Effects And Molecular Mechanism Of PLOD2 On 5-FU Resistance In Gastric Cancer Cells

Background: Gastric cancer(GC)is one of the most common cancers.5-Fluorouracil(5-FU)is a commonly used drug for the treatment of advanced GC.5-FU is often used in combination with cisplatin and daunorubicin to increase the therapeutic effect.However,due to the resistance of tumors,a large proportion of patients will eventually fail treatment.At present,more and more evidence shows that tumor invasion and metastasis not only depend on the habit of tumor cells,but also have a close relationship with tumor microenvironment(TME).Extracellular matrix(ECM)is an important component of TME and plays an important role in tumor progression.Collagen is one of the main components of ECM and is considered to be the most abundant scaffold protein in ECM.Hydroxylysine residues provide attachment sites for carbohydrates and provide tensile strength and mechanical stability for collagen fibers.Lysine hydroxylase 2(LH2)/ Procollagenlysine 2-xoglutarate 5-dioxygenase 2(PLOD2),a protein encoded by the PLOD2 gene,is a key enzyme that mediates the formation of stable collagen cross-links.PLOD2 is reported to increase the invasion and migration of several GC cell lines.However,the role of PLOD2 in drug resistance in GC remains unclear.This study aimed to determine whether PLOD2 can affect 5-FU resistancet in GC.Methods: Western blot was used to detect PLOD2 protein expression levels in MKN45,MKN28,MGC803,SGC7901,HGC27,and BGC823,and in normal human gastric mucosal cell line GES-1.Construction of PLOD2 overexpression plasmid and PLOD2 short hairpin interference RNA(sh RNA).Lentiviral transfection technology was used to construct stable transfected cell lines and control groups in MGC803 cells with relatively low expression of PLOD2 and BGC823 cells with relatively high expression of PLOD2,named OE-PLOD2-MGC803,OE-Control-MGC803,sh PLOD2-BGC823,sh Control-BGC823.The fluorescence efficiency of transfection was detected by FACS,and the protein level and m RNA expression level of PLOD2 in the above cells were detected by Western blot and RT-q PCR.CCK-8 proliferation test to detect the effect of 5-FU on cell proliferation;Transwell cell experiment to verify the change of 5-FU on gastric cancer cell migration and invasion ability;Flow cytometry to detect the effect of 5-FU on apoptosis.A xenograft nude mouse model was established.The tumor growth curve and histological observation were used to analyze the change of 5-FU inhibition of tumor growth after PLOD2 knockdown.Immunohistochemistry was used to verify the expression of PLOD2 in tumor tissues of nude mice.The protein levels of P-gp(MDR1),MRP1,BCRP(ABCG2),Bax and Bcl2 were analyzed by Western blot to explore the mechanism by which PLOD2 affects resistance of 5-FU to chemotherapy in GC.Result: The expression protein level and m RNA level of PLOD2 in OE-PLOD2-MGC803 cells were significantly higher than those of NC-MGC803 and OE-Control-MGC803;The expression protein level and m RNA level of PLOD2 in sh PLOD2-BGC823 cells were significantly lower than those of NC-BGC823 and sh Control-BGC823(P < 0.05).The IC50 of 5-FU on OE-PLOD2-MGC803 cells is higher than that of NC-MGC803 and OE-Control-MGC803,while the IC50 of 5-FU on sh PLOD2-BGC823 cells is lower than that of NC-BGC823 and sh Control-BGC823(P < 0.05).With or without 5-FU,OE-PLOD2-MGC803 cells had higher migration and invasion ability than NC-MGC803 and OE-Control-MGC803;sh PLOD2-BGC823 cells had lower migration and invasion ability than NC-BGC823 and sh Control-BGC823(P < 0.05).5-FU-induced apoptosis of OE-PLOD2-MGC803 cells is significantly lower than NC-MGC803 and OE-Control-MGC803;5-FU-induced sh PLOD2-BGC823 apoptosis rate is significantly higher than NC-BGC823 and sh Control-BGC823(P < 0.05).In vivo experiments,5-FU inhibited tumor growth in the sh PLOD2-BGC823 group more significantly.Our mechanism studies show that cells overexpressing PLOD2 resist the 5-FU therapeutic effect in GC cells by up-regulating BCRP,and that PLOD2 affects 5-FU-induced apoptosis of GC cells by affecting Bax and Bcl2 protein expression.Conclusion: PLOD2 contributed to increasing resistance of gastric cancer cells to 5-fluorouracil by upregulating BCRP and inhibiting apoptosis.