肿瘤抑制基因ARHI的生物学功能及研究进展

肿瘤是一种基因病，抑癌基因在抵御肿瘤发生、发展中起着重要作用。ARHI(aplysia ras homolog I)／NOEY2是一个新发现的抑癌基因．是ras/rap超家族成员之一．与ras家族有50％～60％的同源性．位于人染色体1p31，属小GTP结合蛋白，是该家族第1个被报道的肿瘤抑制基因。ARHI基因编码的蛋白在人类多种组织表达，其中正常卵巢的ARHI表达最高。ARHI是一个印迹基因．印迹机制可能与其CpG岛的差异甲基化有关。ARHI参与细胞周期调控可能作用于cyclin D1，使其不能与CDK结合形成活性激酶，从而使细胞停止于G1期。ARHI可能通过依赖caspase和calpain两条途径参与信号通路传导诱发细胞凋亡。该基因的异常表达跟多种肿瘤的发生、发展有关。ARHI基因参与了乳腺癌的发生和发展，该基因的表达缺失可能与乳腺癌的转移机制有关。卵巢癌、乳腺癌存在广泛的1p31缺失，其中ARHI基因是最常见的一个缺失区域。ARHI基因和蛋白在胰腺癌组织中有较高比例的缺失，提示该基因和蛋白在胰腺癌的发生中起一定作用。ARHI基因在膀胱癌、肝癌、前列腺癌等其他肿瘤中也有不同程度的表达异常。     

抑癌基因ARHI研究进展

ARHI（aplasia ras homologue member I）/NOEY2是1999年新发现的一个母源性抑癌印迹基因,位于人染色体1p31,编码一个相对分子量为26kD的小GTP结合蛋白。ARHI属ras/rap超家族成员,与该家族成员有50%～60%的同源性并且两者具有相似的GTP/GDP结合域,但与该家族其它成员不同,ARHI发挥抑癌基因作用,是该家族第一个被报道的肿瘤抑制基因。ARHI基因编码的蛋白在人类多种组织表达,而该基因在人卵巢癌、乳腺癌、胰腺癌、肝癌等多种肿瘤中表达下调或缺失,提示其与上述肿瘤的发生、发展密切相关。ARHI可能通过作用于cyclin D1,使其不能与CDK结合形成活性激酶,从而使细胞停止于G1期来参与细胞周期调控;可能通过依赖caspase和calpain两条途径参与信号通路传导诱发细胞凋亡;另外,该基因可通过抑制STAT3的激活而发挥抑癌基因功能,也可以调节自体吞噬和肿瘤细胞休眠。目前研究显示,ARHI基因的表达缺失主要通过遗传事件和表观遗传学机制发生,包括DNA甲基化异常、杂合性丢失,乙酰化组蛋白的低水平表达及基因突变有关,但有待进一步深入研究。可以预见,ARHI基因的深入研究必将为早期肿瘤的基因诊治提供新的思路和理论依据。      

ARHI在肿瘤发生和发展中的作用与机制研究进展

ARHI是1999年发现的母源性印迹基因,属于小G蛋白Ras超家族,是该家族第1个被报道的抑癌基因。ARHI的功能包括抑制小鼠生长和生殖发育,抑制细胞增殖、迁移,参与周期调控和凋亡,近年来研究发现ARHI还具有调控肿瘤细胞自噬、抑制肿瘤转移的作用。ARHI蛋白在人体卵巢、乳腺等多种组织中表达,在卵巢癌、乳腺癌、甲状腺癌、胰腺癌、肝癌组织中表达下调,ARHI基因高表达可提示卵巢癌、胰腺癌的预后良好。ARHI表达调控机制包括杂合性丢失、DNA甲基化、组蛋白去乙酰化、转录因子调节、miRNA和突变,应用表观遗传学技术调节ARHI表达可能在一些肿瘤中具有抗肿瘤临床应用价值。ARHI基因参与调节肿瘤细胞休眠过程,有望成为诱导肿瘤细胞休眠、抗肿瘤转移复发的关键点。     

抑癌基因ARHI与全身各系统肿瘤相关性及机制的研究进展

抑癌基因 ARHI（aplasia ras homologue I）是1999年新发现的母源性印迹基因。最近研究发现ARHI 具有抑制肿瘤细胞增殖、转移、侵袭,调控肿瘤细胞自噬等作用。另外,学者们还发现 ARHI 不仅与卵巢癌、乳腺癌、胰腺癌密切相关,而且还与其他生殖、消化、内分泌等全身各系统肿瘤具有相关性。本文就最近几年国内外对 ARHI 与全身各系统肿瘤的相关性及其机制的最新研究进展进行综述。     

ARHI基因与肿瘤的关系

近年来研究表明，一种新的抑癌印迹基因ARHI(NOEY2)的异常参与了一些肿瘤的发生。该文就ARHI(NOEY2)的结构、功能及其与某些肿瘤的关系进行综述。     

新型肺癌候选抑癌基因RASSF1A研究进展

Ras相关区域家族1A(RASSF1A)是最近从3p1.3上发现的新型候选抑癌基因,在肺癌、乳腺癌和结肠癌等十几种恶性肿瘤中均出现高频率表达失活和异常高甲基化.将该基因转染入肺癌、肾癌等肿瘤细胞株中均可显著抑制癌细胞生长,逆转其恶性表型,提示该基因的失活可能在肺癌等多种肿瘤的发生发展中发挥重要作用.     

ARHI基因对人胰腺癌组织血管生成的影响

目的 探讨ARHI基因与胰腺癌组织血管密度和临床病理的相关性.方法 收集23例胰腺癌石蜡标本作为实验组,27例正常胰腺石蜡标本作为对照组.然后采用免疫组化方法检测两组标本ARHI、CD34蛋白表达情况;分析ARHI、CD34蛋白表达与胰腺癌的关系;分析ARHI与MVD的相关性.结果 对照组和实验组标本中ARHI阳性率分别为88.9%(24/27)、21.7%(5/23),实验组标本中ARHI基因表达下降,两组比较差异具有统计学意义(P<0.05);对照组MVD值显著低于实验组,差异具有统计学意义(P<0.05);ARHI表达与胰腺癌分化程度呈负相关(P<0.05),其中肿瘤分化程度越低,ARHI表达缺失概率越大;实验组标本中CD34阳性表达较高,多数具有管腔结构,且MVD值明显高于对照组标本,差异具有统计学意义(P<0.001);胰腺癌组织中,MVD与ARHI的表达无相关性(P>0.05);而所有统计病例中,ARHI阴性者比阳性者的MVD值高,且MVD与ARHI的表达呈负相关(P<0.001).结论 ARHI可能通过抑制胰腺癌微血管的生成进而抑制胰腺癌生长.     

p73基因与肿瘤

癌基因的激活和抑癌基因的失活是人类恶性肿瘤发生和发展的重要原因。p53基因是与人类肿瘤相关性最高的基因,近半数的恶性肿瘤与p53基因的突变有关,而p73基因是1997年发现的p53基因家族的一个成员,近年来对其结构、功能、分布和表达情况都有了深入的研究。我们就p73基因的研究现状以及其在人类肿瘤中的表达情况作一综述。p73基因定位于1p36.33,由13个外显子和12个内含子组成,表达相对分子质量为73×103的蛋白,在结构上与p53蛋白有同源性。p73过表达时能抑制细胞生长和诱导细胞凋亡,但是病毒原癌蛋白却与p73没有相互作用。C-Ab1激酶可以活化p73基因并参与凋亡反应。p73基因敲除的小鼠没有肿瘤易患现象发生,但却有严重的发育异常。p73基因在神经母细胞瘤中不常发生突变,在肺癌、食管癌、肝癌、卵巢癌、乳腺癌表达增高,在淋巴瘤和某些白血病表达降低。与p53基因有明显的不同,p73基因有自己独特的功能。对于野生型p73基因在多种人类肿瘤中过表达的一种解释是,p73基因的过表达可能是一种p53基因失活的代偿反应,虽然这种作用可能无法完全替代p53基因的作用。但是目前研究已经证实p73基因很少突变,而且在肿瘤组织中存在异常表达,增加其表达可能诱导肿瘤细胞的凋亡,并且在p53基因突变时起抑制肿瘤细胞增殖的作用。因此增强p73基因表达也许是恶性肿瘤治疗的一种新方法。     

p73基因与消化系统肿瘤

p73基因是肿瘤抑制基因p53家族的新成员,在编码蛋白的结构、功能等方面两者很相似,但在其它方面有非常显著的差异。许多研究发现p73基因异常与神经母细胞瘤、黑色素瘤、前列腺癌、肺癌有关。近年来还发现p73基因可能参与了一部分消化系统肿瘤的发生发展。本文综述了p73基因的研究现状及其与消化系统肿瘤的发生、发展、预后的关系及表达情况,从而认为,对p73基因的深入研究将有助于人们进一步认识肿瘤的本质,为肿瘤基因治疗提供新的靶点。     

p16基因与肺癌关系研究进展

肿瘤抑癌基因p16对于肺癌的早期诊断非常重要,启动子的异常甲基化、第二号外显子的纯合子缺失、基因点突变、蛋白及mRNA的缺失表达都与肺癌的发生关系密切,且与肺癌的基因治疗、药物靶向治疗有关.     

Effects of ARHI on breast cancer cell biological behavior regulated by microRNA-221

The aplysia ras homolog member I (ARHI) is a tumor suppressor gene and is downregulated in various cancers. The downregulation of ARHI was regulated by miR-221 in prostate cancer cell lines. However, it has not been reported whether ARHI is regulated by miR-221 in breast cancer. Here, we reported that the ARHI protein level was downregulated in breast cancer tissues and breast cancer cell lines. The overexpression of ARHI could inhibit cell proliferation and invasion and induce cell apoptosis. To address whether ARHI is regulated by miR-221 in breast cancer cell lines, the results in this study showed that a significant inverse correlation existed between ARHI and miR-221. MiR-221 displayed an upregulation in breast cancer tissues and breast cancer cell lines. The inhibition of miR-221 induced a significant upregulation of ARHI in MCF-7 cells. To prove a direct interaction between miR-221 and ARHI mRNA, ARHI 3′UTR, which includes the potential target site for miR-221, was cloned downstream of the luciferase reporter gene of the pMIR-REPORT vector to generate the pMIR-ARHI-3′UTR vector. The results confirmed a direct interaction of miR-221 with a target site on the 3′UTR of ARHI. In conclusion, ARHI is a tumor suppressor gene that is downregulated in breast cancer. The overexpression of ARHI could inhibit breast cancer cell proliferation and invasion and induce cell apoptosis. This study demonstrated for the first time that the downregulation of ARHI in breast cancer cells could be regulated by miR-221.     

MicroRNAs 221/222 and genistein-mediated regulation of ARHI tumor suppressor gene in prostate cancer

ARHI is an imprinted tumor suppressor gene and is downregulated in various malignancies. However, ARHI expression, function, and mechanisms of action in prostate cancer have not been reported. Here, we report that ARHI mRNA and protein levels were downregulated in prostate cancer tissues compared with adjacent normal tissues. Overexpression of ARHI inhibited cell proliferation, colony formation, invasion, and induced apoptosis. Further studies on a new mechanism of ARHI downregulation showed a significant inverse relationship between ARHI and miR-221 and 222, which were upregulated in prostate cancer cell lines. Transfection of miR-221 and 222 inhibitors into PC-3 cells caused a significant induction of ARHI expression. A direct interaction of miR-221 or 222 with a target site on the 3'UTR of ARHI was confirmed by a dual luciferase pMIR-REPORT assay. Finally, we also found that genistein upregulates ARHI by downregulating miR-221 and 222 in PC-3 cells. In conclusion, ARHI is a tumor suppressor gene downregulated in prostate cancer, and overexpression of ARHI can inhibit cell proliferation, colony formation, and invasion. This study demonstrates for the first time that prostate cancer cells have decreased level of ARHI which could be caused by direct targeting of 3'UTR of ARHI by miR221/222. Genistein, a potential nontoxic chemopreventive agent, restores expression of ARHI and may be an important dietary therapeutic agent for treating prostate cancer.     

The expression of ARHI in pT2a and pT2b stage gastric cancer and its clinical significance

ARHI is a novel tumor suppressor gene located on chromosome 1p31. Downregulation of ARHI expression has been detected in many types of cancer. However, the effects of ARHI in gastric cancer remain unclear. The aim of this study was to identify the relationship between ARHI expression and gastric cancer clinicopathological features. In this study, 81 pT2 stage gastric cancer specimens were subclassified by pT2a and pT2b stage. ARHI mRNA and protein levels were evaluated by real-time PCR and western blot analysis, respectively. Methylation plays an important role in suppressor gene silencing. We utilized methylation-specific PCR to identify the status of CpG islands in the ARHI gene. We used immunohistochemistry to determine the expression of the protein and analyzed clinicopathological features. The levels of ARHI mRNA in gastric cancer were lower compared to normal tissues (P<0.01). Similarly, the levels of ARHI protein in the cancer specimens were lower (P<0.05). DNA hypermethylation was identified in 79.1% of gastric cancer specimens without ARHI expression. Immunohistochemistry results were significantly correlated with the pT2 category (P<0.05). The cumulative survival rate of patients with ARHI expression was significantly higher compared to those without ARHI expression (P<0.05). ARHI as a suppressor is not only an important factor in the pathogenesis of gastric cancer, but also a potential factor for tumor aggravation. ARHI expression in gastric cancer can be employed to indicate favorable prognosis for the disease.     

Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway

ARHI, an imprinted putative tumor suppressor gene, encodes a M(r) 26,000 GTP-binding protein that is 60% homologous to ras and rap but has a dramatically different function. ARHI expression is down-regulated in a majority of breast and ovarian cancers. Using a dual adenovirus system, we have reexpressed ARHI in ovarian cancer and breast cancer cells that have lost ARHI expression. Reexpression of ARHI inhibited growth, decreased invasiveness, and induced apoptosis. At 5 days after infection with ARHI adenovirus, 30-45% of MDA-MB-231 breast cancer cells and 5-11% of SKOv3 ovarian cancer cells were apoptotic as judged by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay and by Annexin V staining with flow cytometric analysis. Although poly(ADP-ribose) polymerase could be detected immunohistochemically in the nuclei of apoptotic cells, no activation of the effector caspases (caspase 3, 6, 7, or 12) or the initiator caspases (caspase 8 or 9) could be detected in cell lysates using Western blotting. When gene expression was analyzed on a custom cDNA array that contained 2304 known genes, infection with ARHI adenovirus up-regulated 15 genes relative to control cells infected with LacZ adenovirus. The greatest degree of mRNA up-regulation was observed in a Homo sapiens calpain-like protease. On Western blot analysis, calpain protein was increased 2-3-fold at 3-5 days after infection with ARHI adenovirus. No increase in calpain protein was observed after LacZ adenovirus infection. Calpain cleavage could be detected after ARHI reexpression, and inhibitors of calpain, but not inhibitors of caspase, partially prevented ARHI-induced apoptosis. Consequently, reexpression of ARHI in breast and ovarian cancer cells appears to induce apoptosis through a caspase-independent, calpain-dependent mechanism.     

ARHI is the center of allelic deletion on chromosome 1p31 in ovarian and breast cancers

In our previous work, we had characterized ARHI as an imprinted putative tumor-suppressor gene in ovarian and breast cancers. ARHI is expressed in primary breast and ovarian cell lines but largely absent from the corresponding malignant tumors. Moreover, the non-imprinted functional allele is typically deleted in malignant cells. Since ARHI had been mapped to 1p31, a common deletion site in breast and ovarian cancer and male germ-cell tumors, in this study, we set out to define precisely the physical location of ARHI at 1p31 and to determine if this location lies within the smallest common region of deletion in breast and ovarian cancers. To this end, we first carried out radiation hybrid mapping of ARHI and surrounding markers, followed by a high-resolution study of loss of heterozygosity at 1p31 in 49 ovarian and breast cancers. Combining a radiation hybrid map and a physical map of the region encompassing ARHI, 3 discrete regions of minimal deletion were found at 1p31 in breast and ovarian cancers. ARHI is the most common deletion region at 1p31. Two other less common regions of deletion were found centromeric to this gene. One of them centered on D1S207 and the other one included and was proximal to D1S488. We also confirmed the preferential loss of non-imprinted functional allele in 7 of 9 tumor specimens. These data support the possibility that ARHI is a tumor-suppressor gene and suggest that additional tumor-suppressor genes may lie proximal to ARHI at 1p31. The data obtained from our study should aid in the identification and characterization of genes in this novel imprinted region.     

Expression of the tumor suppressor gene ARHI in epithelial ovarian cancer is associated with increased expression of p21WAF1/CIP1 and prolonged progression-free survival.

PURPOSE: ARHI, an imprinted putative tumor suppressor gene, is expressed in normal ovarian epithelial cells, but its expression is down-regulated or lost in most ovarian cancer cell lines. Reexpression of ARHI in cancer cells induces p21(WAF1/CIP1), down-regulates cyclin D1 promoter activity and inhibits growth in cell culture and in heterografts. To determine the relevance of these observations to clinical cancer, we have now measured ARHI expression in normal, benign and malignant ovarian tissues using immunohistochemistry and in situ hybridization. EXPERIMENTAL DESIGN: Paraffin embedded tissues from 7 normal ovaries, 22 cystadenomas and 42 borderline lesions were analyzed using standard immunoperoxidase and in situ hybridization techniques to assess ARHI expression. In addition, immunohistochemistry against ARHI was performed on a tissue microarray containing 441 consecutive cases of ovarian carcinoma. RESULTS: Strong ARHI expression was found in normal ovarian surface epithelial cells, cysts and follicles using immunohistochemistry and in situ hybridization. Reduced ARHI expression was observed in tumors of low malignant potential as well as in invasive cancers. ARHI expression was down-regulated in 63% of invasive ovarian cancer specimens and could not be detected in 47%. When immunohistochemistry and in situ hybridization were compared, ARHI protein expression could be down-regulated in the presence of ARHI mRNA. ARHI expression was correlated with expression of p21(WAF1/CIP1) (P = 0.0074) but not with cyclin D1 and associated with prolonged disease free survival (P = 0.001). On multivariate analysis, ARHI expression, grade and stage were independent prognostic factors. ARHI expression did not correlate with overall survival. CONCLUSIONS: Persistence of ARHI expression in epithelial ovarian cancers correlated with prolonged disease free survival and expression of the cyclin dependent kinase inhibitor p21(WAF1/CIP1).     

ARHI/NOEY2 inactivation may be important in breast tumor pathogenesis

Allelic loss frequently occurs on the short arm of chromosome 1 in human breast carcinoma, suggesting that the ARHI/NOEY2 gene, an imprinted putative tumor suppressor gene, is involved in the pathogenesis of the tumor entity. To clarify the clinical importance of ARHI/NOEY2 mRNA in breast cancer, we studied whether ARHI/NOEY2 inactivation might contribute to tumors arising in the breast. An ARHI/NOEY2 message was detected by real-time PCR analysis in all noncancerous breast tissues, but was not detected in 2 of 26 breast cancer tissue samples. In 10 of 26 breast cancer tissue samples ARHI/NOEY2 mRNA was substantially reduced. ARHI/NOEY2 expression was lost or markedly reduced in 12 of 26 (46.15%) breast cancer tissue samples. In summary, we conclude that decreased ARHI/NOEY2 mRNA expression may play an important role in the pathogenesis of breast cancer.