| Backgrand Hepatocellular carcinoma(HCC)is one of the most common malignancies.It is estimated that there are about 740,000 new HCC cases annually,and it was responsible for 70 million HCC mortality cases worldwide,while approximately half cases appeared in China.Because of the high incidence and high mortality of HCC,HCC as one of malignant tumors is related to the overall cause of the second death rate has become a global and serious public health problem.The occurrence and development of HCC is a complex biological process.To improve the therapeutic effect and prognosis of patients,we need to further understand the underlying molecular mechanisms of HCC development and explore new strategies for early diagnosis and targeted therapy of HCC incidence.At present,with the rapid development of the next generation sequencing technology,increasing studies have shown that long non-coding RNA(Lnc RNA)play an important role in life activities,and has become one of the hot fields.Long noncoding RNA(linc RNA)is a class of RNA with a length of 200-100000 nt,which does not encode a protein but is involved in a variety of intracellular processes.linc RNA can be used to regulate gene expression in a variety of ways,including exogenous silencing and the expression of a regulated gene.Although recent studies have shown that linc RNA has a specific regulatory mechanism in a variety of tumors,the current research on noncoding transcripts is still unexplored.In addition,most of the clinical significance and mechanism of action of linc RNA in the development and progression of hepatocellular carcinoma remains largely unknown.Therefore,in-depth study of linc RNA function and mechanism of action for the early diagnosis and treatment of liver cancer to provide a new direction.Based on the human Lnc RNA database GSE55191 and GSE580435,we found that the long-chain non-coding RNA 524346(long intergenic non-coding RNA 524346,linc RNA 524346)was significantly reduced in hepatocellular carcinoma(HCC)The problem).However,there is little research on linc RNA 524346,and its low expression in hepatocellular carcinoma is not a significant functional significance.The purpose of this study is to invsetigate the expression of linc RNA 524346 in hepatocellular carcinoma and its clinical significance.Firstly,the expression of linc RNA 524346 in 61 cases of hepatocellular carcinoma tissue and liver normal cell line LO2 and hepatocellular carcinoma cell lines SMMC-7721,Hep G2 and Huh7 were analyzed by q RT-PCR.Then,we analyzed the relationship between the expression level of linc RNA 524346 and clinical pathological features in HCCpatients.Next,the performance of linc RNA 524346 in hepatocellular carcinoma was evaluated by receiver operating characteristics(ROC)in the early diagnosis of hepatocellular carcinoma.Finally,we analyze the relationship between the expression of linc RNA 524346 and the prognosis of patients with hepatocellular carcinoma.Materials and methods(1).Clinical sample collection and cell lines All samples were obtained in accordance with the agreement of the same Hospital Ethics Committee.All patients with HCC who participated in this study had signed informed consent.61 patients with hepatocellular carcinoma were enrolled in this study.61 fresh HCC sample were treated with the first hepatocarcinoma resection from December 2013 to December 2015.The samples were stored at nitrogen within30 min after tissues were separated.All specimens were collected without any antitumor therapy,including chemotherapy,surgery and radiotherapy,postoperative specimens were pathological results have been confirmed by a professional pathologist for hepatocellular carcinoma.The control group of specimens from the patient with the adjacent tissue(at least 3cm away from the surgical margin),postoperative pathological examination did not find cancer cells.A randomized selection of the included HCC samples was performed and the patients were followed for more than one year.The extent of tumor differentiation was determined by the Edmondson-Steiner classification criteria,which were determined according to the criteria for the seventh edition of the American Joint Committee on Cancer(AJCC)2010.The normal hepatocyte cell line LO2 and hepatocellular carcinoma cell lines SMMC-7721,Hep G2 and Huh7 were cultured in DMEM medium containing 10% fetal bovine serum and cultured in a cell incubator at 37 ? 5% CO2.By the 302 Hospital Clinical Management Center to provide.2.RNA extraction,reverse transcription and real-time quantitative PCR Total RNA was extracted from tissue samples and cell lines using Trizol(Life Technologies,USA)reagents and was transfected with RNA kit Transcriptase Kit Trans Script First-strand c DNA Synthesis Super Mix(Beijing,China)according to tmanufacturer's instructions.The reverse transcription was carried out using a Veriti TM thermal cycler to reverse transcribe RNA into c DNA.Reaction parameters:25 ? 10 min,42 ? 60 min,85 ? 5s.Real-time fluorescence quantitative PCR was performed using Step One Plus ? real-time fluorescence quantitative PCR system.The reaction parameters were 50 ? for 2 min;95 ? for 10 min;95 ? for 10 s,60 ? for30 s and 72 ? for 30 s for 40 cycles.The primer specificity and efficiency of all q RTPCR were tested.The change of multiple is calculated by the relative quantitative method of 2-? Ct.3.Statistical analysis SPSS18.0 statistical software for data analysis.Nonparametric Wilcoxon rank sum test was used to compare the expression levels of linc RNA524346 in cancer tissues and matched paracancerous tissues and cell lines.The expression of linc RNA524346 in LO2 cells and hepatocarcinoma cell lines was analyzed by t test.ROC curve was used to evaluate the sensitivity and specificity of linc RNA524346 in the diagnosis of hepatocellular carcinoma.The diagnostic ability of linc RNA 524346 was evaluated using the area under the ROC curve(AUC);95% confidence interval was used.Survival analysis was performed using survival analysis and Log-rank test.The factors influencing survival were assessed by single factor and multivariate Cox regression model.Univariate analysis was used to identify the factors with statistical differences,and then further evaluated by multivariate analysis The value of independent prognosis of liver cancer,P <0.05 for the difference was statistically significant.Results1.linc RNA 524346 expression is down-regulated in HCC cancer tissues and cells The expression of linc RNA 524346 in 61 cases of HCC was detected by q RT-PCR.The expression of linc RNA 524346 in 83.6%(51/61)HCC was significantly downregulated.(P <0.01).AUC was 0.74(95% CI: 0.6435-0.8429,P <0.0001),which was higher than 0.7,indicating that the predicted value was moderate,the cutoff value was0.009,the sensitivity was 65.12%,the specificity was 63.83 % And Jordanian index of0.29,which indicated that linc RNA 524346 had certain diagnostic value for hepatocellular carcinoma.Compared with the normal hepatocyte LO2,linc RNA524346 was low in three kinds of hepatocellular carcinoma cell lines,the difference was statistically significant(P <0.01);in SMMC-7721 cells,the lowest level of expression.2.The relationships between linc RNA 524346 expression and HCC patients' clinicopathological features The expression of linc RNA 524346 was significantly correlated with pathologic stage,number of tumors,PVTT,cirrhosis and microvascular infiltration,and the expression of linc RNA 524346 was significantly correlated with the expression of linc RNA524346 and clinicopathological features in 61 patients with hepatocellular carcinoma.Tumor size,serum AFP level,tissue differentiation level,drinking,smoking no significant statistical difference.Meanwhile,we analyzed the the expression of linc RNA 524346 in 61 patients with HCC and the survival time of patients with hepatocellular carcinoma and found that the low expression of linc RNA 524346 was related with the recurrence-free survival time of HCC patients.Conclusion1.linc RNA 524346 expression was significantly decreased in hepatocellular carcinoma tissues compared with paired paracancerous tissue.2.linc RNA 524346 expression was significantly decresed in HCC cell lines(SMMC-7721?Hep G2 ? Huh7)compared with normal liver cell lines LO2.3.linc RNA 524346 expression was significantly correlated with pathologic stage,number of tumors,PVTT,cirrhosis and microvascular infiltration.4.Decreased linc RNA 524346 expression predicts poor prognosis in HCC patients.5.linc RNA 524346 might be used as potential prognostic factor for the prognosis of HCC patients... |
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