| Background:High incidence rate and high mortality rate of Primary liver cancer are the common causes leading to cancer death,which seriously endangers human health.As the most common pathological type of primary liver cancer,hepatocellular carcinoma(HCC)accounts for more than 90%of primary liver cancer cases[1].At present,although various treatment methods are in the ascendant,for early liver cancer,surgical treatment is still the first choice in the current guidelines for the treatment of liver cancer at home and abroad.However,the biggest problem in the treatment of HCC is that most of the tumors are found in the middle and late stages,and the opportunity for effective surgery is lost.For advanced liver cancer,although new treatment methods such as targeted therapy and immunotherapy have appeared and achieved encouraging results,the mechanism of HCC has not been fully clarified,and the combined application of multiple treatment methods is still the mainstream of the treatment of advanced HCC.Therefore,it is still of great significance to find new potential molecular mechanisms and new treatment strategies for HCC.MicroRNAs(miRNAs)are small noncoding RNAs,usually 18-23 endogenous oligonucleotides in length,that are widely expressed in various tissues and cells of the body.They can be used as proto-oncogenes or tumor suppressor genes to specifically bind to the 3’-UTRs of downstream target genes to regulate tumor cell proliferation,apoptosis,invasion and other tumorigenesis and development processes.They are currently the most studied non-coding RNAs.miR-4461 is a newly discovered mitochondria-related non-coding small RNA.Studies have shown that mir-4461 inhibits the occurrence and development of some tumors such as rectal cancer and renal cell carcinoma.COPB2 expression in colorectal cancer inhibits tumorigenesis;miR-4461 inhibits renal cell progression by targeting and downregulating PPPLR3C;in addition,Studies have shown that mir-4461 can target PTEN to enhance the malignant biological behavior of ovarian cancer cells.However,the biological function of miR-4461 in HCC remains unclear,therefore,this research mainly discusses the mechanism of miR-4461 in HCC..Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1(NUCKS1)is a highly conserved DNA-binding protein associated.A large number of studies indicate that NUCKS1 is up-regulated in various cancers and is involved in tumor growth and metastasis.Studies have indicated that NUCKS1 in HCC tissues is higher than that in paired adjacent tissues,However,the research on its mechanism is still insufficient.Therefore,NUCKS1 is a potential target for the treatment of liver cancer.Inhibiting the expression of NUCKS1 may be of great significance for the treatment of HCC.In this study,we used biological information analysis,clinicopathological data,in vivo and in vitro experiments as research approaches to explore how miR-4461 affects the biological behavior of HCC through the regulation of NUCKS1,in order to find a new method to treat HCC.Part 1 miR-4461 expression in hepatocellular carcinomaPurposes:To explore the expression of miR-4461 in liver cancer tissues and paired adjacent tissues and its relationship with tumor microvascular invasion and metastasis.Methods:1.HCC tissue samples and paracancer normal liver tissue samples from 53 HCC patients were collected.Quantitative real-time Polymerase Chain Reaction(qRT-PCR)was used to detect the expression of mir-4461 in hepatocellular carcinoma tissues and their adjacent tissues.2.Fifty-three patients were classified as non-microvascular invasive according to the presence of vascular invasion and distant metastasis.non-MVI group(N=23),microvascular invasive(MVI)group(N=19)and intrahepatic metastasis(IHM)group(N=11),The relationship between miR-4461 expression level and the three groups was analyzed.Results:1.The expression level of miR-4461 in liver cancer tissue was lower than that in adjacent tissue,and the difference was statistically significant(P<0.05).2.There was significant difference in the expression level of miR-4461 among non-MVI,MVI and IHM groups(P<0.05).The expression level of miR-4461 was the highest in non MVI tissue,lower in MVI tissue and the lowest in IHM tissue.Conclusions:The expression of miR-4461 in HCC was significantly lower than that in normal adjacent tissues;With the progress of HCC,we can see that the level of miR-4461 in non-MVI,MVI and IHM tissues decreased step by step,suggesting that the decrease of miR-4461 may promote the evolution of HCC,resulting in the occurrence of malignant biological behavior of microvascular invasion and metastasis.Part 2 Regulation of miR-4461 on the malignant biological behavior of hepatocellular carcinoma cellsPurposes:The effects of miR-4461 on the biological functions(proliferation,clone formation,invasion,migration and apoptosis)of HCC cells were detected by in vivo and in vitro experiments.Methods:1.The expression of miR-4461 in normal hepatocytes HL7702 and HCC cell lines BEL-7402,Hep3B,HepG2,SMMC-7721,97H,HCCLM3 and Huh7 was detected by real-time quantitative PCR,and two cell lines were screened for followup experiments.2.Construct miR-4461 overexpression,knockdown and control stably transfected hepatoma cells by lentivirus technology,or transfect cells with miR-4461 mimics for miR-4461 overexpression.3.The overexpression and inhibitory effect of miR-4461 in the selected liver cancer cells were detected by qRT-PCR.The CCK-8 method was used to detect the cell proliferation at 24,48,72,and 96 hours;Cell apoptosis was detected by flow cytometry.4.The effects of overexpression and inhibition of miR-4461 on the migration and invasion of hepatoma cells were detected by transwell assay and pre-gel transwell assay.5.The constructed miR-4461 overexpression,knockdown and control cells were inoculated into nude mice subcutaneously,6 in each group,to detect the effect of miR-4461 on the proliferation of hepatoma cells in nude mice,4 weeks later.Tumor nudes were collected and weighed,and the collected tumor nudes were subjected to HE staining and immunohistochemical staining for Ki-6 7.miR-4461 knockdown and control stable cells were injected into the tail vein.Lung tissues were collected at 8 weeks,and lung metastasis of tumor was observed by HE staining.6.The correlation between miR-4461 expression and Ki-67 expression(expression intensity of Ki-67 in clinicopathological report)in clinical HCC tissue samples was analyzed.Results:1.Compared with normal hepatocytes HL7702,the expression of miR-4461 in seven hepatoma cell lines decreased,while the expression of miR-4461 in HCCLM3 and Huh7 cell lines was the lowest.Therefore,HCCLM3 and Huh7 cell lines were selected for follow-up experiments.2.After miR-4461 overexpression lentivirus infected HCCLM3 and Huh7 cells,the expression of miR-4461 was significantly increased,while after infection with knockdown lentivirus,the expression of miR-4461 was significantly decreased(P<0.05),indicating that the overexpression and knockdown stable cells were successfully constructed.After miR-4461 mimics were transfected into cells,the expression of miR-4461 increased significantly(P<0.05),indicating the successful establishment of transient cells3.Compared with the control group,the proliferation,invasion and migration of HCCLM3 and Huh7 cells decreased significantly after overexpression of miR-4461(P<0.05),while the proliferation,migration and invasion of HCCLM3 and Huh7 cells increased significantly after inhibition of miR-4461(P<0.05).Moreover,overexpression of miR-4461 by mimics could significantly promote the apoptosis of HCCLM3 and Huh7 cells(P<0.05).4.Compared with the control group,overexpression of miR-4461 could significantly reduce the weight and volume of transplanted tumor(P<0.05),and the positive rate of Ki-67 immunohistochemical staining was reduced;Inhibiting the expression of miR-4461 could significantly increase the weight and volume of transplanted tumor(P<0.05);The positive rate of Ki-67 immunohistochemical staining was increased.5.Linear analysis showed that there was a negative correlation between the expression of miR-4461 and Ki-67 in clinical HCC samples.Conclusions:1.In vitro,inhibition of miR-4461 can reduce the proliferation,invasion and migration of HCCLM3 and Huh7 cells;Overexpression of miR-4461 can upregulate the proliferation,invasion and migration of HCCLM3 and Huh7 cells,and promote the apoptosis of HCC cells.It is suggested that miR-4461 can inhibit the proliferation,invasion and migration of HCC cells in vitro,and promote the apoptosis of HCC cells2.When inhibiting the expression of miR-4461 in HCCLM3 cells in nude mice,the subcutaneous tumorigenesis and lung metastasis of HCCLM3 cells were strengthened,while the subcutaneous tumorigenesis effect of HCCLM3 cells overexpressing miR-4461 was significantly reduced in nude mice,suggesting that miR-4461 plays a negative regulatory role in the proliferation,migration and invasion of liver cancer cells in vivo.Part 3 miR-4461 inhibits malignant biological behavior of HCC cells by targeting NUCKS1Purposes:To explore the molecular mechanism of miR-4461 on malignant biological behaviors such as proliferation,cloning,invasion,migration and apoptosis of liver cancer cells.Methods:1.Predict the target genes of miR-4461 on Targetscan,TarBase,miRmap,and miRbase,find out the predicted common target genes of miR-4461,and combine the previous proteomics results in the laboratory and GEPIA online tumor database for further Screening to preliminarily determine the target genes of miR-4461.2.To verify the direct interaction between miR-4461 and NUCKS1,construct wild-type NUCKS1 wild-type(NUCKS1-WT)and NUCKS1 mutant(NUCKS1-Mut)dual-luciferase reporter vectors,and transfer the constructed vectors into miRs respectively In HCCLM3 and Huh7 cells overexpressed or knocked down by-4461,the fluorescence intensity of cells was detected by dual-luciferase reporter gene detection kit.3.qRT-PCR and Western Blot were used to detect the expression of NUCKS1 after overexpression or inhibition of miR-4461 in HCCLM3 and HUH7 cells,respectively.Correlation analysis was performed based on the mRNA expression of miR-4461 and NUCKS1 in human HCC tissues.4.Via qRT-PCR,WB and IHC the expression of NUCKS1 in clinical HCC tissues and paired adjacent tissues was detected.5.Three kinds of NUCKS1 siRNA sequences(si-1,si-2,si-3)were constructed by small interfering RNA(siRNA)interference technology,and three siNUCKS1 sequences were used to knock down the expression of NUCKS1 in HCCLM3 and Huh7 cells,the inhibitory efficiency of NUCKS1 was detected by qRT-PCR and WB technology,and the siRNA with the highest inhibition efficiency was selected for follow-up experiments6.HCCLM3 and HUH7 cells were transfected with the most efficient siRNA(siNUCKS1)and control siRNA(si-NC)for silencing NUCKS1,respectively.CCK-8 was used to detect HCCLM3 and Huh7 cells at 24,48,72,and 96 hours after transfection.Plate cloning assay was use to reveal the proliferation of HCCLM3 and Huh7 cells;the migration and invasion abilities of HCCLM3 and Huh7 cells after transfection were detected by transwell assay and pre-gel transwell assay.7.The siRNA with the highest NUCKS1 silencing efficiency(siNUCKS1)and the control siRNA(si-NC)were used to transfect miR-4461 to knockdown and stably transfect HCCLM3 and Huh7 cells,respectively.CCK-8 was used to detect the expression of HCCLM3 and Huh7 cells after transfection.Cell proliferation at 24,48,72,and 96 hours;plate cloning method was used to detect the difference of cell clone formation ability;transwell experiment and pre-gel transwell experiment were used to detect the change of migration and invasion ability of HCCLM3 and Huh7 cells after transfection.Results:1.The intersection of Targetscan,TarBase,miRmap and miRbase was analyzed to obtain 33 downstream target genes that can interact with miR-4461.Compared with the proteomic data of our laboratory and 33 target genes,NUCKS1 and SET were two candidate genes with significant difference in expression(P<0.05).The analysis of GEPIA website showed that,NUCKS1 showed significant difference between cancer and adjacent tissues in TCGA HCC database(P<0.05),but SET did not show significant difference.Therefore,NUCKS1 was selected as the target gene for further research.2.According to the detection results,the fluorescence activity in control cell of NUCKS1-WT was significantly higher than that in miR-4461 knockdown HCCLM3 and Huh7 cells;the fluorescence activity of NUCKS1-WT was significantly increased in miR-4461 overexpressed HCCLM3 and HUH7 cells.significantly decreased in cells.However,the fluorescent activity of NUCKS1-Mut was not significantly altered in both HCCLM3 and Huh7 cells either inhibited or overexpressed by miR-4461 activity.3.Compared with the control group,the expression levels of NUCKS1 mRNA and protein in HCCLM3 and Huh7 cells increased when the expression of miR-4461 was inhibited,while the expression levels of NUCKS1 mRNA and protein in HCCLM3 and Huh7 cells were significantly inhibited after the overexpression of miR-4461(P<0.05).The correlation analysis of the mRNA expression of miR-4461 and NUCKS1 in human HCC showed that there was a significant negative correlation between miR-4461 and NUCKS1(P<0.05).4.qRT-PCR,WB and IHC detection showed that the level of NUCKS1 in human HCC tissues was significantly upregulated than that in normal tissues(P<0.05).5.Compared with the cells transfected with si-NC,the proliferation,clone formation,invasion and migration of HCCLM3 and Huh7 cell lines decreased significantly after transfection of siNUCKS1 and silencing NUCKS1(P<0.05)6.Compared with the control group,the proliferation ability of HCCLM3 and Huh7 cells increased after inhibiting miR-4461,and decreased after transfection of siNUCKS1.However,in miR-4461 knockdown cells,transfection of siNUCKS1 reversed the promotion of miR-4461 knockdown on cell proliferation,clone formation,migration and invasion.Conclusions:NUCKS1 is a downstream target of miR-4461 in HCC,and miR-4461 can inhibit the expression of NUCKS1 at gene and protein levels;It is suggested that miR-4461 can affect the biological function of HCC cells by negatively regulating the expression of NUCKS1. |
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