| Hepatocellular carcinoma(HCC)is a worldwide cancer with high occurrence and mortality.According to International Agency for Research on cancer(IARC)published,five-year survival rate of liver cancer is mere 6.9%,there will be 909,742 deaths in2020 years due to liver cancer.China has the highest rate of occurrence and mortality in liver cancer,with more than half HCC patients in the world which create great financial burden and threaten to people's life and healthy severely.The occurrence of HCC is a long and complicated process involved in many events,such as growth factors and oncogenes activating,tumor suppressor inactive.Up to now,there is no effective drug for HCC patients,so it's very urgent to investigate the specific mechanism of liver cancer to provide new target for early screening and therapy.In our previous studies,we found that HBV could promote the migration and invasion of liver cancer cells.Mi R-340-5p is a small noncoding RNA,which functions as a tumor suppressor and plays an important role in cancer progress including cervical cancer,colon cancer and breast cancer.So,we further explored the role of mi R-340-5p in liver cancer cells' proliferation and apoptotic progress.In this study,we demenstrated that HBV increased the proliferation and inhibited the apoptosis of Huh7 cells through mi R-340-5p by CCK8 assay,EDU assay and apoptosis assay.Furthermore,we explored the mechanism of HBV-mi R-340-5p inducing HCC.Putative target genes for mi R-340-5p were predicted using the Target Scan human computationalmethod,then Luciferase reporter assay was used to identify the target gene,results confirmed activating transcription factor 7(ATF7)as one of target gene of mi R-340-5p;meanwhile,HBV-mi R-340-5p promoted proliferation and inhibited apoptosis through ATF7.By protein immune coprecipitation experiment and mass spectrometry analysis,we found that 70 KD heat shock protein 1 B(HSPA1B)interacted with ATF7;Besides,HSPA1 B participated in the carcinogenesis process of HBV-mi R-340-5p inducing.We investigated the expression of ATF7 and HSPA1 B in human liver cancer tissues and TCGA database,results shown that ATF7 and HSPA1 B were higher expression in liver cancer samples compared with the adjacent non-tumor tissues,which indicated their important function in HCC occurrence.Sox(sry-related HMG box)proteins belong to HMG-box superfamily which involved in the regulation of diverse developmental processes including nervous system,pluripotent stem cells,sex determination,hemopoiesis and chondrogenesis.Sox3 is a transcription factor of Sox B1 subfamily(including Sox1,Sox2 and Sox3)which expressed predominantly in central nervous system,intestine and gonad.Studies confirmed its important function in X-linked congenital generalized hypertrichosis(CGH),hypopituitarism and neural tube defects.Recently,results proved that Sox3 could function as oncogene and promote cancer progress in cervical cancer and endometrial carcinoma,but there is bare evidence to describe its function in liver cancer progress.In this manuscription,we investigated its function in liver cancer.Using q RT-PCR assay,we found that HBV increased the m RNA levels of Sox3 to further investigate whether Sox3 could affect on HBV-HCC progress,we constructed the stable expression cell lines which is high expression of Sox3.When Sox3 was overexpressed in Hep G2 cells,proliferation,clone formation,migration and invasion o f cells were promoted,more cells entered into S phase;The results were consistent in Huh7 cells and LO2 cells in which Sox3 expression was knockdown.Secondly,we investigated the proliferational mechanism of Sox3 in hepatocellular carcinoma.We detected that Sox3 could promoted mi R-203a-3p expression,CCK8 and EDU staining assay confirmed that Sox3 promoted the growth of liver cancer cells through mi R-203a-3p;Furthermore,luciferase reporter gene assay identified GAS2 as a target gene of mi R-203a-3p.Previous study reported that GAS2 could inhibit growth through p53-induce apoptosis in osteosarcoma.So,we hypothesis that Sox3 promoted growth of liver cancer cells through mi R-203a-3p/Gas2/p53 axis.we checked the protein expression of GAS2 and p53 in Hep G2 and Huh7 cells,western blotting resultsshowed that Sox3 inhibited the expression of GAS2 and p53 through mi R-203a-3p;Proliferational assays results showed that GAS2 suppression resumed the inhibition effect on growth of HCC cell lines when Sox3 was overexpressed meanwhile knockdown the expression of mi R-203a-3p,which indicated that Sox3 acted as an oncogene and drived liver cancer cells growth through mi R-203a-3p/GAS2/p53 pathway.Thirdly,we tested how Sox3 driving metastasis.we found that Sox3 increased the m RNA and protein expression of AKT1 obviously.Previous numberous studies reported that AKT/m TOR signaling pathway involved the metastasis of cancers including colorectal cancer,breast cancer,lung cancer and cervical cancer,So we hypothesis that Sox3 promote migration and invasion through AKT1/m TOR signaling pathway.si RNA and small molecule inhibitors against AKT1 and m TOR were used to knockdown the endogenous expression of AKT1 and m TOR in Hep G2 cells.Migration and invasion assays results showed that Sox3 promoted metastasis of HCC cells through AKT1/m TOR signaling pathway to regulate the protein expression of EMT markers including E-cadherin and MMP-9.Finally,we performed nude mouse tumorigenicity experiment,HE staining and IHC assays to demonstrate that Sox3 promoted tumorigenesis and metastasis of HCC cells in vivo.In this work,we discovered two new mechanism of HBV promoting HCC occurrence:(1)we proved that HBV promoted proliferation and suppressed apoptosis of liver cancer cells through mi R-340-5p-ATF7-HSPA1 B axis.(2)we demonstrated that Sox3 acted as a noval oncogene,promoting growth through mi R-203a-3p/Gas2/p53 pathway and dirving metastasis through AKT1/m TOR signaling pathway in liver cancer.We revealed new molecular mechanisms in liver cancer,meanwhile provided potential biological marker or target for Hdiagnosis,therapy and prognosis. |
PDF Full Text Request