Effect Of RNAi-mediated Knockdown RPL34 Genne On Proliferation And Cell Cycle Of Gastric Cancer SGC-7901 Cells

Objective: Gastric cancer is one of the most common gastroinestinal cancers.The occurrence and development of gastric cancer is a complicated process involving multiple genes.RPL34 gene plays an important role in the occurrence and development of a variety of malignant tumors,which involves in the regulation of differentiation,proliferation and apoptosis of tumor cells.This study constructed a using lentivirus vector mediated RNA interference technology to knockdown RPL34 gene,seting the negative control group and interference group,to detect interference effect of two groups of cells by real time PCR technology,to detect cell proliferation rate by MTT,and to measure cell cycle distribution with flow cytometer.Through this experiment lays the foundation for the further study of RPL34 gene therapy on tumor gene,and provide a new strategy for the treatment of gastric cancer.Methods:1.Lentivirus infect gastric cancer SGC-7901 cells The gastric cancer SGC-7901 cells were divided into two groups.The experimental group and negative control group were transfected by RPL34-si RNA lentiviral vector and si RNA inegative control lentiviral vector,respectively.Before infection,the gastric cancer cells were inoculated in 6 well plates and cultured 24 h,adding 20 ul lentiviral concentrate,after 72 h,observed the expression of GFP under a fluorescence microscope and calculated the infection efficiency.2.To detect RPL34 gene expression by using Real-time PCR Trizol method was used to extract cell RNA,using Oligo(d T)primers and random primers for reverse transcription,with each group of cells c DNA as template,each set 3complex holes for Real-time quantitative PCR detection.3.MTT method detect the cell proliferation rate of RPL34 gene Take 2 groups of logarithmic growth phase cells were seeded in 96 well plates,each set three complex hole,after the inoculation of 1,2,3,4 and 5 days,per hole was added10 ul of 5g / L MTT solution respectively,discard the supernatant for 4 hours of culture,adding DMSO 100 ul to each hole,crystallization fully dissolved,enzyme mark instrument in the wavelength of 490 nm absorbance(A)value,record results,and draw the cell growth curve.4.To detect cell cycle with flow cytometry Trypsin digest logarithmic growth phase cells which was infected by lentivirus,the cells completely decentralized variable to a single cell,with precooling of PBS washing cells 3times,15000 rpm centrifuge 10 min and then collect cells,supernatant,add 1ml precolding PBS cell suspension was accession PI(1:100)and RNase(1:100)to avoid light ice bath for 10 min,then the cell cycle was detected by flow cytometry.Results: The concentration of the recombinant lentiviral infection of human gastric cancer cell line SGC-7901,after 48 h under fluorescence microscope observed green fluorescence cells expressed higher level,the cells were in good condition,indicating that lentivirus has a stable infection in gastric cancer cells.Real time fluorescence quantitative PCR detection showed that compared with negative control group,the expression level of RPL34 in the experimental group was significantly inhibited(P<0.05),suggesting that RPL34-si RNA can effectively silence RPL34 gene in human gastric cancer cell line SGC-7901.The MTT experimental results suggest that comparing with the control group, the overall growth rate of the experimental group is reduced(P<0.05),suggesting that targeted inhibition of RPL34 gene expression can significantly reduce gastric cancer cell proliferation.To detect the cell cycle experiments by flow cytometry indicated that the experimental group of cells in S phase decreased significantly(P < 0.05),while the G2/M phase cells increased(P<0.05),indicating that downregulation RPL34 gene expression,cell mitosis slows significantly,cell proliferation was inhibited and apoptosis.Conclusion:1.Lentivirus mediated RNAi can effectively down expression of RPL34 gene.2.After inhibiting the expression of RPL34 gene,the proliferation rate and mitosis of gastric cancer SGC-7901 cell were significantly slowered..3.RPL34 gene plays an important role in maintaining the biological function of human gastric cancer cells and may offer new ideal for gene target treatment of gastric cancer.