The Experimental Research Of CLN3-siRNA On The Growth And Apoptosis Of HCT116Cells

Objiective The aim of present study is to employ the sequence-sepcific siRNAsilencing the CLN3gene and investigate its growth and apoptosis on colorectal cancer lineHCT116,which provides theoretical basis and experimental basis for the gene therapy ofcolorectal cancer.Methods1,Structure of CLN3mRNA was analysed by software to select targetedinterference sites. The CLN3-siRNA was transfected into colorectal cancer line HCT116by positive ion liposome Lipofectamine2000.Transfect efficiency was assessed by flowcytometry.2, After human colon cancer cell line HCT116was transfected by CLN3small interfering RNA (siRNA)，the mRNA and protein of CLN3was determined byRT-PCR and Western blot assay respectively.3, Testing the cell growth inhibition byMTT after transfecting cell line HCT116by CLN3small interfering RNA (siRNA)24h,48h and72h.4, Apoptosis and cell cycle were measured by flow cytometry after48h whenhuman colon cancer cell line HCT116was transfected by CLN3small interfering RNA(siRNA).Results1Negative control FAM-siRNA was successfully transfected into humancolorectal cancer cell line HCT116.The transfection efficiency was about93.9%detected by FCM with cell density at1.0×105/well and siRNA concentration with80pmol in6well play.2, After CLN3-siRNA were successfully transfected into HCT116cell24h, detected by RT-PCR，CLN3mRNA levels in cells was significantly inhibited.Contrasted with negative control and blank control, the transfected control was statisticallysignificant difference (p﹤0.05), but negative control and blank control was no significantdifference of statistical significance (p﹥0.05). Contrasted with negative control and blank control, the expression of CLN3protein was obviously decreased48h after-siRNAtransfection. Contrasted with negative control and blank control, the transfected controlwas statistically significant difference (p﹤0.05), but negative control and blank controlwas no significant difference of statistical significance (p﹥0.05).3, CLN3-siRNAcould effectively inhibit the growth of HCTl16cells.4, CLN3-siRNA could accelerateapoptosis in vitro but it could not affect cell cycle.Conclution1.CLN3-siRNA is successfully constructed and efficiently transfectedinto HCT116cells. CLN3-siRNA can effectively silence the overexpression of CLN3incolon cancer cell lines HCT116.2.Using CLN3-siRNA transfection could inhibit thegrowth of HCT116cells3. Using CLN3-siRNA transfection could accelerate HCT116cells apoptosis,but it could not change cell cycle.