MiR-335-3p Promotes Proliferation And Migration Of Colorectal Cancer Cell By Targeting APPL1 And Modulating P-Akt Signaling Pathway

Colorectal cancer(CRC)is one of the most common malignant tumors in the world.Statistics of cancers in China(2015)showed that CRC ranks 4th in the incidence of cancers in our country.And its mortality rate ranks 5th,which shows an increasing trend.It is a serious threat to the physical and mental health of our citizens.The occurrence and development of CRC is affected by many factors.It involves the abnormal expressions of many oncogenes and tumor suppressor genes,and the abnormality of many signal pathways.Therefore,in-depth studies of molecular mechanisms of tumor cell growth,proliferation,and metastasis are helpful to identify potential biotherapy targets in CRC.miRNA,as a small single strand RNA molecule,has been a hot topic in CRC researches in recent years.A large number of studies show that it plays an important role in the development of CRC.Some studies have shown that miR-335 plays an important role in many tumors’tumorigenesis and progression,which including CRC.However,at present,the studies of miR-335 are mainly focused on miR-335-5p,and there is no report on miR-335-3p.As the another important component of mature miR-335,the expression and function of miR-335-3p in CRC are unclear.Therefore,we focused miR-335-3p as a potential regulatory factor involved in the pathogenesis of CRC and carried out an in-depth research.Methods1 The expression of miR-335-3p in CRC tissuesThe expression levels of miR-335-3p in CRC tissues and their paired normal tissues were detected by real-time fluorescence quantitative PCR(q-PCR).2 The effects of miR-335-3p on the biological characteristics of CRC cells1)In vitro cell experiments:CCK8 cell proliferation assays,plate clone formation assays,transwell cell migration assays,cell wound healing assays,flow cytometry were used to detect the proliferation and migration ability changes of SW480 and HT-29 transfected with miR-335-3p mimics or inhibitors.2)In vivo cell experiments:The stable overexpression cell line of miR-335-3p was constructed by lentivirus transfection technique.The successfully constructed stable overexpression cells of miR-335-3p were injected into nude mice subcutaneously.After the subcutaneous tumor model was established,the effect of miR-335-3p on the proliferation of CRC cells was observed.Immunohistochemical technique was used to detect the expression of Ki67 in subcutaneous implanted tumors in nude mice.3 Prediction and confirmation of downstream target gene of miR-335-3pOnline prediction databases,DAVID pathway enrichment analysis and double-luciferase reporter assays were performed to determine the target gene of miR-335-3p.4 The expression of APPL1 in CRC and its effects on the biological characteristics on CRC cellsAfter construction of APPL1-overexpressed plasmid,cell function assays were used to detect the proliferation and migration ability changes of SW480 and HT-29 overexpressed with APPL1.5 Evaluation of the molecular mechanism of miR-335-3p involved in the regulation of malignant progression of CRCDAVID bioinformatics analysis and Western blot techniques were used to detect the expression changes of the associated signaling pathway proteins in SW480 and HT-29 which were overexpressed or inhibited of miR-335-3p.Result1 The expression of miR-335-3p was up-regulated in CRC tissues.The expression levels of 86 pairs of CRC tissues and their matched normal adjacent tissues were detected by q-PCR.The results showed that the expression of miR-335-3p in CRC group was significantly higher than that in normal group(P<0.01).In addition,the expression of miR-335-3p in CRC showed in TCGA database was also significantly higher than that in normal intestinal mucosa(P<0.01),which was consistent with the results of colon tissues.2 Up-regulation of miR-335-3p may be associated with metastasis of CRC and may be an important index in evaluating the condition of patients with CRC.The clinical data of 86 patients with CRC were statistically analyzed.The results of X2 test showed that the expression of miR-335-3p was closely associated with the important clinical features such as lymph node metastasis(P = 0.002)and AJCC stage(P = 0.003).The results suggested that the up-regulation of miR-335-3p may be associated with metastasis of CRC and may be an important index in evaluating the condition of CRC patients.3 Overexpression of miR-335-3p promoted the proliferation and migration of CRC cells,but inhibition of miR-335-3p suppressed the proliferation and migration.4 miR-335-3p promoted G1/S phase transformation of cell cycle in CRCThe results of flow cytometry showed that both the numbers of G1/G0 phase cells in the miR-335-3p overexpressed group of SW480 and HT-29 were significantly lower than that in the control group(P<0.05)and the numbers of S phase cells were significantly increased(P<0.05),whereas the results in the inhibitor group were opposite(P<0.05).Western blot results showed the expression of CCND1、CDK2、CDK4、CDK6、pRb were up-regulated in miR-335-3p over-expressed group,inhibited group were opposite.5 miR-335-3p inhibited cell apoptosis in CRC.The results of flow cytometry showed that the number of apoptosis cells in over-expressed miR-335-3p mimcis group was significantly lower than that in control group,while the number of apoptosis cells in miR-335-3p inhibitors group was significantly higher than that in control group.6 APPL1 was the target gene of miR-335-3p.Double luciferase reporter gene assays were used to confirm that APPL1 was a target gene of miR-335-3p.7 The expression of APPL1 was down-regulated in CRC and overexpression of APPL1 inhibited the proliferation and migration of CRC cells(P<0.05).8 miR-335-3p promotes proliferation and migration of colorectal cancer cell by targeting APPL1 and modulating p-Akt signaling pathway.The results of Western blot showed that the expression level of p-Akt was up-regulated and the expression of p21,p27 and Cleaved-casp9 were down-regulated after over-expressed with miR-335-3p,but the effect was weakened after over-expression of APPL1.The results suggested that miR-335-3p promoted the proliferation of CRC cells by regulating p-Akt-p27/p21 via targeting APPL1,inhibted cell apoptosis by regulating p-Akt-Casp9,and promoted the migration of CRC cells through APPL1-p-Akt pathway.Conclusion:In this study,we systematically studied the expression and roles of miR-335-3p and its target gene APPL1 in CRC,and the mechanism of miR-335-3p as a tumor promoter in promoting the proliferation and migration of CRC cells by APPL1-p-Akt signaling pathway.It provided a new idea for the study of pathogenesis in CRC and new clues for treatments in the future.