Dynregulation Of MiR-330-3p Effect On Malignant Behavior Of CcRCC And Its Clinical Significance

Background:RCC is the most common form of kidney cancer in adults,Clear cell renal cell carcinoma(ccRCC)is the most common subtype,accounting for 70%~80%of all kidney cancer.Renal cancers are more common in males compared to females(1.7:1 ratio),making it the 9th most common cancer in men,and 14th in women.Surgical treatment is still the most effective treatment,but the postoperative recurrence of patients is about 20%to 40%,the average survival time of renal cancer patients was about 26 months.Hence,a means of identifying patients with a poor prognosis,and who may benefit from aggressive treatment,is greatly needed.MicroRNAs(miRs)are a class of small(~22 nucleotide)noncoding RNAs that regulate post-transcriptional gene expression epigenetically,through RNA interference.This is usually mediated by their direct interaction with the 3?-UTR of complementary mRNA target transcripts,which facilitates their degradation or inhibits their translation.MiRs are involved in a variety of biological functions,including cellular proliferation and cell cycle control,apoptosis,angiogenesis,tissue invasion,and metastasis,suggesting that they have a vital role in the development and progression of different cancers.Correspondingly,recent studies show that miR-330-3p profiling may discriminate between different subgroups of tumors.However,to our knowledge,there was no published study evaluating the relationship of miR-330-3p and ccRCC.Objective:1.To determine the levels of miR-330-3p in ccRCC tissues and paracancerous tissues,analysis the correlation between the expression of miR-330-3p and Clinic pathological parameters.2.To investigate the effects of over-expression miR-330-3p on the malignant behaviors of 786-O and ACHN.3.To explore the effect of miR-330-3p down-regulation on the malignant behaviors of 786-O and ACHN.4.To determine mi R-330-3p effect on 786-O through signaling pathway and the relative protein expression in ccRCC tissues.Method:1.qRT-PCR was used to detected the expression level in 42 paired ccRCC tissues and unpaired Student's t-test or One-way ANOVA analysis was used to evaluated the correlation miR-330-3p expression and Clinic pathological parameters.2.Using Lipofetamin~TMM 2000 transfection reagent,miR-330-3p mimic and miR-330-3p inhibitor,mimic negative control(NC),inhibitor NC,respectively,was transiently transfected into 786-O,and the expression of mi R-330-3p were detected by qRT-PCR.3.Using Lipofetamin~TMM 2000 transfection reagent,mi R-330-3p mimic and mimic negative control(NC),respectively,was transiently transfected into 786-O and ACHN,MTT assay determined the effects of over-expression miR-330-3p on the proliferation ability of 786-O and ACHN,respectively;Transwell assay and cell wound scratch assay measured the effects of over-expression mi R-330-3p on the migration and invasion capabilities of 786-O and ACHN,respectively.4.Using Lipofetamin~TMM 2000transfection reagent,miR-330-3p inhibitor and miR-330-3p inhibitor(NC),respectively,was transiently transfected into 786-O and ACHN,MTT assay determined the effects of down-regulation miR-330-3p on the proliferation ability of 786-O and ACHN,respectively;Transwell assay and cell wound scratch assay measured the effects of down-regulation mi R-330-3p on the migration and invasion capabilities of 786-O and ACHN,respectively.4.Used western blot evaluated the expression of P53,MMP2,p-Akt,p-Mek in the ccRCC tissues and corresponding normal tissues.Further investigated the expression of P53,MMP2,p-Akt,p-Mek in 786-O cells after transfected with mi R-330-3p mimic,miR-330-3p inhibitor,mimic NC,inhibitor NC,respectively.Results:1.Compared with normal tissues,the expression of miR-330-3p was up-regulated in 29 ccRCC tissues(69.05%),down-regulated in 13 ccRCC tissues(30.95%),and the overall level was raised at a rate of 138.24%(p=0.0229).The expression of miR-330-3p was positive correlate with Fuhrman grade.Compared with patients diagnosed in grade 1-2,the expression levels of mi R-330-3p of ccRCC patients in Fuhrman 3-4 grade increased by 147.2%(P=0.0477).2.To examine whether overexpression of miR-330-3p affected the proliferation,migration and invasion capacity of ccRCC cells,MTT and transwell assays were introduced.We observed that miR-330-3p overexpression enhanced proliferation,migration ability of 786-O and ACHN compared with mi R-330-3p mimic NC group.In parallel,a Matrigel invasion assay was also performed to study the effects of miR-330-3p on the invasion of ccRCC cells,the result clearly reveal that overexpression of miR-330-3p also increased invasion ability of 786-O and ACHN compared with miR-330-3p mimic NC group.3.We also found the opposite effects of anti-miR-330 exerted on both cell lines compared with anti-miR-con group,the results show that miR-330-3p down-regulation suppressed proliferation,migration and invasion ability of 786-O and ACHN compared with miR-330-3p inhibitor NC group.4.Western blot results indicated that,overexpression of mi R-330-3p increased the protein level of MMP2,p-Akt,p-Mek,respectively and down-regulation the P53 level.Interestingly,we also found the P53protein level was lower,the MMP2,p-Akt,p-Mek,protein level was higher in the ccRCC tissues,respectively,compared the paracancerous tissues.Conclusion:1.miR-330-3p was overexpression in the ccRCC tissues compared with the paracancerous tissues and positive correlated with Fuhrman grade,no clear correlations were determined for the miR-330-3p expressions in ccRCC among the other clinic pathological.2.Up-regulate miR-330-3p enhanced proliferation,migration and invasion ability of 786-O and ACHN compared with miR-330-3p mimic NC group,respectively.3.Down-regulate miR-330-3p decreased proliferation,migration and invasion ability of 786-O and ACHN compared with miR-330-3p inhibit NC group,respectively.4.miR-330-3p affects the expression of P53,MMP-2,p-Akt,p-Mek,thereby affecting 786-O's proliferation,migration and clinical progression of ccRCC.