1, RTN4B Pentapeptide ( ₁₇₈ 182 ) Inhibition Of Angiogenesis In Hepatocellular Carcinoma 2, And The Application Of Betaine To Increase The High GC Content Of The Trinucleotide Repeat Sequence DNA Fragment Sequencing Identification The Be

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Annually newly reported HCC cases and death case are0.74and0.7million, half of them emerged in China, especially in the southeast coast region. Surgical resection is the only effective method to cure HCC currently, however5-year survival prognosis is rare and recurrence rate is high.Proliferation of capillary differ the hepatocellular carcinoma tissue from normal tissue. It is the result for the cancer cells to modulate angiogenesis by promoting proliferation and migration in endothelial cells in response to hypoxia condition.Nogo-B (RTN4B) was cloned by our research group in1998, which is a member of reticulon (RTN) protein family localizing in the endoplasmic reticulum. Lisette Acevedo reported in Nature Medicine that amino terminal of Nogo-B played an important role in angiopoiesis in2004.2years later he papered on PNAS that NgBR-the specific receptor of amino terminal of Nogo-B-played an important role in promoting vessel endothelial cells chemotaxis and migration. Besides, Yasuko Iwakiri reported in Hepatology that amino terminal of Nogo-B could promote the mice cirrhosis, the cirrhosis is the intermediate link between hepatitis and HCC. Taken together, Nogo-B is closely related with angiopoiesis during the process of HCC. Clarification the mechanism of Nogo-B promoting the angiopoiesis during the process of HCC is of great significance to proposing the new potential therapeutic targets and declining the rate of recurrence.Previous work has found that:(1) RTN4B is universally expressed in15kinds normal tissues except liver but remarkable highly expressed in hepatocellular carcinoma tissues;(2) over-expression of RTN4B in hepatoma cells accelerate both tumor formation and angiopooesis in tumor;(3) knockdown of endogenous RTN4B could inhibit both tumor formation and angiopoiesis in tumor;(4) The observation experiment confirmed that RTN4B’s pro-angiogenic activity was Integrinαvβ3dependent, ELISA assay revealed that RTN4B directly bind to lntegrinαvβ3. So Integrinαvβ3is likely to be a functional receptor of RTN4B.Most Integrin interact with extracellular matrix and molecules by the RGD tripeptide complex. But we do not find the RGD tripeptide complex in RTN4B. And we find the RRGSS pentapeptide from the178th to182th of RTN4B, which has structural similarity with RGD tripeptide, hence we conjecture that the RRGSS pentapeptide could be the interact region with lntegrinαvβ3. After chemical synthesis of the RRGSS pentapeptide,we found that it could competitive bind Integrinαvβ3against RTN4B and inhibit the adhesion of RTN4B to vascular endothelial cell in vitro and inhibit both tumor formation and angiopoiesis in tumor in vivo. The RRGSS pentapeitide has a real potential to be an anti-cancer drug. To investigate the optimum concentration of betaine to improve the resolution of sequencing result of G-C rich DNA with abundant trinucleotide repeats by applying concentration gradients of betaine into Sanger sequencing system. Concentration gradients of betaine were introduced into the sequencing system by taking the5’ terminal of Nogo-B cDNA (Am-Nogo-B) whose G-C%was72%and was without trinucleotide repeats and the5’ terminal of Huntingtin cDNA (Am-HTT) whose G-C%was74%and was abundant of CAG/CCG repeats for examples. Compared the sequencing results’ resolution by length, quality and spica. The optimum concentration of betaine for sequencing Am-Nogo-B differed obviously from that for sequencing the CAG/CCG repeats of Huntingtin. Result of sequencing Am-Nogo-B obtained the best quality when the concentration of betaine was between0.8M to1.2M whereas the result of sequencing Am-HTT obtained the best quality when the concentration of betaine was between1.6M to2.4M. These results were reproducible. G-C rich DNA of similar G-C%required different concentrations of betaine in sequencing system due to different base pair compositions. Betaine of0.8M to1.2M is suitable for improving the resolution of sequencing result of G-C rich DNA without trinucleotide repeats whereas betaine of1.6M to2.4M is necessary to improve the resolution of sequencing result of G-C rich DNA abundant in trinucleotide repeats. The concentration of betaine investigated in this study to improve the resolution of sequencing result of G-C rich DNA with abundant trinucleotide repeats could be as reference for the analogous cases.