Experimental Study On The Effect Of Circ MBNL3(hsa_circ₀091570)on The Sensitivity Of Human Hepatocellular Carcinoma Cell Line HepG2 To Chemotherapy

Background and Purpose Liver malignant can be classified into primary and secondary types.Primary liver cancer originates from the epithelium or mesenchymal tissue of the liver.The former is known as primary liver cancer,specifically refers to the malignant tumor of liver cells or intrahepatic bile duct epithelial cells.It is a high-incidence and highly harmful malignant tumor in China,including hepatocellular carcinoma,bile duct cell carcinoma and mixed hepatocellular carcinoma.Hepatocellular Carcinoma(HCC)accounts for more than 85% to 90% of primary liver cancer.The latter,called sarcoma,is rare compared with primary liver cancer.Secondary or metastatic liver cancer refers to a malignant tumor originating in multiple organs of the body that extends to the liver.Generally,it is more common in liver metastasis of malignant tumors of the stomach,biliary tract,pancreas,colorectum,ovary,uterus,lung,breast and other organs.Compared with primary liver cancer,secondary liver cancer is more common,with a ratio of 2:1 4:1 in China and as high as 20:1 in western countries.Because the early symptoms and signs of liver cancer are not obvious,most patients are already in the advanced stage of cancer or even have metastatic cancer when diagnosed clinically,and chemotherapy drugs are needed for adjuvant treatment regardless of whether the operation is performed.Chemotherapy resistance is a common problem in almost all tumors during chemotherapy,especially in liver cancer.Chemotherapy resistance has become one of the main causes of liver cancer treatment failure.Therefore,whether the molecular mechanism of chemotherapy resistance in HCC can be solved effectively has become the key to the success of the treatment of HCC.Gemcitabine(Gem),as a chemotherapy drug of pyrimidine nucleoside analogs,mainly acts on tumor cells in the S phase(DNA synthesis phase),inhibits DNA synthesis and repair,and induces autophagy and apoptosis.Gem is used to treat a variety of cancers,including pancreatic,non-small cell lung,ovarian and breast cancer.Gem is an FDAapproved chemotherapeutic agent for HCC.circRNA is a kind of closed circular non-coding RNA produced by backsplicing.After years of research and development,the regulation of circ RNA in cells has different modes,in which miRNA can be regulated through selective adsorption(sponging),formi-ng the interaction network of circ RNA-miRNA-m RNA,forming the endogenous compet-ing RNA(ce RNA)mechanism.circ RNA regulates the expression level of downstream target genes through the ce RNA mechanism.circ MBNL3(hsa_circ₀091570)is a newly discovered circ RNA that is significantly down-regulated in HCC and may inhibit the process of HCC through the ce RNA mechanism.Based on bioinformatics analysis,we found that there was a binding site for miR-19b-1-5p on circ MBNL3,which may play a role as sponge for absorbing miR-19b-1-5p.In addition,bioinformatics analysis showed that TCF4(Gene ID: 6925)was a potential target Gene of miR-19b-1-5p.It has been reported that in breast cancer,the mutation or loss of TCF4 may alter the regulation of cell cycle and make cancer cells acquire resistance to chemotherapy.Cell models showed that silenced TCF4 increased the tolerance of Cal-51 and MDA-MB-436 to either cisplatin or olaparib.By TCGA data analysis,it was found that the expression level of TCF4 was negatively correlated with several DNA repair pathways.Based on the above background,we hypothetically assumed that in HCC treated with circ MBNL3 combined with Gem,the up-regulation of circ MBNL3 could down-regulate the level of intracellular free miR-19b-1-5p by adsorbing free miR-19b-1-5p.Thus,the inhibitory effect of miR-19b-1-5p on TCF-4 was weakened,and the expression of TCF-4 was up-regulated,which increased the sensitivity of HCC cells to chemotherapy.Therefore,this study studied the regulation and influence mechanism of circ MBNL3 in liver cancer,and further investigated whether the above mechanisms can improve the sensitivity of Gem chemotherapy,and looked for targets to increase the sensitivity of liver cancer cells to chemotherapy.This research is divided into four chapters:Chapter 1: Gem inhibits the proliferation of human hepatoma HepG2 cells Objective: To determine the killing concentration and working time of Gem on HepG2 hepatoma cellsMethods: Cell Counting Kit-8(CCK8)experiment was used to detect the inhibition of proliferation activity of HepG2 cells by Gem with different concentrations in different incubation periods,so as to calculate the proliferation rate and inhibitory rate of HepG2 cells and the inhibition of proliferation activity.Results: The inhibitory effect of Gem on the proliferation of HepG2 cells was dependent on drug concentration and incubation time.Conclusions: Gem can inhibit the proliferation of HepG2 cells,and cells were treated with 1.25mg/L Gem for 48 h in subsequent experiments.Chapter 2: Determining the binding of miR-19b-1-5p to circ MBNL3 and the binding of TCF4-3 'UTR terminal and further investigating whether circ MBNL3 regulates the expression of TCF4 by adsorbing miR-19b-5p as a ce RNAObjective: To clarify the targeting relationship between circ MBNL3 or TCF4 and miR-19b-1-5p.Methods: 1)Based on GEO database,bioinformatics and TCGA data analysis,it was predicted that miR-19b-1-5p could bind to circ MBNL3 and TCF4 might be its target gene.Luc-circ MBNL3-MUT,Luc-circ MBNL3-WT,Luc-TCF4-MUT,Luc-TCF4-WT and Luc-NC were constructed by the vector,and then the above vectors were detected by double luciferase;2)HepG2 was divided into four groups: si-NC,si-circ MBNL3,si-circ MBNL3 +anti-miR-NC and si-circ MBNL3 +anti-miR-19b-1-5p.Q-PCR and Western Blotting(WB)were used to detect the above groups.Results: 1)The interaction between miR-19b-1-5p and circ MBNL3 in HepG2 cells showed that the binding of miR-19b-1-5p with circ MBNL3 WT was statistically significant,but not with circ MBNL3 MUT.Studies on the target genes of miR-19b-1-5p in HepG2 cells showed that the overexpression of miR-19b-1-5p inhibited the expression of TCF4,and when the binding sites of miR-19b-1-5p on TCF4 m RNA 3 UTR were mutated,the expression of TCF4 was no longer inhibited by miR-19b-1-5p;2)circ MBNL3 silencing decreased the levels of TCF4 m RNA and protein in HepG2 cells,but this trend was partially reversed after miR-19b-1-5p inhibition.Conclusion: circ MBNL3 regulates the expression of TCF4 by sponging miR-19b-1-5p.The third chapterPart I: Determine whether circ MBNL3 regulates the sensitivity of miR-19b-1-5p to chemotherapyObjective: To demonstrate that circ MBNL3 enhances the sensitivity of chemotherapy by regulating miR-19b-1-5p through cell experiments.Methods: HepG2 cell lines overexpressing circ MBNL3 were constructed by plasmid,and the subjects were divided into 7 groups,namely HepG2 group.Gem+HepG2 group;Gem+empty vector(circ RNA overexpression and no load)group;Gem+NC(miRNA noload)group;Gem+circ MBNL3-OE(circ MBNL3 overexpression)group;Gem+miR-19b-1-5p inhibitor(miR-19b-1-5p inhibitor)group;Gem+circ MBNL3-OE+miR-19b-1-5p mimic(miR-19b-1-5p mimic)group;Then CCK-8 assay,clone formation and flowcytometry assay were used to detect the drug sensitivity of HepG2 in different groups to Gem,and the corresponding proliferation and inhibition rates,clone formation and cell cycle changes were calculated.Results: There were different changes in proliferation rate,inhibition rate,clone formation and cell cycle of HepG2 in different groups.Conclusions: circ MBNL3 can improve the sensitivity of Gem to HepG2 chemotherapy by negatively regulating miR-19b-1-5p.Part II: To determine the mechanism of circ MBNL3's increased chemotherapy sensitivity mediated by negative regulation of miR-19b-1-5pObjective: To determine the changes of circ MBNL3 related molecules and downstream pathway proteins mediated by miR-19b-1-5p chemosensitivity increase through cell experiments.Methods: TCF4,RB1 and CDK2 were detected in HepG2 cells of different groups by QPCR and WB.Results: 1)Compared with untreated HepG2 cells,the expression of RB1 and CDK2 were up-regulated in Gem treated HepG2 cells,which inhibited the cell cycle progression of cancer cells,inhibited the cell entering into S phase,and inhibited the cell line division and proliferation ability;2)In Gem-treated HCC cells,TCF4 was up-regulated to a certain extent,suggesting that TCF4 may be involved in the inhibition of cell cycle;3)circ MBNL3 overexpression combined with Gem treatment can significantly up-regulate the expression level of TCF4 and further up-regulate the expression level of RB1;4)Inhibition of miR-19b-1-5p or transfection of miR-19b-1-5p mimic could up-regulate and inhibit the expression level of TCF4,respectively.The regulation effect of miR-19b-1-5p on TCF4 m RNA translation was verified at the protein expression level.In the circ MBNL3 overexpression group,TCF4 protein expression level was also up-regulated.Conclusions: circ MBNL3 can act as a molecular sponge of miR-19b-1-5p,negatively regulate the expression of miR-19b-1-5p,and up-regulate the expression of TCF4 by miR-19b-1-5p,thus improving the sensitivity of Gem to HepG2 cells.Chapter 4: Evaluation of the effect of circ MBNL3 on Gem chemotherapy in HCC cells using a subcutaneous xenograft tumor model in nude miceObjective: To further verify the effect of circ MBNL3 on Gem chemotherapy in hepatoma cells by animal experiments and the mechanism of action.Methods: Nude mice were inoculated with human liver cancer cell line HepG2.After tumor formation,different cell groups(experimental group,control group and blank group)in the first part of Chapter 3 were injected.The length diameter and short diameter of the tumor body were measured with vernier calipers every 7 days,and the tumor volume was calculated: V= A * B2/2.After 28 days,the nude mice were sacrificed,the tumor tissues were stripped,and the tumor tissues were stained by HE and immunohistochemistry.The tumor cells were detected by Q-PCR and WB for TCF4,RB1 and CDK2.Results: Based on the monitoring data of tumor body,Gem had a certain inhibitory effect on tumor growth(no matter in terms of volume or mass).Overexpression of circ MBNL3 further enhanced the inhibitory effect of Gem,and miRNA inhibitor showed the same synergistic inhibitory effect on Gem as circ MBNL3.However,miRNA mimics can reverse the tumor suppressants of circ MBNL3,which can restore the tumor volume growth and mass proliferation to a certain extent.Conclusion: Animal experiments further suggested that circ MBNL3 could enhance the sensitivity of Gem to chemotherapy in liver cancer cells,and verified that circ MBNL3 could act as a molecular sponge of miR-19b-1-5p to interfere with miR-19b-1-5p expression and up-regulate the expression of TCF4 after transcription.These results provide a theoretical basis for circ MBNL3 to enhance HCC chemotherapy on Gem.