New Mechanism Of Non-coding RNA Regulating TGF-? Signal Pathway In Gastric And Colorectal Cancer

Cancers raised from gastric and colorectum are the most common malignant tumors in China with the characteristics of high mortality,therefore,those cancers seriously threaten human health.TGF-?(transforming growth factor-beta)signaling pathway can accelerate the progression of various cancers in humans,and it has been confirmed in many animal models of different cancer species that blocking TGF-?can inhibit the metastasis of tumor cells.On the contrary,TGF-? can accelerate the process of a variety of human cancers,and is closely related to the occurrence and development of gastric and colorectal cancer.Three subtypes of TGF-?(?1,?2,and?3)are elevated in many tumor types and are associated with poor prognosis of cancer.Current studies have shown that non-coding RNAs including microRNAs(miRNA),long non-coding RNAs(lncRNAs)and circular RNAs(circRNAs)play various vital regulatory roles in the occurrence and progression of gastric cancer and colorectal cancer.MEG3(maternally expressed gene 3)is the first lncRNA expressed as a tumor suppressor gene.Expression of MEG3 is down regulated in gastric cancer and colorectal cancer,especially in the advanced stage of them,and participates in the process of tumor proliferation,invasion and metastasis.MEG3 as a regulatory RNA targets the TGF-? pathway by down-regulating the expression of TGFBR1,TGFB2 and SMAD2 genes,leading to the inhibition of TGF-? signal pathway.Hence,it is crucial to find out how MEG3 crosstalk with TGF-? signaling pathway for the betterunderstanding of the molecular mechanism of gastric and colorectal cancers.MiRNAs are endogenous non-coding RNA with a length of 18-25 nt.MiR-21 is involved in almost the whole process of cancer cell growth,invasion,metastasis,angiogenesis,metabolism and apoptosis.The expression of miR-21 can be up-regulated by phosphorylation of Smad3 via TGF-? signaling pathways.The expression level of miR-21 can be decreased by inhibiting the phosphorylation of Smad3.As a result,miR-21 is a vital marker closely related to the occurrence and development of gastric cancer and colorectal cancer.However,the detail mechanism how miR-21 crosstalks with TGF-? pathway in human cancer remains unknown.Hence,in this project,gastric cancer and paracancerous tissue specimens were obtained.The differential expression profiles of lncRNAs and circRNAs were established by using gene microarray and bioinformatics technology.We screened out lncRNAs and circRNAs with significant expression differences in gastric cancer tissues to find out the biological behaviors that they may be involved in,and to select target genes for subsequent studies.The GO and KEGG pathway analysis were performed on significantly different lncRNAs and circRNAs to confirm TGF-? signal pathway in gastric cancer.Subsequently,functional and mechanism studies were conducted to examine how MEG3 crosstalked with genes of TGF-? signal pathway and how miR-21 related to TGF-? pathway interacts with multi-target drug in vitro models.At first,a new computer model(SVMR statistical regression model)was established.Then,the remote homologous genes of MEG3 and TGF-? signal pathway was tested by this model.As a result,it was found that MEG3 has specific recognition ability and high affinity for homologous genes.Secondly,we developed a new computer model to simulate in vitro how the multi-target drug Regorafenib interacts on the precursor of miR-21,thereby blocking the formation of mature miR-21.The ultimate aim of our study is to provide a new research method for the development of new targeted drugs for gastric and colorectal cancer in the future.Part ? Establishment and bioinformatics analysis of differential expression profiles of lncRNA and circRNA in gastric cancer Method1.6 specimens of fresh gastric cancer tissues and corresponding paracancerous tissues were collected.Her2 receptor statues were tested on all specimens by immunohistochemistry.LncRNA,mRNA and circRNA expression profiles were investigated on these 12 specimens by Agilent Human lncRNA V6 gene microarray.The screening criteria were p<0.05 and FC>2.0.The differential expressions of lncRNA,mRNA and circRNA relevant to gastric cancer were obtained by volcanic map and cluster analysis.2.GO(Gene Ontology)analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway were used to analyze the differential mRNA,lncRNA target genes and circRNA target genes of gastric cancer and their paracancerous tissues.The networks diagram between lncRNA-TF(transcription factor)and lncRNA-mRNA-TF were constructed by cytoscape software.Differential expressions of lncRNA and circRNA in relation to biological function were collected.3.The qRT-PCR method was used to further verify the expression level of 5lncRNAs up-regulated and 5 lncRNAs down-regulated in gastric cancer tissues.4.The qRT-PCR method was used to compare the expression levels of miR-21 in6 pairs of gastric cancer tissues and adjacent gastric tissues.Result1.The gene chip sequencing technique screened a total of 1217 lncRNAs with significantly different expressions(up 751,down 466).A total of 49 circRNAs(up 23,down 26).2.GO and significantly enriched lncRNA target genes and circRNA parent genes were obtained.KEGG pathway analysis revealed that TGF-? pathway was an important signal pathway related to gastric cancer.3.The results of qRT-PCR showed that the expression of 4 up-regulatedlncRNAs was abnormally increased and the expression of 5 down-regulated lncRNAs was decreased,which was highly consistent with the sequencing results of the chip.4.The results of qRT-PCR verified that expression of miR-21 was significantly increased in gastric cancer tissues,which also confirmed that miR-21 is an oncogene of gastric cancer.ConclusionMEG3 and TGF-? signaling pathways play an important regulatory role in gastric cancer.Further study on the mechanism of action between them is of great significance to determine whether MEG3 is an ideal target for cancer control.MiR-21 is a vital target in gastric cancer.Novel therapeutics that target miR-21 is of great value for the treatment of gastric cancer.Part ? Interaction mechanism between MEG3 and TGF-?signal pathway MethodThe molecular mechanism of MEG3 binding to its cognate and noncognate DNA partners was investigated systematically via an integrative chemometric approach.We applied a spatial information characterization technique to capture the structural and energetic features of DNA-RNA triplex architecture.A genome-wide statistical analysis strategy was used to quantitatively evaluate the inter-molecular interaction between DNA and RNA in a high-throughput manner.The binding potency and recognition specificity of MEG3 with its interacting TGF-? DRE(distal regulatory elements)partners TGFBR1?TGFB2 and SMAD2 were also evaluated.ResultA new computer model(SVMR statistical regression model)was established to evaluate the affinity and specificity of RNA and bound homologous andnon-homologous DNA.SVMR statistical regression model verified the specificity and affinity of MEG3 binding to the TGFBR1,TGFB2 and SMAD2,DREs of TGF-?pathway.ConclusionThe TGF-? pathway interacts directly with MEG3 in gastric cancer.MEG3 and TGF-? pathway are potential targets for gastric cancer treatment,and they can regulate a variety of cellular biological events.Part ? Interaction between Regorafenib and TGF-?-miR-21 precursor MethodIn order to explore the interaction between Regorafenib and TGF-?-miR-21,an in silico-in vitro integrated investigation of the direct inter molecular interaction between Regorafenib and TGF-?-miR-21,miR-21 pre-element was performed by using active pocket identification,molecular dynamics simulation,binding energetic analysis,and fluorescence-based assay.ResultWe observed that Regorafenib could bind to the major groove-like stem region of miR-21 pre-element through three geometrically hydrogen bonds as well as a number of hydrophobic forces and ?-? stacking.The mutation of two base pairs(G6C,C12G)and(A13U,U4A)related to H bond decreased the binding force of Regorafenib on miR-21 precursors.ConclusionRegorafenib can directly act on miR-21 precursors.Combined with previous study results and relevant literature reports,miR-21 is a crucial oncogene for gastriccancer and colorectal cancer,that is associated with the TGF-? signaling pathway.Hence,the binding model established in this experiment lays a foundation for the development of targeted drugs for other targets of TGF-?.