The Mechanism Of Isoliensinine Against Oral Squamous Cell Carcinoma And The Synergistic Anticancer Effect Combined With MiR-7-5p

Research background and purpose:Oral squamous cell carcinoma（OSCC）is a common malignant tumor in head and neck cancer,and its incidence is increasing.Natural active small molecular compounds from plants or dietary materials have high efficiency and low toxicity antitumor activity,which is an important source for the development of anti-tumor drugs.Isoliensinine,a bis-benzylisoquinoline alkaloid isolated from the Nelumbo Nucifera Gaertn,has the biological activities of protecting blood vessels,antiarrhythmic and antitumor.Isoliensinine has been found to have inhibit the proliferation of breast cancer,cervical cancer,liver cancer and other tumor cells by regulating the activation NF-κB,PI3K/AKT,JNK,p38 and other signaling pathway.However,there have been no studies elucidating the biological effect of Isoliensinine on OSCC,nor have there been reports on the involvement of ERK signal pathway in the anti-tumor activity of Isoliensinine.Therefore,this study takes OSCC cell lines HSC-3 and HSC-4 as the research objects to explore the inhibitory effect of Isoliensinine on the proliferation of OSCC cells and explain the molecular mechanism that Isoliensinine activates p38 and JNK signaling pathways,and regulates ERK signaling pathways to inhibit proliferation.The low five-year survival rate of most OSCC patients is mainly related to the reduced sensitivity of OSCC patients to therapeutic agents.The emergence of tumor drug resistance can significantly reduce the therapeutic efficacy of existing drugs.micro RNA（miRNA）is a kind of short-stranded non-coding RNA with the length of about 22nt,which can regulate gene expression.Some risk factors can induce the disordered expression of miRNA,and the disordered expression of miRNA has been found to be closely related to tumorigenesis and tumor drug resistance.Therapeutic approaches to regulate miRNA disorder expression have shown excellent research prospects in the field of tumor therapy.Previous studies have found that miR-7-5p is lowly expressed in tumor cells.Exogenous transfection of miR-7-5p mimics can upregulate the expression of miR-7-5p in tumor cells,inhibit the proliferation of colorectal cancer,lung cancer and other tumor cells,and improve the sensitivity of tumor cells to chemotherapeutic drugs.Clinically,the occurrence and development of OSCC are the result of the combined stimulation of complex regulatory network.There are some limitations to the long-term effective inhibition of tumor cell proliferation by single chemotherapy drugs.In order to prevent the occurrence of drug resistance and improve the therapeutic effect,drug combination strategy is adopted in clinic.Based on the experimental results in Chapter 1 that Isoliensinine can inhibit the proliferation of OSCC cells,it is ready to treat the OSCC cell line HSC-3 with a combination of Isoliensinine and miR-7-5p mimics.It is expected to observe the synergistic inhibitory effect of Isoliensinine combined with miR-7-5p on the proliferation of HSC-3 cells,which provides support for the synergistic anti-OSCC strategy of natural products combined with miRNA.Materials and Methods:Chapter 11.The effect of Isoliensinine on the proliferation of OSCC cell lines HSC-3 and HSC-4 was detected by CCK-8 method.2.The differential expression of genes in HSC-3 cells was analyzed by RNA-seq at the m RNA level after Isoliensinine treatment.3.The effects of Isoliensinine on HSC-3 cells and HSC-4 cells cycle ratios,apoptosis,mitochondrial membrane potential（MMP）and reactive oxygen species（ROS）were examined using PI,Annexin V-FITC/PI,JC-1,DCFH-DA fluorescent dyes and Flow cytometry（FCM）.4.Western blotting（WB）was used to detect the effects of Isoliensinine on the expression of cyclin,mitochondrial pathway,and MAPK signal pathway-related proteins in OSCC cells.Chapter 21.The differential expression of miRNA in HSC-3 cells was analyzed by RNA-seq after Isoliensinine treatment.2.Quantitative PCR（q PCR）was used to detect the expression of miRNA in HSC-3 cells and HSC-4 cells after exogenous transfection of miRNA mimics,and to detect the effect of Isoliensinine on the expression of miR-7-5p in HSC-3 cells.3.CCK-8 method was used to detect the effects of miR-7-5p mimics on the proliferative of OSCC cell lines HSC-3 and HSC-4,and the effect of Isoliensinine combined with miR-7-5p mimics treatment on the proliferation of HSC-3 cells.4.OMIM,Gene Cards and Disgent databases were used to obtain OSCC target information,and miRWalk and Target Scan8.0 databases were used to screen miR-7-5p targets.The OSCC target and the miR-7-5p target are intersected to obtain the"miR-7-5p-OSCC"common target.STING database was used to analyze the interaction network of"miR-7-5p-OSCC"common targets,and Cytoscape3.9.0 software was used for visual analysis and core target screening.GO and KEGG analysis of"miR-7-5p-OSCC"common target using DAVID database.5.The effects of Isoliensinine combined with miR-7-5p mimics treatment on ROS levels,MMP and apoptosis in HSC-3 cells were detected by DCFH-DA,JC-1,Annexin V-FITC/PI fluorescent dye and FCM.Result:Chapter 11.CCK-8 assay results showed that Isoliensinine inhibited the proliferation of OSCC cell lines HSC-3 and HSC-4 in a time-dependent and concentration-dependent manner.After treated with Isoliensinine for 24 H and 48 H,the IC₅₀of HSC-3 cells was39.50μM and 21.10μM,and the IC₅₀of HSC-4 cells was 57.28μM and 43.31μM,respectively.2.RNA-seq analysis showed that 1831 known genes were significantly differentially expressed in HSC-3 cells after Isoliensinine treatment,among which 1202were up-regulated and 629 were down-regulated.GO analysis and KEGG analysis of differentially expressed genes showed that Isoliensinine may regulate the activation of MAPK,p53,PI3K/AKT and other signaling pathways and the expression of tumor-associated miRNA.3.After HSC-3 cells and HSC-4 cells were treated with Isoliensinine,the results of FCM detection showed that Isoliensinine induced cell cycle arrest in G2/M phase,apoptosis,MMP decreased and ROS level increased.4.WB results showed that Isoliensinine increased the expression of p-cdc25c,p-cdc2,Bax,Bid,cleaved caspase-3/-9,cleaved PARP,p-p38,and p-SPAK/JNK in HSC-3 and HSC-4 cells,decreased the expression of p-ERK1/2.Chapter 21.RNA-seq analysis showed that after Isoliensinine treatment,the expression of 52known and unknown miRNA in HSC-3 cells was significantly different,among which18 were up-regulated and 34 were down-regulated.GO analysis and KEGG analysis were performed on the target genes of differentially expressed miRNA,and the analysis results showed that the target genes of differentially expressed miRNA may be related to chemokines,inflammatory mediators,and MAPK signaling pathway.2.The results of q PCR analysis showed that the effect of Isoliensinine on miR-7-5p expression in HSC-3 cells was not statistically significant.Exogenous transfection of miR-7-5p mimics significantly increased the expression level of miR-7-5p in HSC-3cells and HSC-4 cells.3.The results of CCK-8 detection showed that miR-7-5p high expression could inhibit the proliferation of HSC-3 cells and HSC-4 cells,and further studies found that Isoliensinine combined with miR-7-5p mimics treatment had a synergistic inhibitory effect on the proliferation of HSC-3 cells.4.928 OSCC targets and 1669 miR-7-5p targets were obtained from the database.The intersection was used to obtain 70"miR-7-5p-OSCC"common targets.Cytoscape3.9.0 software analysis results show that the core targets of"miR-7-5p-OSCC"include EGFR,BRCA1,PIK3CA,EGF,and NF1.GO analysis and KEGG analysis showed that the"miR-7-5p-OSCC"common target was involved in regulating DNA damage,drug resistance,Fox O,Erb B,HIF-1 and other signaling pathways.5.The results of FCM analysis showed that Isoliensinine combined with miR-7-5p mimics treatment significantly increased ROS levels,decreased MMP and induced apoptosis in HSC-3 cells.Conclusion:1.Isoliensinine inhibited the proliferation of OSCC cell lines HSC-3 and HSC-4.The potential molecular mechanism may be that Isoliensinine arrest OSCC cell cycle in G2/M phase and induces apoptosis through ROS-mediated MAPK signal pathway and mitochondrial pathway.2.The Isoliensinin induced the differential expression of 52 miRNAs in HSC-3cells.The effect of Isoliensinine on miR-7-5p expression in HSC-3 cells was not statistically significant.Isoliensinine combined with miR-7-5p mimics showed synergistic inhibitory effect on the proliferation of HSC-3 cells.The possible molecular mechanism was related to ROS mediated apoptosis.