Study On The Mechanism Of LncRNA Lnc712 In Hepatoma Cell And Its Correlation With Liver Cancer After Ablation

Part Ⅰ:The expression of LncRNALnc712 in hepatoma cell and studies on the mechanism of miR-142-3p/Bach-1 targeted by LncRNA Lnc712 to promote the proliferation,migration and epithelial-mesenchymal transformation in liver cancer.Objective:To explore the expression of LncRNALnc712 in hepatoma cell and the mechanism of liver cancer by targeting miR-142-3p/bach-1 axis.Materials and Methods:The BEL-7402 cells of liver cancer were selected as the research object.LncRNA Lnc712,miR-142-3p and Bach-1 overexpressed plasmids were designed for cell transfection,and the specific groups were as follows:Control group(liver cancer cells were not treated);OE-LncRNA Lnc712 group(overexpression of LncRNA Lnc712 in HCC cells);SiRNA-LncRNALnc712 group(silenced expression of lncRNA Lnc712 in HCC cells);Mimic group Of OE-LncRNA Lnc712+miR-142-3p mimic(LncRNA Lnc712 and miR-142-3p overexpression in liver cancer cells);In the group of OE-LncRNALnc712+miR-142-3p+Bach-1(LncRNA Lnc712,miR-142-3p,and Bach-1 overexpression in hepatocellular carcinoma cells);The targeted relationship between LncRNA Lnc712 and miR-142-3p and miR-142-3p and Bach-1 was analyzed by double luciferase gene detection.The expression levels of LncRNA Lnc712,miR-142-3p and Bach-1 in each group were detected by RT-PCR.The proliferation,apoptosis,migration and invasion of common cells were detected by functional assay.Western blot was used to observe the changes of markers of EMT transformation in each group;The tumorigenicity of Lnc712 was verified by subcutaneous injection of silenced Lnc712 hepatoma cell line in nude mice.T test or MW test was used for comparison between groups.Correlation analysis adopts Pearson correlation and other statistical analysis data to draw the conclusion.Results:(1)Expression of LncRNALnc712,mir-142-3p and Bach-1 in each hepatoma cell group were detected by RT-PCR:LncRNALnc712 is highly expressed in the hepatoma cell;LncRNA Lnc712 was overexpressed or partially silenced successfully in cancer cell groups.The addition of mir-142-3p and Bach-1 had little effect on the expre ssion of LncRNALnc712,and the LncRNALnc712 reduced mir-142-3p’s expressi on and promoted Bach-1’s expression.mir-142-3p inhibited the expression of Bach-1.(2)The verification of LncRNA Lnc712 targeting mir-142-3p’s relationship:TargetScan software predicted that the mRNA 3’non-coding region of LncRNA Lnc712 had a binding site of mir-142-3p;The binding ability of LncRNA Lnc712 to mir-142-3P-UTR was identified by co-transfection experiment.(3)Verification of the relationship between mir-142-3p targeting Bach-1:In the prediction results of TargetScan software,it was found that the mRNA 3’non-coding region of mir-142-3p had a binding site of Bach-1;wild type was significantly lower than that of control group,;we did not find that the mRNA 3’non-coding region of LncRNA Lnc712 predicted the binding site of Bach-1.(4)MTT detection of cell proliferation in each group:the tumor cells of LncRNA Lnc712 overexpressed in hepatoma cells showed obvious proliferation;The intervention of mir-142-3p can reduce the proliferation trend of tumor cells and has obvious tumor inhibition.The overexpression of Bach-1 again promoted the proliferation of tumor cells.(5)Annexin V-FITC detect the apoptosis of each group:the apoptosis ability of hepatoma cells decreased after overexpression of LncRNA Lnc712,whereas the apoptosis ability increased.The intervention of mir-142-3P can promote the apoptosis of hepatoma cells,and the tumor inhibition is obvious.The overexpression of Bach-1 increased the anti-apoptotic activity of the cells again.(6)Scratch/Transwell assay was used to detect the migration and invasion ability of cells in each group:the overexpression of LncRNA Lnc712 in hepatoma cells significantly enhanced the invasion and invasion ability of tumor cells,whereas it decreased on the contrary.mir-142-3p was overexpressed,and its invasion ability was decreased.The ability of bach-1 with overexpression was enhanced again.(7)Analysis of Western-blot:Overexpression of LncRNA Lnc712 in hepatoma cells significantly enhanced the EMT process of cells;otherwise,the EMT process decrease d;Overexpression of mir-142-3p can reduce the EMT process.The overexpression of Bach-1 made the EMT ability of the cells improve again.(8)Effects of SiRNA-LncRNA Lnc712 in nude mice:Tumor growth was restricted in the sirNA-LncRNA Lnc712 hepatocarcinoma cell group in nude mice,further confirming the oncogenic characteristics of lncRNA Lnc712.Conclusions:LncRNA Lnc712 was highly expressed in hepatoma cells,LncRNA Lnc712 and Bach-1 showed oncogenic characteristics,and mir-142-3p showed tumor suppressive ch aracteristics.Overexpression of LncRNA Lnc712 significantly inhibited the expression of mir-142-3p and promoted the expression of Bach-1,and LncRNA Lnc712 regulated the proliferation,apoptosis,migration,invasion and EMT process of hepatoma cells by targeti ng mir-142-3p/Bach-1 axis..Part Ⅱ Analysis of the efficacy after microwave ablation in patients with large-diameter hepatocellular carcinoma and afflect on the change of LncRNALnc712 in various tissues and serum by ablation.Objective:To evaluate the efficacy of microwave ablation in the treatment of large-diameter liver cancer and understand the effect of microwave ablation on the expression level of LncRNA Lnc712 in the tissues and serum of patients with liver cancer and to explore the correlation between the expression level of LncRNALnc712 and the prognosis of HCC treated by microwave ablation.Materials and Methods:A retrospective analysis was made of 110 patients diagnosed with hepatocellular carcinoma(10cm≥Maximum tumor diameter≥5cm)and treated with microwave ablation in our center from January 2015 to December 2020.After microwave ablation treatment,relevant indicators were evaluated and analyzed:Analysis of patients’ ablation rate:primary and secondary complete ablation rate and factors influencing the ablation efficacy;Safety analysis of side effects and complication rate after treatment was conducted by referring to WHO score for side effects such as pain and expert consensus complication score of 2013 version of liver tumor ablation.General conditions during operation such as operation time,blood loss and other indicators statistics;Prognostic evaluation:Kaplan-Meier survival curve and COX model were used to analyze patients’ progression-free survival(PFS),overall survival(OS)and their influencing factors.40 patients with single large liver cancer were selected from the above 110 patients,35 patients with small hepatocellular carcinoma(tumor diameter<5 cm)who underwent microwave ablation at the same time were matched as control.The levels of LncRNA Lnc712 in peripheral blood of these patients were detected by RT-PCR before treatment,1 week after ablation and 1 month after ablation and the effect of microwave ablation on LncRNA Lnc712 was analyzed,The difference of serum LncRNA Lnc712 expression in patients with large and small liver cancer after ablation was compared.Meanwhile,30 cases of seru m of normal healthy persons were selected as control.The COX model was used to analyze the influencing factors of local recurrence of lesions after ablation.According to the level of Lnc712’s expression in the preoperative serum,the patients were divided into high and low expression groups to compare the prognosis.Preoperative tumor tissues and paracancer tissues were obtained by intraoperative biopsy,and paracancer tissues were punctured immediately after ablation in the 75 patients.The expression levels of LncRNA Lnc712 in the above three tissues were detected by RT-PCR,and the expression differences between large and small liver cancers were compared and analyzed.Results:(1)Analysis of indicators during microwave ablation:In 110 patients with liver cancer,the total number of tumor lesions was 164,the average duration between operations was 42.0±16.1 minutes,Outside the skin permeability of the amount of blood loss was 24.2±10.1 ml,and the average length of hospital stay was 6.1±1.9days.All patients underwent sucessful operation without any sudden or unexpected events.(2)Safety evaluation:liver function related indicators such as ALT,AST,TBil and DBil showed an increasing trend Postoperative day 1.Comparison of data among each group,P<0.05.Comparison of blood routine indexes before and after treatment:The changes of RBC and Hb in blood routine indexes before and 1 day and 1 month after microwave treatment were small,and WBC increased significantly.The proportion of toxic and side effects after the operation was about 31.8%,8.2%,13.6%,31.8%,11.8%and 1.8%for abdominal pain,fever,malignancy,vomiting,fatigue and diarrhea.The overall proportion was low,which was improved after symptomatic treatment.The symptoms of most patients disappeared or gradually recovered after discharge without affecting their normal life.Incidence of Postoperative complicat ions:1 case of abdominal bleeding,2 cases of obstructive jaundice,1 case of intestinal perforated,2 cases of renal failure,10 pleural effusion,and 1 osevere infection.Most of the complications were recovered or improved after symptomatic treatment,without affecting the prognosis of the patients.(3)Efficacy evaluation:In 110 patients,164 tumors were ablated,the total(Complet ely ablation first)CA1st was 86.5%,(Completely ablation second)CA2nd was 93.7%,and the residual rate after secondary ablation was 6.3%.The influence of tumor size,tumor site and tumor number on the tumor ablation effect was analyzed.Data analysis showed that tumor size had statistical significance on the first complete ablation rate.CA1st was 91.2%for patients with 5cm≤maximum tumor diameter<7cm,while CAst was 59.5%in larger ones.CA2nd showed no significant difference.However,there was no statistically significant difference in ablation caused by tumor location and tumor number,which may be related to the small sample size.After the microwave ablation,the expression level of AFP was gradually decreased.(4)Results of short-term prognostic survival analysis:Based on the results of univariate analysis of PFS in 110 patients with liver cancer after ablation,alkaline phosphatase and the rate of complete ablation one month after ablation were correlated with the prognosis of PFS,P<0.05.The lower the alkaline phosphatase,the higher the rate of complete ablation one month after surgery,and the longer the progression-free survival.At the level of 0.1,y-GGT was also associated with PFS,which may be related to the low sample size.Through multivariate analysis of COX model,it was found that whether the patients were completely ablated one month after ablation was closely related to their prognosis.(5)Expression analysis of LncRNA Lnc712 in the tissues of patients:LncRNA Lnc712 was highly expressed in liver cancer tissues and low in paracancer tissues.LncRNA Lnc712 increased in paracancer tissues before and after ablation,but the difference was not significant.The expression of LncRNA Lnc712 in paracancer tissues of large liver cancer after ablation was higher than that of small liver cancer,but the difference was not statistically significant(P>0.05).(6)Expression analysis of LncRNA Lnc712 in the serum of patients:The expression of LncRNA Lnc712 in healthy control group was always at a low level,while the expression level of LncRNA Lnc712 in liver cancer patients was at a high level before ablation,but after microwave ablation,the expression of LncRNA Lnc712 decreased gradually,There was no significant difference in serum changes between large and small HCC patients,However,the expression of LncRNA Lnc712 in patients with larger liver cancer after ablation was slightly higher than small ones.(7)The analysis of LncRNALnc712’s expression and survival in all the patients:According to the expression of LncRNA Lnc712 in patients’ serum before ablation,we divided into the high expression group of LncRNA Lnc712(43),and the low group(32)depend on the median expression value.The corresponding midian survival time was 28 and 32 months,respectively,The difference was statistically significant.Cox model was used to analyze the influencing factors of tumor recurrence after microwave ablation.Gender,underlying diseases,liver cirrhosis,liver and kidney function and other factors had poor correlation with local recurrence after microwave ablation,with P values of 0.122,0.102,0.278 and 0.113,respectively,and the difference was not statistically significant.The initial tumor size,the degree of microwave ablation and the expression of LncRNA Lnc712 afterablation were all correlated with whether the lesion recurred after microwave ablation,with P values of 0.040,0.013 and 0.030,respectively,and the differen ces were statistically significant.Conclusions:(1)Microwave ablation for liver cancer is safe and effective,and can be used as an effective method for local tumor control in clinic.It also has a good effect on the large liver cancer,which can effectively control the local lesions and prolong the survival time.(2)LncRNA Lnc712 showed a tendency of high expression in tissues and serum of HCC patients and most expression levels of LncRNA Lnc712 in blood decreased after ablation,the expression of LncRNA Lnc712 in patients with larger liver cancer after ablation was slightly higher than small ones,but there was no significant difference in them.There was little change in the expression of LncRNA Lnc712 in the paraca ncer tissues of patients after ablation,but the change of the expression of LncRNA Lnc712 in the paracancer tissues of patients with large liver cancer was slightly higher than that of small liver cancer,but there was no significant difference in them and patients with high expression of LncRNA Lnc712 were prone to recurrence of local lesions.(3)LncRNA Lnc712 in adjacent tissues and serum of larger HCC was slightly increas ed than small ones after thermal ablation,which provides some inspiration to the clinic:For example,Drugs targeted to the mechanism of LncRNALnc712 can be used to improve the therapeutic effect of liver cancer or circulating tumor cells can be controlled by combining PD-I or CAR-T immunotherapy after ablation due to the release of tumor antigen and liver cancer can be locally controlled and then treated in a systemic way as"blood tumor".