Dicer Enhances The Anti-Tumor Effect Of Lenvatinib Through Interleukin-8 In Hepatocellular Carcinoma And The Relationship Between Interleukin-8 And Immune Related Adverse Events

Part One Dicer suppresses hepatocellular carcinoma via interleukin-8Objective: Our previous studies confirmed that Dicer inhibits the growth,migration,and invasion of hepatocellular carcinoma(HCC)cells,and promotes apoptosis.Elevated interleukin-8(IL-8)levels can lead to poor prognosis of tumors.Therefore,this study investigated the interaction between Dicer and IL-8 and the impacts on HCC.Methods:The HCC cell line SMMC-7721 was transfected with green fluorescent protein(GFP)labeled Dicer overexpression lentivirus(p CMVDicer)and negative control lentivirus(p CMV-NC)for subsequent experiments.Western blot was used to verify the expression of Dicer protein after transfection.The effects of overexpression of Dicer on the proliferation,migration,and invasion of HCC cells were evaluated through Cell Counting kit-8(CCK8),wound healing and transwell assay.The levels of IL-8 was detected by Flow immunofluorescence microsphere assay.Results:1.The proliferation,migration,and invasion abilities of Dicer overexpressed cells were significantly decreased compared to the SMMC-7721 and negative control cells,suggesting that Dicer inhibits the proliferation,migration,and invasion of HCC cells.2.Compared with the negative control group,the level of IL-8 secreted by Dicer overexpressed cells was significantly reduced,indicating that Dicer inhibited the secretion of IL-8 levels in HCC cells.3.The addition of recombinant human interleukin 8(rhIL-8)promotes the proliferation,migration,and invasion of negative control and Dicer overexpressed cells,indicating that IL-8 promotes the proliferation,migration,and invasion of HCC cells,and reduces the inhibitory effect of Dicer overexpression on the proliferation,migration,and invasion of HCC cells.4.The addition of reparixin inhibits the proliferation,migration,and invasion of negative control and Dicer overexpressed cells,indicating that reparixin inhibits the proliferation,migration,and invasion of HCC cells,and enhances the inhibitory effect of Dicer overexpression on the proliferation,migration,and invasion of HCC cells.Conclusions: Dicer inhibits the secretion of IL-8 in HCC cells and inhibits the proliferation,migration,and invasion of HCC cells through the regulating of IL-8.Part Two Dicer enhanced the anti-tumor effects of lenvatinib by regulating the interleukin-8Objectives: Our previous studies have confirmed that Dicer inhibits the proliferation,migration,and invasion of HCC cells by regulating IL-8.So,we will further explore the relationship between Dicer,IL-8,and the anti-tumor effect of lenvatinib.Methods: Cell Counting kit-8(CCK-8),clone formation assay,wound healing,and transwell were used to evaluate the effects of lenvatinib on HCC cell proliferation,clone formation,migration,and invasion.To evaluate the effect of Dicer and lenvatinib on the growth of HCC cells in vivo through subcutaneous xenograft tumor in nude mice.Immunohistochemistry was used to detect the expression of CD31 to measure the microvessel density(MVD)of the transplanted tumor,and cytometry immunofluorescence bead assay was used to detect the level of IL-8 in HCC cells.Results:1.The addition of lenvatinib inhibits the proliferation,clone formation,migration,and invasion of negative control and Dicer overexpression cells,indicating that lenvatinib inhibits the proliferation,cloning formation,migration,and invasion of HCC cells,and enhances the inhibitory effect of Dicer overexpression on the proliferation,clone formation,migration,and invasion of HCC cells.In addition,the inhibitory effect of lenvatinib on Dicer overexpressed cells is more enhanced than that of negative control cells,indicating that Dicer enhances the inhibitory effect of lenvatinib on the proliferation,clone formation,migration,and invasion of HCC cells.2.Cocultured of lenvatinib with SMMC-7721 cells,the IL-8 levels in the supernatant were significantly reduced than that without lenvatinib,indicating that lenvatinib inhibited the secretion of IL-8 levels in HCC cells.The level of IL-8 in the supernatant was detected by cocultured of lenvatinib with negative control and Dicer overexpression cells.The results showed that the level of IL-8 secretion in Dicer overexpressed cells was significantly decreased compared with negative control cells,suggesting that Dicer enhanced the inhibition of lenvatinib on IL-8 secretion in HCC cells.Therefore,Dicer enhance the antitumor effect of lenvatinib through inhibiting the secretion IL-8.3.It was confirmed by subcutaneous tumor formation experiments in nude mice that lenvatinib inhibited the xenografts growth of negative control and Dicer overexpressed cells in vivo,and the inhibitory effect on Dicer overexpressed cell xenografts showed a more obvious trend,indicating that the Dicer enhanced the inhibition of lenvatinib on the xenograft tumor growth of HCC cells.4.Immunohistochemical results of CD31 showed that lenvatinib reduced the microvascular density(MVD)of the negative control and Dicer overexpressed cell xenografts,and the MVD decrease of Dicer overexpressed cell xenografts was more obvious than that of the negative control cells,which suggested that Dicer enhanced the inhibition of lenvatinib on the microangiogenesis of xenograft tumor in HCC cells.Conclusions:1.Dicer enhances the inhibition of lenvatinib on HCC cells proliferation,clone formation,migration,and invasion by regulating IL-8.2.Dicer enhanced the inhibition of lenvatinib on tumor growth and the microangiogenesis of xenograft tumors in HCC cells.Part Three Relationship between circulating interleukin-8 levels and immune related adverse events in solid tumor patientsObjectives: To investigate the relationship between circulating IL-8 levels and immune related adverse events(ir AEs)in tumor patients treated with Anti PD-1/PD-L1 therapy.Methods: Retrospective analysis was conducted on tumor patients receiving PD-1/PD-L1 treatment at the Fouth Hospital of Hebei Medical University from November 2019 to May 2022.Peripheral blood was collected and IL-8 levels were measured using Flow immunofluorescence microsphere assay.Chi-square test was used to analyze the relationship between IL-8 levels and clinical data of tumor patients and the occurrence of ir AE.The follow-up date was as of March 1st,2023.Results:1.A total of 71 solid tumor patients treated with anti-PD-1/PD-L1 therapy were included in this study,and circulating blood was collected for IL-8 level detection.Among them,49 patients were tested for IL-8 levels before the first application of anti PD-1/PD-L1 treatment(baseline IL-8),23 patients were tested for IL-8 levels at the time of ir AE,and 13 patients were tested at any time.Among them,40 patients developed ir AE and 31 patients did not.The incidence of ir AE was 56.3%.A total of 33 patients with grade 1-2 ir AE occurred,and 7patients with grade 3-4 ir AE.The main ir AEs occurred were endocrine diseases,cardiac toxicity,skin damage,colitis/ diarrhea,pneumonia,liver function damage,myalgia,elevated creatinine,thrombocytopenia,and conjunctivitis.Grade 3-4 ir AE mainly occurs in skin injury,colitis/diarrhea,liver function damage,elevated creatinine,and thrombocytopenia.2.There was no significant relationship between the occurrence of ir AE and gender,age,clinical stage,number of treatment lines,ECOG score,and neutrophil lymphocyte ratio(NLR).The occurrence of ir AE is correlated with baseline IL-8 levels,and patients developed ir AE have lower baseline IL-8levels.Longitudinal monitoring of the changes in IL-8 levels in 9 patients with ir AE,and it revealed an upward trend in IL-8 levels in 8 patients and a downward trend in 1 patient.Conclusions: Solid tumor patients treated with anti PD-1/PD-L1 therapy usually have lower baseline IL-8 levels in patients with ir AE,and their IL-8levels show an upward trend when ir AE occurs.Conclusions:1.Dicer inhibits the secretion of IL-8 in HCC cells and inhibits the proliferation,migration,and invasion of HCC cells through the IL-8 pathway.2.Dicer enhances the inhibitory effect of lenvatinib on the proliferation,clone formation,migration,and invasion of HCC cells through IL-8.3.Dicer enhanced the inhibition of lenvatinib on tumor growth and the microangiogenesis of xenograft tumors in HCC cells.4.Solid tumor patients treated with anti PD-1/PD-L1 therapy usually have lower baseline IL-8 levels in patients with ir AE,and their IL-8 levels show an upward trend when ir AE occurs.