Piperlongumine Achieves Antitumor Effects by Targeting Angiogenic Genes that Have Predictive Value for the Diagnosis, Prognosis and Recurrence of Hepatocellular Carcinoma

Abstract

Background: In the context of the high recurrence rate and poor clinical prognosis of hepatocellular carcinoma (HCC), and considering the indispensable effect of angiogenesis in the development and metastasis of tumors, we analyzed HCC-related RNA sequence data to identify angiogenic genes closely associated with the prognosis of HCC. We used the identified genes to establish diagnostic, prognostic, and recurrence models to help clinicians effectively develop individualized treatment strategies for HCC. Method: We comprehensively analyzed 132 angiogenic genes and HCC-related RNA sequence data from the TCGA and the ICGC databases by Cox regression and least absolute shrinkage and selection operator(LASSO) regression to identify angiogenic genes; the genes were used to establish prognosis, recurrence and diagnostic models and to construct corresponding predictive nomograms. Pharmacological analysis was performed to detect target angiogenic drugs. Subsequently, in vitro cell experiments and western blot experiments were used to explore the anti-tumor effect and possible molecular mechanism of an angiogenesis-targeting target drug. Results: Four angiogenic genes (ENFA3, EGF, MMP3 and AURKB) were used to establish prognostic, relapse and diagnostic models. Through the prognostic model, we found that the overall survival (OS) of the high-risk group in the training cohort (P < 0.001, HR = 0.33) and validation cohort (P < 0.001, HR = 0.33) were significantly worse than those of the low-risk group. The recurrence model also demonstrated that in the training cohort (P<0.001, HR= 0.42) and validation cohort (P=0.01, HR=0.5), the recurrence rate of the high-risk group was significantly higher than that of the low-risk group. In addition, angiogenic genes were an independent predictor of prognosis and recurrence (P < 0.05). The diagnostic model has been validated in terms of correctly distinguishing between HCC and normal samples, as well as between HCC and proliferative nodule samples, with high sensitivity and high specificity. Through pharmacological analysis, we identified piperlongumine as a drug for targeting angiogenesis. Subsequent targeted drug cell experiments suggested that the anti-tumor effect of piperlongumine was achieved by inhibiting angiogenesis via the EGF/EGFR axis. Conclusion: We identified and validated the predictive properties of the diagnostic, recurrence, and prognostic models of four key angiogenic genes and confirmed the value of these four genes for constructing models. Piperlongumine was confirmed to inhibit HCC cell proliferation and angiogenesis via the EGF/EGFR axis. These findings may help to provide new individualized treatment ideas for HCC patients. Funding Statement: This study was supported by National Natural Science Foundation of China (Nos. 81803778), and The Key Research and development Project of Zhejiang Province (No. 2018C0302), and Public Welfare Project of Zhejiang Province (Nos. LGF19H180010, LGD19H160002 and LGF19H180009), and Medical and Health Care Key Project of Zhejiang Province (Nos. 2016146810, 2018KY197 and 2018KY932). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Since these mRNA-sequencing data were obtained from TCGA and ICGC databases and were freely available to the public, and as this study also strictly followed the publication guidelines and access policies of these databases, this study does not require ethical review and approval from an Ethics Committee.