The Role And Mechanism Of Long-chain Noncodeing RNA SNHG16 In Early Recurrence After Hepatocellular Carcinoma

Background Hepatocellular carcinoma(HCC)is one of the common malignancies in China.The prognosis of patients is poor,and the recurrence rate after complete resection remains high.Some patients with liver cancer recurred within 3 months after tumor resection.The prognosis of this relapsed patient is poor and we call it early recurrence.This may be because we know less about the occurrence and development mechanism of liver cancer.However,with the breakthrough in sequencing technology,the abnormal expression of non-coding RNA in liver cancer has proved to be an important factor affecting HCC.Long non-coding(lnc RNA)is a non-coding RNA with a length of more than 200 nucleotides.It basically does not have the function of transcription and encodes proteins.However,lnc RNA is now found in various tumors such as liver cancer,lung cancer,and gastric cancer.Abnormal expression,lnc RNA may play an important role in the growth and reproduction of tumors.At present,the research of lnc RNA in hepatocellular carcinoma is still relatively few,and most lnc RNA regulatory mechanisms and specific functions are still not very clear.Therefore,it is extremely important to find lnc RNAs that are associated with abnormal expression of liver cancer.Our group used RNA-Seq to detect the difference in lnc RNA expression profiles of hepatocellular carcinoma tissues from patients with tumor recurrence within 3 months after surgery and patients with no recurrence more than 3 years,and screened lnc RNAs associated with liver cancer through bioinformatics.Functional and mechanism research.Purpose RNA-Seq technology was used to screen lnc RNAs that were significantly differently expressed in patients with early postoperative recurrence of HCC,and the biological functions and regulatory mechanisms of the lnc RNAs in hepatocellular carcinoma and hepatoma cells were explored.Methods 1.RNA-Seq technique was used to detect the difference of lnc RNA expression profiles in 3 cases of hepatocellular carcinoma with tumor recurrence within 3 months and patients with no recurrence greater than 3 years,including expression level,lnc RNA length,complete lnc RNA sequence of database,and GO enrichment.The differentially expressed lnc RNAs associated with hepatocellular carcinoma were screened for KEGG pathway analysis and other methods.Real-time quantitative PCR(q RT-PCR)was used to verify partially differentially expressed lnc RNAs in hepatocellular carcinoma cell lines and tissues,and finally SNHG16 was determined as the subsequent study subject.2.The position of SNHG16 expression in cells was determined by FISH.3.Collect 20 cases of early recurrence of hepatocellular carcinoma and corresponding paracancerous tissues in patients undergoing hepatocellular carcinoma resection,and use the q RT-PCR method to detect the expression of SNHG16.4.Collect 102 patients with hepatocellular carcinoma who underwent hepatectomy.Analyze the relationship between SNHG16 expression and clinicopathological features and prognosis.5.Prediction,validation and functional studies of SNHG16 target genes: In combination with gene mapping and bioinformatics predictions,it is speculated that SNHG16 may compete with mi R-17-5p for the regulation of P62,and luciferase assays are used to verify P62,mi R-17-Relationship between 5p and SNHG16.6.The molecular mechanism of SNHG16 regulation: using q RT-PCR,Western-blot,detection of m TOR pathway.Results 1.Compared with tumor tissue that has not relapsed in 3 years,lnc RNAs with high or low level expression and more than 2 fold change in the recurrence of hepatocellular carcinoma within 3 months are considered to be of significance for differential expression.The bioinformatics analysis showed that these differentially expressed lnc RNAs are involved in cell metabolism,apoptosis,cell cycle and other related genes,and some may also be involved in signal pathway regulation such as m TOR,NF-KB,MAPK,JAK-STAT,and Wnt.2.Through sequencing and tissue screening,we found that lnc RNA SNHG16 is highly expressed in early recurring HCC tissues.After q RT-PCR validation,lnc RNA SNHG16 was up-regulated simultaneously in hepatoma cells and early recurrent HCC tissues.3.SNHG16 overexpression was significantly associated with BCLC stage and tumor number.Kaplan-Meier analysis showed that high SNHG16 expression was associated with low survival and high tumor recurrence.4.FISH localization analysis revealed that SNHG16 is mainly expressed in the cytoplasm and a small amount is expressed in the nucleus.5.The results of luciferase assay report showed that SNHG16 competitively binds mi R-17b-5p through the ce RNA mechanism and regulates the expression of P62.6.After q RT-PCR/Western-blot showed that the expression of SNHG16 was up-regulated,P62 and P-m TOR were significantly up-regulated.After SNHG16 was down-regulated,P62 and P-m TOR were down-regulated.Conclusion 1.By RNA-Seq,we found that lnc RNA SNHG16 is highly expressed in hepatocellular carcinoma and early recurrent hepatocellular carcinoma.The expression of SNHG16 is related to the pathological features of liver cancer patients and can predict the prognosis of liver cancer patients.2.Up-regulation of SNHG16 expression promotes proliferation,invasion and apoptosis of hepatoma cells.SNHG16 regulates the expression of target gene P62 by mi RNA-17b-5p through ce RNA competition mechanism,and activates the downstream m TOR pathway to promote the proliferation,invasion and inhibition of apoptosis in hepatoma cells.Therefore,there may be a regulatory mechanism of SNHG16-P62-m TOR in liver cancer,which can promote the occurrence and development of liver cancer through affecting cell proliferation and apoptosis.This will provide new ideas for further research on the molecular mechanism and clinical treatment of liver cancer.