MicroRNA-455 Suppresses The Oncogenic Function Of HDAC2 In Human Colorectal Cancer

Background:Colorectal cancer(CRC),also known as bowel cancer and colon cancer,is one of the common cancers with high morbidity and mortality.CRC is the third most common cancer worldwide,and it is the fourth leading cause of cancer-related death.The incidence of this cancer is increasing year by year,and according to statistics,there are 1.2 million new cases in 2008 worldwide.The etiology of CRC is multifactorial and genetic,and this cancer is one of the results of long-term formation of lesions.Early discovery,diagnosis and treatment are critical for improving the cure rates of CRC.But,CRC is difficult to be detected early,because of its hiding onset.Many patients with CRC have no apparent symptoms,they are not diagnosed until the disease reaches an advanced stage.The risk factors of CRC are multiple,including family history of this cancer,smoking,excessive intake of red meat,and etc.The incidence of CRC is also closely related with age.Low incidence is observed in people under 50 years;while high incidence is observed in elder people.Growing evidence demonstrated that,the incidence and mortality of CRC in lowincome countries are increasing rapidly.Although,the prognosis of CRC patients has been largely improved recent years,the 5-year survival rate of patients in low-income countries is less than 50%.Thus,studies for developing novel and effective therapeutic targets are urgently needed.In this regard,micro RNAs(mi RNAs)have attracted a lot of attention,since they play pivotal roles in regulating their targeted m RNAs.MiRNAs,a kind of non-coding RNAs,with approximately 22 nucleotide.They are widely existed in eukaryotes,and contribute in numerous of biological process by binding to the 3'untranslated regions(UTRs)of its target m RNAs and thereby repressing their expression.Previous studies have reported that mi RNAs are widely existed in various species and tissues,and are involved in the modulation of cell growth,differentiation,apoptosis,and even involved in the controlling of cancer cell growth.Several researches confirmed that mi RNAs can act as tumor suppressive gene or oncogene to directly participate in the tumorigenesis,such as lung cancer,liver cancer,breast cancer,and etc,implying the pivotal roles of mi RNAs in the occurrence and development of cancers.Ithas been established that differentially expressed mi RNAs can alter the occurrence and development of CRC by targeting cancer-related genes.However,the detailed functions of mi RNAs and their target genes have not been fully revealed,which still need further investigations.Histone deacetylases(HDACs)are a class of enzymes that remove acetyl groups(O=C-CH3)from histone substrates,allowing the histones to wrap the DNA more tightly.HDACs play pivotal roles in regulation of gene expressions and modification of chromosomes.In general,HDACs decondense chromosomes,inhibiting gene expressions;and of course,there also include tumor suppressive genes.HDACs are divided into four classes,I,II,III,and IV.HDAC2 belongs to class I,and it is a key regulator in multiple diseases.In gastric cancer,CRC,and prostatic cancer,high expression of HDAC2 was closely related with poor outcome of patients.There is evidence suggests that several mi RNAs exert tumor suppressive or tumor promoting actions via modulation of HDAC2.Thus,exploration for specific mi RNAs which can bind with HDAC2,will provide us a better understanding for the role of HDAC2 on the pathogenesis of CRC,and will provide theoretical support for gene therapy of CRC.Object:This study aimed to reveal the functions of HDAC2 and mi R-455 on CRC cells growth,and to reveal the targeting relationship between HDAC2 and mi R-455.Firstly,the expression of HDAC2 in CRC tissues and adjacent non-tumor tissues was compared.Secondly,the expression of HDAC2 in HCT116 cells was knocked down,the effects of HDAC2 silence in cell proliferation was evaluated.Thirdly,we explored the relationship between HDAC2 and mi R-455.Meanwhile the effects of mi R-455 overexpression on HCT116 cells proliferation and apoptosis were detected.Finally,it was revealed that mi R-455 could inhibit the carcinogenesis of colorectal cancer by HDAC2.Methods:Firstly,20 pairs of tumor and adjacent non-tumor tissue samples were collected from CRC patients who underwent surgical treatment in the Affiliated Hospital of Qingdao University,between March,2014 and December,2014.Tissue samples were immediately placed in liquid nitrogen and stored at-80°C until use.No patient involved in this study was received chemotherapy or radiotherapy before surgery.Quantitative reverse transcription PCR(q RT-PCR)was performed to assess the expression levels of HDAC2 in20 pairs of CRC tissues and adjacent non-tumor tissues.To compare whether there exists significant difference in HDAC2 expression between these two tissue samples.Human CRC cell line HCT116 was used in this study for cell culture.For silencing HDAC2,HDAC2 targeted short hairpin RNA(sh RNA)were cloned into lentivirus(GV118),and transfected this lentivirus into HCT116 cells.Transfection efficiency was verified by detection of HDAC2 expression using q RT-PCR and Western blot analysis.Then,MTT assay was performed to determine cell proliferation at day 1 to day 5 posttransfection.By screening Target Scan and microrna databases,the site in 3'UTR of HDAC2 which can be bind with mi R-455 was predicted.The binding sequence was inserted into pmir GLO vector,and the vector were co-transfected with mi R-455 mimics into cell.After48 h of transfection,luciferase activity was assayed by using Dual-Luciferase Reporter Assay System,to verify that HDAC2 3'UTR was directly bind with mi R-455.In addition,q RT-PCR and Western blot were performed to assess the m RNA and protein levels of HDAC2 in cells transfected with mi R-455 mimics,to explore the regulatory relationship between HDAC2 and mi R-455.HCT116 cells were transfected with mi R-455 mimics,and then cell proliferation was measured at day 1 to day 5 post-transfection by using MTT assay.Also,cell apoptosis was detected by flow cytometry detection after transfection.To compare whether there exists significant difference between transfected cell and non-transfected cell.Results:The expressions of HDAC2 in CRC and its effects on CRC cells proliferation:HDAC2 was highly expressed in CRC tissues resected from 20 CRC patients,when compared with adjacent non-tumor tissues(P< 0.05).Lentivirus encoding sh RNA targeting HDAC2 was transfected into HCT116 cells.q RT-PCR and Western blot analytical results showed that HDAC2 expressions were significantly knocked down by transfection with HDAC2 sh RNA,indicating HDAC2 was successfully silenced in HCT116 cells.Thereafter,MTT assay results indicated that cell proliferation was significantly reduced at day 3,4,and 5 post-transfection(P< 0.05,P< 0.01 or P< 0.001).The targeting relationship between HDAC2 and mi R-455: By screening Target Scan and microrna databases,a 7-mer sequence(UGGACUG)in the 3'UTR of HDAC2 was predicted as binding site which can directly bind with mi R-455.This binding site wasinserted into pmir GLO vector and this vector was co-transfected with mi R-455 mimics into cell.The results showed that luciferase activity was significantly reduced after transfection(P< 0.01).q RT-PCR and Western blot analytical results showed that the m RNA and protein levels of HDAC2 were low expressed in cell transfected with mi R-455mimics(P< 0.01),indicating HDAC2 was negatively regulated by mi R-455.The effects of mi R-455 on CRC cells proliferation and apoptosis: MTT assay results showed that,cell proliferation was significantly reduced after HCT116 cells transfected with mi R-455 mimics for 3,4,and 5 days(P< 0.05,P< 0.01,or P< 0.001).Flow cytometry detection showed that apoptotic cell rate was significantly increased after transfection with mi R-455 mimics(P< 0.001).Conclusions:1.HDAC2 was highly expressed in CRC tissues,indicating HDAC2 might contributein the occurrence and development of CRC;2.HDAC2 silence reduced the proliferation of human CRC HCT116 cells,implyingthe tumor promoting activity of HDAC2 in CRC;3.HDAC2 was a target gene of mi R-455,and it was negatively regulated by mi R-455;4.The mi R-455 overexpression inhibited HCT116 cells proliferation,whilepromoted apoptosis,indicating the tumor suppressive role of mi R-455 in CRC;5.The mi R-455 suppressed the oncogenic function of HDAC2 in human CRC.