| ?Background and Objective?Hepatocellular carcinoma?HCC?is one of the most common cancers in China.Cancer statistics in 2015,which was published in CA Cancer J Clin and reported by Wan-Qing Chen[1],et al from Chinese National Cancer Registry,indicated that HCC is the 4th most commonly diagnosed cancers among men in China.In recent years,prevention of hepatitis B virus?HBV?infection through vaccination,a reduction in the consumption of corn contaminated with aflatoxins and safer injection practices have made the prevention of HCC effective.Therefore,the rate of HCC decreased in the last ten years.However,the malignant degree of HCC is still to be reckoned with.Cancer statistics in 2015 also reported that HCC is the third leading causes of cancer death among both men and women in China.The estimated new cancer cases of HCC in 2015 were 466.1 thousand,while the death was 422.1 thousand.Its mortality to incidence ratio ranked first among all the cancers.The reasons for HCC with high mortality include the difficulty of early diagnosis,the tendency of recurrence and metastasis as well as the lack of effective therapy for advanced HCC.Hence,molecular mechanism investigation is urgently needed for the development of novel therapeutic targets for HCC.microRNAs?miRNAs?are a class of endogenous single-stranded non-coding RNA molecules with 18-22 nucleotides which regulate posttranscriptional events by complementing the 3?untranslation region of the target gene mRNA.Therefore,the roles of miRNAs in many cancers including HCC have become a research hotspot.Because of their stability in serum and tissues,miRNAs may serve as good diagnostic markers,especially as noninvasive serological markers.With the development of drug delivery system,miRNA-related therapeutic drugs have been used in some clinical trials,indicating their important roles in the diagnosis and treatment of HCC[2].miR-541 is located within the mi RNA cluster of DLK1/DIO3 imprinting gene region on human chromosome 14.Many miRNAs in this cluster have been reported to be involved in cancer pathogenesis.Our previous studies demonstrated that hepatocyte nuclear factor4?,which inhibited the malignant biological behavior of hepatocellular carcinoma cells,can regulate transcription of the miR-379-656 cluster of the DLK1/DIO3 imprinted gene region,including miR-541[3].Further studies showed that miR-541 had the strongest inhibitory effect on the proliferation of various hepatoma cell lines among 28 miRNAs regulated by HNF4?,manifesting its vital roles in the development of HCC.Previous report about miR-541 is few,particularly its effects on tumors.One study reported that mi R-541 inhibited the proliferation of breast cancer cells in vitro[4]and another one demonstrated that miR-541 suppressed the progression of non-small cell lung cancer cells[5].However,its role in HCC has not been repoted.In this study,we will investigate the effect of miR-541 on HCC cells in vitro and in vivo and elucidate the in-depth molecular mechanism by seeking its downstream targets and the related signal transduction pathways in the progression of HCC,which will provide a new prospect in the clinical treatment of HCC.?Methods?1.Effect of miR-541 on the malignant behaviours of HCC cells1)Effects of miR-541 on the proliferation of HCC cells in vitroHCC cells were transfected with mi R-541 mimic and negative control?NC?or mi R-544 inhibitor and inhibitor NC.Cell counting kit-8?CCK-8?was used to detect the proliferation of the HCC cells.2)Effects of miR-541 on the Colony-formation of HCC cells in vitroHCC cells were transfected with miR-541 mimic or miR-544 inhibitor.Colony-formation assay was used to detect the colony-formation ability of the HCC cells.3)Effect of miR-541 on the migration of HCC cells in vitroTranswell assays were performed in YY-8103 cells or PLC/PRF/5 cells transfected with miR-541 mimic or miR-541 inhibitor to access the migration of both of the two cells.4)Effect of miR-541 on the invasion of HCC cells in vitroTranswell assays were performed to access the invasion of HCC cells transfected with mi R-541 mimic or inhibitor.5)Effect of miR-541 on drug-sensitivity of HCC cells in vitroDifferent concentration gradient of Sorafenib and Adriamycin were added in YY-8103 cells or PLC/PRF/5 cells transfected with miR-541 mimic or miR-541 inhibitor.Cell counting kit-8?CCK-8?was used to detect the proliferation of the HCC cells.IC50was used to access sensitivity of theses drugs.6)Effect of miR-541 on tumorigenesis of HCC cells in vivoYY-8103?2×106?cells infected with Ad-miR-541 or control adenovirus Ad-MAX in200?l of serum-free medium were injected subcutaneously into both flanks of Balb/c nude mice?n=6?.Tumor formation and size were evaluated periodically and all mice were sacrificed 3 weeks after innoculation.Comparison of the xenograft size and weight was performed in both groups.2.The mechanism of miR-541 in HCC1)Prediction of miR-541 targetsTargetScan?http://www.targetscan.org/?,miRDB,microRNA.org,DIANA MICROT,TARGETMINER and RNA22-HAS were performed to screen for putative targets of miR-541.2)Effect of mi R-541 on potential targetsReal-time PCR and Western Blot were used to detect the expression of miR-541targets in HCC cells transfected with miR-541 mimic or inhibitor.Reporter assays were performed to detect the effect of miR-541 on the wild type luciferase reporter plasmid and mutated luciferase reporter vector to prove the direct binding of miR-541 to the 3?UTR of potential targets.3)Effect of potential targets on HCCsSmall interfere RNAs?siRNAs?targeting the targets of miR-541 were used to down-regulated the expression of the targets,and then the proliferation level of HCC cells was evaluated.Cotranstected with miR-541 inhibitor and siRNAs,the proliferation and migration of HCCs were evaluated.4)Effect of mi R-541 on autophagy of HCC cellsWestern Blot was carried out to detect the expression of LC3 in HCC cells transfected with miR-541 mimic and cells with starvation or rapamycin treatment.PLC/PRF/5 cells infected with Ad-mRFP-GFP-LC3 and transfected with miR-541 mimic or inhibitor were used to evaluate autophagy level.Small interfere RNAs?siRNAs?targeting the targets of miR-541 were used to down-regulated the expression of the targets,and then the autophagy level of HCC cells was evaluated.4.Statistical analysesStatistical analyses were performed with SPSS software?18.0 version?,with a P value<0.05 considered significant.For experiments involving only two groups,data were analyzed with two tail Student's t test or Mann-Whitney U test.?Results?1.miR-541 inhibits the malignant phenotypes of HCC cells in vitro and in vivo1)Overexpression of miR-541 significantly suppressed,while inhibition of miR-541enhanced the proliferation of HCC cells.2)Up-regulation of miR-541 inhibited colony-formation of HCC cells and down-regulation miR-541 increased colony-formation of HCC cells.3)miR-541 mimic markedly reduced while miR-541 inhibitor increased the migration and invasion of PLC/PRF/5 cells and YY-8103 cells in vitro.4)Overexpression of miR-541 remarkably increased the sensitivity of Sorafenib and Adriamycin,however inhibition of mi R-541suppressed the sensitivity of both drugs.5)YY-8103 cells infected with Ad-mi R-541 or Ad-MAX were subcutaneously transplanted into two flanks of Balb/c nude mice.Small xenografts were detected in 100%?6/6?of the group infected with Ad-MAX as early as day 3,while only one small xenograft in mice receiving YY-8103 cells infected with Ad-miR-541 were observed on day 3,and four small nodules were identified in 66.7%?4/6?of the mices until the mices were sacrificed on week 3.Compared with the Ad-MAX group,the size and weight of xenografts were significantly smaller in the Ad-miR-541 group.2.miR-541 inhibits HCC by targeting ATG2A and RAB1B1)All of the six softwares have identified ATG2A and RAB1B,two important autophagy-related genes,as potential downstream targets for mi R-541.Both mRNA and protein levels of ATG2A and RAB1B were decreased in HCC cells transfected with mi R-541 mimic and increased in cells transfected with miR-541 inhibitor.2)Reporter assays revealed that overexpression of miR-541 significantly decreased the luciferase activity of wild-type ATG2A 3?UTR and wild-type RAB1B 3?UTR.Point mutation of the targets sequence diminished the impact of miR-541 on the target genes,indicating that ATG2A and RAB1B are two direct downstream targets for miR-541.3)The proliferation level of HCC cells were inhibited by siRNAs targeting ATG2A and RAB1B.Proliferation and migration of HCCs were increased by miR-541 inhibitor,which were reversed by siRNA.4)Western blot analysis showed that LC3?/?was downregulate in HCC cells transfected with miR-541 mimic,which was more obviously in cells treated with starvation or rapamycin.Autophagy level was lower in PLC/PRF/5 cells dealed with Ad-mRFP-GFP-LC3 and miR-541 mimic than in the control group,which was highter in PLC/PRF/5 cells dealed with Ad-mRFP-GFP-LC3 and miR-541 inhibitor than in the control group.The autophagy was inhibited in HCC cells transfected with siRNAs targeting ATG2A or RAB1B.?Conclusion?1.miR-541 inhibits the malignant phenotypes of HCC cells in vitro and in vivo.2.ATG2A and RAB1B are direct targets of miR-541.3.miR-541 may supress the progression of HCC,at least partially,by inhibiting autophagy by down-regulating these two autophagy-related genes. |
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