Effects Of Hsa_circ₀000524 On Hepatocellular Carcinoma Cell Viability And Migration Through Regulating The Expression Of MiR-138

The Encyclopedia of DNA Elements（ENCODE）project revealed that 70%⁹0% sequences in human genomes possess the ability of transcription but only less than 2% sequences are capable of encoding proteins.Those non-coding sequences can form non-coding RNA（ncRNA）through transcription,thus regulating biogenic activities.There exists a complex RNA-RNA network relationship between RNAs,via which they can regulate each other’s expression level.Based on it,biologists proposed the hypothesis of competing endogenous RNA（ceRNA）.The transcript of many RNAs can integrate with the response element（MRE）of miRNA,reducing the inhibition of miRNA on target genes and thus improving the expression level of target genes,within which the RNA transcript and target genes are referred to as ceRNA.This regulation method of ceRNA has been found in different biogenic activities;however,there are few studies involving the genes which exert ceRNA function on tumors.Primary hepatocarcinoma is one of the most common clinical malignant tumors with high mortality,among which the mortality of men with hepatocarcinoma ranks the second in tumor-related mortalities.At present,the molecular mechanism of hepatocarcinoma remains unclear.Currently,studies on genes mainly focus on miRNA,lncRNA,circRNA and mRNA,and the study on ceRNA regulation of circRNA-mRNA in the occurrence and development of hepatocarcinoma has become a hotspot.miRNA plays a key role in ceRNA,and it is a kind of small single-stranded non-coding RNA with the length of 22 nt.Mature miRNA can be rapidly integrated into miRNA-induced silencing complex and will inhibit RNA translation through complementary integration with mRNA’s 3’ non-coding area,thus negatively regulating the expression of mRNA.The expression disorder of miRNA is the vital factor leading to a series of tumors including hepatocarcinoma.Some miRNAs participate in the regulation of cell cycles,so their expression disorder may lead to the dysfunction of cell cycle and induce the formation of tumors.A lot of studies concerning the regulation of cell cycle-related miRNA on hepatocarcinoma cells have proved that the expression disorder of miRNA plays a vital role in the formation of tumors.circRNA is a kind of RNA molecule with important biological functions but has long been neglected.As early as in 1976,the first circRNA was found in viruses.It is typically a kind of circular RNA consisted of exons.In recent years,some studies have shown that circRNA contains a large number of MRE of miRNA and can exert ceRNA function,hence becoming a new member of ceRNA.With constant studies on and explorations into circRNA,its role in the occurrence of tumors has been gradually acknowledged by people.The latest study showed that hsa-circ-0005075 could interact with the miRNAs of target genes,including miR-23b-5p,miR-93-3p,miR-581 and miR-23a-5p,to remove or mitigate their inhibition effect,thus promoting the progress of hepatocarcinoma.By testing the expression of miRNA in hepatocarcinoma cells,Wang et al.discovered 10 up-regulated miRNAs and 10 down-regulated miRNAs,in which,compared with the mir-138 in adjacent non-tumor tissues,the expression of mir-138 in 77.8% hepatocarcinoma tissues is down-regulated.Howerver,a recent circRNA profiling study revealed that the expression of hsa-circ-0000524 in hepatocarcinoma tissues was up-regulated.Therefore,in this study,we proposed that the down-regulation of hsa_circ₀000524 in hepatocarcinoma tissues can regulate the miR-138 mediated signal pathway through promoting miR-138 expression,and further promote tumorigenesis.Part One Expression of hsa_circ₀000524 in liver tissues and different HCC cell lines Methods:1.RT-PCR determination of the expression of hsa_circ₀000524 in HCC tissues.2.RT-PCR determination of the expression of hsa_circ₀000524 in HCC cell lines.Results:1.The expression of hsa_circ₀000524 was significantly up-regulated in HCC tissues when compared with the adjacent liver tissues（P<0.01）.2.The expression of hsa_circ₀000524 was significantly up-regulated in HCC cell lines,including HepG2,HHCC,HUH7,and BEL-7402,when compared with the normal liver cell line HL-7702（P<0.01）.Part Two Down-regulation of hsa_circ₀000524 inhibits HepG2 cell growth and migration Methods:1.Knowdown of hsa_circ₀000524 by the tranfection of hsa_circ₀000524 siRNAin HepG2 cells.2.Cell viability determination by CCK-8 and EdU.3.Cell cycle analysis with DNA dye（PI）.4.Cell migration ability was detected by wound healing assay and Transwell cell migration assay.Results:1.The expression of hsa_circ₀000524 was significantly reduced（P<0.01 versus the NC group）when transfected with hsa_circ₀000524siRNA.2.SiRNA transfected HepG2 cells became significantly less viable than the control 72 h post transfection（P<0.01）.3.Hsa_circ₀000524 siRNA transfection,compared with the control,reduced the percentage of S phase cells（P<0.01 versus the control group）.4.Down-regulation of hsa_circ₀000524 inhibits HepG2 cell migration.Part Three Down-regulation of hsa_circ₀000524 promotes miR-138 expression Methods:1.Binding sites predication between hsa_circ₀000524 and miR-138 byTargetScan.2.RT-PCR determination of the expression of miR-138 in HCC tissues.3.RT-PCR determination of the expression of miR-138 in HCC cell lines.4.Knockdown of hsa_circ₀000524 by the tranfection of hsa_circ₀000524 siRNA and RT-PCR analysis of the expression of miR-138 in HepG2 cells.5.Western blot analysis of the miR-138 mediated signal pathway related proteins.Results:1.TargetScan prediction indicates that hsa_circ₀000524 can bind with miR-138.2.The expression of hsa_circ₀000524 was significantly down-regulated in HCC tissues when compared with the adjacent liver tissues（P<0.01）.3.The expression of hsa_circ₀000524 was significantly down-regulated in HCC cell lines,including HepG2,HHCC,HUH7,and BEL-7402,when compared with the normal liver cell line HL-7702（P<0.01）.4.Down-regulation of hsa_circ₀000524 significantly promoted the miR-138 expression in HepG2 cells（P<0.01 versus the control group）.5.The expressions of vimentin and CCND3 were decreased which were inversely correlated with the miR-138 expression.Part Four Effects of microRNA-138 on the HepG2 cell viability and migration Methods:1.Co-trnsfections of miR-138 mimic or inhibitor with hsa_circ₀000524 siRNA or onlytransfection of hsa_circ₀000524mimicinto HepG2 cells.2.Cell viability determination by CCK-8 after co-transfections of miR-138 mimic or inhibitor and hsa_circ₀000524 siRNA or.3.Cell migration ability was detected by wound healing assay and Transwell cell migration assay.4.Western blot analysis of the miR-138 mediated signal pathway related proteins after tranfections.Results:1.Hsa_circ₀000524 mimic wAS transfected separately to HepG2 cells.Hsa_circ₀000524 siRNA and miR-138 mimic,or miR-138 inhibitor were co-transfected to HepG2 cells.The miR-138 was significantly overexpressed or suppressed,respectively,detected by RT-PCR（P<0.01 versus the control group）.2.The si-hsa_circ₀000524can inhibit the viability HepG2 cells than hsa_circ₀000524 mimics only transfection.The miR-138 mimic transfection withsi-hsa_circ₀000524can further inhibit the viability HepG2 cells than hsa_circ₀000524 mimics only transfection.On the other hand,miR-138 inhibitor transfectionwithsi-hsa_circ₀000524 turned to significantly enhance the viability of HepG2 cells 72 h post transfection（P<0.01）.3.In both of the wound healing and transwell chamber assays,si-hsa_circ₀000524 transfectionsignificantly inhibited the wound closure and weaken the ability of migration compared with the hsa_circ₀000524 mimics only transfected group（P<0.01）.In addition,miR-138 mimic transfection significantly inhibited the wound closure and displayed a lower ability of migration compared with the hsa_circ₀000524 mimics only transfected group（P<0.01）,while a promoted wound closure rate and a higer ability of migration were observed after miR-138 inhibitor transfection（P<0.01）.4.The expressions of vimentin and CCND3 were inversely correlated with the miR-138 expression.Part Five Down-regulation of hsa_circ₀000524 inhibits HCC progression Methods:1.HepG2 cells were inoculated to 6 nude mouse to establish the subcutaneous transplanted model.2.The hsa_circ₀000524 shRNAs were tranfected to the transplanted tumor tissues by lentivirus.3.The tumorigenesis in nude mouse as well as the expressions of Ki67 protein in the HCC tissues were examined by hematoxylin and eosin（HE）staining and immunohistochemical stainining.4.RT-PCR determination of the expression of miR-138 in HCC tissues of the nude mouse.5.Western blot analysis of the miR-138 mediated signal pathway related proteins in the transplanted tumor tissues.Results:1.More than 80% of the HepG2 cells were tranfected by hsa_circ₀000524 shRNA when the MOI=200。2.Down-regulation of hsa_circ₀000524 by shRNAs tranfection significantly lower down the tumor size in the mouse.3.The miR-138 expression was up-regulated when trafected by hsa_circ₀000524 shRNA.4.Down-regulation of hsa_circ₀000524 also showed inhibitive effect on the tumorigenesis in nude mouse as well as the expressions of Ki67 protein in the HCC tissues.5.The expressions of vimentin and CCND3 in the transplanted tumor tissues of nude mouse were inversely correlated with the miR-138 expression.Conclusions:With all these results,we proposed that the down-regulation of hsa_circ₀000524 in hepatocarcinoma tissues can regulate the miR-138 mediated signal pathway through promoting miR-138 expression,and further promote tumorigenesis.Studying the mechanism of miRNA and circRNA participating in the progress of hepatocarcinoma in an in-depth manner will provide the basis for future clinical treatment of hepatocarcinoma and usher in a new horizon for people to conquer hepatocarcinoma.