Influence Of UBAP2L On The Growth And Metastasis Of Hepatocellular Carcinoma And Its Potential Molecular Mechanism

Primary liver cancer is one of the most common malignant gastrointestinal tumors.It currently ranks fourth in the incidence of cancer in our country and has the third highest mortality rate,which seriously threatens the health of the people.Hepatocellular carcinoma(HCC)is the main pathological type in primary liver cancer.Nowadays,hepatectomy and liver transplantation remain the most important treatment methods for HCC patients to acquire the radical and long-term survival opportunities.However,in view of the low rate of early diagnosis of HCC patients,most of them are diagnosed as advanced stage and difficult to obtain radical surgery.Meanwhile,traditional radiotherapy or systemic chemotherapy is ineffective.And even if they could receive operation,postoperative recurrence and metastasis rate is also high,which has become a major obstacle to the long-term survival.In recent years,a large number of studies have shown that angiogenesis plays an important role in promoting the proliferation,growth,invasion and metastasis of HCC.Therefore,anti-angiogenic therapy has also become one of the most promising strategies in patients with advanced HCC.However,in clinical practice,it has been confirmed that effective anti-angiogenic molecular targeted drugs,such as sorafenib and regorafenib,have a limited therapeutic effect.Therefore,exploring new molecular targets to predict patients' prognosis,preventing tumor recurrence and metastasis,and developing potentially effective anti-angiogenic targeted therapies have now become the major research fields in HCC.Ubiquitin associated protein 2-like(UBAP2L)can bind ubiquitin and is a new member of the ubiquitin-related protein family,which can participate in a variety of cellular pathophysiological processes.In recent years,relevant research on UBAP2L in tumors has gradually increased.Studies have found that UBAP2L was highly expressed in prostate cancer,glioma,colorectal cancer,lung adenocarcinoma and HCC,which was closely related to the cell proliferation,apoptosis and invasion and metastasis of malignancies.However,the biologic function and molecular mechanism of UBAP2L in HCC are still unclear.In view of HCC characterized by hypervascularity and high rate of recurrence and metastasis,and the above reports on UBAP2L in varieties of tumors,therefore,we carried out the present study of the topic under the support of Anhui Province Natural Science Foundation(No.1708085QH177),to deeply explore the influence of UBAP2L on the growth and metastasis of HCC and its underlying mechanisms,and reveal the clinical significance between UBAP2L expression level and the prognosis of HCC patients.The study will provide a theoretical basis for finding new targets and strategies for the treatment of HCC.Part 1:Expression level of UBAP2L in hepatocellular carcinoma and its prognostic significanceObjective:Recently,accumulating studies have shown that ubiquitin associated protein 2-like(UBAP2L)is overexpressed in many kinds of malignant tumors,which is closely associated with the tumor growth and metastasis.However,the correlations of UBAP2L expression with clinicopathological factors and prognosis of hepatocellular carcinoma(HCC)patients still remain unclear.Therefore,we intended to conduct this study to explore the above issues.Methods:Bioinformatics database(GEO and TOGA)and our own experimental results(including using by immunohistochemical staining,western blot and real-time PCR)were analyzed to validate the expression levels of UBAP2L in HCC.Further,Kaplan-Meier univariate and Cox multivariate survival analyses were performed to demonstrate the associations of UBAP2L expression with clinicopathological factors and prognosis of HCC patients.Additionally,the potential underlying mechanisms associated to angiogenesis were also explored preliminarily.Results:Compared to the normal control groups,UBAP2L was significantly highly expressed in HCC cell lines and tissues.Kaplan-Meier univariate survival analysis revealed that patients with high UBAP2L expression level had dramatically less survival time than those with low UBAP2L expression level(P =0.000).Moreover,Cox multivariate survival analysis showed that UBAP2L high expression was an independently unfavorable prognostic parameter for OS of HCC patients(P =0.000).Additionally,Pearson correlation analysis revealed that the relationship between UBAP2L expression and VEGF or MVD was significantly positive,respectively(r=0.460,P =0.000 and r =0.387,P =0.000).Conclusion:UBAP2L was overexpressed in HCC and patients with high UBAP2L expression had unfavorable prognosis.UBAP2L will be a new potential therapeutic target for HCC in the future.Part 2:Influence of UBAP2L on the growth and metastasis of hepatocellular carcinoma and its potential molecular mechanismObjective:Based on the results of the first part of the study,the effects of ubiquitin associated protein 2-like(UBAP2L)on the growth and metastasis of hepatocellular carcinoma(HCC)and its potential molecular mechanism were further explored.Methods:UBAP2L gene expression in SMMC-7721 was knocked down by lentivirus-mediated RNA interference.Then,the effects of UBAP2L knockdown on hepatoma cell function(including cell proliferation,cell cycle,apoptosis,colony formation,wound healing,transwell assay and angiogenesis)were examined.Besides,a subcutaneous xenograft tumor model was established,using by SMMC-7721 transfected cell line with UBAP2L knockdown,to observe the effect of UBAP2L knockdown on HCC growth in vivo.Finally,the whole genomic microarrays were used to screen and analyze the potential mechanism of UBAP2L involved in regulating the biological function of HCC.Simultaneously,western blot was used to verify the key molecules in the significant enrichment pathway.Results:Compared with those in the control group,the numbers of cell proliferation and clone formation were significantly reduced,cell cycle was arrested in G2/M phase,the number of apoptotic cells was remarkably increased and the abilities of vascular formation and cell migration and metastasis were dramatically weakened in shUBAP2L group(All P<0.05).Besides,UBAP2L knockdown could significantly suppress the tumor growth of HCC in the xenograft nude model in vivo.Moreover,Global gene expression profiling showed that a total of 320 genes changed significantly after UBAP2L knockdown,among which 159 genes were up-regulated and 161 genes were down-regulated(All P<0.05).Then,gene enrichment analysis in David website revealed that PI3K/AKT and P53 signal pathway was the most significant in the top 10 enrichments.Finally,western blot verified that UBAP2L knockdown caused the increase of P21 and PTEN,and decrease of CDK1,CCNB1,P-PI3K and p-AKT.Conclusion:UBAP2L played an oncogenic role in HCC and knockdown of its expression could significantly inhibit the growth and metastasis of HCC,the potential mechanism of which might be related to the regulation of PI3K/AKT and P53 signaling pathways by UBAP2L.