| Background:It is well-known that colorectal cancer is one of the most common malignant tumor. In recent years due to the treatment that combined surgical resection, radiotherapy and chemotherapy, the5-year survival rate of colorectal cancer has been improved.But nearly50%patients eventually developed tumor metastasis which was the leading cause of death in these patients.Based on the limited role of traditional treatment in colorectal cancer,targeted therapies to colorectal cancer has emerged as the times require to improve efficacy and reduce drug toxicity.Epidermal growth factor receptor (EGFR) is one of the members of the family of receptor tyrosine kinase,and plays an important role in a variety of important signaling pathways. EGFR were abnormal expression in a variety of tumors such as head and neck cancer, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, etc. EGFR when binding to the ligand will trigger a variety of signaling pathways, including Ras/Raf-1/MAPK, PI3K/AKT which promote cell proliferation, cell cycle and cell anti-apoptosis. EGFR was overexpression in82%of colorectal cancer patients。Cetuximab,which is targeting the epidermal growth factor receptor (EGFR) monoclonal antibody,can inhibit cell growth, proliferation and metastasis. A study shows that cetuximab combined cytotoxic chemotherapy drugs can improve treatment efficacy on KRAS wild-type metastatic colorectal cancer patients. But cetuximab only applies to the wild type of KRAS colorectal cancer patients. However there are still40%of the KRAS wild-type patients with colorectal cancer which cetuximab is invalid or the resistance. So exploring new biomarkers for early diagnosis and target molecular for treatment in colorectal cancer is imminent.MicroRNAs (miRNAs) which is a kind of endogenous non-coding small single-stranded RNA molecules negatively regulate gene expression through targeting3’-UTR of mRNA.MiRNAs play an important physiological role in many biological processes including cell cycle, differentiation, proliferation, apoptosis, or aggressive.Many miRNAs act as oncogenes and tumor suppressor genes and play an importment role in the process of tumor formation,and also affect the efficacy on the antitumor treatment. So, MicroRNAs could become a powerful drugs or therapeutic targets.It is reported that miR-21is overexpressed in a variety of malignant tumors, including esophageal cancer, breast cancer, cancer of the stomach, bile duct cancer, liver cancer, pancreatic cancer, colorectal cancer, ovarian cancer, etc. So miR-21is considered to be carcinogenic micrornas. Studies have found that miR-21promotes the formation of tumors through mediating EGF/RAS signaling pathway. MiR-21involved in many biological processes, such as cell proliferation, apoptosis, invasion and metastasis, and also played an important role in the aspect of anti-tumor drug resistance by mediating RAS signaling pathway. Therefore we will explore the effects of miR-21on colon cancer cell biological function and cetuximab drug sensitivityAim:By constructing up-and down-regulation of miR-21lentiviral vectors and transfecting RKO colon cancer cell, detecting the effects of up/down regulation of miR-21on colon cancer cell biological function and cetuximab drug sensitivity. And then exploring the role of miR-21in the development of colon cancer and possible role in drug resistance. It will provide new ideas for further elucidating molecular mechanisms in the development of colon cancer and provide the theory basis for early diagnosis and treatment of new targets. Method:1.The upstream and downstream primers of hsa-miR-21and hsa-miR-21-inhibition was synthesized. Target fragment of double-stranded DNA was obtained by primer annealing. Target fragment and vectors were taken to proceed enzyme digestion reaction by EcoRI or AgeI/EcoRI,for37℃1h. The product proceeded gel electrophoresis. The fragment and vector which had preceeded enzyme digestion reaction were recycled by gel recovery kit(TIANGEN BIOTECH Co.Ltd): Collect and dissolve the single purpose band of agarose gel for50℃10min,then the solution was transfered to the adsorption column CA2, and was centrifuged, then abandoned waste liquid.Then the eluent was join in to elute DNA. ligase detection reaction by using T4DNA Ligase was proceeded for22℃,1h. Transformating fresh competent cells e. coli cells DH-5a and connection product: Ice for30min,42℃90seconds for heat shock, and then rapidly cooled in the ice for3-5min. The cells adding LB liquid medium were put in37℃water for30min, in order to make sure the bacteria returned to normal growth state.After the transformation is done,we took some product to culture16-24h in37℃.We picked dental plaque as the template to perform PCR reaction:95℃3min—(94℃30s—60℃30s—72℃30s) x30cycles—72℃6min.We then picked PCR products of positive colony as a template to undergo sequence with BigDyeTerminator v3.1kit. Take expression plasmid, packaging plasmid and POLOdelivererTM3000Transfection Reagent to transfect293T cells for lentivirus packaging. The supernatant fluid of293T cells which was collected was centrifuge to concentrated virus solution. Gradient dilution method is used to determine the virus drops. The purpose cell RKO cell were transfected by lentivirus transfection method. The expression of fluorescent was observed by the inverted fluorescence microscope. And to preliminarily estimate the transfection efficiency.2. The cell of RKO was purchased from Shanghai cell bank. The culture condition:MEM-a that contained10%of Australian fetal bovine serum at37℃under an atmosphere which contains5%CO2for sterility. The total RNA of RKO cells which growed in good condition and in logarithmic growth phase,which were transfected with hsa-miR-21、hsa-miR-21-inhibition and negative control, were extracted by Trizol(Life Technologies). The highly purified RNA was chosen as template to synthesized cDNA by reverse transcription. Reaction conditions:RNA degeneration:70℃,10min;2min on ice;cDNA:42℃,60min;70℃,10min. After a cycle, we started fluorescence quantitative PCR reaction.Each sample was examined in quadruplicate and the amount of product was normalized relative to that of U6. Samples were underwent amplification and melting curve analysis by quantitative PCR instrument at the same time under the optimum reaction conditions. PCR reaction conditions:95℃30s—(95℃10s—60℃20s—72℃30s)×45cycles. We confirmed the Real Time PCR amplification curve and melting curve at the end of the reaction,and got the CT value. Quantitative values were calculated according to the flowing formula: Quantitative values=2-ΔΔCT, in which the ACT value for each cell was calculated by subtracting the average CT value of the target gene from average CT value for the U6gene. AACT value was calculated by subtracting the ACT value of experimental group from ACT value of the control group.3. RKO cells which growed in good condition and in logarithmic growth phase,which were transfected with hsa-miR-21、hsa-miR-21-inhibition and negative control were digested by trypsin enzyme and then counted. Cells that up/downregulated miR-21, negative control cells, and control cells were seeded (1×104cells,100μL per well) in a96-well plate and incubated at37℃under an atmosphere which contains5%CO2for sterility for24h. As a parallel test, a blank control group containing only medium without cells was established. Each sample was analyzed six times. All the cells were seeded in four plates each to be incubated for24h,48h,72h, and96h, respectively. At the end of each incubation period, MTT solution (5mg/mL,20μl/well) was added to the plates and incubated at37℃for4h, and then the medium supernatant was carefully discarded and replaced with150μl of dimethylsulfoxide. After shaking for10min, the optical density (OD) value was determined at490nm.4. Sterile low-melting point agarose of1.2%and0.7%was prepared and maintained in a dissolved state at40℃. Two milliliters per well of a mixture containing1.2%agarose and2×complete medium at a ratio of1:1was added in6-well plates for solidification. Next, a mixture containing0.7%agarose and2X complete medium at a ratio of1:1was mixed in the sterile tubes.RKO cells that up/downregulated miR-21, negative control cells, and blank control cells were digested by trypsin enzyme and then counted,and made into cell suspensions of10,000cells/mL. One milliliter of the mixture and0.1mL of the single-cell suspension (1,000cells/well) were poured into the above-mentioned6-well plate and incubated at room temperature for20min, followed by37℃for10days. Clone formation was then assessed by observation and counting.5.Proteins were extracted from RKO cells that up/do wnregulated miR-21, negative control cells, and blank control cells by RIPA. The extracted proteins which contained loading buffer were degenerated for5-10min at95-100℃water. Samples of the total protein lysates (10ul) and Marker were underwent SDS-PAGE electrophoresis to isolate protein:60V for30min;120v60min. Then proteins were transferred to PVDF membranes at300mA for3h. The PVDF menbranes were closed by skim milk powder solution. Polyclonal anti-AKT(1:1000,CST), anti-p-AKT(1:2000,CST),anti-Raf-1(1:1000, Abcam) and anti-p-actin (1:2000, Abcam) antibodies were used as primary detection reagentst at4℃for overnight.After membrane rinsing, the horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2000,CST) was used as the secondary detection reagent for1h. After membrane rinsing, immunoreactive proteins were visualized using an enhanced chemiluminescence (ECL, Invitrogen, USA) system. Then the film were subjected to expose, develop, fix and scan for the analysis of band density.6. Cells that up/downregulated miR-21, negative control cells, and control cells were digested by trypsin enzyme and counted, then seeded (1×104cells per well) in a96-well plate and incubated at37℃for24h. Cells were then incubated with300μg/mL of cetuximab for24h. Each sample was analyzed in triplicate. After discarding the medium,100μl of serum-free medium and10μl of CCK-8were added to each well, and the mixtures were incubated at37℃for0.5-4h. The OD value at450nm per0.5h was determined until the OD value of the wells did not contain cetuximab (at OD values of1.0to1.2).7.Statistical analysisThe description of the continuous variable in normal distribution was used (x±s).The statistical significance of the differences among different transfected groups in cell growth were assessed by the repearted measures. The statistical significance of the differences among different transfected groups in colony formation were assessed by the one-way ANOVA. The statistical significance of the differences among different transfected groups in drug sensitivity were assessed by the one-way ANOVA. When the homogeneity of variance was meeted,the difference between groups were calcμLated by LSD test. When the homogeneity of variance was not meeted,the difference between groups were calculated by Dunnett T3test.All P-values<0.05were deemed to be statistically significant. All statistical tests were two-sided. The SPSS13.0software was used for the statistical analysis.Result:1. The construction, packaging and transfection of hsa-miR-21, hsa-miR-21-inhibition lentivirus vector.We constructed hsa-miR-21, hsa-miR-21-inhibition lentivirus vector by enzyme digestion, agarose gel electrophoresis(AEG), purification, links, transformation of DH-5a cells and coated plate, PCR identification.Fnally we chosed positive clone to detect sequence identification, the result showed that the result of sequence identification is consistent with the DNA sequence that experiment was required. Then package virus. Lentivirus interference plasmids were transfected into293T cells, and recombinant lentivirus particles were generated by packaging. More than90%of the cells showed green fluorescence. The lentivirus titers of LV-miR-21、 LV-anti-miR-21、LV-miR-21NC、LV-anti-miR-21NC cells were3.0E+9TU/mL、2.0E+9TU/mL、6.0E+8TU/mL、8.0E+8TU/mL respectively. RKO colon cancer cells were infected with the virus particles, and the infection efficiencies were over80%.2. Expression of miR-21in RKO colon cancer cells transfected with different lentiviral vectors The expression of miR-21was significantly different among the LV-miR-21, LV-anti-miR-21and control groups (P<0.05).The relative expression of miR-21in LV-miR-21、LV-anti-miR-21cells were significantly different from negative control groups and control group(P<0.05).The expression of miR-21in LV-miR-21cells was higher than that in LV-anti-miR-21cells(P<0.05).There was no statistically significant difference between the negative control groups and the blank control group(P>0.05)3. The changes in the cell proliferation ability among human colon cancer cells transfected with different lentiviral vectorsThe cell proliferation ability was significantly different among the LV-miR-21, LV-anti-miR-21and control groups at24h、48h、72h、96h(P<0.01).Over time, the cell proliferation capacity was decreased in the LV-anti-miR-21group and increased in the LV-miR-21group.4. The clone-forming ability of cells transfected with different lentiviral vectorsThe clone-forming ability was significantly different among the LV-miR-21, LV-anti-miR-21, and control groups (P<0.05).The clone-forming ability of the LV-miR-21group was significantly higher than that of the LV-miR-21NC, control, and LV-anti-miR-21groups (P<0.05). By contrast, the clone-forming ability of the LV-anti-miR-21group was lower than that of the negative control and control groups(P<0.05).5. The expression of protein in signaling pathways downstream in cells transfected with different lentiviral vectors.The expression of p-AKT、Raf-1was significantly different among the LV-miR-21, LV-anti-miR-21, and control groups (P<0.05).The expression of p-AKT、 Raf-1in LV-miR-21group was higher than that in LV-anti-miR-21group(P<0.05).6. DownregμLation of miR-21enhanced the sensitivity of the targeted therapy drug cetuximabCCK8assay results showed that the cell-inhibition rate of the LV-anti-miR-21group was higher than that of LV-miR-21group at300μg/mL of cetuximab, and the difference was statistically significant (P<0.05). There was no statistically significant difference between the negative control group and the blank control group(P>0.05)Conclusion:Downregulation of miR-21can inhibited the activation of the RAS signaling pathways, inhibited the cell proliferation and clone forming ability of colon cancer cells, and enhance the sensitivity to cetuximab。Mir-21may play an important role in the development of colon cancer and drug resistance by activating the RAS signaling pathways... |
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