| Objective: Gastric cancer is a common malignant tumor in the world.The incidence and mortality of gastric cancer ranked fifth and third among malignant tumors.In China,the incidence and mortality of gastric cancer are also among the highest.In recent years,with the development of diagnosis and treatment,the clinical diagnosis and treatment effect of gastric cancer have been improved.However,the five-year survival rate of gastric cancer patients is still lower than some malignant tumors,such as breast cancer,prostate cancer and so on.Therefore,it is of great scientific significance to explore the molecular mechanism of gastric cancer and find new diagnostic markers.As a common non-coding RNAs,miRNAs have an important biological role.By binding to the 3 'UTR region of the downstream target gene,it can regulate its expression,affect the activity of the downstream pathway,lead to the phenotypic changes of tumor cells,and finally regulate the occurrence and progression of tumor.MiRNAs with the same seed sequence are classified as a miRNA family,and members of the family have similar roles.The miR-148/152 family includes miR-148 a,miR-148 b and miR-152,and has shown anticancer effects in malignant tumors.In our previous works,we confirmed that the expression of miR-148/152 family members in gastrointestinal cancer tissues were significantly lower than adjacent non-tumor tissues,and the low expression was associated with the malignant phenotype of tumors.In addition,cell functional experiments confirmed that miR-148 b could also inhibit the proliferation of gastrointestinal cancer cells.Based on this,we constructed the single and three gene knockout mice,and gastric cancer model using these mice,stimulated the occurrence and development of gastric cancer in vivo.We want to confirm the role of miR-148/152 family on regulating gastric cancer,and explore the molecular mechanism.Methods: Based on the Cre/loxp system,the flox modification was conducted by homologous recombination of fertilized ovum,and then mated with mice specifically expressing Cre recombinant enzyme to obtain miR-148 a,miR-148 b and miR-152 singlegene knockout mice.Next,the single-gene knockout mice were cross-bred to construct three-gene knockout mice.After the PCR amplification,agarose gel electrophoresis was used to identify the genotypes of the mice in each group.In situ hybridization and real-time PCR were used to detect the expressions of miR-148 a,miR-148 b and miR-152 in the gastric mucosa of each group mice.Combined with the genotype identification results,it was finally confirmed that the single-and three-gene knockout mice were successfully constructed.The changes of organs were observed and stained with H&E after dissection.The expression of related biomarkers in the gastric mucosa of mice were detected by immunohistochemistry.Following,the gastric cancer model was induced by MNU,and the gastric cancer induction was confirmed by H&E staining.The expression changes of miR-148/152 family members in the process of gastric cancer induction were detected by in situ hybridization.Bioinformatics analysis preliminarily screened the potential target genes of the miR-148/152 family,and real-time PCR and western blotting assay were used to detect the expression of potential target genes in the miR-148/152 family members overexpressed gastric cancer cell lines and mice gastric mucosa in each group.The dual luciferase assay was performed to detected the binding of miR-148/152 family members to the 3 'UTR region of potential target genes,and confirmed the existence of targeted regulation.Gastric cancer cell lines MGC-803 and HGC-27 were used for in vitro validation of the target genes and downstream pathways.The expression of key genes in the regulatory pathway was interfered with in vitro transfection,respectively.The expression of pathway marker proteins in cell lines treated with different treatments was detected by western blotting to verify the regulatory role of the miR-148/152 family on the predictive pathway.Finally,immunohistochemical experiment was used to verify the relevant mechanisms,and clarify the regulatory effect of miR-148/152 family on gastric cancer.Results: ?.Construction of transgenic mice and observation under physiological conditionsMiR-148 a,miR-148 b and miR-152 single-gene knockout mice were constructed by Cre/loxp system,and three-gene knockout mice were obtained by mouse hybridization.After the PCR amplification,agarose gel electrophoresis was used to identify the genotypes of the mice in each group.In situ hybridization and real-time PCR were used to detect the expressions of miR-148 a,miR-148 b and miR-152 in the gastric mucosa of each group mice,and confirmed the successful construction of gene knockout mice.Mice were fed normally,and no significant differences of the gross and organs were observed between knockout mice and wild-type(WT)mice.Immunohistochemical results showed that the expression of proliferation markers Ki67 and PCNA in the gastrointestinal mucosa of mi R-148/152 family knockout mice were higher than that of WT mice,while the other indicators showed no positive changes.?.Knocking out miR-148/152 family significantly promoted the formation of gastric cancer in mice,and Wnt1 and Wnt10 b were target genes of miR-148/152 familyMNU was used to induce gastric cancer in mice,and in situ hybridization confirmed that miR-148/152 family members were significantly decreased in gastric cancer tissues.H&E staining confirmed the successful construction of gastric cancer.We found that knocking out miR-148/152 family significantly promoted gastric cancer formation by comparing tumor formation rate and number of nodules,and no significant difference exists between the single-gene knockout mice and three-gene knockout mice.Bioinformatics analysis preliminarily screened Wnt1 and Wnt10 b as the common potential target genes of the mi R-148/152 family.Real-time PCR and western blotting results confirmed that the overexpression of the mi R-148/152 family could significantly inhibit the expression levels of Wnt1 and Wnt10 b in gastric cancer cell lines.Besides,the expression of Wnt1 and Wnt10 b in miR-148/152 family knock out mice were significantly higher than WT mice.Luciferase experiments confirmed that miR-148/152 family members could bind to the 3 'UTR regions of Wnt1 and Wnt10 b,and targeted regulating their expression.Further,we detected the expression of Wnt1 and Wnt10 b in gastric cancer induced tissues of miR-148/152 knockout mice and WT mice through immunohistochemical experiments,and found that the expression of Wnt1 and Wnt10 b in knockout mouse gastric cancer induced tissues was significantly higher than that of the wild type.These results confirmed Wnt1 and Wnt10 b are target genes of miR-148/152 family regulating the progression of gastric cancer ?.MiR-148/152 family inhibited the activity of mTORC1 pathway by Wnt/GSK3?/TSC2,and regulated the progression of gastric cancerAfter literature studying and website searching,we found that Wnt could regulate mTORC1 pathway activity by inhibiting downstream GSK3? and TSC2.Based on this,we inhibited the expression of key factors in Wnt/ mTORC1 pathway,including GSK3? and TSC2 separately in miR-148/152 family member overexpressed gastric cell lines.We confirmed that miR-148/152 family could activate GSK3? and TSC2 by targeting Wnt1 and Wnt10 b,inhibiting the activity of downstream mTORC1 pathway.Similarly,using immunohistochemical,we confirmed that the expressions of mTORC1 pathway markers pS6 and pS6 K in gastric cancer tissues of miR-148/152 family knockout mice were significantly higher than those of WT group.In conclusion,we concluded that the miR-148/152 family could inhibit the activity of mTORC1 pathway by targeting Wnt1 and Wnt10 b,ultimately inhibiting the formation of gastric cancer.Conclusion: MiR-148/152 family could target Wnt1 and Wnt10 b to regulate the activity of the downstream mTORC1 pathway,inhibiting the occurrence and progression of gastric cancer. |
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