Studies Of Clinicopathological Factors And Long Noncoding RNA HNF1A-AS1 Affecting The Sensitivity Of Neoadjuvant Chemotherapy In Gastric Cancer

Part 1 Clinicopathological factors affecting the effect of neoadjuvant chemotherapy in patients with gastric cancerBackground and objective:Neoadjuvant chemotherapy is an important part of comprehensive treatment for advanced gastric cancer(GC).The effect of neoadjuvant chemotherapy is crucial to the prognosis of GC patients.The degree of pathological response of tumor tissue after neoadjuvant chemotherapy can represent the effect of neoadjuvant chemotherapy.However,studies about the correlation between clinicopathological factors and the effect of neoadjuvant chemotherapy in GC are rarely reported.In this part,through retrospective research,we first intend to compare the efficacy between neoadjuvant chemotherapy plus surgery plus adjuvant chemotherapy and surgery plus adjuvant chemotherapy to clarify the effect of neoadjuvant chemotherapy.Further,we analyze the clinicopathological factors that affect the effect of neoadjuvant chemotherapy in GC patients and make suggestions for improving the effect of neoadjuvant chemotherapy of GC.Methods:Firstly,the effects of neoadjuvant chemotherapy plus surgery plus adjuvant chemotherapy and surgery plus adjuvant chemotherapy on the prognosis of patients with advanced GC from 2012 to 2016 in Peking Union Medical College Hospital were retrospectively analyzed.In addition,GC patients who underwent radical surgery after neoadjuvant chemotherapy with SOX regimen in Peking Union Medical College Hospital from 2016 to 2019 were retrospectively analyzed and the clinicopathological data of the patients were collected and their influences on the effect of neoadjuvant chemotherapy were analyzed.The effect of neoadjuvant chemotherapy was judged by the College of American Pathologists(CAP)grading.Chi-square test or t-test was used for univariate analysis.Logistic regression was used for multivariate analysis.Receiver operating characteristic curve(ROC)was used to determine the cut-off value of variables which significantly influenced the effect of neoadjuvant chemotherapy.Results:In the comparison of neoadjuvant chemotherapy plus surgery plus adjuvant chemotherapy and surgery plus adjuvant chemotherapy,218 and 232 patients with advanced GC were included in the two groups respectively.The results showed that the neoadjuvant chemotherapy group had higher R0 resection rate(P=0.014),lower postoperative pathological T stage(P=0.002)and lymph node metastasis rate(P<0.0001),and improved the 5-year overall survival rate of GC patients(59.6%vs 50.9%).In addition,in the analysis of clinicopathological factors affecting the effect of neoadjuvant chemotherapy in GC,203 patients with advanced GC were included.Univariate and multivariate analysis showed that patients<60 years(OR=1.840[1.016-3.332],P=0.044),histological type of poor differentiated adenocarcinoma or signet ring cell carcinoma(OR=2.606[1.321-5.140],P=0.006),and weight loss during neoadjuvant chemotherapy(OR=2.110[1.161-3.834],P=0.014)had poorer neoadjuvant chemotherapy effect.They were independent risk factors affecting the effect of neoadjuvant chemotherapy in GC patients.The ROC analysis of weight change and neoadjuvant chemotherapy effect showed that the area under the curve(AUC)was 0.593(P=0.024),and the cut-off value of weight change was-2.95%,indicating that patients with weight loss>2.95%during neoadjuvant chemotherapy had worse chemotherapy effect.Chi-square test showed that patients without weight loss during neoadjuvant chemotherapy had a higher proportion of oral nutritional supplement(ONS)than patients with weight loss(82.3%vs 70%,P=0.039).Conclusions:Neoadjuvant chemotherapy plus surgery plus adjuvant chemotherapy can improve the prognosis of patients with advanced GC.Patients<60 years old,histological type of poor differentiated adenocarcinoma or signet ring cell carcinoma,and weight loss during neoadjuvant chemotherapy are independent risk factors affecting the effect of neoadjuvant chemotherapy in GC patients.Patients with weight loss>2.95%during neoadjuvant may have a worse chemotherapy effect.Timely nutritional support such as ONS to maintain the patient’s weight plays an important role in improving the effect of neoadjuvant chemotherapy.Part 2 The expression of HNF1A-AS1 in human gastric cancer tissues and cells and its relationship with chemotherapy effectBackground and objective:GC is one of the leading causes of cancer-related deaths worldwide,and chemoresistance is an important reason for the poor prognosis of GC patients.Studies have found that long noncoding RNA(lncRNA)plays an important regulatory role in drug resistance.In this section,we will explore the expression of IncRNA HNF1A-AS1 in GC tissues and GC cells,and further analyze the expression of HNF1AAS1 in 5-fluorouracil(5-FU)sensitive tissues and 5-FU resistant tissues.The possible association between HNF1A-AS1 and 5-FU resistance in GC is sought to lay the foundation for the follow-up research.Methods:GC tissues were collected from GC patients by gastroscopic biopsy before neoadjuvant chemotherapy,and normal gastric mucosal tissues were collected from healthy subjects by gastroscopic biopsy.GC cell lines used in the study were HGC-27,MKN-45,AGS and NCI-N87 and the normal gastric mucosa cell line was GES-1.The neoadjuvant chemotherapy regimen is 5-FU based SOX regimen.The neoadjuvant chemotherapy effect of GC patients was judged by pathological CAP grading.Real-Time PCR was used to detect the expression level of HNF1A-AS1 in GC tissues and GC cells.Differences of HNF1A-AS1 expression between different groups were compared using ttest or one-way analysis of variance.Results:The expression level of HNF1A-AS1 in GC tissue was significantly higher than that in normal gastric mucosa tissues,and the expression level of HNF1A-AS1 in 5-FU resistant GC tissues was significantly higher than that in 5-FU sensitive GC tissues.The expression level of HNF1A-AS1 in the four GC cell lines were significantly higher than that in the normal GC cell line GES-1,and the expression level of HNF1A-AS1 was highest in MKN-45 and lowest in HGC-27.Conclusions:HNF1A-AS1 was significantly overexpressed in GC tissues and GC cells,and was more elevated in 5-FU resistant GC tissues,suggesting that HNF1A-AS1 may be associated with 5-FU resistance of GC.Part 3 Functional researches of HNF1A-AS1-induced 5-FU resistance in gastric cancer cellsBackground and objective:5-FU-based chemotherapy is an important part of GC chemotherapy.5-FU resistance is an unfavorable factor affecting the prognosis of GC patients.More and more studies have found that lncRNA plays an important regulatory role in 5-FU resistance.Studies from part 2 have shown that lncRNA HNF1A-AS1 may promote 5-FU resistance of GC.In this part,we will explore the effect of HNF1A-AS1 on 5-FU resistance of GC cells through a series of in vitro and in vivo functional experiments.Methods:Lentiviral infection was used to construct HNF1A-AS1 knockdown stable cell lines(sh-HNF1A-AS1)in MKN-45 cells,and HNF1A-AS1 overexpressing stable cell lines(LV-HNF1A-AS1)was constructed in HGC-27 cells.The effect of HNF1A-AS1 on proliferation of GC cells treated with 5-FU was detected by Cell Counting Kit-8(CCK-8)assays and plate cloning assays.The effect of HNF1A-AS1 on apoptosis of GC cells treated with 5-FU was detected by flow cytometry.Nude mice xenograft experiments were performed to verify the effect of HNF1A-AS1 on tumor formation treated with 5-FU.Results:CCK-8 assays and plate cloning assays showed that knockdown of HNF1A-AS1 inhibited the proliferation of MNK-45 stable cell lines under 5-FU treatment.On the contrary,overexpression of HNF1A-AS1 promoted the proliferation of HGC-27 stable cell lines under 5-FU treatment.Flow cytometry showed that knockdown of HNF1A-AS1 promoted the apoptosis of MNK-45 stable cell lines under 5-FU treatment.Inversely,overexpression of HNF1A-AS1 inhibited the apoptosis of HGC-27 stable cell lines under 5-FU treatment.Nude mice xenograft experiments showed that knockdown of HNF1AAS1 inhibited the tumorigenic ability of MNK-45 stable cell lines with the treatment of 5FU.On the contrary,overexpression of HNF1A-AS1 promoted the tumorigenic ability of MNK-45 stable cell lines with the treatment of 5-FU.Conclusions:In vitro and in vivo functional studies showed that high expression of HNF1A-AS1 could promote the proliferation,inhibit the apoptosis and promote xenograft tumors growth in the presence of 5-FU,indicating that HNF1A-AS1 reduces the sensitivity of gastric cancer cells to 5-FU and promotes drug resistance.Part 4 Mechanism researches of HNF1A-AS1-induced 5-FU resistance in gastric cancer cellsBackground and objective:After the studies of part 2 and part 3,we found that HNF1AAS1 could induce 5-FU resistance of GC.However,the mechanism about how HNF1AAS1 works has not been reported.In this section,we will explore the specific molecular mechanism of HNF1A-AS1-induced 5-FU resistance,and find potential therapeutic targets for overcoming 5-FU resistance in GC.Methods:The competitive endogenous RNA(ceRNA)mechanisms among HNF1A-AS1,miR-30b-5p,and EIF5A2 were explored through dual-luciferase reporter gene assays and RNA immunoprecipitation(RIP)assays;Rescue experiments were performed by salvaging the expression of miR-30b-5p to explore the effect on proliferation,apoptosis and proteins levels in stable GC cell lines with knockdown or overexpression of HNF1A-AS 1;Western Blot and immunohistochemical(IHC)staining were used to detect the expression levels of EIF5A2,E-Cadherin,Vimentin,N-Cadherin in GC cells and nude mice xenografts.Results:Luciferase reporter gene assays confirmed the binding effect between HNF1AAS1 and miR-30b-5p,also miR-30b-5p and EIF5A2.RIP assays further demonstrated the binding of HNF1A-AS1 and miR-30b-5p.Rescue experiments indicated that salvaging the expression of miR-30b-5p could reverse the effects of HNF1A-AS1 on proliferation,apoptosis and 5-FU resistance in GC cells.Western Blot and IHC suggested that HNF1AAS1 promoted epithelial-mesenchymal transition(EMT)by upregulating the expression of EIF5A2.Western Blot showed that EMT was reversed after salvaging the expression of miR-30b-5p.Conclusions:HNF1A-AS1 promotes EIF5A2-mediated EMT process by competitively binding to miR-30b-5p,thereby inducing the resistance to 5-FU in GC cells.Our studies suggest that HNF1A-AS1/miR-3 0b-5p/EIF5A2/EMT pathway plays an important regulatory role in 5-FU resistance of GC,and HNF1A-AS1 may become a therapeutic target to reverse 5-FU resistance in GC.