| Objective: Hepatocellular carcinoma(HCC)is one of the common malignant tumors,which seriously threatens human health.Due to the complexity of the disease,many factors should be considered when determining the best treatment plan for HCC.Among them,radiofrequency ablation(RFA)is one of This relatively new technology has become a widely used method for early HCC treatment and palliative treatment of advanced liver cancer due to its precise therapeutic effect,high safety,cost-effectiveness and minimal surgical invasiveness.Radiofrequency ablation hopes to achieve the required complete treatment result with one ablation.However,due to the proximity of blood vessels or special anatomical parts during the ablation process,it is necessary to choose Intermittent radiofrequency ablation to achieve the ideal ablation result.However,it is unclear whether the cumulative effect of intermittent ablation at different time intervals is equivalent to the result of a single complete ablation.If the intermittent ablation fails to deliver enough energy,the internal temperature of the tumor tissue does not meet the necrosis standard,resulting in incomplete ablation,not only cannot completely destroy the tumor tissue,but also leads to the rapid progress of residual HCC,which greatly affects the therapeutic effect of this technology.Therefore,it is of great significance to study the effect of intermittent ablation and possible incomplete ablation on the growth and proliferation of hepatocellular carcinoma and its mechanism.At present,many studies have been conducted on the mechanism of abnormal tumor proliferation caused by incomplete ablation,but it is still not fully elucidated.S100A4 is a member of the S100 calcium binding protein family and is highly expressed in a variety of malignant tumors.The high expression of S100A4 in HCC is associated with poor prognosis.GDF15(Growth differentiation factor 15,GDF15)is a member of the TGF-βsuperfamily,which is obviously related to the proliferation and metastasis of a variety of tumor tissues,and is highly expressed in a variety of tumor tissues.Studies have shown that GDF15 can promote the survival,invasion and angiogenesis of HCC.The above results suggest that S100A4 and GDF15 play an important role in the occurrence and development of HCC and other tumors.The previous research of our group showed that GDF15 is an important downstream gene of S100A4,and S100A4 can regulate GDF15 and affect the biological characteristics of gastric cancer cells.However,in HCC after radiofrequency ablation,whether S100A4 gene regulates the expression of GDF15 and thus affects the abnormal proliferation of HCC is still unclear.This study intends to adopt the methods of simulated ablation of pig liver in vitro,simulated ablation of transplanted tumors in nude mice,and heating of HCC in vitro to explore how intermittent ablation affects the growth and proliferation of HCC and its possible molecular mechanism,that is,whether intermittent ablation affects the S100A4 gene.In turn,it regulates the expression of GDF15 and affects the growth and proliferation of HCC.2.Methods1.Experimental materialsThe HCC-LM3 liver cancer cells,immunodeficient mice were used for related experiments.2.Experimental method:(1)Establish a single complete ablation condition for radiofrequency ablation.1)Preliminary establishment of a single complete ablation condition for the simulated ablation experiment of pig liver in vitro2)Experimental verification of a single complete ablation condition of transplanted tumor in nude mice(2)The effect of intermittent ablation and single complete ablation on the growth of liver cancer transplanted tumor in nude mice1)In vitro porcine liver simulated ablation experiment to compare the effects of intermittent ablation and a single complete ablation2)Experiments on transplanted tumors in nude mice verify the effect of intermittent ablation and single complete ablation on the proliferation of liver cancer(3)Real-time fluorescent quantitative PCR and Western blot were used to detect the m RNA and protein expressions of S100A4 and GDF15 in transplanted tumors in nude mice after ablation.(4)Heat treatment of HCC at different temperatures in vitro,and test the cell proliferation ability,and select a heat treatment temperature that simulates incomplete ablation.(5)Detect the m RNA and protein expression of S100A4 and GDF15 in heat-treated cells.(6)Transfect S100A4-si RNA into heat-treated cells,and detect the expression of S100A4 and GDF15(the method is the same as above).(7)GDF15-sh RNA was transfected into heat-treated cells to detect cell proliferation ability.(8)Co-transfect S100A4-si RNA and GV161-GDF15 expression vectors in heat-treated cells to detect GDF15 expression and cell proliferation(the method is the same as above).3.Results:(1)Experimental results of simulated ablation of pig liver in vitro show that there is a linear correlation between ablation width and ablation time(y=0.0667x+2.8955,R2=0.9646),a single ablation time increases by 30 s,and the injury width has a significant difference.In the curve,9～10mm is selected as the target width of complete ablation.It can be seen that a single ablation at 70°C for 120 seconds can achieve the goal of complete ablation,while a single ablation at 70°C for 90 seconds cannot achieve a complete ablation result.(2)The experimental verification results of the transplanted tumor in nude mice showed that the maximum diameter,volume and weight of the tumor in the 70°C and 120 seconds ablation group were significantly smaller than those in the control group,while the corresponding indicators in the 70°C and 90 seconds group were significant It is larger than the control group(P<0.05),which proves that a single ablation at 70°C for120 seconds can achieve a complete ablation result,and a single ablation at 70°C for 90 seconds leads to incomplete ablation.(3)According to the results of the in vitro simulated ablation experiment,compared with the single complete ablation group,the width of the injury in the intermittent60-second ablation group was significantly reduced(P<0.05).(4)Intermittent ablation of transplanted tumors in nude mice proved experimental results.It can be found that the maximum growth diameter,tumor volume and tumor weight of the tumor in the 60-second intermittent ablation group were significantly higher than those in the control group(P<0.05),suggesting intermittent ablation May cause incomplete ablation.(5)Intermittent 60 S ablation significantly increased the m RNA and protein expression of S100A4 and GDF15 in the tumor(P<0.01).(6)Heat treatment of liver cancer cells at different temperatures.Compared with the control group,the 48℃ treatment significantly enhanced the cell proliferation ability(P<0.05),and the 50℃ treatment significantly reduced the cell proliferation ability(P<0.05).Follow-up heat treatment of liver cancer tumor cells with 48 degrees and used as a heat treatment group.(7)Heat treatment significantly increased the m RNA and protein expression levels of S100A4 and GDF15 in HCC(P<0.05).(8)After transfection with S100A4-si RNA,the expression levels of S100A4 m RNA and protein of abnormally proliferated heat-treated cells decreased significantly,and the m RNA and protein expression levels of GDF15 also decreased significantly(P<0.05).(9)After silencing the expression of GDF15 in the heat-treated cells,the cell proliferation ability was significantly reduced(P<0.05).(10)Co-transfected S100A4-si RNA and GDF15 expression vector(GV161-GDF15)in heat-treated cells,the GDF15 m RNA expression level was significantly increased,the protein expression level was also significantly increased,and compared with the control group,its proliferation ability was also significantly improved.(P<0.05)4 Conclusion:(1)Through the simulated liver ablation method in vitro and verified by nude mouse transplantation tumor experiments,the conditions required for a single complete ablation,namely 70°C,120 seconds,are established.(2)The simulated liver ablation experiment in vitro indicates that the width of the intermittent ablation injury is insufficient and the complete ablation effect cannot be achieved.(3)Experiments on transplanted tumors in nude mice proved that intermittent ablation with an interval of 60 seconds resulted in incomplete ablation,which not only failed to inhibit the tumor but promoted tumor growth,and the m RNA and protein expression levels of S100A4 and GDF15 in tumor tissues increased significantly after intermittent ablation.(4)Incomplete ablation significantly promotes the proliferation of liver cancer cells by up-regulating the expression of S100A4/GDF15. |
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