| Background and ObjectivesColorectal cancer(CRC)is one of the most common malignant tumors.Its morbidity and mortality rank the 3rd and 2nd in all malignant tumors,respectively,and it shows an increasing trend year by year in China.Lynch syndrome is the most common hereditary colorectal cancer syndrome,which is caused by a genetic mutation of mismatch repair genes.It often presents with High Microsatellite Instability(MSI-H)and loss of mismatch repair function(DMMR deficiency).A large number of studies have confirmed that solid tumor patients with MSI-H/DMMR status can benefit significantly from immunotherapy represented by PD-1/PD-L1 inhibitors,but they also face the challenge of drug resistance.Ras gene mutation has been proved to be able to up-regulate the expression of PD-L1 in tumor cells and act on PD-1,enhance the stability of mRNA and enable cancer cells to have a stronger ability of immunosuppression.Therefore,between the PD-1 treatment and the RAS mutant subtype of Lynch syndrome with both DMMR and RAS mutation characteristics,whether different categories of non-coding RNAs can play a role in the related drug resistance mechanism will be the focus of this study.Based on the analysis of transcriptome characteristics,clinicopathology and prognosis of Lynch syndrome patients,we will analyze and excavate the signaling pathways and target genes associated with RAS mutation subtypes of Lynch syndrome and PD-1 drug resistance by taking the pathways and target genes associated with RAS mutation subtypes of Lynch syndrome and PD-1 as entry points.To further elucidate the potential molecular mechanism between RAS mutant subtypes of Lynch syndrome and PD-1 drug resistance,in order to find molecules that can be used to improve the immunotherapy resistance of Lynch syndrome,which will contribute to the formulation of individualized immunotherapy methods for solid tumors of MSI-H/DMMR,and provide a theoretical basis for the response strategy of immunotherapy resistance.Methods2.1 Characterization of lncRNA transcriptome in Lynch syndromeAfter matching the clinical baseline data,3 cases of Lynch syndrome were selected as experimental group,6 cases of sporadic colorectal cancer were selected as the positive control group and the distal normal intestinal mucosal tissues adjacent to the cancer were selected as the negative control group.The expression of lncRNA in the tissues was detected by high-throughput sequencing,and the differentially expressed lncRNA in the tissues were screened.Functional annotation and enrichment analysis of the screened target genes with differentially expressed lncRNA were conducted to comprehensively and systematically analyze the transcriptome characteristics of lncRNA in Lynch syndrome.Combined with transcriptional characteristics,lncRNA target genes and related pathways related to RAS gene mutation and PD-1 resistance were mined.2.2 Characterization of circRNA transcriptome in Lynch syndromeMethods The transcriptome characteristics of circRNA in Lynch syndrome were analyzed comprehensively and systematically,and the circRNA target genes and related pathways related to RAS mutation and PD-1 resistance were excavated.2.3 Characterization of miRNA transcriptome in Lynch syndromeMethods The transcriptome characteristics of miRNA in Lynch syndrome were analyzed comprehensively and systematically as before,and the miRNA target genes and related pathways related to RAS mutation and PD-1 resistance were mined.2.4 Characterization of mRNA transcriptome in Lynch syndromeMethods The mRNA transcriptome characteristics of Lynch syndrome were analyzed comprehensively and systematically as before,and the mRNA target genes and related pathways related to RAS gene mutation and PD-1 resistance were mined.2.5 Clinicopathological analysis of mutant subtypes of RAS in Lynch syndromeThe clinicopathological factors and prognosis of Lynch syndrome patients with RAS mutant subtypes were retrospectively analyzed.Bioinformatics analysis was used to mine target genes linking PD-1 and RAS signaling pathways,and combined with the results of transcriptome characteristics analysis of Lynch syndrome patients in the previous period,the pathways and target genes related to the drug resistance mechanism of PD-1 of Lynch syndrome RAS mutant subtype were searched.3.1 Characterization of lncRNA transcriptome in Lynch syndromeLncRNA accounted for 41%of the total RNA expression,and was mainly expressed on chromosome 14 in Lynch syndrome.Compared with normal intestinal mucosal tissue,1178 up-regulated and 2242 down-regulated lncRNA were screened for Lynch syndrome,and 1749 and 1,961 up-regulated lncRNAs and 1734 and 1,901 down-regulated lncRNA were screened for sportive colorectal cancer in the two groups,respectively.Through trend analysis,it was found that 24 signaling pathways were only regulated by lncRNA with specific differential expression in Lynch syndrome,and 11 signaling pathways were regulated by lncRNA with specific differential expression in Lynch syndrome and sportive colorectal cancer,among which apoptosis pathway and HTLV-1 infection pathway ranked in the top 10 of significance,respectively.In the analysis of lncRNA related to RAS mutation and PD-1 resistance,it was found that the MAPK signaling pathway enriched by differentially expressed lncRNA and the proteoglycan pathway in cancer were related to one of the target genes of PD-1 and RAS signaling pathway.3.2 Characterization of circRNA transcriptome in Lynch syndromeCircRNA accounts for 4%of the total RNA expression,and is mainly expressed on chromosome 6 in Lynch syndrome.Compared with normal intestinal mucosal tissue,112 circRNA were up-regulated and 324 circRNA were down-regulated in Lynch syndrome,149 circRNA were up-regulated and 273 and 261 circRNA were down-regulated in sporadic colorectal cancer in both groups.Two signaling pathways,namely RAP1 signaling pathway and Fanconi anemia,were regulated by differential expression of circRNA specific to Lynch syndrome by trend analysis.Four signaling pathways are regulated by circRNAs that are differentially expressed in common between Lynch syndrome and sporadic colorectal cancer.The analysis of circRNA related to RAS gene mutation and PD-1 drug resistance revealed that the PI3K-Akt signaling pathway and VEGF signaling pathway had statistical differences among the different circRNA-enriched signaling pathways in all cancer tissues,among which the target genes of PI3K-Akt signaling pathway were ITGA7 and PTK2.The target gene of VEGF signaling pathway is PTK2.3.3 Characterization of miRNA transcriptome in Lynch syndromeMiRNA accounts for 4%of the total RNA expression,and is mainly expressed on chromosome 7 in Lynch syndrome.Compared with normal intestinal mucosal tissue,143 up-regulated and 147 down-regulated miRNAs were screened in Lynch syndrome,90 and 100 up-regulated miRNAs and 95 and 90 down-regulated miRNAs in sporadic colorectal cancer in the two groups,respectively.Through trend analysis,four signaling pathways were regulated by miRNAs that were uniquely differentially expressed in Lynch syndrome.Forty-six signaling pathways were regulated by differentially expressed miRNAs common to Lynch syndrome and sporadic colorectal cancer,among which MAPK signaling pathway and adhesion plaque pathway ranked in the top ten of significance.In the analysis of miRNA related to RAS gene mutation and PD-1 drug resistance,it was found that all miRNA enriched in RAS signal pathway,PI3K-Akt signal pathway and VEGF signal pathway with different expression had statistical significance.In addition,among the statistically significant pathways enriched by miRNAs differentially expressed in Lynch syndrome,the target genes of five pathways,namely RAS signaling pathway,PI3K-Akt signaling pathway,RAP1 signaling pathway,cancer pathway and cancer central carbon metabolism pathway,all contain genes linking RAS mutation and PD-1.In addition,there are MAPK,regulation of actin cytoskeleton,regulating pluripotency of stem cells,and target genes of four pathways in prostate cancer,including FGFR1 and FGFR2.One of the three target genes is found in 6 signaling pathways including anti-EGFR tyrosine kinase inhibitor,proteoglycan in cancer,Adherens node,melanoma,bactericidal action and acute myeloid leukemia.3.4 Characterization of mRNA transcriptome in Lynch syndromeThe mRNA expression level of total RNA was 51%.The mRNA expression of Lynch syndrome was mainly concentrated on chromosome 6.Compared with normal intestinal mucosal tissue,1498 mRNA up-regulated and 2594 mRNA down-regulated in Lynch syndrome were screened,and 1280 mRNA up-regulated and 1579 mRNA up-regulated and 1758 mRNA down-regulated and 1951 mRNA down-regulated in the two groups of scattered colorectal cancer respectively.The RAS signaling pathway and the target gene-related pathway linking PD-1 and RAS signaling pathway in the Lynch syndrome group were not statistically significant.3.5 Clinicopathological analysis of mutant subtypes of RAS in Lynch syndromeThe mRNA expression level of total RNA was 51%.The mRNA expression of Lynch syndrome was mainly concentrated on chromosome 6.Compared with normal intestinal mucosal tissue,1498 mRNA up-regulated and 2594 mRNA down-regulated in Lynch syndrome were screened,and 1280 mRNA up-regulated and 1579 mRNA up-regulated and 1758 mRNA down-regulated and 1951 mRNA down-regulated in the two groups of scattered colorectal cancer respectively.The RAS signaling pathway and the target gene-related pathway linking PD-1 and RAS signaling pathway in the Lynch syndrome group were not statistically significant.Conclusions(1)lncRNA,circRNA,miRNA and mRNA were differentially expressed in Lynch syndrome and sporadic colorectal cancer,and the differences were more significant in Lynch syndrome patients.(2)Differential genes specifically expressed in Lynch syndrome are related to different pathway changes at different RNA levels:lncRNA level may be related to apoptosis pathway;The circRNA level may be related to Fanconi anemia and RAP1 signaling pathway.At the miRNA level,it may be related to reverse nerve signaling pathways,fatty acid metabolism pathways,sphingolipids metabolism pathways,and calcium reabsorption pathways regulated by endocrine and other factors.(3)The differentially expressed miRNA in Lynch syndrome was significantly correlated with RAS mutation and PD-1 resistance,with circRNA having a moderate correlation and lncRNA having a low correlation.However,in the differentially expressed mRNAs,no pathway related to RAS signaling pathway or pathway linking it to PD-1 target gene was found.(4)The molecular mechanism of PD-1 resistance of Lynch syndrome RAS mutant subtypes may be that specific circRNA or lncRNA acts as the miRNA cavernosum or protein binding on mRNA to regulate PD-1 immune response.Due to the influence of RAS mutation on the downstream mRNA,there is no effective target and signal pathway on mRNA,so that mRNA remains stable and has stronger ability of immunosuppression,leading to drug resistance of PD-1.(5)Based on the above research results,to explore non-coding RNA targets such as miRNA and circRNA,is expected to find an effective way to deal with drug resistance of MSI-H/dMMR immunotherapy in solid tumors. |
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