Paqr4 Sensitizes The Killing Effect Of Apatinib On Hepatocellular Carcinoma Through PI3K/AKt Pathway

Objective:Hepatocellular carcinoma(HCC)is the most common histological subtype of liver cancer and the third leading cause of cancer-related death.Surgical resection and radiofrequency ablation are the first and most effective treatment options for patients with early-stage liver cancer.Due to the strong invasiveness and insidious nature of liver cancer,most patients are diagnosed as intermediate and advanced stage.However,the existing therapies for advanced liver cancer still have limitations,and the improvement of patient prognosis is unsatisfactory.Therefore,the discovery of biomarkers and molecular targets for early diagnosis may open up new avenues for the treatment of liver cancer.The progesterone and fat receptor(PAQR)family is a novel membrane receptor family consisting of 11 members(PAQR1 to PAQR11).Many researchers have demonstrated that PAQR proteins influence metabolism and tumorigenesis.For example,PAQR3 has been identified as a tumor suppressor in many cancers.In addition,PAQR4 has been found to be highly expressed in a variety of solid tumors,including non-small cell lung cancer,breast cancer,prostate cancer,and gastric cancer,and promote the progression of these cancers.In addition,it has been documented that upregulation of PAQR4 is associated with poor prognosis in several cancers.However,the role of PAQR4 in HCC and its regulatory mechanism remain unclear.Phosphatidylinositol 3-kinase/protein kinase B pathway(PI3K/AKT)regulates the normal physiological activity of cells.On the other hand,accumulating evidence confirmed that abnormal activation of PI3K/AKT pathway is involved in the tumorigenesis and progression of malignant tumors.Deregulation of this pathway contributes to proliferation,invasion,migration,and therapeutic resistance of cancer cells,and inhibits cell apoptosis.Of note,PI3K/AKT pathway plays an important role in the progression of HCC.Previous study has shown that inhibition of PI3K/AKT signaling via pharmacological targeting of APLN decreases cell proliferation and triggers cell apoptosis in HCC.Ma et al.demonstrated that PI3K/AKT pathway is crucial for CD73 to exert its oncogenic function in HCC.Vascular endothelial growth factor(VEGF)is considered to be a potent factor in angiogenesis.Studies have shown that vascular endothelial growth factor and its receptor(VEGFR)can be highly expressed in tumor tissues and are involved in tumor progression and metastasis.Apatinib is an oral small-molecule tyrosine kinase inhibitor targeting VEGFR-2.It can inhibit angiogenesis by blocking multiple signal transduction pathways downstream of VEGFR-2,thereby controlling the occurrence and development of tumors.And it can make drug-resistant tumor cells sensitive to chemotherapeutic drugs and improve the effectiveness of conventional chemotherapeutic drugs.Current studies have shown that apatinib can prolong the median overall survival and progression-free survival of patients with clinical hepatocellular carcinoma,but its criticized complications and uncertain clinical efficacy remain to be studied,so even if it may become a How to reduce the dose and improve the efficacy of drugs for second-line and above treatment of liver cancer is still a topic of clinical research today.In the early stage of this study,by searching the bioinformatics database,it was found that the expression of paqr4 was abnormally elevated in hepatocellular carcinoma.However,the role of paqr4 in the development of hepatocellular carcinoma has not been reported,and the effect of paqr4 on hepatoma cells is unclear.Therefore,this topic intends to study the effect of PAQR4 on hepatocellular carcinoma cells and its combined effect with apatinib,so as to find potential targets for the treatment of hepatocellular carcinoma.Methods:1.The expression of paqr4 in hepatocellular carcinoma tissues and cells was quantitatively detected by immunofluorescence,and its correlation with prognosis was analyzed.2.The expression of target protein was detected by Western blotting,and the effects of paqr4 on proliferation,cycle and apoptosis of hepatoma cells were detected by flow cytometry.3.The invasiveness of tumor cells was detected by scratch test.4.To construct a nude mouse xenograft tumor model for subsequent experimental verification.5.RT-q PCR was used to detect the m RNA expression of the target gene.6.HE staining was used to observe the pathological changes of tumor tissues in each group.7.Western blot detection of PAQR4,Ki-67,PARP,cleaved PARP,caspase3,cleaved caspase3,PI3K-p85,p-p85(Tyr467/199),AKT,p-AKT(Ser473)protein expression in tumor tissues of each group.8.Immunohistochemical detection of protein expression in allogeneic tumor tissue.Results: Analysis of GEPIA database indicated that PAQR4 was highly expressed in HCC samples,and the m RNA level of PAQR4 was negatively correlated with overall survival of HCC patients.Knockdown of PAQR4 in Hep3 B cells suppressed cell proliferation by hindering G1/S transition of cell cycle as shown by the flow cytometry analysis.PAQR4 knockdown also expedited the cell apoptosis.Knockdown of PAQR4 repressed the migratory and invasive potential of Hep3 B cells.PAQR4 knockdown sensitized Hep3 B cells to apatinib-based chemotherapy.PAQR4 knockdown blocked the activation of PI3K/AKT pathway,as reflected by the reduced phosphorylation of AKT and p85.Conversely,overexpression of PAQR4 exerted opposite effects in Huh-7 cells.PI3 K inhibitor LY294002 could eliminate the effects of PAQR4 on proliferation,apoptosis,and invasion.In tumor xenograft model,knockdown of PAQR4 suppressed tumor growth in vivo,while PAQR4 overexpression promoted tumor growth.Knockdown of PAQR4 can increase the antitumor effect of apatinib.Conclusion: The expression of PAQR4 in liver cancer tissue is correlated with the survival time of patients.Apatinib,as a domestic targeted drug,can effectively inhibit the development of liver cancer.PAQR4 plays a role in promoting hepatocarcinogenesis in the development of liver cancer,and plays a role through PI3 K / Akt signal pathway.Inhibiting PAQR4 can further enhance the sensitivity of patients to apatinib.