Study On The Effect Of Sh-ASIC3 Lentivirus On Metastatic Cutaneous Cancer Pain In Mice

Objective:To construct an acid-sensing ion channel 3(ASIC3)gene interference plasmid,verify the interference efficiency of recombinant plasmid,and obtain sh-ASIC3 lentivirus.Then establish a mouse model of cutaneous cancer pain and to investigate the effect of ASIC3 on metastatic cutaneous cancer pain in mice by intrathecal injection of sh-ASIC3 lentivirus.Methods:Using GenBank to obtain the ASIC3 gene sequence to synthesize the corresponding interference sequence,then the ASIC3 gene fragment was ligated to the circular Plko.1-Puro vector by T4 DNA ligase to obtain a recombinant interference ASIC3 plasmid(sh-ASIC3).The neuronal cells(PC12,N2a,BV2)were verified by Quantitative Real-time PCR(RT-PCR)and Western blotting to verify the interference efficiency of sh-ASIC3 plasmid.Then the recombinant plasmid was transfected into293T cells to obtain sh-ASIC3 lentivirus,and the titer of sh-ASIC3 lentivirus was detected by RT-PCR.The model of metastatic cutaneous cancer pain in mice was established by subcutaneous injection of approximately 2×10~5 mouse breast cancer cells(4T1)in the left toe of healthy female Balb/c mice.After successful modeling,mice were divided into groups by the method of complete random number table.Paw Withdrawal Thermal Latency(PWTL)was measured every other day after 4T1inoculation in each group.On the 9th day after successful inoculation of 4T1 cancer cells,intrathecal and local injections of lentivirus were performed according to the experimental group,followed by PWTL test every other day to compare the changes of PWTL threshold before and after lentivirus treatment.After 13 days of lentiviral treatment,mice were sacrificed by spinal cord amputation to obtain L4-5 spinal dorsal root tissue,the changes of ASIC3 in DRG were detected by immunohistochemistry.RESULTS:The results of the bacterial solution PCR indicated that the ASIC3interference sequence was inserted into the vector successfully.Sequencing results showed that the insertion sequence of the interfering plasmid ASIC3 was identical with the Genbank standard sequence.The results of RT-PCR and Western blotting showed that the mRNA transcription level and protein expression level in three kinds of nerve cells of sh-ASIC3 lentivirus infection group were significantly lower than those of sh-EGFP lentivirus infection group,the difference was statistically significant(P<0.05).After 4T1 cancer cells were injected subcutaneously into the left posterior toe of the mice,the redness and activity disorder appeared on the inoculation side.The PWTL test threshold was shorter than that of the normal group,the results of RT-sqPCR and Western blotting showed that the expression of ASIC3 in DRG of mice inoculated with4TI cancer cells was significantly higher than that of the normal group.The PWTL test threshold of mice with cutaneous cancer pain after intrathecal injection of sh-ASIC3interferes with lentivirus was significantly higher than that of local injection of sh-ASIC3 interference with lentivirus and intrathecal injection of sh-EGFP treatment.The results of ASIC3 immunohistochemistry in DRG were also consistent with PWTL results.Conclusion:The sh-ASIC3 lentiviral interference plasmid was successfully constructed.The PWTL of female mice subcutaneously injected with breast cancer cells was significantly shorter than that of normal mice,and the expression level of ASIC3 was also increased,indicating that the skin cancer pain model mice were successfully established,also indicating that ASIC3 mediated the production of skin cancer pain in mice.Intrathecal injection of sh-ASIC3 lentivirus increased the PWTL threshold in cancer pain mice.The results of immunohistochemistry further confirmed that the mechanism may be related to the down-regulation of ASIC3 expression.