| Hepatocellular carcinoma?HCC?is the most common histologic type of liver tumors,accounting for about 80%.About 95%of the new cases worldwide end up dying.In the past,we have focused more on the coding genes that can eventually be translated into proteins,rather than non-coding genes.Long non-coding RNA?lncRNA?is a kind of RNA which does not have protein coding function and is larger than 200nt in eukaryotes.Studies have shown that it is mainly through the impact of chromatin remodeling,transcriptional activation,mRNA stability and protein translation to achieve epigenetic,transcriptional and post-transcriptional regulation.The regulatory mechanism of competitive endogenous RNA?ceRNA?mainly suggests that transcriptional products such as lncRNA and mRNA compete with miRNA,through miRNA response element?MRE?to regulate each other's expression levels.LncRNA SNHG16?hereinafter referred to as SNHG16?is a newly discovered lncRNA,belonging to the host gene family of non-protein coding small nucleolus RNA?snoRNA?.At present,there are few reports on SNHG16 at home and abroad.Studies have shown that SNHG16 plays an important role in the occurrence and development of malignant tumors and ceRNA mechanism is involved.However,it is not clear whether SNHG16 and ceRNA mechanism play an important role in the occurrence and development of liver cancer.We target SNHG16 for in-depth study.The purpose of this study was to explore its role and mechanism in the occurrence and development of hepatocellular carcinoma?HCC?.The completion of this project is expected to further clarify the role of SNHG16 in the occurrence and development of liver cancer,and has important theoretical and clinical application value in evaluating the malignant degree of liver cancer,judging the curative effect,judging the prognosis and further elucidating the pathogenesis of liver cancer.Also,it would provide a new theoretical and experimental basis for the treatment of liver cancer.In the first part,qRT-PCR and CISH?chromogenic in situ hybridization?were used to detect the expression of SNHG16 in human tissues in order to determine its expression status in hepatocellular carcinoma?HCC?.At the same time,Kaplan-Meier and log-rank methods were used to analyze the relationship between the expression of SNHG16 and the overall survival?OS?and disease-free survival?DFS?of HCC patients,and draw the survival curve.Then the COX proportional risk regression model was used to evaluate whether SNHG16 can be used as a factor for the poor prognosis of HCC patients.The effect of SNHG16 on the malignant biological behavior of hepatocellular carcinoma cells in vitro was analysed mainly in the second part.In the first section of the third part,bioinformatics was used to predict the ceRNAs of SNHG16 and MLXIP was selected as the ceRNA molecule.Next the miRNAs which can be bound by both SNHG16 and MLXIP were predicted and miR-4458 was selected as the downstream miRNA molecule after literature analysis.Then qRT-PCR and luciferase report experiments were used to analyze the mutual regulation and targeting binding of SNHG16 and miR-4458.Finally,the rescue experiment showed that the effect of SNHG16 on the proliferation and sorafinib resistance of hepatocellular carcinoma cells depended on miR-4458.In the second section,the relationship between SNHG16 and MLXIP in liver cancer was analyzed by qRT-PCR,western blot and IHC?immunohistochemical method?,and then the regulatory relationship between miR-4458 and MLXIP was verified by experiments.Finally,it was discussed whether the regulatory effect of SNHG16 on MLXIP could be antagonized or counteracted by miR-4458.Objective:1.To investigate the expression of SNHG16 in hepatocellular carcinoma and paracancerous tissues and its correlation with the prognosis of patients with hepatocellular carcinoma.2.To investigate the effects of SNHG16 on malignant biological behaviors such as proliferation,invasion and metastasis and sorafinib resistance of liver cancer cells.3.To explore the regulatory mechanism of SNHG16 promoting proliferation and sorafinib resistance of liver cancer cells from the point of view of competitive endogenous RNA.Methods:1.Detection of SNHG16 expression in Hepatocellular carcinoma.Ten fresh liver cancer tissue samples and matched paracancerous tissues were collected.According to the instructions of Trizol Reagent reagent,the total RNA was extracted;and the cDNA was synthesized by reverse transcriptional reaction according to the instructions of PrimeScript RT reagent kit.The synthesized cDNA was used as the template.The expression level of SNHG16 in tissues was detected by fluorescence quantitative PCR reaction according to the instructions of SYBR Premix Ex Taq?kit,and the expression level of SNHG16 in paraffin sections of 61 cases of hepatocellular carcinoma was detected by chromogenic in situ hybridization?CISH?.2.Detection of SNHG16 expression in liver cancer cells.The normal liver cell line HL-7702 and four cancer cell lines SK-Hep-1,Huh7,Hep3B and HepG2 were extracted according to the instructions of Trizol Reagent reagent;The cDNA was synthesized by reverse transcription reaction according to the instructions of PrimeScript RT reagent kit;The cDNA was used as a template,and the expression level of SNHG16 in cells was detected by fluorescence quantitative PCR according to the instructions of SYBR Premix Ex Taq?kit.3.Cell transfection.The cells were transferred according to the instructions of lipofectamine2000.After48 hours of transfection,the RNA or protein of the cells were extracted or subsequently treated.4.Cell proliferation and sorafenib resistance test.Cell proliferation and drug resistance to sorafinib were detected by MTT assay.After the cells were digested into single cell suspension,the cells were inoculated in96-well plate.The number of cells inoculated in each hole was about 1×103 cells,and there were 3 double holes in each group.At 37?,the cells were cultured in the cell incubator containing 5%CO2 for 1,2 and 3 days,respectively,and then abandoned the culture medium.About 200?l of culture medium without serum and about 20?l of tetrazolium salt?MTT?with 5mg/ml concentration were added to each well,and then cultured in the cell incubator for 4 hours.Discard the excess liquid,add dimethyl sulfoxide?DMSO?150?l per hole,and then wrap the culture plate in aluminum foil and incubate it at room temperature for 10 minutes.At room temperature,the microoscillator is used to oscillate the culture plate for about 15 minutes,and attention should be paid to avoiding light during the operation.Finally,the OD value of 570nm was detected by enzyme-linked immunoassay.For the sorafinib resistance experiment,the cells were inoculated after 96 well plates and divided into six dose groups of 0,2,4,8,16 and 32?m,with three double holes in each dose group.After 72 hours of culture,the survival of cells was detected by MTT method.5.Cell migration and invasion experiments.The Transwell chamber was used to detect the migration and invasion of the cells.The transfected cells were made into cell suspension.In the migration experiment,the cell suspension containing 1×105 tumor cells was planted into the upper chamber.In the invasion experiment,the cells left in the upper layer of the residual chamber were wiped off with cotton swabs after being incubated on the basement membrane of the chamber with 20-50?g/cm2 matrix glue and incubated in an incubator at 37?for 24hours.The cells below the chamber were fixed with methanol and stained with crystal purple.Five visual fields were randomly selected under 200-fold microscope to count the migration and invasion of cells.6.Western blot experiment.The total protein was extracted by RIPA and the protein concentration was determined.5×SDS-PAGE loading Buffer was added for protein denaturation.After electrophoresis,membrane transfer and blocking,the first antibody and the second antibody were incubated and developed by ECL method.7.Luciferase reporter gene detection.SNHG16 containing miR-4458 hypothetical binding site and luciferase reporter plasmid containing mutant SNHG16 and miR-4458 or negative control were co-transfected into HCC cells.48 hours after transfection,the luciferase activity of the sample was detected according to the operation instructions of Dual-luciferase reporter gene detection system,and the luciferase activity of firefly was used as the internal reference.The inhibitory effect of miR-4458 on SNHG16 was analyzed according to the fluorescence ratio of sea kidney firefly to firefly?Renilla/Firefly?.8.Detection of MLXIP protein expression in tissues by immunohistochemical staining.The paraffin specimens of HCC and paracancerous tissues were collected and fixed,embedded and sectioned with 4%paraformaldehyde to detect the expression of MLXIP in HCC tissues.9.Rescue experiment design.PcDNA3.1,pcDNA3.1-SNHG16,miRNA-4458 mimics and NC mimics were co-transfected into HCC cells in a certain order to verify the effect of SNHG16 on the proliferation of HCC cells,the effect of sorafinib resistance and the regulation of MLXIP dependent on miR-4458.10.Bioinformatics analysis.A variety of database analysis tools such as starBase,Targetscan and DIANA TOOLS were used to predict and screen the downstream miRNA molecules and ceRNA molecules of SNHG16,and then the expression of ceRNA molecules and their relationship with prognosis were examined by ulcan and the Human protein Atlas.11.Statistic analysis.The statistical analysis was made by using SPSS 16.0 and GraphPad Prism6.0software.The differential expression of SNHG16 in paraffin sections and the correlation between SNHG16 expression and clinicopathological parameters were analyzed by X2 test?chi-square test?.The relationship between the expression of SNHG16 and the survival time of patients with hepatocellular carcinoma was analyzed by Kaplan-Meier and log-rank statistics.To determine which indicators were risk factors for overall survival and disease-free survival in patients with liver cancer,we used Cox proportional risk regression model for univariate and multivariate analysis.The differences between two or more groups of data were compared by using the statistical methods of t-test of independent samples or one-way analysis of variance,respectively.The data were represented by mean±standard deviation,and each experiment was repeated at least 3 times.*P<0.05 was statistically significant,and there were significant differences in the three levels of*P<0.05,**P<0.01 and***P<0.001.Result:1.Expression of SNHG16 in hepatocellular carcinoma and paracancerous tissues?fresh tissues?.The results of qRT-PCR detection showed that the expression level of SNHG16 in HCC tissues was significantly higher than that in corresponding paracancerous tissues for seven of the matched cancer and paracancerous tissues,and the expression level of SNHG16 in paracancerous tissues was almost the same as that in HCC tissues for the other three groups of matched cancers and paracancerous tissues.Therefore,the difference of SNHG16 expression between fresh HCC and paracancerous tissues was statistically significant?*P=0.033?.2.Expression of SNHG16 in 61 cases of hepatocellular carcinoma and 42 cases of paracancerous tissues?paraffin specimens?.The results of chromogenic in situ hybridization?CISH?showed that the positive expression of SNHG16 was mainly located in cytoplasm,and strongly positive in liver tumor tissues,and negative or weakly positive in paracancerous tissues.Further statistical analysis showed that among the sixty-one paraffin specimens of hepatocellular carcinoma,thirty-five cases showed high expression of SNHG16,with a high expression rate of 57.4%,while in forty-two cases of paracancerous tissues,only seven cases showed high expression of SNHG16.The high expression rate was 16.7%?***P<0.001?.3.Relationship between SNHG16 expression and clinicopathological parameters.The results showed that the expression of SNHG16 was significantly correlated with tumor size?*P<0.031?,metastasis?**P<0.007?,AFP level?***P<0.001?and portal vein thrombus?***P<0.001?.There was no significant correlation with age,sex,clinicopathological stage,liver cirrhosis and so on.4.The relationship between the expression of SNHG16 and the prognosis of patients with hepatocellular carcinoma.Kaplan-Meier and log-rank analyzed the relationship between SNHG16 expression and overall survival?OS?or disease-free survival?DFS?in HCC patients.The results showed that compared with patients with lower SNHG16 expression,DFS?***P<0.001?and OS?***P<0.001?were shorter in patients with higher expression of SNHG16,and the difference was statistically significant.5.Further evaluation of whether the level of SNHG16 expression may be used as a factor for poor prognosis in patients with HCC.COX proportional risk regression model for univariate and multivariate analysis,Univariate analysis showed tumor size?***P<0.001?,AFP level?***P<0.001?,PVTT?***P<0.001?.metastasis?***P<0.001?and SNHG16 expression?***P<0.001?were poor prognostic factors in HCC patients,respectively.Multivariate analysis showed that SNHG16 expression?HR=4.985,95%CI=1.451-17.132,*P=0.011?,AFP level?HR=7.078,95%CI=2.465-20.36,***P<0.001?,PVTT?HR=12.082,95%CI=3.660-39.890,***P<0.001?and metastasis?HR=6.179,95%CI=2.188-17.444,***P<0.001?were important independent risk factors for poor prognosis in patients with HCC.6.Expression of SNHG16 in liver cancer cells and normal liver cells.The expression of SNHG16 in four liver cancer cell lines and one normal cell linewas detected by qRT-PCR.The results showed that the expression of SNHG16 in tumor cells Hep3B?15.18±1.675?,HepG2?9.90±0.8381?,SK-Hep-1?5.533±0.7095?and Huh7?6.233±0.8145?was significantly higher than that in normal cells?1.000±0.8145?.The difference was statistically significant?*P=0.011?.7.Effect of SNHG16 on proliferation of liver cancer cells.The results of MTT assay showed that after interfering with the expression ofSNHG16 in Hep3B and HepG2,the proliferation ability was significantly lower than that of the control group?**P<0.01?.Similarly,after we overexpressed SNHG16 in SK-Hep-1 and Huh7,compared with the control group,the proliferation ability was significantly enhanced.The difference was statistically significant?**P<0.01?.8.Effect of SNHG16 on invasion and metastasis of liver cancer cells.The results of transwell experiment showed that it could significantly improve the migration ability of hepatocellular carcinoma cells.The migration ability of HCC cells was significantly decreased after SNHG16 small interference RNA was transfected into HCC cell line Hep3B?**P<0.01?.9.Effect of SNHG16 on sorafenib resistance in liver cancer cells.With the increasing concentration of sorafenib,the cell survival of Hep3B and HepG2 cells transfected with SNHG16 was significantly lower than that of the control group,and the difference was statistically significant?**P<0.01?;while the cell survival of SK-Hep-1 and Huh7 cells transfected with plasmid vector pcDNA3.1-SNHG16 was significantly higher than that of the control group,the difference was statistically significant?**P<0.01?.10.Prediction and screening validation of ceRNA and miRNA of SNHG16.The ceRNA column in the database"Database starBase 2.0."is used to predict the mRNA,of coding genes that may compete with SNHG16.We selected five alternative ceRNA molecules and used the RT-RCR method to verify the analysis.After predictive screening,we chose miR-4458 and MLXIP as downstream molecules and ceRNA molecules.11.qRT-PCR experiment shows that SNHG16 and miR-4458 can regulate each other.The results showed that the expression of miR-4458 was significantly increased after interference with SNHG16 in Hep3B and HepG2 cell lines?**P<0.01?,while the expression of miR-4458 showed a significant decrease after overexpression of SNHG16 in hepatoma cell lines SK-Hep-1 and Huh7?**P<0.01?.At the same time,according to the principle of ceRNA mechanism,we transfected miR-4458 mimics into liver cancer cells and found that the expression of SNHG16 was significantly decreased after miR-4458 was overexpressed in Hep3B and HepG2 cells?**P<0.01?.12.Direct targeting binding of SNHG16 to miR-4458 verified by luciferase experiment.The results showed that the luciferase activity of the wild-type SNHG16 vector was significantly down-regulated by miR-4458?***P<0.001?,while the luciferase activities of the mutant plasmid pmirGLO-SNHG16-mut?miR-4458?and the blank plasmid pmirGLO were not weakened.13.Expression of miR-4458 in hepatocellular carcinoma and its correlation with SNHG16 expression.qRT-PCR was used to detect the expression of miR-4458 in ten fresh liver cancer tissues and adjacent tissues.The results showed that the expression of miR-4458 in liver cancer tissues was significantly lower than that in adjacent tissues?***P=0.0004?.The correlation between the expression of miR-4458 in HCC and the expression of SNHG16 was analyzed,and the method of pearson correlation analysis was used.The results showed that there was a significant negative correlation between the expression of SNHG16 and miR-4458?r=-0.4976,*P=0.0227?.14.Effects of miR-4458 on proliferation of hepatocellular carcinoma cells and drug resistance to sorafinib.After overexpression of miR-4458 in hepatocellular carcinoma cell line Hep3B with miR-4458mimics,MTT assay showed that the proliferation of tumor cells was significantly inhibited compared with the control group?***P<0.001?;In addition,when the increasing concentration of sorafenib was added,the survival number of HCC cells whose miR-4458 have been overexpressed was significantly lower than that of the control group?***P<0.001?.15.Effect of MLXIP on proliferation of hepatocarcinoma cells and resistance to sorafenib.The small interfering siRNA of MLXIP was used to down-regulate the expression of MLXIP in hepatocellular carcinoma cell line Hep3B.It was found that compared with the control group,the proliferation activity of liver cancer cells interfered with MLXIP was significantly decreased?**P<0.01?,while in the drug resistance of sorafenib,the survival of cells decreased significantly with the increase of the concentration gradient of sorafinil after the interference of MLXIP?**P<0.01?.16.Regulatory effect of miR-4458 on MLXIP in hepatoma cells.To verify whether miR-4458 regulates MLXIP in hepatoma cells,we transfected miR-4458 mimics in hepatoma cells Hep3B and HepG2,and detected the expression level of MLXIP by qRT-PCR.The results showed that the expression level of MLXIP was significantly down-regulated after overexpression of miR-4458 in hepatoma cells?***P<0.001?.17.SNHG16 can regulate the expression of MLXIP in hepatoma cells.Plasmid pcDNA3.1-SNHG16 and plasmid pcDNA3.1?empty transfer as control group?were transfected into Huh7 to observe the changes of MLXIP protein expression.The results showed that overexpression of SNHG16 in HCC cells could significantly increase the expression of MLXIP protein?*P=0.0117?;At the same time,we also observed the expression of MLXIP protein after siRNA transfection in hepatocellular carcinoma cell line Hep3B.The results showed that the expression of MLXIP protein was significantly decreased?*P=0.0158?.18.Immunohistochemical detection of MLXIP protein expression in hepatocellular carcinoma and the analysis of its relationship with SNHG16 expression.The protein expression of MLXIP was found to be positively expressed in cytoplasm in liver cancer tissues,and the expression level was significantly higher than that in adjacent tissues?***P<0.001?.Further,spearman rank correlation test was used to analyze the relationship between SNHG16 expression and MLXIP protein expression.The results showed a significant positive correlation?r=0.574,***P<0.001?.19.Rescue experiment shows that the regulatory effect of SNHG16 on MLXIP can be attenuated by miR-4458.After the plasmid pcDNA3.1-scramble,pcDNA3.1-SNHG16,NC mimics and miR-4458 mimics were co-transfected into hepatocellular carcinoma cells Huh7 cells in a certain order,we used qRT-PCR to detect the changes of mRNA expression of MLXIP.The results showed that the positive regulatory effect of SNHG16 on MLXIP could be weakened by miR-4458?***P<0.001?.20.Rescue experiment shows that the ability of SNHG16 to promote the proliferation of hepatocellular carcinoma cells and its resistance to sorafinib depends on miR-4458.Plasmid pcDNA3.1-scramble,pcDNA3.1-SNHG16,NC mimics and miR-4458mimics were co-transfected into HCC cells in a certain order in Huh7 cells.It was found that the ability of SNHG16 to promote the proliferation of hepatocellular carcinoma cells and its resistance to sorafinib was decreased by the overexpression of miR-4458?**P<0.01?.Conclusion:1.SNHG16 has the attribute of oncogene in hepatocellular carcinoma and promotes the proliferation,migration and sorafinib resistance of hepatocellular carcinoma cells.2.SNHG16 could be used as a molecular indicator of poor prognosis.3.SNHG16 can bind to the downstream molecule miR-4458 and down-regulate each other.4.MiR-4458 has the attribute of tumor suppressor gene in hepatocellular carcinoma,which can inhibit the proliferation of HCC cells and the drug resistance of sorafenib.5.MiR-4458 can regulate the expression of MLXIP.6.MLXIP has the attribute of oncogene in hepatocellular carcinoma,which promotes the proliferation of HCC cells and the drug resistance of sorafenib.7.SNHG16 competes with MLXIP for binding to miR-4458,which is a ceRNA. |
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