| Objective Hepatocellular carcinoma(HCC),the most common type of primary liver cancer,is one of the most prevalent malignant tumors in our country.It is characterized by an insidious onset,short disease duration,and a high mortality rate.The molecular mechanism behind the occurrence and development of HCC is complex and not yet fully understood,but many studies have proven the involvement of miRNA in the biological behavior of HCC,contributing to the disease development.Therefore,in this study,we screened for differentially expressed miR-1266 in HCC tissues through TCGA databases,explored the correlation between miR-1266,target gene ATG7,and their clinicopathological characteristics,and explored the effect of the overexpression and inhibition of miR-1266 on HCC biological behaviors,specifically on the cell proliferation,cell cycle,and apoptosis of HepG2 HCC cells.This research provides the theoretical basis for the early diagnosis and targeted treatment of liver cancer.Methods 1.Bioinformatics analysis :(1)Downloaded the gene expression data of HCC from the TCGA database(the Cancer Genome Atlas),screened for the differentially expressed miRNAs,and selected a miRNA which has not been previously reported in HCC and is involved in tumor-related biologicalfunctions.miR-1266 was chosen as the candidate gene for this study after literature retrieval;(2)The miRNA target gene prediction website(TargetScan,RNA22,miRwalk,miRtarbase)was used to predict candidate target genes of miR-1266.The correlation between miR-1266 and the candidate mRNA was calculated using the expression data of miR-1266 and mRNA in the TCGA database.The negative correlation between miR-1266 and the candidate mRNA was used as the final miRNA target gene,and ATG7 was selected as the target gene of miR-1266 in this study.(3)The clinical information data of HCC in the TCGA database was downloaded to analyze the relationship between miR-1266 and the clinicopathological characteristics of HCC.The Kaplan-Meier analysis method was used for survival analysis to analyze the effect of miR-1266 on the prognosis of liver cancer.2.Verified the gene expression in the population: Sixty cases of liver cancer tissues and adjacent tissues from HCC patients in The Cancer Hospital of Guangxi Medical University were collected,and the clinicopathological information of the patients were also retrieved.The real-time fluorescent quantitative PCR method was used to detect the expression of miR-1266 and ATG7 in liver cancer tissues and adjacent tissues samples.It was also used to calculate the correlation of miR-1266 and ATG7,and to analyze the relationship between mir-1266,ATG7,and the clinicopathological characteristics of HCC.3.The expression of miR-1266 was verified in liver cancer cell lines :Real-time fluorescence quantitative PCR was used to detect the expression of miR-1266 in BEL-7404,SK-HEP-1,SMCC-7721,Hep 3B,Huh7,and HepG2 cells.The HepG2 cell line was selected to construct an overexpression and inhibition expression system and to verify the expression of miR-1266 and ATG7.4.Cell function test:Overexpressed and inhibited miR-1266 expression lentiviruses were constructed and transfected into HepG2 cells.The correlation between the expression of miR-1266 and clinical features,as well as the function of miR-1266 in HCC was explored,proving the theoretical basis for HCC in early diagnosis,targeted therapy,and prognosis analysis.Results 1.Bioinformatics analysis showed:(1)The TCGA database provided information on 377 HCC patients with an average age of 59,of which209 patients had clinical data that matched the gene expression data.Among the377 cases,377 samples of liver cancer tissue and 50 samples of adjacent liver tissue were sequenced.(2)A total of 98 differentially expressed miRNAs were screened,including 27 upregulated and 71 down-regulated miRNAs.In this study,miR-1266 was selected as the target gene.(3)A total of 60 mRNAs were screened from 4 miRNA target gene prediction sites,of which GRINA,PCSK2,RAB15,PTDSS2,EIF5AL1,MUTYH,and ATG7 were found to be negatively correlated with miR-1266(P<0.05).After analysis,ATG7 was selected as the target gene of miR-1266.(4)Analysis of the relationship between miR-1266 and the clinicopathological features of HCC: There was a statistically significant difference in the expression level of miR-1266 in the tumor grade,alpha-fetoprotein(AFP)level,and age(P<0.05).No statistically significant difference was found in the expression level of miR-1266 in the gender,family history,MVI(Microvascular invasion),tumor status,T stage,and survival status distribution(P>0.05).(5)The Kaplan-Meier model analysis showed no statistical correlation between the expression level of miR-1266 and the survival time of HCC patients(P>0.05).2.The results of gene expression analysis on human liver cancer:(1)The expression levels of miR-1266 and ATG7 were verified by real-timefluorescence quantitative PCR.miR-1266 was found to have a higher ex pression level in liver cancer tissues than in adjacent tissues(t=3.402,P=0.001),and ATG7 was found to have a lower expression level in liver ca ncer tissues than in adjacent tissues(t=-2.452,P=0.001).(2)miR-1266 wa s negatively correlated with ATG7 in cancer tissues(r=-0.324,P=0.023)(3)Statistically significant differences were found in the expression level of miR-1266 in the degree of tumor differentiation and AFP(P<0.05).Th ere was no statistically significant difference found in the gender,age,occ upation,portal hypertension,family history,HBsAg infection,tumor size,MVI,capsule invasion,extrahepatic metastasis,ascites,BCLC stage,and p ortal vein tumor thrombus(P>0.05).(4)There was a statistically significa nt difference in the expression level of ATG7 and the tumor size and BC LC stage(P<0.05).There was no statistically significant difference found in the gender,age,occupation,portal hypertension,family history,HBsAg infection,AFP,degree of tumor differentiation,MVI,capsule invasion,ex trahepatic metastasis,ascites,and portal vein tumor thrombus(P>0.05).3.The results of gene expression analysis on liver cancer cells:(1)miR-1266 was found to have a low expression level in BEL-7404,SK-HEP-1,and SMCC-7721 cancer cells,and a high expression level in Hep 3B,Huh7,and HepG2 cancer cells.Due to the fact that the expression level of miR-1266 is the highest in HepG2 cells,HepG2 cells were chosen for subsequent trials.(2)The expression abundance of the overexpressed miR-1266 group is 417.78 times of the negative control group,and the expression abundance of the inhibited miR-1266 expression group is 0.79 times of the negative control group.(3)The expression of ATG7 decreased when there is overexpression of miR-1266(P<0.05).On the other hand,there is no statistically significant difference foundin the expression of ATG7 when the expression of miR-1266 is inhibited(P>0.05).4.Cell function test:(1)CCK8 experiment: The ability of cell proliferation in HepG2 cells increased with the overexpression of miR-1266(P=0.001).The ability of cell proliferation in HepG2 cells decreased with inhibited expression of miR-1266(P = 0.037).(2)Cell cycle test: The duration of the S phase increased(P<0.05),and the duration of the G2/M phase decreased in the miR-1266 overexpression group(P<0.05).The duration of the G2/M phase increased in the inhibited miR-1266 expression group(P<0.05).(3)Apoptosis experiment: The rate of apoptosis in HepG2 cells increased with the overexpression of miR-1266(P=0.046),but the rate of change is not obvious.No statistically significant difference was observed in the rate of apoptosis in HepG2 cells with the inhibited miR-1266 expression(P=0.740).Conclusions 1.The expression level of miR-1266 was up-regulated in liver cancer tissues,and the expression level of ATG7 was down-regulated in liver cancer tissues;2.miR-1266 and ATG7 were associated with the clinicopathological features of HCC;3.The expression level of miR-1266 was negatively correlated with ATG7 in liver cancer tissues;4.Overexpression and inhibition of miR-1266 can affect the cell proliferation and cell cycle of HCC.This suggests that miR-1266 may promote the biological behavior of HCC by regulating the expression of ATG7. |
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