The Role Of ANXA2 And Related MiRNAs In The Occurrence And Development Of Colorectal Cancer And Their Relationship With Targeted Therapy For Liver Cancer

Background and Purpose:Colorectal cancer(CRC)and Hepatocellular cancer(HCC)are common malignant tumors in the world,and their morbidity and mortality are high in China.Annexin A2(ANXA2)is upregulated in various tumor tissues and promotes the malignant development of tumors such as proliferation,invasion and metastasis of cancer cells.The study found that ANXA2 is also upregulated in CRC and HCC.ANXA2 may be one of the key gene expression alienation events during the CRC progression.There may be an ANXA2 signaling pathway.ANXA2 is upregulated in 29.5%of the investigated CRC cases and is closely related to tumor growth and metastasis.Activation of ANXA2 is the basis of its signal transduction.The reference indicates that the important functional domain of ANXA2 activation is located at its N-terminus.However,the mechanism of key N-terminal amino acid sites in oncogenesis and progression is rarely reported.Further research on the key N-terminal sites of ANXA2 is of great significance for understanding the regulation of its signaling pathway and its relationship with oncogenesis and progression.Based on the previously constructed ANXA2 human colorectal cancer knockout cell line(ANXA2-/-caco2),we constructed the single-point and multi-point mutant expression vectors of its key N-terminal sites using the Site directed mutation and Multiple sites directed mutation.The expression vectors were expressed in ANXA2-/-caco2 cells to explore the regulatory role of key ANXA2 N-terminal sites mutation in the malignant phenotypes and behaviors of cells and to evaluate the relationship between the molecular structure of ANXA2 and the malignancy of tumor cells.The miRNAs that may have a targeted relationship with ANXA2 were predicted and 3 miRNAs(miR-1-3p,miR-206 and miR-613)that bind to the ANXA2 3’UTR with the highest score were selected.We demonstrated that miR-206 had the best targeting relationship with ANXA2 by the dual luciferase reporter assay and further studied the regulation of miR-206 on the malignant phenotypes and behaviors of caco2 cells.The incidence of HCC is concealed,the surgical cure rate is low and the chemotherapy effect and prognosis are not ideal.The currently widely used liposome doxorubicin(Lipo-DOX)is a commonly used broad-spectrum anticancer first-line chemotherapeutic drugs,but Lipo-DOX lacks targeting specificity and the toxic side effects are still large,accompanied by rapid formation of Mutiple drug resistance(MDR).Therefore,the current clinical chemotherapy of tumors faces two major bottlenecks:the lack of targeting specificity and the formation of MDR of drugs.The two bottlenecks have led to poor cancer chemotherapy,high side effects,recurrence and metastasis rates and high mortality.It is extremely urgent to develop new anticancer drugs with sufficient tumor targeting specificity and MDR inhibition or even reverse.The 12-peptide HCSP4 which targeted to HCC with specificity and sensitivity has good HCC cell/tissue binding specificity and sensitivity.It is found that miR-101 is extremely low in HCC and may target ANXA2 and MDR-related genes by the references and database analysis.It is speculated that miR-101 has the potential to inhibit MDR against chemotherapeutic drugs.Based on the above-mentioned research on the regulation of ANXA2 signaling pathway in the oncogenesis and progression of CRC,HCSP4-Lipo-DOX-miR101 liver cancer targeted drug delivery system was prepared by using HCSP4 and miR-101 as drug targeting and MDR inhibitory elements.The targeting specificity and anti-MDR effects of the drug delivery system were studied in vitro(the other postgraduate research in our lab)and in vivo.The possible role of ANXA2 gene expression in the in vivo treatment of the drug delivery system was analyzed.Methods:1.Analyze the variation of ANXA2 in CRC and HCC clinical samples and its role in clinical development with databases such as cBio Cancer Genomics Portal and GEPIA.2.Using colon cancer and liver cancer clinical tissues and tissue chips as materials,the expression level of ANXA2 was studied by immunofluorescence.3.Design and construct the single-point,multi-point mutant expression vectors of ANXA2 key N-terminal sites and transfect ANXA2-/-caco2 cells.4.ANXA2+/+caco2,ANXA2-/-caco2 and ANXA2-/-caco2 transfected with NM plasmids as control groups,ANXA2-/-caco2 transfected with S1,S11,S25,Y23,S1-Y23,S11-Y23 and S25-Y23 as the experimental groups,the methods of MTT,wound healing,Transwell,flow cytometry and immunofluorescence staining were used to detect the effects of ANXA2 key N-terminal sites on caco2 cell proliferation,cell cycle,motility,apoptosis,overall cell skeleton and motility assosiated with microstructures.5.Predict miRNAs that have the targeting relationships with ANXA2 by the authoritative websites.The targeting relationships were confirmed by the dual luciferase reporter assay.6.The miR-206 expression vector was transfected into wild-type caco2 cells and the inhibition rate of ANXA2,the related behavior and morphology of tumor cells and the expression level of EMT-related genes were detected.7.The miR-101 expression vector was designed and constructed by the analysis of ANXA2 and MDR-related gene targeting relationships.8.The liposomes were prepared by thin film ultrasonic dispersion method.The drug was loaded by ammonium sulfate pH gradient method.miR-101 were linked to liposomes by the positive polarity of cationic liposomes interacting with negatively charged DNA.HCSP4-Lipo-DOX-miR101 was obtained.9.The characteristics and drug encapsulation efficiency of HCSP4-Lipo-DOX-miR101 were detected by fluorescence spectrophotometer,laser particle size analyzer,scanning electron microscope and transmission electron microscope.10.Optimize the transfection efficiency of the HCSP4-Lipo-DOX-miR101 and evaluate the targeting specificity.11.Establish the nude mouse model of HCC xenografts and treat them with HCSP4-Lipo-DOX and HCSP4-Lipo-DOX-miR101(tail vein injection).The therapeutic effect of HCSP4-Lipo-DOX-miR101 in vivo was comprehensively evaluated from the aspects of tumor growth curve,tumor volume,tumor weight and lung metastasis.Results:1.The expression of ANXA2 was highly correlated with the degree of colon cancer malignancy,especially upregulated in the poorly differentiated adenocarcinoma at the T3N0Mx stage,which was highly consistent with the results of the database analysis.2.The expression of ANXA2 was highly correlated with the degree of liver cancer malignancy,especially upregulated in the moderate-poorly differentiated hepatocellular carcinoma,which was highly consistent with the results of the database analysis.3.The activation levels of ANXA2 can be differentially inhibited by the mutation of key ANXA2 N-terminal sites.4.The malignant behaviors of caco2 cells can be differentially inhibited by the mutation of key ANXA2 N-terminal sites.5.Motility-associated microstructures of caco2 cells can be remodeled by the mutation of key ANXA2 N-terminal sites.6.miR-206,miR-1-3p and miR-613 can target ANXA2.miR-206 had the best targeting relationship.7.miR-206 negatively regulated ANXA2 expression by targeting ANXA2.8.The forced expression of miR-206 inhibited the proliferation and migration of caco2 cells,promoted apoptosis and inhibited tumor EMT in vitro.9.HCSP4-Lipo-DOX-miR101 liver cancer targeted drug delivery system was successfully constructed with good characteristics.10.The transfection efficiency of HCSP4-Lipo-DOX-miR101 was high.The system can target to HepG2 cells and inhibit the expression of ANXA2.11.HCSP4-Lipo-DOX-miR101 inhibited the growth of xenografts in nude mice,especially in the resistant cell line HepG2/ADR,and inhibited the expression of ANXA2 in xenografts.Conclusions:1.The expression of ANXA2 is highly correlated with the degree of colon cancer and liver cancer malignancy.2.The expression of ANXA2 and its 4 key N-terminal sites,especially Y23,are essential for maintaining the malignant phenotypes of caco2 cells.3.miR-206 targets ANXA2 and negatively regulates its expression.The forced expression of miR-206 inhibits the malignant phenotypes of caco2 cells in vitro.4.HCSP4-Lipo-DOX-miR101 has obvious HCC targeted therapeutic effects in vivo and MDR inhibitory property.Its mechanism is closely related to the inhibition of ANXA2 expression by miR-101.