The Role And Mechanism Of Tieng In Promoting The Proliferation And Metastasis Of Hepatocellular Carcinoma By Activating PI3K/Akt/mTOR Pathway Through CSTF

ObjectiveHepatocellular carcinoma(HCC)is a kide of highly invasive malignant tumor with a poor prognosis and the pathogenesis remains unclear.Previous reports have shown that TINAG is closely related to kidney-related diseases,but there has been no research on the role of TINAG in the development of HCC.Therefore,this study used TCGA database to analyze the expression of TINAG in HCC,to detect the expression level of TINAG in HCC samples and its relationship with clinicopathological data,and to study the effect of TINAG on the biological activity of HCC cells by in vitro cell experiments and internal experiments,such as proliferation,invasion,and proliferation.Then we initially explore its mechanisms.Methods(1)First using TCGA and KEGG database,using DAVID information analysis platform to analyze the differentially expressed genes in HCC,screening Pudmed on the literature report number less than 300,and selecting the research target by logFC value ranking,selecting significantly up-regulated genes for correlation analysis as research target.(2)After collecting the clinical specimens,we detect the expression of TINAG in HCC tissues and paracancer tissues,and analyze the relationship between clinical pathological parameters and survival rate of patients with TINAG and HCC.(3)By molecular biology technology,we detected TINAG expression in HCC cell lines(HepG2,HB611,HHCC and Hep3B),while human normal liver line(HL-7702)using cytology technology,The expression of TINAG in HCC cells was designed and synthesized by si-TINAG knockdown,and the corresponding control group(si-con)was designed and synthesized.Si-TINAG and si-con were transfected into HCC cells respectively to detect TINAG mRNA and protein in cells.Expression;CCK-8 method was used to detect the proliferative activity of the transfected cells,and the colony forming ability of each group was detected.The scratching test and transwell chamber method were used to research the migration and invasion ability of each group after transfection;Apoptosis and cell cycle were detected by flow cytometry.(4)Using plasmid infection knocking TINAG HCC cell line construct naked mouse skin micro-tumor model,Through the internal experiment,TINAG were infected with plasmid and divided into two groups(shcon group and shTINAG group).The tumor volume were observed.The tissue sections proliferation were detected with Ki-67 staining.The apoptosis-related indicators were detected with TUNEL staining:(5)After Transfected HepG2 and Hep3B cells toTINAG silence,each groups of cells were detected with Western Blot method.The protein expressions of p-PI3K,PI3K,p-Akt,Akt,p-p7026k and p7026k were detected;The changes of p-PI3K,PI3K,p-Akt,Akt,p-p7026k and p7026k proteins were observed by adding LY294002,an inhibitor of PI3K/Akt pathway.(6)The changes of other genes in HCC cells after knocking down TINAG were analyzed by sequencing and the possible enriched signal pathways were predicted by GESA;The phosphorylation expressions of p-Akt,p-PI3K,and P-70S6K in si-TINAG group and control group were detected by Western blot.The significant correlation between TINAG and other genes was analyzed by the TCGA database.(7)si-TINAG and si-NC were transfected into HCC cells including HepG2 and Hep3B.Respectively,the protein expression of CSTF2 was detected by western blot.The cells of HepG2 and Hep3B were respectively transfected with si-TINAG#1,si-con,si-TINAG#1+PC-DNA3.1NC,and si-TINAG#1+PC-DNA CSTF2.The expressions of PI3K\AKT signaling pathway proteins such as p-PI3K,PI3K,p-AKT,AKT,p-mTOR,and mTOR were detected by western blot.(8)Detecting the expression of CSTF2 in HCC tissues and HCC cells,and analyze its survival rate.(9)CSTF2 was overexpressed in Huh7 cells,and CSTF2 was knocked down in HCCLM3 cells to detect the colony formation ability of cells in each group by clone formation assay.EDU assay was used to detect cell proliferation.Scratch assay and transwell chamber assay were used to detect the migration and invasion ability of the transfected cells.Flow cytometry was used to detect cell apoptosis,and Western blot was used to detect the expression of cleaved caspase-3,Bax,Bcl2 and EMT-related proteins,as well as the phosphorylation of pathway proteins PI3K,Akt and mTOR.(10)After transfection of si-CSTF2#2,si-NC and si-CSTF2#2+740Y-P(PI3K activator)into HCCLM3 cells,colony forming ability of cells in each group was detected by clone formation assay.EDU assay was used to detect cell proliferation.Scratch assay and transwell chamber assay,were used to detect cell migration and invasion.Flow cytometry was used to detect cell apoptosis,and western blot was used to detect the expression of EMT-related proteins.After PC-DNA CSTF2,PC-DNA 3.1NC and PC-DNA CSTF2+Wortmannin(PI3K inhibitor)were transfected into Huh7 cells,the colony forming ability of each group was detected by clone formation assay.EDU assay was used to detect cell proliferation.Scratch assay were used to detect the migration.ability Rranswell chamber assay were used to detect the invasion ability of the transfected cells.Flow cytometry was used to detect cell apoptosis,and western blot was used to detect the expression of EMT related proteins.Results(1)After differential gene screening,3,682 significant differentially expressed genes were obtained,of which 3403 were up-regulated and 279 were down-regulated.The number of reported cases in Pudmed was less than 300,and the research target was selected by log FC value.The genes with significant up-regulation were selected for correlation analysis.Finally,the differentially expressed TINAG gene in HCC was screened out.There are 29 literatures on TINAG currently included in pubmed.By analyzing the data of liver cancer patients obtained,TINAG has high expression in liver cancer patients,which is related with lymph node metastasis of liver cancer,and with the high expression of TINAG,the survival rate of liver cancer patients is reduced,and TINAG can be used as an independent predictor of liver cancer prognosis.(2)The expression level of TINAG mRNA in HCC tissues was significantly higher than that in the paracancer tissues(p<0.0001).TINAG is associated with pathological staging and distant metastasis of lymph node metastasis in patients with HCC.There was a remarkbale difference in survival between the TINAG high expression group and the low group(P=0.006).The samples were analyzed for univariate and multivariate according to the level of TIGAN expression,age,gender,grade,lymphatic metastasis,and distant metastasis.The results show that TINAG can be used as an independent predictor of liver cancer prognosis.(3)Compared with HL-7702,there was no significant difference in the expression level of TINAG in HB611 cells.The expression level of TINAG in HepG2,HHCC and Hep3B cells,was noteworthly higher than that in HL-7702 cells(HepG2 and Hep3B cells p<0.001,HHCC)Cells p<0.05).The mRNA and protein expression levels of TINAG in HepG2 and Hep3B cells transfected with si-TINAG were significantly lower than those in si-con group(P<0.001).In HepG2 and Hep3B,compared with si-con group,the growth of cells was inhibited(P<0.001),the cell migration ability and invasion ability were decreased(P<0.001),the colony forming ability was significantly decreased(P<0.001),and apoptosis ability was increased(P<0.001)in si-TINAG group;Cell cycle was arrested in G1 and M phases.(4)The growth of subcutaneous tumor in nude mice was observed,after subcutaneous injection of plasmid infected HCC cells from day 6.On the 21st day,the volume of subcutaneous tumor in shTINAG group was significantly smaller than that in the control group.And with the extension of time,the difference between the two groups gradually increased.The results of HE staining,Ki67 staining and tunel staining further showed that the proliferation of lung cancer cells was significantly inhibited and the apoptosis rate was significantly increased.(5)In HepG2 and Hep3B,the ratio of p-PI3K/PI3K and p-Akt/Akt in si-TINAG group were significantly decreasedcompared with si-con group(P<0.001).Tthe expression level of p-70s6k protein was significantly decreased(P<0.001),but there was no significant difference in the total expression levels of Akt,PI3K and Akt.After intervention with LY294002,the p-PI3K/PI3K,p-Akt/Akt,p-p60s6k/p60s6k ratios of si-TINAG and LY294002 groups were significantly lower than those of si-con group;The p-PI3K/PI3K,p-Akt/Akt and p-p60s6k/p60s6k ratios in si-TINAG+LY294002 group were significantly lower than those in si-TINAG and LY294002 groups.(6)The results of sequencing analysis showed that CSTF2 gene changed significantly in HCC cells after knocking down TINAG,and PI3K/Akt/mTOR pathway was enriched by GSEA analysis.Western blot results showed that compared with the control group,the ratio of p-Akt/AKT,p-PI3K/PI3K and p-70S6K/GAPDH in si-TINAG group was significantly decreased,and the degree of phosphorylation was significantly decreased(P<0.01).A significant correlation between TINAG and CSTF2 was found in TCGA database(P<0.001).(7)The expression of CSTF2 in the si-TINAG group was significantly decreased compared with the control group(P<0.01);The results showed that the phosphorylation of PI3K,Akt and mTOR decreased after knocking down TINAG alone,while knockdown of TINAG and overexpression of CSTF2 simultaneously could reverse the pathway inactivation caused by knockdown of TINAG alone.(8)The expression of CSTF2 in HCC tissues was analyzed by TCGA-HCC database.The expression level of CSTF2 in HCC tissues was significantly higher than that in normal tissues(P<0.05);Compared with lower CSTF2,the expression The TCGA database showed that patients with high CSTF2 expression had a lower overall survival rate(P<0.001);The results of qRT-PCR and Western blot showed that expression of CSTF2 in HCC cells were higher than the normal liver tissue cells at the transcription and protein level(P<0.01).(9)qRT-PCR and western blot showed that CSTF2 was successfully overexpressed in Huh7 cells,and CSTF2 was successfully knocked down in HCCLM3 cells(P<0.01);Colony formation assay detects that CSTF2 overexpression significantly promoted colony formation of Huh7 cells(P<0.01);EdU assay showed that the number of positive cells increased significantly after overexpressing CSTF2 in Huh7 cells(P<0.01);Apoptosis assay showed that compared with the control group,overexpression of CSTF2 reduced the apoptosis rate(P<0.01);Western blot results further showed that overexpression of CSTF2 could reduce the expression of apoptosis-related cleavedcaspase-3 and Bax proteins(P<0.01),the expression of Bcl-2 increased(P<0.01);Compared with the control group,the results of cell invasion assay showed overexpression of CSTF2 remarkbalely ideveloped the number of cell invasion and migration(P<0.01);Western blot analysis detected that overexpression of CSTF2,and EMT markers Claudin 1 and ZO-1,decreased in Huh7 cells(P<0.01).The expressions of MMP-9,MMP-2,Vimentin and N-cadherin increased(P<0.01).The ratios of p-Akt/AKT,P-PI3K/PI3K and P-mTOR/mTOR developed remarkly after overexpression of CSTF2,and the phosphorylation of AKT,PI3K and mTOR increased significantly(P<0.01).However,the result of knockdown of CSTF2 was opposite to the above experiment.(10)Western blot results showed that the phosphorylation degree of PI3K/AKT/mTOR pathway was decreased and the pathway was inactivated in,si-CSTF2#2 group.However,,si-CSTF2#2+740Y-P group could significantly reverse the inactivation of PI3K/AKT/mTOR triggered by CSTF2 knockdown(P<0.01).The results of clone formation assay showed that 740Y-P could significantly reverse the low colony formation in si-CSTF2-HCCLM3 cells(P<0.01).EDU test results detected that the number of positive cells increased significantly after adding 740Y-P compared with CSTF2 knockdown(P<0.01);Apoptosis assay showed that 740Y-P significantly eliminated the phenomenon of increased apoptosis rate caused by CSTF2 knockdown(P<0.01);In addition,740Y-P treated cells reversed the inhibition of invasion and migration in si-CSTF2-HCCLM3 cell group(P<0.01);Western blot assay drteceded that 740Y-P could reverse the expression of EMT related proteins triggered by CSTF2.Transfection of PC-CSTF2,PC-DNA 3.INC and PC-DNA CSTF2+Wortmannin(PI3K inhibitor)in Huh7 cells could also reverse the process of malignant development of CSTF2 to hepatocellular carcinoma cell phenotype.Conclusion(1)TINAG is overexpressed in HCC.(2)TINAG is related to hepatocarcinoma invasion,patient survival and poor forward positively correlation.(3)TINAG can develop proliferation,invasion,and migration of liver cancer cells,while inhibit apoptosis.(4)TINAG affects the biological behavior of liver cancer by activating the PI3K/Akt signal transduction pathway.(5)CSTF2 overexpression in hepatocellular carcinoma is negatively correlated with the survival time and poor prognosis of patients.(6)There was a significant correlation between TINAG and CSTF2(7)Overexpression of CSTF2 aggravates the malignant phenotype of HCC cells(8)TINAG promotes the malignant progression of HCC cell phenotype by regulating PI3K\AKT signaling pathway through CSTF2.