CircRPS16 Promotes Proliferation And Metasitasis Of HCC Via Mir-876-5p And Upregulation Of SPINK1

BackgroundPrevious studies have found that circRNAs play important roles in the development of several malignancies,including HCC.Nevertheless,the mechanisms of circRNAs in hepatocellular carcinoma remain to be explored.Therefore,an in-depth study of circRNAs may provide effective protocols for early detection,effective treatment and prognosis improvement of HCC patients.In the present study,we found that patients with abnormally high expression of SPINK1 in hepatocellular carcinoma tissues compared to hepatocellular carcinoma paracancer tissues and high expression of SPINK1 had a poorer prognosis.Therefore,we hypothesized that SPINK1 may play a role as a pro-cancer factor in the proliferation,invasion and metastasis of hepatocellular carcinoma.Through bioinformatic analysis,we predicted that CircRPS16 may sponge miRNAs and further regulate SPINK1 expression.ObjectiveTo explore the molecular mechanisms by which circRPS16 regulates SPINK 1 expression,and to provide molecular targets for the diagnosis and treatment of hepatocellular carcinoma patients to improve their prognosis.Method and materials1.By analyzing the GEO database（GSE14520）,we examined the expression levels of SPINK 1 in paired hepatocellular carcinoma and paracancer groups;analyzed the relationship between the abnormal expression of SPINK1 and the prognosis of hepatocellular carcinoma patients using TCGA;analyzed the immunohistochemical results in The Human Protein Atla to further examine the SPINK1 expression levels in hepatocellular carcinoma tissues.2.25 pairs of HCC and paraneoplastic tissues were selected to detect the expression level of SPINK1 by qRT-PCR assay.SPINK1 was knocked down or overexpressed in hepatocellular carcinoma cells,and the regulatory effect of SPINK 1 on proliferation was detected by CCK-8 and EdU assays;the regulatory effect of SPINK1 on cell cycle was detected by flow cytometric analysis,and the regulatory effect of SPINK1 on invasion ability was detected by Transwell assay.3.Through raw signal analysis of miRanda,Targetscan,miRwalk and StarBase,we found that circRPS16（hsa_circ₀050997）may regulate SPINK1 expression by binding miR-876-5p.And we further examined the expression levels of circRPS16 and miR-876-5p in liver cancer tissues.4.To further validate the molecular mechanism of circRPS16 regulating SPINK1 through miR-876-5p,we examined the co-localization of circRPS16 and miR-876-5p in HCC cells by using fluorescence in situ hybridization（FISH）technique.The binding sites of circRPS16 to miR-876-5p and miR-876-5p to SPINK1 were verified by dual luciferase reporter gene assay.The expression level of SPINK1 in transfected hepatocellular carcinoma cells was detected by Western blot assay5.Functional reversion assay to verify that circRPS16 regulates hepatocarcinogenesis through miR-876-5p.Stable transfected cell lines with low expression of circRPS16,low expression of miR-876-5p and co-low expression of circRPS16 and miR-876-5p were constructed and negative NC groups were set up,respectively.Further,the phenotypic changes of hepatocellular carcinoma cells such as proliferation and cell cycle invasion ability were detected.6.A subcutaneous tumorigenic model of circRPS16 low-expressing hepatocellular carcinoma in nude mice was constructed.After 20 days,the tumor tissues were separated and removed intactly after executing the nude mice,and the weights of tumorigenic tissues in different groups were recorded and statistically analyzed.The tumor-forming tissues were made into paraffin sections,and the expression levels of SPINK1 in the tumor-forming tissues of different subgroups were detected by immunohistochemistry（IHC）.7.Statistical analysis was performed using GraphPad Prism 8.2.1 software.Quantitative data were analyzed using paired or independent t-test,and data were presented as mean ± standard deviation（mean±SD）.p-values<0.05 were considered statistically different,*,p<0.05;**,p<0.01;***,p<0.001.Conclusion1.SPINK1 was aberrantly highly expressed in hepatocellular carcinoma tissues and acted as an oncogenic factor.2.CircRPS16 is aberrantly highly expressed in hepatocellular carcinoma tissues,and upregulates SPINK1 expression via miR-876-5p,thus promoting the development of hepatocellular carcinoma.3.This study is the first to report the important roles of circRPS16/miR-8765p/SPINK1 axis in the progression of HCC,which is expected to provide a novel molecular therapeutic target for hepatocellular carcinoma treatment.The results of this study are divided into the following six parts:Part Ⅰ:SPINK1 is abnormally up-regulated in HCC tissues and strongly associated with poor prognosisBy analyzing the GSE14520 dataset we found SPINK 1 mRNA levels were abnormally upregulated in the HCC tissues compared to the adjacent noncancerous tissues.Heat map analysis showed that the abnormally high SPINK 1 expression was significantly enriched in HCC tissues.To further verify the expression level of SPINK1 in hepatocellular carcinoma tissues,we selected 25 pairs of paired HCC and and paraneoplastic tissues and examined the expression level of SPINK1 by qRT-PCR assay,and the results also confirmed that SPINK 1 was significantly highly expressed in HCC tissues.To verify whether SPINK1 is also highly expressed at the protein level,we analyzed the IHC results of human normal and hepatocellular carcinoma samples from the public database named The Human Protein Atlas.We found that,at the protein level,SPINK1 was also abnormally highly expressed in the HCC tissues,while it was barely detected in normal tissues.To verify the correlation between the expression level of SPINK 1 and the prognosis of hepatocellular carcinoma patients,we analyzed the clinical data of HCC patients in the TCGA database,and the results confirmed that patients with high SPINK1 expression had a poorer prognosis.In conclusion,SPINK 1 is aberrantly highly expressed in hepatocellular carcinoma tissues and is closely associated with poor prognosis of hepatocellular carcinoma patients.Part Ⅱ:SPINK1 regulates the proliferation,cell cycle and invasive ability of hepatocellular carcinoma cells in vitro.To verify the effect of SPINK1 on the biological function of hepatocellular carcinoma cells,we constructed small interfering RNA（siRNA）of SPINK1 and overexpression plasmid of SPINKI.We used SPINKI siRNA to transfect hepatocellular carcinoma cells.Then,the transfection efficiency was confirmed by qRT-PCR assay.The experimental results confirmed that SPINK1 siRNA could significantly decrease the expression level of SPINK 1.Similarly,we confirmed that SPINK 1 overexpression plasmid could significantly upregulate the expression level of SPINK1.The CCK-8 assay confirmed that inhibition of SPINK1 expression inhibited the proliferation of HCC cells,while overexpression of SPINK 1 promoted the proliferation of HCC cells.EdU assay exhibited that inhibition of SPINK1 expression could inhibit the DNA synthesis of HCC cells,and the overexpression of SPINK 1 promoted DNA synthesis in HCC cells.Flow cytometry analysis showed that downregulation of SPINK1 induced cell cycle arrest,while SPINK1 overexpression accelerated the cell cycle.And Transwell assay showed that inhibition of SPINK1 expression inhibited the invasive ability of HCC cells,while SPINK1 overexpression showed the opposite result.In conclusion,the above experimental results suggest that SPINK1 significantly promotes the proliferation,cell cycle and invasive ability of HCC cells.Part III:CircRPS16 regulates SPINK1 expression via miR-876-5pPrevious studies have shown that CircRNAs act as sponge for miRNAs,which further regulate the activity of miRNA target genes.Therefore,we conducted a preliminary exploration of whether SPINK 1 aberrant expression is regulated by circRNAs.First,we used four databases,miRanda,Targetscan,Starbase and miRwalk,to predict the potential upstream miRNAs of SPINK 1.Together,these four databases predicted that miR-876-5p is the most potential miRNA regulating SPINK 1.Therefore,we served miR-876-5p as the potential molecule target of our study.Next,The StarBase database was used to predict the potential upstream circRNAs of miR876-5p.Among the many potential circRNAs,we found that circRPS16（circBase ID is hsa_circ₀050997）was the most potential one to binding miR-876-5p which was validated by 32 Argonaute（AGO）CLIP-seq experiments.Next,we validated the expression levels of circRPS16 and miR-876-5p in HCC and adjacent noncancerous tissues by qRT-PCR experiments.And the results confirmed that circRPS16 was significantly highly expressed in HCC tissues;miR-876-5p was significantly decreased in HCC tissues,and the difference was statistically significant,p<0.05.Further,the binding relationship between circRPS16 and miR-876-5p,miR-876-5p and SPINK1 was verified by mechanistic experiments.FISH experiments confirmed that circRPS16 and miR-876-5p were colocalized in the cytoplasm of HCC cells suggesting that circRPS16 and miR-876-5p play post-transcriptional regulatory roles.Further,we verified the binding sites between circRPS16 and miR-876-5p,miR-8765p and SPINK 1 using dual luciferase reporter assays.The experimental results confirmed the direct binding relationship between circRPS16 and miR-876-5p,miR876-5p and SPINK1.Finally,we used Western blot experiments to verify the effects of circRPS16 and miR-876-5p on SPINK1 expression and whether circRPS16 regulates SPINK1 expression through miR-876-5p.The results confirmed that overexpression of miR-876-5p inhibited SPINK1 expression,while inhibition of miR-876-5p promoted SPINK1 expression;inhibition of circRPS16 inhibited SPINK1 expression,while circRPS16 overexpression promoted SPINK1 expression.Furthermore,rsscue experiments confirmed that simultaneous inhibition of miR-8765p expression blocked the inhibitory effect on SPINK1 expression induced by circRPS16 downregulation,while simultaneous overexpression of miR-876-5p reversed the promoting effect on SPINK1 expression induced by circRPS16 overexpressing.Taken together,the above experimental results confirm that circRPS16 can regulate SPINK1 expression via sponging miR-876-5p.Part Ⅳ:miR-876-5p regulates HCC Cell proliferation,cell cycle,and invasion.To explore the regulatory effects of miR-876-5p on HCC cells,we constructed miR-876-5p mimics and miR-876-5p inhibitor.We transfected hepatocellular carcinoma cells with miR-876-5p mimics and miR-876-5p inhibitor to overexpress or inhibit miR-876-5p expression,respectively.CCK-8 experiments confirmed that overexpression of miR-876-5p inhibited the proliferation,while inhibition of miR876-5p expression promoted the proliferation of HCC cells.EdU experiments demonstrated that overexpression of miR-876-5p expression inhibited DNA synthesis,whereas inhibition of miR-876-5p expression promoted DNA synthesis in HCC cells.Flow cytometry analysis showed that overexpression of miR-876-5p induced cell cycle arrest,while inhibition of miR-876-5p accelerated the cell cycle.Transwell assay showed that overexpression of miR-876-5p could inhibit the invasive ability of liver cancer cells,while inhibition of miR-876-5p expression could show the opposite results.In conclusion,the above experimental results indicate that miR-876-5p significantly inhibited the proliferation,cell cycle and invasion ability of HCC cells.Part V:CircRPS16 regulates HCC cell proliferation,cell cycle,and invasion via miR-876-5p.To confirm whether circRPS16 exerts the biological function through miR-876-5p,we performed the functional rescue assays.CircRPS16 siRNA was transfected to knock down the expression of circRPS16,and then the miR-876-5p mimics were cotransfected.Next,the changes of HCC cellular phenotypes such as proliferation,cell cycle,and invasion ability were observed.CCK-8 assay confirmed that inhibition of circRPS16 could inhibit the proliferation of HCC cells;EdU assay confirmed that inhibition of circRPS16 could inhibit the synthesis of DNA of HCC cells;flow cytometry analysis showed that inhibition of circRPS16 expression induced cell cycle arrest;Transwell assay found that circRPS16 siRNA inhibited HCC cells’ invasive ability;while simultaneous inhibition of miR-876-5p could reverse the inhibitory effect induced by circRPS16 inhibiton.In summary,circRPS16 regulates the proliferation,cell cycle and invasion ability of HCC cells via sponging miR-876-5pPart VI:Knockdown of circRPS16 inhibits Tumor Growth by Suppressing SPINK1 In Vivo.Firstly,we constructed stable circRPS16 low-expressing cell lines by lentiviral transfection.Then,the cells were injected to the back of nude mice to construct the subcutaneous tumor model.The experimental results confirmed that inhibition of circRPS16 expression could inhibit the growth of HCC in vivo.Immunohistochemical（IHC）experiments of the tumor confirmed that SPINK1 expression was significantly decreased in the circRPS16 low expression group compared to NC group.Thus,our results confirm that inhibition of circRPS 16 expression can inhibit SPINK1 expression and HCC growth in vivo.