MiRNA-22Suppresses Colon Cancer Cell HCT116Proliferation, Migration And Invasion By Inhibiting The Expression Of TIAM1, VEGF, MMP2and MMP9

Background:Colorectal cancer is a significant health burden worldwide, accounting for over1,2million new cancer cases and more than600,000cases of cancer-related mortalityannually. However, incidence of this type of cancer has recently decreased in severalWestern countries, largely due to increased awareness, early detection and preventionmeasures. In addition, advancements in treatments have improved the survival ofpatients with this disease. To date, surgery is curative for the early stages of colorectalcancer, while chemotherapy plus surgery is used to treat the later stages of the disease.However, most patients eventually develop metastatic disease and succumb tocolorectal cancer. Thus, novel approaches for the early detection, treatment andsuppression of tumor progression are required to effectively control this disease.microRNAs (miRNAs) are a group of naturally-occurring, small non-codingRNAs,21-25nucleotides long, that regulate target gene expression by specificallyinhibiting the translation of mRNA or inducing mRNA degradation. Accumulatingevidence has demonstrated that miRNAs play a critical role in regulating a variety ofphysiological and pathological processes in the human body, such as cell differentiation, morphogenesis and tumorigenesis. In human cancer, miRNAs can function either asoncogenes or tumor suppressor genes in regulating tumor development and progression,depending on the target gene. Thus, identifying and understanding the molecular targetsof miRNAs by using computational in silico analysis with a specific algorithm, such asTargetScan, could elucidate the signaling cascades involved in colorectal cancerprogression.Because of the high efficiency, energy-saving, rapid adjustment, reversible andother characteristics in MicroRNA regulation process, we have full interest in it. As isknown to all, tumor formation is a complex and mysterious process. Although with thedevelopment of science, people gradually mastered some signal pathway and actionsites, but it is difficult to achieve good results by blocking a single site or pathway.Obviously, MicroRNA bring us a new research direction. It can regulate multipletargets at the same time, have a conserved sequence and can be acquired easily. Itplays an important role not only in physiological and pathological process, but also inthe diagnosis and treatment results. It was recently shown that miR-22is dysregulatedand functions as a tumor suppressor gene in several types of cancer. In addition, itsaltered expression is associated with the poor prognosis of colon cancer. We chooseTiam1,VEGF,MMP2and MMP9as the target gene by the analysis result of predictionsoftware. Furthermore, we want to know more about the directly or indirectlyregulation function of MicroRNA22in colon cancer from those action sites which playan important role in tumorigenesis and tumor development, and observe the regulationrole from some different point of view such as the cytoskeleton changes, the formationof new blood vessels, and gelatinase generation. In addition, although previous studyshowed that MicroRNA22can regulate hypoxia signaling in colon cancer cells bysuppressing the expression of hypoxia inducible factor-1α (HIF-1α), in aerobiccondition the regulation mechanism and target gene of MicroRNA22in colon cancerstill remain unknown. Objective:Analysis the variation in proliferation, invasion and migration of colon cancercells HCT116after overexpressed MicroRNA22, find out the target gene ofMicroRNA22under aerobic environment, and try to discover the regulation ability ofMicroRNA22towards Tiam1, VEGF, MMP2and MMP9.Method：The HCT-116human colorectal cancer cell line was purchased from The ChineseAcademy of Sciences tissue bank. hsa-miR-22precursor sequences(5’-AGUUCUUCAGUGGCAAGCUUUA-3’) were got from miRBase, and synthesizedby invitrogen. The plasmid encoding the miR-22precursor was labeledSD13-hsa-miR-22. Use Chromas to conform the high expression vector wassuccessfully established. Finish plasmid extraction according to the plasmid extractionkit steps. Briefly, cells plated at70%confluence in24-well plates were transfected with0.8μg SD13-hsa-miR-22or control plasmid and re-fed with fresh medium4h aftertransfection. The expression of green fluorescent protein (GFP) was monitored in cellsas an indicator of transfection efficiency and total RNA was isolated48h aftertransfection to verify gene expression.Total RNA was isolated using RNAiso for Small RNA (Takara, Japan), accordingto the manufacturer’s instructions, and then reverse transcribed into cDNA using OneStep PrimeScript miRNA cDNA Synthesis kit (Perfect Real Time, Takara, Japan).The newly synthesized cDNA was then used as a template for detection of miR-22andother genes. Specifically,100ng cDNA was mixed with SYBR Premix Ex TaqTMII(Perfect Real Time) in a20-μl reaction. The primer of has-miR-22and U6weredesigned and synthesized by Guangzhou RiboBio(RiboBio, China). The relativeexpression of mRNA was analyzed using7300System SDS software. After made surethe standard curve and melting curve, we compare the miRNA-22expression amongmiRNA-22transfected group, NC group and untreated control cell group, to confirm the overexpression model was constructed successfully. We use MTT cell viabilityassay to make Cell proliferation curves of the3groups. In vitro tumor cell migrationand invasion assay were performed with a transwell system.To predict miR-22target genes, we performed online searches of two differentGenbank databases; i.e., miRNA database; http://www.sanger.ac.uk/Software/Rfam/mirna and TargetScan algorithm (http://www.targetscan.org). Design primersspecific for miR-22, β-actin, matrix metalloproteinases2and9(MMP-2and MMP-9),TIAM1and vascular endothelial growth factor (VEGF) and All reactions were run intriplicate with the ABI PRISM7300Real-Time PCR system (Applied Biosystems,Foster City, CA, USA). To detect changes in gene expression in HCT-116cells in theabsence or presence of miR-22transfection, we performed immunocytochemistry usinga streptavidin peroxidase method. Western blot analysis was performed to determinethe effects of miR-22on the regulation of expression of different genes. The film wasscanned and analyzed with Gel Image System software (Simon, ProteinSimple, SantaClara, CA, USA).Results:In this study, we used qRT-PCR to examine miR-22expression in HCT-116colorectal cancer cells and found that it was weakly expressed. Our data showed thatthe expression level of transfected miR-22was17.65±0.98-fold higher than that of NCor parental cells. Next, we assessed the effect of miR-22on colon cancer cell prolif-eration and found that miR-22significantly inhibited cell viability. Specifically, theoptical absorbance rates of parental and NC cells were>2-fold higher than those ofmiR-22-transfected HCT-116cells (P<0.05). By contrast, there was no significantdifference between parental and NC control cells during the7days of culture. NC andmiR-22-expressing HCT-116cells were subjected to transwell migration and invasionassays. We found that the number of cells that migrated through the pores in miR-22-expressing cells was59±3.75, which was significantly lower than that of NC(81±4.65) or parental cells (78±3.97)(P<0.05; Fig.3A), suggesting that miR-22expression suppressed the migration of colon cancer cells. Similarly, the invasion assayshowed that the number of cells that invaded the Matrigel and migrated through thepore in the membrane was markedly lower in tumor cells expressing miR-22(29±1.75)compared to parental (42±2.65) or NC cells (38±2.32). Collectively, these resultssuggest that induction of miR-22expression significantly inhibits the migration andinvasion of colon cancer cells.We then verified our finding in miR-22-transfected HCT-116cells. As shown,levels of TIAM1mRNA in negative control shRNA-transfected HCT-116cells were8.05±0.64-fold higher than those of miR-22-transfected cells. Western blot analysisshowed that TIAM1protein expression was>4-fold higher in both the parental or NCcells than in miR-22-transfected cells. Immunocytochemistry confirmed our westernblotting data. Collectively, these data demonstrate that miR-22is able to suppressTIAM1mRNA and protein expression and, consequently, to contribute to thesuppression of colon cancer cell migration and invasion. Furthermore, we analyzedexpression of matrix MMP-2and MMP-9, which play critical roles in cancer cellinvasion and metastasis, as well as the expression of pro-angiogenic protein VEGF inthese cells. The expression of MMP-2, MMP-9and VEGF was well associated withmiR-22expression at both the transcriptional and translational levels. In particular, inparental and NC cells, the level of MMP-2mRNA expression was17.27±0.64and17.74±2.53-fold higher, respectively, than that in miR-22-transfected cells, while levelsof MMP-9mRNA in parental and NC cells were15.76±0.85and16.04±0.23-foldhigher, respectively, than those in miR-22-transfected cells. Similarly, VEGFexpression levels in parental and NC cells were12.14±0.61and11.78±0.82-fold higher,respectively, than those in miR-22-transfected cells. A similar trend was observed atthe protein level. Conclusions:Colon cancer cell proliferation ability significantly decline after transfectedmiR-22vector. Induction of miR-22expression significantly inhibits the migration andinvasion of colon cancer cells. In aerobic conditions microRNA22can also regulate thecolorectal cancer cells as a tumor suppressor. Tiam1is the target gene of microRNA22in colon cancer. Mir-22shows its antitumor effect by inhibiting the expression ofTiam1. Mir-22can inhibit VEGF expression to decrease colorectal tumor angiogenesis.Mir-22can inhibit MMP2and MMP9expression in HCT116colon cancer cells torestrain the invasion and migration of colon tumor cells.The innovation of this research: We firstly proved in this dissertation thatTiam1is a target gene of mir-22in colon cancer cells. Recent study havedemonstrated that in hypoxia condition mir-22could inhibit HIF-1a translation andexerts its tumor suppressor role in colorectal cancer cells.However, this studyconfirmed in oxygen condition mir-22can also inhibit colorectal cancer cells inproliferation, invasion and migration, through the regulation of Tiam1, VEGF, MMP2and MMP9. Moreover, the tumor suppressor effect which is shown by overexpressmicroRNA22gene, reveals that microRNA22has enormous potential in the therapyapplication.