MiR-335 Regulates The Malignant Biological Behaviors Of Gastric Cancer Cells And Its Mechanism

Gastric cancer is the fourth common cancer and causes the second cancer related death worldwide with estimated 950,000 new stomach cancer cases and 720,000 deaths in 2012.Although there are greater understandings in gastric cancer epidemiology,pathology,molecular mechanisms,in therapeutic options and strategies over recent years,patients with gastric cancer are mostly diagnosed at advanced stage and have poor survival.Hence,it is of significant value to screen a biomarker for diagnose and develop therapeutic strategies.MiRNAs consisting of 20-22 nucleotides are a kind of non-coding RNAs that inhibit gene expression by targeting the 3’-untranslated region.Mi RNAs function as tumor suppressors and oncogenes in various cancer progression and work in cancer cell proliferation,apoptosis,invasion,metastasis and stem-like properties.MiRNAs are suitable choice for early diagnose biomarker and therapeutics.MiR-335 played different roles in different cancers in different cancers and was reported to be downregulated in various cancers,including prostate cancer,breast cancer,ovarian cancer and upregulated in colon cancer.There was little reporter of the effect of miR-335 on gastric cancer cells and its mechanism.Therefore in present study,we explored the role of miR-335 in the regulation of gastric cancer cell malignant biological behaviors and its underlying mechanism.Part Ⅰ The Expression Of Mi R-335 In Gastric Cancer Tissues And Cell Lines.Objective:to explore the expression of miR-335 in gastric cancer and gastric cells.Methods:1.Real-time quantitative RT-PCR was used to detect the expression of miR-335 in gastric cancer tissues and paired adjacent normal gastric tissues.2.Real-time quantitative RT-PCR was used to detect the expression of miR-335 in gastric cancer cell lines.Results:miR-335 was downregulated in gastric cancer tissues and gastric cancer cell lines compared with the gastric tissues and cells.miR-335 was downregulated in T_3-4 stage and metastasis gastric tissues.Part Ⅱ The Effect Of MiR-335 On Gastric Cancer Cell Malignant Biological Behaviors.Objective:to explore the role of miR-335 on gastric cancer cell malignant biological behaviors.Methods:1.miR-335 overexpression and antagonist lentivirus and their negative control lentivirus were transfected seperately to SGC-7901 cell line and BGC-823 cell line.2.CCK-8,EdU assay and subcutaneously implanted tumor model in nude mouse were used to detect the effect of miR-335 on proliferation.3.transwell assay and lung metastatic tumor model in nude mouse were used to detect the effect of miR-335 on metastasis.4.Sphere formation,flow cytometry testing the CD44⁺/EpCAM⁺positive cell rate and subcutaneously implanted tumor model in nude mouse were used to detect the effect of miR-335 on stemness.Results:1.the expression of miR-335 was upregulated in SGC-7901 cell line after transfected with overexpression miR-335 lentivirus and downregulated in BGC-823 cell line after transfected with antagonist miR-335 lentivirus compared with their negative control.2.CCK-8 assay showed the absorbance in overexpression miR-335group was decreased while in antagonist mi R-335 group was increased compared with its negative control.EdU assay showed that the EdU positive cell rate in overexpression miR-335 group was decreased while in antagonist mi R-335 group was increased compared with its negative control.subcutaneously implanted tumor model in nude mouse showed that the tumor volume in overexpression miR-335 group was declined.3.The result of transwell assay showed cell permeating septum was downregulated in overexpression miR-335 group and upregulated in antagonist mi R-335 group.lung metastatic tumor model in nude mouse showed the lung metastasis tumor number was decreased in overexpression miR-335 group.4.Sphere formation experiment showed the sphere formation number in overexpression miR-335 group was decreased and in antagonist miR-335 was increased.CD44⁺/EpCAM⁺positive cell rate was downregulated in in overexpression miR-335 group and upregulated in antagonist miR-335 group detected by flow cytometry assay.Subcutaneously implanted tumor model in nude mouse showed that the tumor formation rate was decreased in overexpression mi R-335 group compared with its negative control group.PartⅢ The Underlyting Mechanism Of MiR-335 On The Regulation Of Gastric Cancer Malignant Biological BehaviorsObjective:to explore the underlying mechanism of miR-335 on the regulation of gastric cancer cells.Methods:1.Predict the target gene of mi R-335 by bioinformation software and dual luciferase reporter assay was used to confirm the target gene.2.Western Blot assay was used to inverstigate the expression of Oct4,AKT and pAKT.Results:1.Oct4 was predicted to be the targeted gene of mi R-335 and dual luciferase reporter assay confirmed miR-335 could target Oct4.2.Western Blot assay showed the expression of Oct4,AKT and pAKT were downregulated in overexpression mi R-335 group and upregulated in antagonist miR-335 group.Conclusion:1.miR-335 was downregulated in gastric cancer cell lines,gastric cancer tissues,T_3-4stage and metastasis tissues.2.mi R-335 could inhibit the proliferation,metastasis and stemness of gastric cancer cells.3.miR-335 might decrease the expression of Oct4 to regulate AKT pathway to inhibit gastric cancer malignant biological behavior.