the BCP group decreased gradually.Compared with sham group,the relative expression of miR-153-5p in BCP group was decreased at 9,12,18 d(P<0.05,0.01).Conclusion MiR-153-5p decreased in the spinal cord of BCP rats,indicating its possible involvement in BCP.Chapter Ⅲ Effect of intrathecal injection of agomiR-153-5p on BCPObjective To explore the possible therapeutic effect on BCP by miR-153-5p.Methods 24 bone cancer pain rats were randomly divided into intrathecal injection of normal saline(BCP + NS)group,intrathecal injection of agomiR-153-5p(BCP + agomir)group and intrathecal injection of agomiR-153-5p negative control(BCP + NC)group,with 8 rats in each group.On the 9th day,each group of rats underwent intrathecal catheterization.After a 3-day repair period,intrathecal injection was performed on day 12,10μl each time,for a continuous injection of 3 days.BCP + agomir group injected 10 ul with a total of 1 nmol agomiR-153-5p,BCP + NC group injected 10μl with a total of 1 nmol agomiR-153-5p NC,while BCP + NS group injected equal volumes of NS according to the same method.AS and PWT of each group were observed respectively before and after injection.Results There was no marked difference in AS and PWT between BCP + NS group and BCP + NC group(P >0.05).Whereas,compared BCP + agomir group with BCP + NC group,after intrathecal injection of agomiR-153-5p,AS gradually decreased from 2.59 ± 0.16 scores to 0.79 ± 0.25 scores and PWT increased from 5.67 ± 0.62 g to 13.96 ± 1.24 g in BCP + agomir group and the differences were statistically significant(P < 0.05,0.01).Conclusion Intrathecal injection of agomiR-153-5p could effectively alleviate BCP,suggesting that miR-153-5p might be involved in BCP process. |
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