| Section I The up-regulated expression of CTB-193m12.5 was positively correlated with the poor prognosis of hepatocellular carcinomaBackground Hepatocellular carcinoma(HCC)is one of the most common malignancies with high mortality and high invasiveness.Because of the high rate of recurrence and metastasis after hepatectomy,the long-term prognosis of hepatocellular carcinoma is still not satisfied.Therefore,it is of great value to search for new targets reflecting the early diagnosis and prognosis of recurrence and metastasis of tumor for treatment and prognosis.Objective To investigate the clinical correlation between Lnc RNA CTB-193M12.5 and hepatocellular carcinoma.Methods We collected 78 cases of HCC tissues and adjacent tissues(>3cm).All patients had not receive preoperative radiotherapy or chemotherapy before surgery.Tissue samples immediately after surgery in vitro with liquid nitrogen frozen and stored in-80 ? for further analysis.Three pairs of HCC and paracancer tissues were sequenced and analyzed.Moreover,DAVID software was used to do function enrichment analysis.Cytoscape software was used to construct the network graph.We detected the relevant Lnc RNAs,and selected the sensitive Lnc RNA for further experiments.RT-PCR was used to detect the differences in the expression levels of CTB-193M12.5 in the HCC and adjacent tissues,and the correlation between the expression levels of ctb-193m12.5 and mi R-195-5p and their pathological stages and prognosis was analyzed.AT the same time,the correlation between CTB-193M12.5 and mi R-195-5p in the same tissue was analyzed.Western blot,RT-PCR and immunohistochemistry were used to detect the differences in HMGA1 expression in HCC and adjacent tissues.Result The bioinformatic analysis indicated that the up-regulated Lnc RNA was CTB-193M12.5,the downstream mi RNA was mi R-195-5p.There was a negative correlation between CTB-193M12.5 and hsa-mi R-195-5p in the same HCC tissue.CTB-193M12.5 was positively correlated with HMGA1,promoting the growth and proliferation of hepatocellular carcinoma.In the tissue samples,the level of CTB-193M12.5 and HMGA1 expressed more highly in the HCC than that in the paracancer tissues,which showed a positive correlation.Conclusion CTB-193M12.5 was negatively correlated with mi R-195-5p,and positively correlated with HMGA1.Patients with higher expression of CTB-193M12.5 in HCC tissue had lower expression of mi R-195-5p,and had a later stage and more poor prognosis.Backgroud As shown in section 1,CTB-193M12.5 was expressed highly in HCC tissues and its expression correlated with tumor developmental stage and prognosis.Further more,the expression of CTB-193M12.5 negatively correlated with the expression of mi RNA mi R-195-5p and positively correlated with HMGA1,suggesting tha CTB-193M12.5 promotes the development of HCC through regulating mi RNA mi R-195-5p and HMGA.However,the exact mechanismremains to be investigated.Objective The test aimed to investigate the specific mechanism by which CTB-193M12.5 promotes the proliferation and progression of hepatocellular carcinoma by regulating the expression of mi R-195-5p.Method According to the results in section I,CTB-193M12.5,mi R-195-5p and target protein HMGA1 were selected for doing further experiments.Two sensitive liver cancer cells(Hep G2,smmc-7721)were selected for functional experiments.2.1 The interference vector of CTB-193M12.5 was constructed by plasmids.After transfection into HCC cells,the interference efficiency of each vector was detected by RT-PCR,and the vector was screened for subsequent experiments.2.2 The proliferation activity of transfected HCC cells was detected by CCK-8 and EDU,and the invasion and migration ability of HCC cells were detected by scratch test and Transwell.The effects of si CTB-193M12.5 on the proliferation,invasion and migration of HCC cells were analyzed.2.3 The subcutaneous tumorigenesis model of nude mice was established to study the inhibitory effect of si CTB-193M12.5 group on the growth of HCC transplanted tumor in nude mice.2.4 RT-PCR was used to select the mi RNA with the closest competitive binding to CTB-193M12.5.2.5 Dual-luciferase assay was used to verify whether CTB-193M12.5 and mi R-195-5p could bind competitively,and analyze the effect of mi R-195-5p on the expression of HMGA1.2.6 At different time,mi R-NC,mi R-195-5p,mi R-195-5p + pc DNA-NC,mi R-195-5p +pc DNA/ CTB-193M12.5 were transfected to SMMC7721 and Hep G2 cell lines respectively,CCK-8 method and EDU method were used to test the proliferation activity of HCC cells,and the invasion and migration ability of HCC cells were studied by scratch test and Transwell.2.7 After mi R-NC,mi R-195-5p,mi R-195-5p+pc DNA-NC,mi R-195-5p+pc DNA/CTB-193M12.5,si RNA-NC,si CTB-193M12.5,pc DNA-NC,pc DNA/CTB-193M12.5 transfected to HCC cells,Western blot was used to detect the expression of HMGA1 in each cell line.2.8 Western blot was used to detect the expression of EMT signaling pathway-related proteins in HCC cell lines.Result We found that: 2.1 the si CTB-193M12.5-1 and si CTB-193M12.5-2 were specific interference vectors for CTB-193M12.5 sequence,which can effectively prevent the off-target effect,and the interference efficiency of si CTB-193M12.5-1 was more than 70%,which could be used for subsequent experiments.2.2 compared with the NC and sirna-nc transfection groups,SMMC-7721 and Hep G2 cell lines showed significantly decreased proliferation activity,invasion and migration ability after transfection of si CTB-193M12.5 for 48 h.2.3 compared with the si RNA-NC group,the tomor growed significantly lower and smaller in sictb-193m12.5 transfection group.2.4 RT-PCR test showed that only mi R-195-5p was significantly higher than other mi RNAs in SMMC7721 and Hep G2 cell lines transfected with si CTB-193M12.5(P<0.01).2.5 compared with the mi R-NC transfection group,the relative luciferase activity of p MIR-CTB-193M12.5-WT in SMMC7721 and Hep G2 cells co-transfected with mi R-195-5p decreased by 56% and 51% respectively,while the relative luciferase activity of p MIR-CTB-193M12.5-Mut showed no significant change.Meanwhile,mi R-195-5p inhibited the expression of HMGA1,a downstream target gene.2.6 in the mir-195-5p transfection group,the ability of proliferation and invasion of liver cancer cell lines SMMC7721 and Hep G2 was significantly decreased.Compared with mi R-195-5p +pc DNA/CTB-193M12.5 group,mi R-195-5p + pc DNA-NC group showed significantly enhanced cell proliferation activity,invasion and migration ability.2.7 compared with mi R-NC or si RNA-NC group,mi R-195-5p transfected SMMC-7721 cells and si CTB-193M12.5 transfected Hep G2 cells significantly reduced HMGA1 m RNA and protein expression(P<0).01);Compared with the pc DNA-NC group,the expression of HMGA1 in pc DNA/CTB-193M12.5 transfected Hep G2 cell line was significantly increased(P<0).01): in addition,compared with mi R-195-5p+pc DNA-NC group,the expression of HMGA1 in SMMC-7721 cell line co-transfected with mi R-195-5p +pc DNA/CTB-193M12.5 was partially enhanced.2.8 Compared with si RNA-NC group,si CTB-193M12.5 transfected Hep G2 cells showed significantly lower expression levels of Snail,Slug,Twist and other proteins(P<0).01);Compared with pc DNA-NC group,the expression levels of Snail,Slug,Twist and other proteins in SMMC7721 cells transfected with pc DNA/ CTB-193M12.5 were significantly higher(P<0).01).In addition,compared with pc DNA/CTB-193M12.5 + si-NC group,pc DNA/CTB-193M12.5 + si-HMGA1 co-transfected Hep G2 and SMMC7721 cells can significantly reduce the expression levels of Snail,Slug,Twist and other proteins,and increase the expression level of E-cad.Conclusion CTB-193M12.5 could act as a ce RNA and competitively bind to mi R-195-5p,and HMGA1 was their downstream target gene.CTB-193M12.5 could reverse the anti-cancer effect of mi R-195-5p,promote the expression of HMGA1,accelerate the progress of EMT,and promote the malignant process of HCC.It may provide guidance for further study of new targets for hepatocellular carcinoma. |
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