| Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in China,and associated with high rates of morbidity and mortality.Surgical treatment of HCC is one of the most important means of long-term survival of HCC patients.However,after five years of hepatectomy,the tumor recurrence and metastasis rates were high as 40% ? 70%.The discovery and utilization of molecules related to the progression and prognosis of HCC is conducive to more accurate,rapid diagnosis of the patient's condition and prognosis.CTHRC1(Collagen triple helix repeat containing 1)is a secreted protein and playing vital role in different biological processes,such as increasing cell migration capacity,promoting vascular and tissue repair processes,preventing liver fibrosis and scar formation,bone formation and bone remodeling,etc.As an oncogene,CTHRC1 is associated with the development of a variety of human cancers.However,the clinical significance of CTHRC1 development in HCC is still unknown,and further studies are required.Hsa-miR-30b is located on human chromosome 8q24 and is a member of the miR-30 family.It functions by interacting with a variety of target genes.The miR-30b is important to multiple biological processes such as cancer invasion and migration,cell cycle regulation and so on.Whether CTHRC1 in HCC is a target of miR-30b,as well as the role,precise mechanism of CTHRC1 in the development and metastasis of HCC is still unclear.The process of tumor metastasis,epithelial-derived malignant tumor cells must first acquire the ability of migration and invasion before they can get into the circulatory system from their primary location.Epithelial-mesenchymal transition(EMT)is one of the main causes of migration and invasion of epithelial-derived malignancies.During EMT,the expression of epithelial phenotypic markers such as E-cadherin decreased,while interstitial phenotypic markers such as N-cadherin and vimentin increased.E-cadherin is an important factor in the maintenance of epithelial phenotype in tumor cells,and its down-regulated or absent expression is a significant feature of EMT transformation.Previous studies on HCC genomics have shown that during the development of HCC,members of the Wnt pathway often undergo mutations or copy number changes,and over 44% of the HCC samples have genetic changes in the Wnt signaling axis.Mutations in members of the Wnt pathway often promote cellular transformation to mesenchyme.However,it is not clear CTHRC1 can induce EMT through the Wnt pathway.The purpose of this study is to collect cancer and adjacent tissues and blood samples from patients with HCC and carry out cytological and zoological experiments.First,to observe the clinical significance,prognostic values of CTHRC1 expression and provide new clues to the prognosis in HCC.Second,the interaction of miR-30b with CTHRC1 and its effect on the biological activity of hepatoma cells,as well as its related mechanisms,which were verified by in vitro experiments and provided a basis of further animal experiments.Third,the inhibition of miR-30b targeted CTHRC1 on hepatoma cells in vivo was examined by tumor-bearing nude mice.This study is divided into the following three parts: Part one Expression levels of CTHRC1 in HCC tissues,serum and blood cells of patients with HCC and their relationship to clinicopathologic characteristicsObjective:The expression of CTHRC1 in HCC tissues,serum and blood cells of patients with HCC was observed,correlation with clinicopathological factors and prognosis was analyzed.Methods:Paraffin embedded pathological specimens were collected from 29 HCC patients undergoing surgical operations in the 980 th Hospital of PLA Joint Logistics Support Force from September 2015 to September 2018;serum and blood cells of HCC patients admitted to the 980 th Hospital of PLA Joint Logistic Support Force from March to May 2019;normal blood serum and blood cells from March to May 2019 in the physical examination center of the 980 th Hospital of PLA Joint Logistic Support Force.The expression levels of CTHRC1 protein and mRNA in HCC tissues,paracancerous tissues,serum and blood cells were detected by immunohistochemical staining,total RNA extraction,real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and ELISA etc.To observe its relationship between clinicopathological factors and prognosis.Results:1.CTHRC1 was highly expressed in HCC specimens from the Oncomine database.The expression of CTHRC1 in cancer tissues and normal tissues was analyzed from a total of 281 unique analyses in the Oncomine database.Of those,66 analyses met the following conditions:(1)gene expression of tumor tissues was two-fold higher than that found in normal tissues.(2)gene rank was in the top 10%,and(3)P= 0.0001.In HCC,two analyses meet these conditions.The expression of CTHRC1 in HCC and normal tissues was also revealed by analysis of four independent data from the Oncomine database.According to Guichard Liver(analysis comparison 1),Guichard Liver2(analysis comparison 2),The Cancer Genome Atlas Liver(analysis comparison 3),and Wurmbach Liver(analysis comparison 4),the median rank for CTHRC1 across afore mentioned analyses was 938.5.Being dependent on the median-ranked analysis,P-value for CTHRC1 was 1.29E-13.Among the four analyses,the Wurmbach Liver demonstrated the highest expression of the CTHRC1 gene.The 25681?at probe was also observed in the Wurmbach Liver analysis.There was a statistically significant difference between the HCC and the normal group(P<0.001).Concerns have been expresssed regarding the gene copy number.Depending on The Cancer Genome Atlas and Guichard Liver analyses,the CTHRC1 gene copy number was increased in HCC tissues versus normal liver tissues.Moreover,Wurmbach Liver analysis,we observed an up regulation in the mRNA levels of CTHRC1 in HCC tissues versus normal tissues.This up regulation was reflected by a 11.156-fold change in mRNA levels,and the piercing expression gene rank was 67(top 1%).Collectively,these observations suggested that CTHRC1 was highly expressed in HCC tissues.2.High expression of CTHRC1 in HCC tissues.We examined the clinical relevance to CTHRC1 in HCC by evaluating its levels and distribution in human HCC tissue arrays through immunohistochemistry.The results showed that all 29 patients expressed CTHRC1,including 7,11,and 11 patients with weakly,moderately,and strongly positive expression,respectively.Among the 29 paracancerous tissues examined,12 were negative,while 13,1,and 3 cases were weakly,moderately,and strongly positive,respectively.The expression of CTHRC1 in HCC tissues was significantly higher than that measured in paracancerous tissues(P<0.001).3.High expression of CTHRC1 in hepatoma cell lines.The consequences from the qRT-PCR showed a higher expression level of CTHRC1 in Huh7,Hep G2,SMMC7721 cell lines versus LO2 and QSG7701 cell lines.The results reported that the expression of CTHRC1 mRNA in cancer cell lines was significantly greater normal cell lines(P<0.01).4.CTHRC1 is highly expressed in the serum of HCC patients.ELISA results showed that the expression level of CTHRC1 in the HCC serum was higher than the control group,with significant difference(P=0.039).5.CTHRC1 is highly expressed in the blood cells of HCC patients.The qRT-PCR results showed the expression level of CTHRC1 mRNA in the blood cells of the HCC patients was 2-9 times that of the control group.6.Elevated expression of CTHRC1 correlated with poor prognosis in HCC.Statistical analysis revealed levels of CTHRC1 were significantly correlated with cirrhosis,tumor size,vascular invasion,TNM stage,and BCLC stage(P<0.05).However,there were no statistically significant correlations identified between the expression of CTHRC1 and other clinicopathological characteristics,including gender,age,alpha-fetoprotein,gamma-glutamyl transferase,alanine aminotransferase,hepatitis B virus infection,and number of tumors.Survival curves for HCC patients indicated elevated expression of CTHRC1 may be associated with poor prognosis.7.The prognostic value of CTHRC1 as a biomarker for HCC.Data from the Gene Expression Profiling Interactive Analysis database and further analysis showed that the expression of CTHRC1 was noticeably higher in 369 patients with HCC versus 160 normal tissues.Additional analysis showed that high expression of CTHRC1 may be associated with poor prognosis.These results showed that CTHRC1 may be used as a biomarker for HCC.Conclusions:1.CTHRC1 was highly expressed in HCC samples of Oncomine database.2.CTHRC1 is highly expressed in HCC tissues,cell lines,serum of HCC patients and blood cells of HCC patients.High CTHRC1 expression of HCC was significantly associated with cirrhosis,tumor size,vascular invasion,TNM stage and BCLC stage.3.In HCC,high expression of CTHRC1 is associated with poor prognosis,and CTHRC1 can be used as a biomarker for the prognosis of HCC.Part two Construction of overexpressed CTHRC1 human hepatoma cell lines and the relationship between miR-30b and CTHRC1Objective: Human hepatoma cell lines of over-expression of CTHRC1 were built to provide a cell model for the subsequent study of the biological roles and interrelationships between miR-30b and CTHRC1 in the processes of proliferation and migration of HCC.Methods: Bioinformatics tools were used to predict the target genes of miR-30b.The expression levels of miR-30b and CTHRC1 in 293 T cells were preliminaries verified by cell transient transfection,qRT-PCR and Western blot.Expression of miR-30b in different hepatoma cell lines was observed.The lentivirus gene delivery system was used to establish stable overexpressed CTHRC1 hepatoma cell lines,which provided a basis of the study of the interaction between miR-30b and CTHRC1,the role of both in the process of hepatoma cell proliferation and migration,and a material basis of the next animal experiments.Results:1.Prediction of the target relationship between CTHRC1 and miR-30b.The basic information about miR-30b was retrieved through Target Scan database(updated in March 2018).The potential target genes of miR-30b were predicted by three databases: miRDB,mir DIP and miRTar Base,respectively.The results predicted that CTHRC1 might be the target gene of miR-30b.2.The expression of miR-30b decreased and the expression of CTHRC1 increased in hepatoma cell lines.QRT-PCR was utilized to detect the expression of miR-30b in three different hepatoma cell lines.The expression levels were SMMC7721,Hep G2 and Huh7 from low to high,which was significantly lower than LO2 and QSG7701 in the control group(P<0.01).The expression level of CTHRC1 in these three cell lines was opposite from the first part.3.Analysis of interaction between CTHRC1 and miR-30b expression level.The plasmid pCTHRC1-IRES2-EGFP was constructed and transiently transfect into 293 T cells to investigate the interaction between CTHRC1 and miR-30b.The results showed that transfection with the miR-30b mimics and the pCTHRC1-IRES2-EGFP plasmid significantly increased the expression levels of miR-30b and CTHRC1 in cells compared with the control group(t=9.96,t=25.238,P <0.05).In 293 T cell,the expression level of miR-30b decreased significantly in the overexpressed CTHRC1 group(t=-3.457,P<0.05).Overexpression of miR-30b significantly reduced the expression of CTHRC1(t=-24.677,P <0.05).4.Preparation of lentivirus supernatant.Sangon Biotech(Shanghai)Co.,Ltd was commissioned to construct the recombinant vector p LV-CTHRC1,which was transfected into 293 T cells after correct sequencing.The results of qRT-PCR showed the expression of CTHRC1 increased significantly after transfection.The CTHRC1 overexpression plasmid was successfully constructed.Lentivirus packaging and titer detection were further carried out.5.The optimal concentration of purinomycin screening was calculated.The optimal concentration of purinomycin was calculated for screening for the infected cells Huh7 and Hep G2.The cell viability tests results showed that 0.6ug/ml of purinomycin could significantly reduce the cell viability of Huh7 and Hep G2,which could be used as the optimal concentration on drug screening.6.Stable cell lines were identified by qRT-PCR,and the expression of CTHRC1 was increased.After the virus supernatant infected Huh7 and Hep G2,0.6ug/ml purinomycin was added to screen the cells,and the expression of CTHRC1 in the cells was detected by qRT-PCR.Compared with the control group,CTHRC1 gene had a significant over expression effect and indicating the stable cell line construction was successful.Conclusions:1.The application of bioinformatics software to predict CTHRC1 may be as a target gene of miR-30b;the results of transient transfection of pCTHRC1-IRES2-EGFP plasmid into 293 T cells showed that the expression of CTHRC1 and miR-30b in cells showed an opposite trend.Accordingly,it can be presumed that miR-30b can be combined with CTHRC1 mRNA 3'UTR region and inhibiting the expression of CTHRC1.2.Lentivirus containing CTHRC1 gene was used to infect Huh7 and Hep G2 cells through the lentivirus gene delivery system to screen stable cell lines.QRT-PCR results showed that stable and highly efficient over-expression of CTHRC1 in hepatoma cell lines Huh7 and Hep G2 were successfully constructed,providing a cell model for the next step to study the effect of CTHRC1 on the proliferation and migration of hepatoma cells.Part Three Related mechanisms of miR-30b targeting CTHRC1 to regulate cell proliferation,migration and cell cycle in hepatocellular carcinomaObjective:In vitro and in vivo experiments were performed to further investigate the role of CTHRC1 in the proliferation,migration and cell cycle of hepatoma cells.Effects on expression of EMT related proteins,Wnt pathway proteins and the regulation of miR-30b on CTHRC1.Methods:Through cell proliferation experiment,scratches,cell cycle detection,immunohistochemical staining,Western blot and small animal in vivo imaging method to explore the miR-30b and CTHRC1 in cell proliferation,migration,the role of cell cycle and expression of EMT related protein and Wnt pathways.To further explore the role of miR-30b targeting CTHRC1 in the progression of HCC and its regulatory mechanism.Results:1.MiR-30b significantly attenuated the proliferation-promoting effect of CTHRC1 on Huh7 and Hep G2.Huh7 and Hep G2 cells consisted of four groups: control group,LV-CTHRC1 group,miR-30b mimics group and miR-30b mimics+ LV-CTHRC1 group.The consequences of CCK-8 experiment showed after 72 hours of culture.The proliferation capacity of LV-CTHRC1 group was significantly greater than control group(P<0.01).In contrast,the proliferation capacity of miR-30b mimics group was significantly lower than control group(P<0.01).However,the proliferation capacity of miR-30b mimics+ LV-CTHRC1 group was significantly lower than LV-CTHRC1 group(P<0.05).The above results indicated over-expression of CTHRC1 increased the proliferation capacity of hepatoma cells,while miR-30b significantly decreased the proliferation promotion of CTHRC1 on Huh7 and Hep G2.2.MiR-30b significantly inhibited migration promoting effect of CTHRC1 on hepatoma cells Huh7 and Hep G2.The scratch test results showed 48 h after the scratch.Scratch healing rate of LV-CTHRC1 group was significantly higher than control group(P<0.001).In contrast,the scratch healing rate of miR-30b mimics group was significantly lower than control group(P<0.001).However,the scratch healing rate of miR-30b mimics+LV-CTHRC1 group was significantly lower than that of LV-CTHRC1 group(P<0.001).The above results showed that over-expression of CTHRC1 increased the migration ability of hepatoma cells,while miR-30b significantly inhibited the migration promotion of CTHRC1 on hepatoma cells Huh7 and Hep G2.3.MiR-30b is under a G2/M blocking effect on the cell cycle of Hep G2.Cell cycles detection results showed the proportion of G1 phase cells in the control group,LV-CTHRC1,miR-30b mimics+ LV-CTHRC1 group and miR-30b mimics group were: 81%,66%,61% and 58%,respectively.The proportion of G2/M cells was 7.75%,19.8%,24.5% and 30.8%,respectively.The difference between the experimental group and the control group was significant(P<0.01).It was therefore proposed that miR-30b had a G2/M blocking effect on the cell cycle of hepatoma cells Hep G2.4.MiR-30b inhibited the promotion of CTHRC1 of EMT and Wnt pathways.In order to examine the role of CTHRC1 in the EMT process,Wnt pathway and the inhibitory effect of miR-30b on CTHRC1,the present experiment used immunohistochemical staining to detect the expression of E-cadherin,Vimentin,and N-catenin in pathological samples.Western blot was used to detect the expression of CTHRC1,E-cadherin,Vimentin and ?-catenin in hepatoma cells.Immunohistochemical results showed that in tissues,positive E-cadherin was located in the cell membrane,positive expression rates of E-cadherin in HCC and paracancer tissues were 34.5%(10/29)and 100%(29/29),respectively,with significant differences(P<0.05).Vimentin positive localization in the cytoplasm,positive expression rates of vimentin in HCC were 100%(29/29),but not expressed in paracancer tissues.?-catenin positive localization in the cytoplasm,positive expression rates of ?-catenin in HCC and paracancer tissues were 62.1%(18/29)and 17.2%(5/29),respectively,with significant difference(P<0.05).Western blot detection results showed that the expressions of CTHRC1,Vimentin,and ?-catenin in the LV-CTHRC1 group were significantly higher than those in the Huh7 control group(P<0.05).In contrast,E-cadherin expression was significantly lower than Huh7 control group(P<0.05).There was no significant difference between miR-30b mimics group and Huh7 control group in terms of CTHRC1,Vimentin,and ?-catenin,but the expression was decreased compared with LV-CTHRC1 group.The expression of E-cadherin in miR-30b mimics group was significantly higher than LV-CTHRC1 group(P<0.05).The above results indicates that miR-30b inhibits the promotion of EMT by CTHRC1 and possibly through the Wnt/ ?-catenin signaling pathway.5.MiR-30b attenuated tumorigenesis of CTHRC1 in mice.Subcutaneous injection was utilized to implant Huh7 cells from different treatment groups into nude mice.No tumor formation was seen in the miR-30b mimics group.The tumor volume of the remaining three groups: the Huh7 control group,LV-CTHRC1 group and the miR-30b mimics+LV-CTHRC1 group was measured every 4d after tumor formation.The experimental results showed the tumor volume of LV-CTHRC1 group increased significantly 24 d after implantation compared with the control group(P<0.001).The volume of transplanted tumor in the miR-30b mimics+LV-CTHRC1 group was significantly inhibited and the differences were significant when compared with the LV-CTHRC1 group or the control group(P<0.01).The average tumor weight of the control group,LV-CTHRC1 group and miR-30b mimics+LV-CTHRC1 group was 0.09 g,2.4g,and 0.97 g,respectively(P<0.01).This suggests that miR-30b attenuates the tumorigenesis of CTHRC1 on hepatoma cells Huh7.Conclusions:1.MiR-30b significantly attenuated the proliferation-promoting effect of CTHRC1 on Huh7 and Hep G2.2.MiR-30b significantly inhibited migration promoting effect of CTHRC1 on hepatoma cells Huh7 and Hep G2.3.MiR-30b has a G2/M blocking effect on the cell cycle of hepatoma cells Hep G2.4.MiR-30b inhibited the promotion of CTHRC1 of EMT and Wnt pathways.5.MiR-30b attenuated tumorigenesis of CTHRC1 in mice.Conclusions:1.CTHRC1 was highly expressed in HCC tissues,serum and blood cells of patients with HCC.The high expression was significantly correlated with cirrhosis,tumor size,vascular invasion,TNM stage,and BCLC stage.2.CTHRC1 may serve as a prognostic biomarker for HCC.3.MiR-30b binds to the 3'UTR region of CTHRC1 mRNA and inhibiting its expression.4.MiR-30b targeting CTHRC1 can inhibit the proliferation and migration of hepatoma cells and cause hepatoma cell cycles to arrest.5.MiR-30b inhibited the promotion of CTHRC1 on EMT and Wnt pathways.6.MiR-30b attenuated the tumorigenesis of CTHRC1 on hepatoma cells Huh7. |
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