Association Of Growth Hormone And Polymorphisms Of Long Non-Coding RNA With Digestive System Cancer

Digestive system tumor is a kind of the most common malignancies in the world.The number of new and dead patients accounts for one quarter and one third of all malignancies.In recent years,the number of digestive system tumors in China has increased every year.The characteristics of such tumors include occult onset,high malignancy,rapid invasion and metastasis,and poor survival time,which have brought a heavy economic and social burden to China and are major public health problems that cannot be ignored.Based on the development status of digestive system tumors and the severe situation of diagnosis and treatment,it is urgent to deeply study its internal molecular mechanism,so as to identify high-risk groups,find new specific early diagnostic markers and effective therapeutic targets for digestive system tumors,and derive relevant molecular targeted drugs,which has far-reaching significance for clinical effective diagnosis and treatment.It is found that the aberrant expression of specific long non-coding RNA(lncRNA)is closely related to the diagnosis or prognosis of patients with digestive system tumors,and participates in targeted therapy and immunotherapy,which may promote the development of prevention strategies and advanced therapies.LncRNA-related single nucleotide polymorphisms play an important role in interfering with the function of lncRNA regulatory genes,which can influence the expression level and biological function of lncRNA.These genes participate in important signaling pathways and carcinogenesis.Recent studies have shown that individual and/or combined genotypes of lncRNA-related polymorphisms can predict cancer risk,clinical and therapeutic outcomes.In recent years,immune checkpoint inhibitors(ICIs)have made breakthroughs in the treatment of digestive system tumors such as esophageal cancer,hepatocellular carcinoma,gastric cancer and colorectal cancer,and have gradually shown good efficacy and significantly prolonged the prognosis of tumor patients,and completely changed the treatment pattern of digestive system tumors.However,the objective response rate of such tumor patients to ICIs is only 10%-20%,which highlights the need to develop reliable biomarkers to screen the beneficiaries.This study is divided into the following three parts:Part One Growth hormone associated with treatment efficacy of immune checkpoint inhibitors in gastric cancer patientsObjective:Immune checkpoint inhibitors(ICIs)combined with chemotherapy has been widely employed to improve the outcome of advanced gastric cancer(AGC).In present study,the impact of post-treatment growth hormone(GH)levels on the treatment efficacy of ICIs for AGC patients was assessed.Methods:1.Seventy five AGC patients treated with anti-PD-1 antibodies at The Fourth Hospital of Hebei Medical University from January 1,2019 to April 30,2022 were involved.2.We divided AGC patients into two groups as high GH group and low GH group based on GH level.Immunotherapy efficacy was assessed in terms of progression-free survival(PFS),overall survival(OS),objective response rate(ORR)and disease control rate(DCR)based on the National Comprehensive Cancer Network Guidelines.3.The enumeration data was compared byχ~2 test.Survival curves were plotted using Kaplan-Meier method,and comparisons between the curves were made using the Log-Rank test.The COX proportional risk model was used for multivariate survival analysis.P<0.05 was considered statistically significant.Results:1.The higher GH levels were associated with lower DCR of ICIs with DCR of 30.0%in high GH group and 53.3%in low GH group(P=0.046).2.The subsequent univariate analysis showed that high GH was associated with both shorter PFS(P=0.016)and shorter OS at the borderline statistical level(P=0.052)in AGC patients treated with ICIs.COX model analysis also proved that the GH level was an independent risk factor for the outcome of AGC patients(PFS:P=0.013,HR,2.424,95%CI,1.202-4.890;OS:P=0.014,HR,3.301,95%CI,1.279-8.519).Conclusions:The post-treatment GH level might be a predictor for ICIs treatment in AGC patients.Part Two The rs6983267 modifies the outcome of hepatocellular carcinoma through mediating CCAT2 expressionObjective:The occurrence and development of hepatocellular carcinoma(HCC)is a complex process involving multiple genes and factors,in which genetic factors play an important role,but the specific molecular biological mechanism has not been clarified.Single nucleotide polymorphisms(SNPs)of long non-coding RNA(lncRNA)involved in various cellular processes including carcinogenesis through mediating the expression of targeting genes.In this study,we evaluated predictive value of SNPs from three cancer associated lncRNA on HCC development.Methods:1.LncRNA SNPs were genotyped using PCR,and the lncRNA expressions were measured by real time quantitative PCR.2.Cell counting kit-8 assay,wound-healing assay and flow cytometry assay were used to measure the proliferation,metastasis and apoptosis of HCC.3.Western blot was used to detect the expression of associated proteins downstream of lncRNA.4.Student’s t-test andχ~2 test were used to analysis measurement data and enumeration data,respectively.The univariate analysis was performed by Kaplan-Meier method and Log-Rank method,the multifactor analysis was done by COX regression model.P<0.05 was considered statistically significant.Results:1.The rs6983267 SNP of colon cancer associated transcript 2(CCAT2)was associated with both cancer risk and overall survival in HCC patients.The GG+GT genotype of rs6983267 increased the risk and shortened the lifespan of HCC by compared with those of TT genotype.2.The GG+GT genotype of rs6983267 produced more CCAT2transcripts than that of the TT genotype.3.The subsequent functional analysis indicated that CCAT2 knockdown could inhibit proliferation and migration as well as promote apoptosis of HCC cells in vitro.4.The MYC under downstream of CCAT2 was also downregulated upon CCAT2 knockdown.Conclusions:Our data demonstrated that the rs6983267 SNP might modify the HCC outcome through mediating the expression of CCAT2 and its downstream target MYC,CCAT2 would be a new target for hepatocellular carcinoma therapy.Part Three Association between single nucleotide polymorphism of longnon-coding RNA PRNCR1 and colorectal cancerObjective:Colorectal cancer(CRC)is a multi-step and multifactorial malignant disease.Although previous studies have found that genetic factors are closely related to carcinogenesis and progression of CRC,the specific molecular mechanism has not been clarified.LncRNA-SNPs can participate in many biological processes of tumorigenesis and development by mediating the expression of target genes.In this part of the study,we evaluated the predictive value and potential biological function of three cancer-related lncRNA-SNPs for CRC.Methods:1.The HCT116 cell line,a colon cancer cell line,was cultured.The expression level of PRNCR1 in HCT116 cells was reduced by shRNA interference technology.2.The lncRNA-SNPs were genotyped by PCR,and the expression level of lncRNA was detected by real-time quantitative PCR.3.Proliferation,migration rate,and apoptosis of HCT116 cells were determined by Cell Counting Kit-8,wound healing assay,and flow cytometry assay,respectively.4.Western blot was used to detect the expression of downstream related proteins of lncRNA.5.Student’s t-test andχ~2 test were used to analysis measurement data and enumeration data.Kaplan-Meier method and Log-Rank method were used for univariate analysis,and COX regression model was used for multiple factor analysis.P<0.05 was considered statistically significant.Results:1.The rs7463708 SNP of prostate cancer non-coding RNA1(PRNCR1)was associated with the risk in CRC patients(P=0.038).Compared with the GT+TT genotype of rs7463708,the GG genotype increased the cancer risk of CRC.2.The rs7463708 SNP was not correlated with the expression level of lncRNA PRNCR1.3.Subsequent functional analysis showed that PRNCR1 knockdown could inhibit the proliferation and migration,and promote its apoptosis of HCT116 cells in vitro.4.Western blot indicated that the expression of ONECUT2 in HCT116cells silenced by PRNCR1 was significantly reduced.Conclusions:The rs7463708 SNP may influence the occurrence and development of CRC by mediating the structure and function of PRNCR1 and the expression of its downstream target ONECUT2.The rs7463708 SNP of PRNCR1 gene would be a new diagnostic marker and a potential target for anti-tumor therapy.