The Inhibititions Of The Growth And The Metastasis Of Human Hepatocellular Carcinoma Cells Via RNA Interfering Respectively Targeting To HTERT And ATP6L

The two main characteristics of tumor cells are uncontrolled proliferation (immortalization) and aggressive behavior.Telomerase is a major contributing factor of immortalization of cells and therefore is a potential target to suppress tumor.Extracellular pH is usually low in metastastic solid tumors,mainly resulted from abnormally active hydrogen pump V-ATPase on plasma membrane.In acidic conditions,activity of proteases that digest extracellular matrix(ECM) are up-regulated,benefiting invasion or metastasis.In our experiments,in order to suppress the growth and metastasis of HCCLM3,a human hepatocellular carcinoma cell(HCC) line with highly metastatic potential, DNA vector-based small interfering RNA was used respectively to knock down hTERT,the subunit of telomerase and ATP6L,the subunit of V-ATPase.siRNA-expressing vector targeting hTERT was constructed and transfected into HCCLM3.Western blot demonstrated that hTERT was inhibited.Assayed by TRAP and ELISA,the activity of telomerase was decreased in transformed cells.MTI cell proliferation curve showed that the proliferation of HCCLM3 with hTERT knockdown was inhibited.HCCLM3 was transfected siRNA-expressing vector targeting ATP6L,screened by G418 and designated as si-A-M3.ATP6L in si-A-M3 was knocked down by～60%, assayed by northern blot.The ability of proton extrusion of si-A-M3 was analyzed by determining the alteration of extracellular pH(pH of culture media) and intracellular pH(pHi) using pH-sensitive fluorescent dyes BCECF/BCECF-AM,real-time monitored on a luminescence spectrometer. The results showed:â‘ The extrusion of proton by long-term(12 hr) cultured si-A-M3 was notably reduced.â‘¡The pHi of si-A-M3 was only marginally recovered from NH₄Cl-prepulsed acidification,contrasting to the full recovery to the resting pHi of the control.The rate of the pHi recovery of si-A-M3 was significantly dropped.^In si-A-M3,at the initial pHi recovery point,the H⁺ effiux(JH+),corrected by intrinsic buffering capacity,was significantly decreased.The above results indicate the inhibition of the activity of V-ATPase in si-A-M3.The secretion and activation of ECM-degenerating proteases are pH-regulated.The expression of matrix metalloproteinase-2 and the gelatinase activity were affected,concomitant with the inhibition of V-ATPase.Western blot showed that the expression of MMP-2 was down-regulated in si-A-M3. The activity of gelatinase of the supernatant of cultured si-A-M3 cells was apparently reduced,further confirmed by zymography of the supernatant and in situ zymography of si-A-M3 xenografts on mice by using FITC-labeled gelatin.In vitro,the invasion ratio of si--A-M3 was decreased remarkably.On the animal model in vivo,the sizes of the xenografts of si-A-M3 implantated into the livers in BalB/c(nu+/nu+) mice were apparently decreased. Intrahepatic and pulmonary metastasis of si-A-M3 group were respectively strikingly decreased.Taken together,The results suggest that the inhibition of V-ATPase function via knockdown of ATP6L expression using RNA interfering technology effectively retarded the cancer growth and suppressed the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity.In conclusion,our experiments confirmed hTERT of telomerase was an effective target to inhibit the proliferation of HCC.As for a target to inhibit the metastasis as well as the growth of HCC,ATP6L was a good candidate.So far as we could know,our experiments firstly reported that the knockdown of ATP6L expression using siRNA technology suppressed the growth and metastasis of HCC.Our results implicate possible mechanisms of carcinogenisis and metastasis of tumor,and furthermore,the potential for cancer treatment.