| Objective:Radiofrequency ablation(RFA)technology is widely used for colorectal cancer liver metastasis(CRLM)of treatment,but local tumor progression(LTP)caused by incomplete ablation seriously affects the prognosis of patients.It has been reported that miR-34a-5p is involved in the metastasis process of colorectal cancer(CRC),and has important value in early diagnosis of CRLM and recurrence monitoring.This study explored the effects of miR-34a-5p and its potential target gene"metastasis-associated protein 2(MTA2)"on the proliferation,migration and invasion of CRC cells.The methods of CRLM animal model establishment and ablation operation suitable for RFA research were explored,and the expression of miR-34a-5p in incomplete ablation tissue specimens was confirmed to be different from that in complete ablation and sham ablation tissue specimens.In addition,the early predictive value of changes in plasma miR-34a-5p expression before and after RFA in CRLM patients for LTP was further evaluated.Methods:1.RT-qPCR was used to detect the expression of miR-34a-5p in 40 pairs of human CRC and corresponding adjacent tissue samples,and to analyze the relationship between the expression of mi R-34a-5p and the clinicopathological characteristics of CRC patients.2.RT-qPCR was used to detect the expression of miR-34a-5p in CRC cell lines and normal colonic epithelial cells.The effect of mi R-34a-5p on CRC cell function was confirmed by cell proliferation assay,wound healing assay and transwell invasion assay in two CRC cell lines.3.TargetScan predicted that MTA2 was the potential target gene of miR-34a-5p,and further confirmed the targeted regulatory relationship between miR-34a-5p and MTA2 by dual-luciferase reporter assay system.The effect of miR-34a-5p on MTA2 mRNA and protein expression was determined by RT-qPCR and western blot(WB).4.We used MTA2 shRNA to interfere with MTA2 mRNA and protein expression,and further explored the effects of inhibition of MTA2 expression on cell proliferation,migration,and invasion.Rescue experiments were performed to confirm whether inhibition of MTA2 expression can restore the effect of anti-miR-34a-5p on cell proliferation,migration and invasion.5.Establishment of Balb/c-nu mice CRLM model and RFA.The subcutaneous tumors were established and cut into small tissue blocks of about l mm~3 as the source of transplanted tumor.Tissue blocks were transplanted to the left lobe of the liver of 15 nude mice by laparotomy,and then raised under SPF conditions for4 weeks.Fifteen Balb/c-nu mice with liver metastases were randomly divided into 3 groups:group 1 was the sham ablation control group,and only the radiofrequency applicator was inserted into the tumor tissue without power;group 2 was the incomplete ablation group,and the radiofrequency applicator was inserted into the edge of the tumor to ensure that the tumor on the side far away from the applicator was not completely damaged;group 3 was the complete ablation group,and the applicator was inserted into the center of the tumor to ensure that the tumor tissue was completely damaged.All nude mice were sacrificed 24 h after RFA,and specimens were collected.Group 1:tumor specimens were completely preserved;group 2:stripped coagulated necrotic tissue,preserved tumor tissue adjacent to coagulated necrosis;group 3:stripped coagulated necrotic tissue,preserved liver tissue around 5 mm of coagulated necrosis.6.The expression of miR-34a-5p in each group was detected by RT-qPCR,and the differences of miR-34a-5p in different groups were compared.7.Forty-two CRLM patients who received RFA treatment in our hospital from January 2016 to December 2017 were selected as the study subjects and followed up to December 2018.Plasma samples were collected before,24 h after and 7 d after RFA,and the expression of miR-34a-5p was detected by RT-qPCR.The relationship between mir-34a-5p and the clinicopathological characteristics of CRC patients was analyzed.8.Enhanced CT examinations were performed at 1,3,and 6 months after RFA and every 3months thereafter to assess tumor progression.According to the LTP situation after RFA treatment,CRLM patients were divided into LTP group and LTP-free group,and the expression of miR-34a-5p in each group was compared at different time points.The Cox regression model was used to analyze independent predictors of local tumor progression-free survival(LTPFS).Results:1.The expression of mi R-34a-5p in CRC tissue samples of 40 patients was lower than that in paracancerous tissue samples(P<0.05).With the progression of TNM stage,miR-34a-5p showed a downward trend.MiR-34a-5p was significantly lower in TNM stage III–IV tumor samples than that in stage I–II(P<0.05).The mean expression of miR-34a-5p in 40 CRC samples was 0.77(range:0.33?1.96)as the cut-off value,and the expression of miR-34a-5p was correlated with tumor differentiation,lymph node metastasis and TNM stage(P<0.05),while not associated with age,gender and tumor size(P>0.05).2.The expression of miR-34a-5p in human CRC cell lines(LoVo,HCT116,HT29,and SW480)was lower than that in normal human colon mucosal epithelial cells(P<0.05).Two CRC cell lines with the lowest miR-34a-5p expression(LoVo)and relatively high miR-34a-5p expression(SW480)were selected.CCK-8 assay,wound healing assay,and transwell invasion assay confirmed that miR-34a-5p inhibited the proliferation,migration,and invasion of CRC cells.3.The dual-luciferase reporting system showed that miR-34a-5p expression upregulation significantly decreased the luciferase activity of wild-type MTA2(P<0.05),but had no significant effect on that of mutant and control group(P>0.05).RT-qPCR and WB results showed that miR-34a-5p could affect the expression of MTA2mRNA and protein.In addition,the expression of MTA2 mRNA in 40 CRC samples was higher than that in paracancerous samples(P<0.05).Correlation analysis of miR-34a-5p and MTA2 mRNA revealed their negative correlation(P<0.05).4.MTA2 shRNA inhibitted MTA2 mRNA and protein expression.After MTA2 was down-regulated,the proliferation,migration and invasion ability of SW480 cells decreased.The rescue experiment results showed that after co-transfection of anti-miR-34a-5p and MTA2shRNA,the proliferation,migration,and invasion ability of SW480 cells decreased compared with the anti-miR-34a-5p and shRNA-NC groups.Based on the above results,miR-34a-5p could affect the proliferation,migration and invasion of CRC cells by targeting MTA2.5.The CRLM models of Balb/c-nu mice were successfully established,and the mean diameter of 15 tumors was 9.3±0.7 mm.According to different grouping principles,the liver metastases were treated with RFA,and the whole experiment went smoothly without the animal death.6.The expression of miR-34a-5p in each group was as follows:sham ablation control group was 1.24±0.15,incomplete ablation group was 0.40±0.08,and complete ablation group was 1.91±0.24.Compared with the sham ablation group and the complete ablation group,the expression of miR-34a-5p in the incomplete ablation group was significantly decreased(P<0.05).7.The mean miR-34a-5p expression was 1.69 before RFA in 42 CRLM patients.The expression of miR-34a-5p was related to the primary tumor differentiation and ablative margin(P<0.05),and was not related to the age,gender,number and size of liver metastases(P>0.05).8.Sixty-eight liver metastases in 42 CRLM patients were followed up,with a median follow-up time of 24months and a median survival time of 34 months.The 1-year and 2-year OS rates were90.5%and 70.9%.A total of 24 patients(57.14%,24/42)presented LTP in 25 lesions(36.76%,25/68).Among them,17 lesions(25.00%,17/68)of 16 patients(38.10%,16/42)presented LTP in the first year after RFA,accounting for 68%(17/25)of the total LTP.Plasma miR-34a-5p expression in 42 CRLM patients at 24 h after RFA was not significantly different from that before RFA(P>0.05);while plasma miR-34a-5p expression at 7 d after RFA was significantly higher than before RFA(P<0.05).Among them,miR-34a-5p in the LTP group(n=24)decreased significantly 7 d after RFA compared with that before RFA(P<0.05),while miR-34a-5p in the LTP-free group(n=18)increased significantly(P<0.05).The ratio of plasma miR-34a-5p 7 d after RFA to that before RFA as"miR-34a-5p change ratio",and the ratio?0.5 was considered as a significant decrease of plasma mi R-34a-5p on 7d after RFA.Cox univariate analysis showed that tumor size,miR-34a-5p change ratio and ablative margin were significant in predicting LTPFS(P<0.05)and entered multivariate analysis.Cox multivariate analysis showed that the ratio of miR-34a-5p 7 d after RFA to that before RFA?0.5(P<0.05)and ablative margin?5 mm(P<0.05)were two independent predicators of short LTPFS.Conclusion:1.MiR-34a-5p inhibits the proliferation,migration and invasion of CRC cells by targeting MTA2,and plays an important role in the occurrence and development of CRC.2.The expression of miR-34a-5p is significantly decreased in the CRLM model of Balb/c-nu mice after incomplete ablation,and it may have the ability to predict LTP after RFA in CRLM patients.3.The dynamic changes of plasma miR-34a-5p before and after RFA and the ablation margin are independent predictors of LTPFS in CRLM patients,which is conducive to early prediction of LTP after RFA,and can provide a basis for clinicians to design personalized follow-up programs and treatment strategies. |
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