| Objective: Ghrelin is an endogenous ligand of growth hormone secretion receptor.Ghrelin is mainly produced by endocrine cells of gastric mucosa,and has many physiological functions,such as promoting metabolism,inducing ingestion,promoting gastric motility.In addition,ghrelin also participates in many physiological processes related to tumor development,such as promoting the proliferation and metastasis of cancer cells,and inhibiting cancer cell apoptosis.Thyroid cancer(TC)is one of the most common head and neck cancers.Studies have shown that there are a large number of ghrelin receptor expression in medullary thyroid cancer.Can ghrelin promote the proliferation of medullary thyroid cancer cells? Is ghrelin related to the development of medullary thyroid cancer? Although there have been a lot of studies on ghrelin promoting cell proliferation,there are few studies on the mechanism of ghrelin promoting cell proliferation,especially on the mechanism of ghrelin promoting thyroid medullary carcinoma cell proliferation.Therefore,in this study,we will observe the effect of ghrelin on the proliferation of medullary thyroid cancer cells or the growth of transplanted tumor in vitro and in vivo,and the potential mechanism,so as to provide new ideas and credible experimental basis for the prevention and treatment of thyroid cancer.Methods: 1.In vitro cell research:(1)The effect of ghrelin on the proliferation of medullary thyroid cancer cells: Use cell culture to observe the effect of ghrelin,LY294002,ghrelin + LY294002,PD98059,ghrelin + PD98059 on the proliferation of TT cells.(2)The effect of down regulated GHSR expression on the proliferation of TT cells: TT cells were transfected with GHSR si RNA or si RNA,then given 0.1ml 5 n M ghrelin,and the effect of down regulated ghrelin on the proliferation of TT cells was observed.(3)The effect of ghrelin on cell proliferation and the signal pathway related to cell proliferation: Western blot was used to observe the expression of Akt,p-Akt,ERK1/2,pERK1/2,PI3 K,p-PI3 K,m TOR and p-m TOR protein in TT cells after 5 n M ghrelin for 72 h.2.In vivo study:(1)TT cell transplantation tumor inoculation: 4-5 weeks BALB/c nude mice,were injected subcutaneously with TT cells on the right back.(2)The effect of ghrelin on the growth of TT cell transplanted tumor and its potential mechanism;after subcutaneous injection of tumor cells in mice for 20 days,5 ?g of GHSR si RNA/50 ?l,5 ?g of si RNA/50 ?l or 5 n M of ghrelin/50 ?l,5 ?g of GHSR si RNA + 5 n M of ghrelin/50 ?l or(5 ?g of si RNA + 5 n M of ghrelin)/50 ?l were injected into the tumor tissues,while in the control group,only 50 ?l NS were injected continuously.Inject for 20 days,once every 5 days.At the end of the experiment,the mice were killed,the tumor tissues were measured and weighed.Western blotting were used to study the effect of ghrelin and GHSR si RNA on the expression of Akt,p-Akt,ERK1 / 2,p-ERK1 / 2,PI3 K,p-pi3 k,m TOR,p-m TOR protein and m RNA.Results: 1.Ghrelin promotes the proliferation of medullary thyroid cancer cells and its mechanism(in vitro experiment)(1)The effect of ghrelin on the proliferation of TT cells was studied.The results showed that TT cells increased significantly after incubation with different concentrations of ghrelin(0.1 n M,1n M,5n M,10 n M)/0.1ml,which was dose-dependent(P < 0.05-0.01).The effect of 5 n M ghrelin should be the largest,so in the follow-up study,5 n M was used as the study dose of ghrelin.(2)The effect of down-regulation of ghrelin on the proliferation of TT cells showed that the expression of GHSR protein in TT cells decreased significantly after transfection of GHSR si RNA for 24 h(P < 0.05);at this time the effect of ghrelin on the proliferation of TT cells was significantly weaker than that in si RNA + ghrelin group(P < 0.05).However,the effect of ghrelin on the proliferation of TT cells could not be changed significantly after pre incubation with GHSR si RNA(P > 0.05).There was no significant difference in the proliferation of TT cells(P > 0.05).(3)The effect of LY294002 on the proliferation of TT cells induced by ghrelin showed that PI3K/Akt signaling pathway plays an important role in cell growth,proliferation and migration.In this study,the effect of ghrelin on the proliferation of TT cells was partially blocked when the TT cells were incubated with LY294002,a PI3K/Akt signal pathway inhibitor(P < 0.05);if LY294002 was given alone,the proliferation of TT cells did not change significantly(P > 0.05).(4)The effect of rapamycin on the proliferation of TT cells induced by ghrelin showed that m TOR,as a downstream effector of PI3K/Akt signaling pathway,plays an important role in the proliferation of a variety of cancer cells.The results showed that the effect of ghrelin on the proliferation of TT cells could be partially blocked after incubation with rapamycin,an inhibitor of m TOR(P < 0.05);if rapamycin was given alone,the proliferation of TT cells had no significant change(P > 0.05).(5)The effect of PD98059 on the proliferation of TT cells induced by ghrelin showed that Ras/Raf/ERK signaling pathway is another important pathway involved in the regulation of cell growth,proliferation and migration.The effect of ghrelin on the proliferation of TT cells was partially inhibited(P < 0.05)by incubation of TT cells with ERK1/2 signal pathway inhibitor PD98059 in advance;if PD98059 was added alone,the proliferation of TT cells was not significantly affected(P > 0.05).LY294002,rapamycin and PD98059 had no significant difference in the inhibitory effect of ghrelin on the proliferation of TT cells(P > 0.05).It is suggested that PI3K/Akt/m TOR or Ras/Raf /ERK signaling pathway is involved in the proliferation of TT cells promoted by ghrelin,and these two signaling pathways have similar effects in the process of ghrelin promoting the proliferation of TT cells.(6)The effect of ghrelin on the activation of PI3K/Akt/m TOR or Ras/Raf /ERK signaling pathway in TT cells showed that the expression of Akt,ERK1/2,PI3 K and m TOR protein in TT cells did not change significantly after incubation with ghrelin(P > 0.05),but the expression of p-Akt,p-ERK1/,p-PI3 K and p-m TOR protein increased significantly(P < 0.05).2.Ghrelin promotes the growth of TT cell transplantation tumor and its potential mechanism(in vivo experiment)(1)The effect of GHSR si RNA on GHSR protein show the expression of GHSR protein in TT cell transplanted tumor showed that the injection of GHSR si RNA into the transplanted tumor could significantly reduce the expression of GHSR protein in TT cell transplanted tumor(P < 0.05).However,microinjection of si RNA into the transplanted tumor had no significant effect on the expression of GHSR(P > 0.05).(2)The effect of ghrelin on the growth of TT cell transplanted tumor showed that the tumor volume and tumor weight increased significantly(P < 0.05)when ghrelin was injected into the transplanted tumor,and decreased significantly(P < 0.05)when GHSR si RNA was injected in advance.(3)The effect of ghrelin on the expression of signal pathway protein related to the growth of TT cell transplantation tumor showed that the expression of p-Akt(P < 0.05),p-ERK1/2(P < 0.05),p-PI3k(P < 0.05)or p-m TOR(P < 0.05)protein and its m RNA in the tumor tissue were significantly increased after intratumoral injection of ghrelin.Further study showed that ghrelin could significantly reduce the expression of p-Akt(P < 0.05),p- ERK1/2(P < 0.05),p-PI3k(P < 0.05)and p-m TOR(P < 0.05)protein by pre injection of GHSR si RNA into the transplanted tumor.It was also found that injection of GHSR si RNA into the transplanted tumor significantly reduced the tumor volume(P < 0.05),tumor weight(P < 0.05),and protein expression of p-Akt(P < 0.05),p-ERK1/2(P < 0.05),p-PI3k(P < 0.05)and p-m TOR(P < 0.05).Conclusion: Ghrelin can promote the proliferation of tumor cells by activating the GHSR signaling pathway,and the PI3K/Akt/m TOR signaling pathway and ERK1/2 signaling pathway may also participate in the regulation of ghrelin's effect on tumor proliferation. |
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